Compendium of Plant Genomes

Schuyler S. Korban Editor

The Pear
Genome
Compendium of Plant Genomes

Series Editor
Chittaranjan Kole, Raja Ramanna Fellow, Government of India,
ICAR-National Research Center on Plant Biotechnology, Pusa,
New Delhi, India
Whole-genome sequencing is at the cutting edge of life sciences in the new
millennium. Since the first genome sequencing of the model plant Arabidopsis
thaliana in 2000, whole genomes of about 100 plant species have been
sequenced and genome sequences of several other plants are in the pipeline.
Research publications on these genome initiatives are scattered on dedicated
web sites and in journals with all too brief descriptions. The individual
volumes elucidate the background history of the national and international
genome initiatives; public and private partners involved; strategies and
genomic resources and tools utilized; enumeration on the sequences and their
assembly; repetitive sequences; gene annotation and genome duplication. In
addition, synteny with other sequences, comparison of gene families and most
importantly potential of the genome sequence information for gene pool
characterization and genetic improvement of crop plants are described.
Interested in editing a volume on a crop or model plant? Please contact
Dr. Kole, Series Editor, at [email protected]

More information about this series at http://www.springer.com/series/11805

Schuyler S. Korban
Editor

The Pear Genome

123
Editor
Schuyler S. Korban
Department of Natural Resources
and Environmental Sciences
College of Agricultural, Consumer
and Environmental Sciences, University
of Illinois at Urbana-Champaign
Urbana, IL, USA

ISSN 2199-4781 ISSN 2199-479X (electronic)

Compendium of Plant Genomes
ISBN 978-3-030-11047-5 ISBN 978-3-030-11048-2 (eBook)
https://doi.org/10.1007/978-3-030-11048-2

© Springer Nature Switzerland AG 2019

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This book series is dedicated to my wife Phullara,
and our children Sourav, and Devleena
Chittaranjan Kole
Preface to the Series

Genome sequencing has emerged as the leading discipline in the plant sci-
ences coinciding with the start of the new century. For much of the twentieth
century, plant geneticists were only successful in delineating putative chro-
mosomal location, function, and changes in genes indirectly through the use
of a number of “markers” physically linked to them. These included visible or
morphological, cytological, protein, and molecular or DNA markers. Among
them, the first DNA marker, the RFLPs, introduced a revolutionary change in
plant genetics and breeding in the mid-1980s, mainly because of their infinite
number and thus potential to cover maximum chromosomal regions, pheno-
typic neutrality, absence of epistasis, and codominant nature. An array of
other hybridization-based markers, PCR-based markers, and markers based
on both facilitated construction of genetic linkage maps, mapping of genes
controlling simply inherited traits, and even gene clusters (QTLs) controlling
polygenic traits in a large number of model and crop plants. During this
period, a number of new mapping populations beyond F2 were utilized and a
number of computer programs were developed for map construction, mapping
of genes, and for mapping of polygenic clusters or QTLs. Molecular markers
were also used in the studies of evolution and phylogenetic relationship,
genetic diversity, DNA fingerprinting, and map-based cloning. Markers
tightly linked to the genes were used in crop improvement employing the
so-called marker-assisted selection. These strategies of molecular genetic
mapping and molecular breeding made a spectacular impact during the last
one and a half decades of the twentieth century. But still, they remained
“indirect” approaches for elucidation and utilization of plant genomes since
much of the chromosomes remained unknown and the complete chemical
depiction of them was yet to be unraveled.
Physical mapping of genomes was the obvious consequence that facili-
tated the development of the “genomic resources” including BAC and YAC
libraries to develop physical maps in some plant genomes. Subsequently,
integrated genetic–physical maps were also developed in many plants. This
led to the concept of structural genomics. Later on, the emphasis was laid on
EST and transcriptome analysis to decipher the function of the active gene
sequences leading to another concept defined as functional genomics. The
advent of techniques of bacteriophage gene and DNA sequencing in the
1970s was extended to facilitate sequencing of these genomic resources in
the last decade of the twentieth century.

vii
viii Preface to the Series

As expected, sequencing of chromosomal regions would have led to too

much data to store, characterize, and utilize with the-then available computer
software could handle. But the development of information technology made
the life of biologists easier by leading to a swift and sweet marriage of
biology and informatics, and a new subject was born—bioinformatics.
Thus, the evolution of the concepts, strategies, and tools of sequencing
and bioinformatics reinforced the subject of genomics—structural and
functional. Today, genome sequencing has travelled much beyond biology
and involves biophysics, biochemistry, and bioinformatics!
Thanks to the efforts of both public and private agencies, genome
sequencing strategies are evolving very fast, leading to cheaper, quicker, and
automated techniques right from clone-by-clone and whole-genome shotgun
approaches to a succession of second-generation sequencing methods. The
development of software of different generations facilitated this genome
sequencing. At the same time, newer concepts and strategies were emerging
to handle sequencing of the complex genomes, particularly the polyploids.
It became a reality to chemically—and so directly—define plant genomes,
popularly called whole-genome sequencing or simply genome sequencing.
The history of plant genome sequencing will always cite the sequencing
of the genome of the model plant Arabidopsis thaliana in 2000 that was
followed by sequencing the genome of the crop and model plant rice in 2002.
Since then, the number of sequenced genomes of higher plants has been
increasing exponentially, mainly due to the development of cheaper and
quicker genomic techniques and, most importantly, the development of
collaborative platforms such as national and international consortia involving
partners from public and/or private agencies.
As I write this preface for the first volume of the new series “Compendium
of Plant Genomes,” a net search tells me that complete or nearly complete
whole-genome sequencing of 45 crop plants, 8 crop and model plants, 8
model plants, 15 crop progenitors and relatives, and 3 basal plants is
accomplished, the majority of which are in the public domain. This means
that we nowadays know many of our model and crop plants chemically, i.e.,
directly, and we may depict them and utilize them precisely better than ever.
Genome sequencing has covered all groups of crop plants. Hence, infor-
mation on the precise depiction of plant genomes and the scope of their
utilization are growing rapidly every day. However, the information is
scattered in research articles and review papers in journals and dedicated
Web pages of the consortia and databases. There is no compilation of plant
genomes and the opportunity of using the information in sequence-assisted
breeding or further genomic studies. This is the underlying rationale for
starting this book series, with each volume dedicated to a particular plant.
Plant genome science has emerged as an important subject in academia, and
the present compendium of plant genomes will be highly useful to both stu-
dents and teaching faculties. Most importantly, research scientists involved in
genomics research will have access to systematic deliberations on the plant
genomes of their interest. Elucidation of plant genomes is of interest not only
for the geneticists and breeders, but also for practitioners of an array of plant
science disciplines, such as taxonomy, evolution, cytology, physiology,
Preface to the Series ix

pathology, entomology, nematology, crop production, biochemistry, and

obviously bioinformatics. It must be mentioned that information regarding
each plant genome is ever-growing. The contents of the volumes of this
compendium are, therefore, focusing on the basic aspects of the genomes and
their utility. They include information on the academic and/or economic
importance of the plants, description of their genomes from a molecular genetic
and cytogenetic point of view, and the genomic resources developed. Detailed
deliberations focus on the background history of the national and international
genome initiatives, public and private partners involved, strategies and
genomic resources and tools utilized, enumeration on the sequences and their
assembly, repetitive sequences, gene annotation, and genome duplication. In
addition, synteny with other sequences, comparison of gene families, and,
most importantly, the potential of the genome sequence information for gene
pool characterization through genotyping by sequencing (GBS) and genetic
improvement of crop plants have been described. As expected, there is a lot of
variation of these topics in the volumes based on the information available on
the crop, model, or reference plants.
I must confess that as the series editor, it has been a daunting task for me
to work on such a huge and broad knowledge base that spans so many
diverse plant species. However, pioneering scientists with a lifetime expe-
rience and expertise on the particular crops did excellent jobs editing the
respective volumes. I myself have been a small science worker on plant
genomes since the mid-1980s and that provided me the opportunity to per-
sonally know several stalwarts of plant genomics from all over the globe.
Most, if not all, of the volume editors are my longtime friends and col-
leagues. It has been highly comfortable and enriching for me to work with
them on this book series. To be honest, while working on this series, I have
been and will remain a student first, a science worker second, and a series
editor last. And I must express my gratitude to the volume editors and the
chapter authors for providing me the opportunity to work with them on this
compendium.
I also wish to mention here my thanks and gratitude to the Springer staff,
Dr. Christina Eckey and Dr. Jutta Lindenborn in particular, for all their
constant and cordial support right from the inception of the idea.
I always had to set aside additional hours to edit books beside my pro-
fessional and personal commitments—hours I could and should have given
to my wife, Phullara, and our kids, Sourav, and Devleena. I must mention
that they not only allowed me the freedom to take away those hours from
them but also offered their support in the editing job itself. I am really not
sure whether my dedication of this compendium to them will suffice to do
justice to their sacrifices for the interest of science and the science
community.

Kalyani, India Chittaranjan Kole

Preface

The pear, belonging to the Pyrus genus and subtribe Malinae of the
Amygdaloideae subfamily within Rosaceae, is the third most important
temperate fruit tree crop, with an annual worldwide production of
*18 million tons (2014 FAOSTAT). The genus Pyrus includes at least 22
known species with over 5000 accessions maintained worldwide. These
accessions display wide variations in morphological and physiological traits
along with broad adaptation to wide agroecological environments. It is
reported that the ancient Pyrus likely arose during the Tertiary period,
between 55 and 65 million years ago (Mya), in the mountainous regions of
southwestern China. From there, it has been dispersed across mountainous
ranges, both toward east and west regions, resulting in the evolution of two
distinct major groups, commonly referred to as European and Asian pears.
Asian pears have been cultivated for about 3300 years ago, while European
pears have been cultivated for more than 2000 years.
While the cultivated European pears predominantly belong to
P. communis, the cultivated Asian pears belong to several major species,
including P. pyrifolia, P.  bretschneideri, P.  sinkiangensis, and
P. ussuriensis. Fruit of European pears is characterized by their typical
pyriform shape (bulbous bottoms and tapering tops), although there are some
with oblate or globose shapes, with soft and fine-grained flesh, few stone or
lignified cells, along with a strong aroma and flavor. Fruit of Asian pears is
predominantly round in shape, although there are some with pyriform shapes,
firm, with a crispy flesh, high sugar, and low acid contents, along with faint
aroma and mild flavor.
The pear tree is cross-pollinated, self-incompatible, and with a long
juvenility period of 5–7 years. However, there are little barriers to inter-
specific hybridization in pear despite its wide geographic distribution.
Although genetic studies are limited, it is well documented that there is a
wide genetic variability in pear. Most commercially grown cultivars have
been selected as chance seedlings and then subsequently maintained through
vegetative propagation, although there are few cultivars that have been
developed from breeding programs via sexual hybridization. There are few
releases of new pear cultivars that have been derived from various breeding
programs from around the world. As with other tree fruit breeding programs,
classical pear breeding is a long-term and expensive effort. Thus, recent
advances in pear genomics are paving the way for a new and promising path
for pear genetic improvement initiatives and efforts.
xi
xii Preface

In recent years, modern genetic and genomic tools have resulted in the
development of a wide variety of valuable resources, including molecular
markers, genetic mapping, genetic transformation, structural and functional
genomics resources, genome sequencing, and genome-wide association
studies, as well as comparative genomic studies. These tools and resources
offer unparalleled opportunities to pursue genetic improvement efforts to
combine fruit quality, high productivity, precocious fruit-bearing, long
postharvest storage life, along with elevated levels of resistance to various
major diseases and insect pests of pear. Furthermore, these new genetic tools
and genomic resources provide unprecedented opportunities to explore and
understand genetic variation, evolution, and domestication of pear, as well as
to better establish population-level relationships among different pear spe-
cies. In the past few years, completion of whole-genome assemblies of
“Dangshansuli”, an Asian pear, and “Bartlett”, a European pear, has enabled
new discoveries in pear, including those of genomic structure, chromosome
evolution, and patterns of genetic variation. All this wealth of new resources
will have a major impact on our knowledge of the pear genome and its
expanding resources. In turn, these resources and knowledge will have sig-
nificant impacts on efforts for genetic improvement of pears.
The Pear Genome book will cover our current knowledge of botanical and
taxonomic classifications; origin, distribution, and early documented distri-
bution of pear; germplasm resources; genetic studies and genetic improve-
ment efforts; genetic linkage maps; molecular genetic and QTL analysis,
along with genomic analysis; whole-genome sequencing strategies and out-
comes; repetitive and regulatory sequences; self-incompatibility; stone cell
development; vegetative budbreak analysis; fire blight genetics and geno-
mics; functional genomic analysis; whole-genome duplication in pear and its
comparisons to apple; and potential opportunities and challenges for future
genetic improvement efforts of pears.
All 16 chapters included in this volume will provide a wealth of infor-
mation and comprehensive overview of the status of early and ongoing
efforts to discern the genetics, breeding, and genomics of the pear. This book
will offer ideas, opportunities, and pathways that will support future research
and discovery efforts that will not only contribute to our expanded knowl-
edge of various traits of this important fruit crop, as well as our under-
standing of the pear genome as a whole, but these will also contribute to
overall advances in genetic enhancement efforts of the pear.

Urbana, USA Schuyler S. Korban

Contents

1 Botany and Taxonomy of Pear . . . . . . . . . . . . . . . . . . . . . . . . . 1

Muriel Quinet and Jean-Pierre Wesel
2 Pear Germplasm Needs and Conservation. . . . . . . . . . . . . . . . 35
Joseph Postman
3 Genetic Diversity and Domestication History in Pyrus . . . . . . 51
Gayle M. Volk and Amandine Cornille
4 Genetics and Breeding of Pear . . . . . . . . . . . . . . . . . . . . . . . . . 63
Lester Brewer and Richard Volz
5 Linkage Mapping in Pear . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Jun Wu and Mengfan Qin
6 Molecular Mapping of Major Genes and QTLs in Pear. . . . . 113
Paolo De Franceschi and Luca Dondini
7 The Genome of Pear . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Jun Wu, Shaoling Zhang and Xiaolong Li
8 Repetitive Sequences in Pear . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Shuang Jiang and Yuanwen Teng
9 Regulatory Sequences of Pear. . . . . . . . . . . . . . . . . . . . . . . . . . 153
Yongping Cai, Muhammad Abdullah and Xi Cheng
10 Self-incompatibility in Pear . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Shaoling Zhang and Chao Gu
11 Stone Cell Development in Pear . . . . . . . . . . . . . . . . . . . . . . . . 201
Xi Cheng, Yongping Cai and Jinyun Zhang
12 Genetic and Genomic Analyses of Vegetative Budbreak
in Response to Chilling Units in European Pear
(Pyrus Communis L.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Gilad Gabay and Moshe A. Flaishman
13 Genetics, Genomics, and Breeding for Fire Blight
Resistance in Pear . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
Richard L. Bell

xiii
xiv Contents

14 Functional Genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

Songling Bai and Yuanwen Teng
15 Whole-Genome Duplications in Pear and Apple . . . . . . . . . . . 279
Hao Li, Chien-Hsun Huang and Hong Ma
16 Future Breeding Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Kamila Łucja Bokszczanin
Abbreviations

4CL 4-coumarate: coenzyme A ligase

AFLP(s) Amplified fragment length polymorphism(s)
CAD Cinnamyl alcohol dehydrogenase
CCR Cinnamoyl-CoA reductase
CR(s) Chilling requirement(s)
CRISPR/cas9 Clusters of regularly interspaced short palindromic
repeats/cas9 associated protein
CU(s) Chilling unit(s)
DIR Dirigent
GE Genotype  environment
GE Genetic engineering
GMO Genetically modified organism
GRF Growth-regulating factor
GS Genomic selection
GSI Gametophytic self-incompatibility
GWAS Genome-wide association study(ies)
HCT Hydroxycinnamoyl-CoA: shikimate/quinate
hydroxycinnamoyltransferase
HSF(s) Heat shock transcription factor(s)
IRAP Inter-retrotransposon amplified polymorphism
LD Linkage disequilibrium
LG(s) Linkage group(s)
LM Linkage mapping
MAB Marker-assisted breeding
MAS Marker-assisted selection
NGS New gene sequencing
NPBT New plant breeding techniques
OA Organic agriculture
OMT O-methyltransferase
POD Peroxidase
QTL(s) Quantitative trait locus/loci
RAPD(s) Random amplified fragment length polymorphism(s)
RBIP Retrotransposon-based insertion polymorphism
RNAi RNA interference
SBP SQUAMOSA promoter binding protein

xv
xvi Abbreviations

SI Self-incompatibility
siRNAs Small interfering RNAs
SNP(s) Single nucleotide polymorphism(s)
SSAP Sequence-specific amplification polymorphism
SSN Sequence-specific nuclease technology
SSR(s) Simple sequence repeat(s)
TE(s) Transposon(s)/able element(s)
TF(s) Transcription(al) factor(s)
VB Vegetative budbreak
VIGS Virus-induced gene silencing
WGD(s) Whole-genome duplication(s)
ZHD Zinc finger homeodomain
 Botany and Taxonomy of Pear
1
Muriel Quinet and Jean-Pierre Wesel

Abstract Oligocene epoch, about 33.35–25.23 Mya. It is

Pear belongs to the Rosaceae family as most of a genus of mainly deciduous trees and shrubs
the cultivated fruit trees. It is the second fruit spread throughout temperate Eurasia, reaching
tree crop in terms of production after apple. the Atlas Mountains in North Africa and
Its production has increased these last decades extending to Japan and South China. Pyrus
to reach a world production of more than species produce generally simple leaves alter-
27 megatons for almost 1,600,000 ha. Pears nately arranged. Leaves are glossy green on
have been cultivated in Europe and in Asia for some species, densely silvery hairy in some
more than 5000 years. Of all known and others. Pyrus flowers are white, borne in
reported pear species and interspecific hybrids, corymbs on short spurs or lateral branchlets
five are mainly cultivated. These include the and are composed of five sepals, five petals,
European pear, Pyrus communis, and the Asian numerous stamens, and usually a five-locular
pears P. pyrifolia, P.  bretschneideri, ovary with free styles. The Pyrus fruit is a
P. ussuriensis, and P. sinkiangensis. Fruits of pseudo-fruit composed of the receptacle or the
European pears are elongated and have a calyx tube, greatly dilated, enclosing the true
full-bodied texture, while those of Asian pears fruit, and consisting of five cartilaginous carpels,
are round and have a sandy texture. The Pyrus known as the core. Morphological characters of
genus belongs to the Amygdaloideae subfamily the leaf, fruit, and calyx are commonly used to
and the Malinae tribe and consists of about 75– differentiate among Pyrus species. There are
80 species and interspecific hybrid species. thousands of pear cultivars over the world with
As several hybridizations are observed among wide diversity for fruit shape, taste, and texture.
Pyrus species, this renders the distinction In this chapter, we have focused on the
among some pear species rather difficult. The description of cultivated Pyrus species and on
origin of the Pyrus genus dates back to the some of the main cultivated cultivars.

M. Quinet (&) 1.1 Introduction

Groupe de Recherche en Physiologie Végétale, Earth
and Life Institute, Université Catholique de Louvain,
Louvain-la-Neuve, Belgium
Two of the main pear species that are cultivated
e-mail: [email protected] include Pyrus communis L. and P. pyrifolia
J.-P. Wesel
(Burm.f.) Nakai (Hedrick et al. 1921).
Flore et Pomone asbl, Jodoigne, Belgium P. communis is native to central and Eastern

© Springer Nature Switzerland AG 2019 1

S. S. Korban (ed.), The Pear Genome, Compendium of Plant Genomes,
https://doi.org/10.1007/978-3-030-11048-2_1
2 M. Quinet and J.-P. Wesel

Europe and to southwest Asia, and it is known as Africa for 3%, and Oceania for less than 1%
European pear or common pear. It is one of the (FAO 2018). The pear tree is the second Rosa-
most important fruits of temperate regions, and it ceous fruit tree crop grown in terms of produc-
is the pear of common cultivation in Europe, tion and the fifth in terms of harvested area
America, Oceania, and Africa (Hedrick et al. (Fig. 1.2). Overall, the main cultivated fruit tree
1921; Bassil and Postman 2010). The cultivation is apple, and pear production is about 30% of
of P. communis makes up about one-third of the apple production. Pear and apple yields average
total pear production (Chagné et al. 2014). While 168,000 hg/ha over the last years, and are the
P. pyrifolia is native to East Asia, and it is mainly best yields among Rosaceous fruit trees (FAO
cultivated in Asia, it is currently also cultivated 2018).
in America, Oceania, and Europe (Bretaudeau
and Fauré 1991; White 2002; Faoro and Orth
2014). Other Pyrus species are also commonly 1.2 Origin and Cultivation of Pear
grown in Asia, including P.  bretschneideri,
P. ussuriensis, and P. sinkiangensis (Wu et al. 1.2.1 Origin of Pear
2013). P. pyrifolia is known by many names
including Asian pear, Chinese pear, Korean pear, The exact origin of the cultivated European pear
Japanese pear, Taiwanese pear, nashi, and sand tree is not known (Hedrick et al. 1921).
pear (Hedrick et al. 1921; Bailey and Bailey According to Debuigne and Couplan (2006), it
1976; Petri and Herter 2002; Lee et al. 2012). may result from the hybridization of several wild
Some of these vernacular names include pear species from Europe and Minor Asia,
other pear species, as some cultivars of including P. communis subsp. pyraster (L.) Ehrh.
P.  bretschneideri and P. ussuriensis are also The wild pear tree of P. communis subsp. pyra-
called nashi pears, or P.  bretschneideri is also ster has likely originated from the mountains of
known as Chinese white pear (Chagné et al. Minor Asia or from Europe (Opoix 1896; Pesson
2014). For the sake of clarity, all these will be and Louveaux 1984; Paris 1996). It could be
collectively grouped and referred to as Asian deemed as a relic of warm oak forests and would
pears. While fruits of European pears are elon- be indigenous of the medio-European flora (Aas
gated and have full-bodied textures, fruits of 1999). It most probably migrated to central and
Asian pears are round and have sandy textures Western Europe 7500 to 4500 years ago during
(Silva et al. 2014). All these Pyrus species are the warm post-glacial period (Aas 1999). The
botanically referred to as pome fruits and belong natural range of the species has not been pre-
to the Rosaceae family, as many other fruit tree cisely identified as it is difficult to distinguish
species including other pome fruits, apple and wild from cultivated P. communis (Aas 1999).
quince, and stone fruits, such as cherry, almond, Currently, the species could be found in large
peach, apricot, plum, and nectarine. areas of temperate regions of Europe, Asia, and
The first landmarks of pear as a cultivated tree America at altitudes of up to 800 m (Pesson and
in Europe were found in ancient Greece Louveaux 1984).
(Hedrick et al. 1921). Pear is currently cultivated In contrast, domestication of Asian pears,
worldwide, and its production has increased over including their centre(s) of origin along with time
the last decades to reach a world production of periods, is clearly documented (Silva et al. 2014).
more than 27 megatons for almost 1,600,000 ha As reported in written Chinese (Shijing) and in
in 2016 (Fig. 1.1a, b) (FAO 2018). China is the other books, the major Asian species, cultivated
largest producer of pear fruits worldwide, pro- for at least 1500 years, are P. pyrifolia and
ducing about 20 times more pears than all other P. ussuriensis (Silva et al. 2014). In Japan, pear
main producers (Fig. 1.1c) (FAO 2018). In seeds dating back to the first century ACN have
2016, Asia contributed for 79% of pear pro- been found during excavations of the Toro Ruins
duction, Europe for 10%, America for 7%, in the Shizuoka prefecture (Saito 2016).
1 Botany and Taxonomy of Pear 3

Fig. 1.1 Evolution of 30

(a)

production (megatons)
worldwide pear cultivation.
a Pear production and b pear 25
harvested areas between 1961
and 2016. c Main countries 20
producing pears in 2016.
Based on FAOSTAT database 15
(FAO 2018)
10

0
2000
harvested area (ha, x 10³)

(b)
1800
1600
1400
1200
1000
800
600
400
1970 1980 1990 2000 2010

(c)
production (megatons)

20,0

10,0
1,0

0,5

0,0
)
nt )

Al rea
th In a

rt a
ut urk y

a ra l
a

No Uk eria
er dia

ut I n
Ko n
Af y

lg n
m
Ja le
Sp ds

K ne

ai ce
A

in F uga

an
ge nd

So T tal
h e
ric

Po ore
in

pa
h ra
Be ai
US

i
iu
Ch

(T n
n

i
rth ra

w
Ar nla

la
I

g
ai
(m

Ne

So
a

Ch
in
Ch

1.2.2 History of Pear Cultivation Bretaudeau and Fauré 1991). By this time, pear
has also been cultivated in both ancient Egypt
In comparison to other fruit tree species, pear and ancient Rome; however, its cultivation in
cultivation has occurred rather late, and this is China would have to go back to 4000 ACN
mainly due to the small fruit size of primitive (Bretaudeau and Fauré 1991).
pears (De Vilmorin and Clebant 1996). Pear In Homer’s Odyssey is the first mention of
domestication has taken place independently in pear cultivation in Greek literature (Royer 1853;
the Far East (China) and in the Caucasus region Hedrick et al. 1921); however, the first definitive
(Ferradini et al. 2017). Pear has been cultivated records of pear cultivation are found in the
in ancient Greece under the name of ‘Achras’ writings of Theophrastus in 370–286 ACN
around 2800 ACN (Hedrick et al. 1921; (Leroy 1867; Hedrick et al. 1921). Theophrastus
4 M. Quinet and J.-P. Wesel

Fig. 1.2 Rosaceous fruit tree (a) (b)

production (a) and harvested
area (b) in 2016. Based on Fruit, stone nes
FAOSTAT database (FAO Fruit, pome nes
2018) Quinces
Plums and sloes
Pears
Peaches and nectarines
Cherries, sour
Cherries
Apricots
Apples
Almonds

0 20 40 60 80 100 0 10 20 30 40 50 60
production (megatons) harvested area (ha x 10 5)

distinguishes between wild and cultivated pears, In Europe, there is no mention of new pear
and he makes reference to four pear cultivars, cultivars during the early Middle Ages, but in the
including ‘Myrrha’, ‘Nardinon’, ‘Onychinon’, eleventh century, Charlemagne has recom-
and ‘Talentiaion’ (Leroy 1867; Hedrick et al. mended planting fruit trees, including pear trees,
1921). He writes about the propagation of pears in Capitulare de Villis (Leroy 1867). Therefore,
from seeds, roots, and cuttings, as well as rec- the credit for establishing the first notable land-
ognizes the necessity for cross-pollination though mark in the history of the pear in France is due to
he does not offer reasons for this practice Charlemagne (Hedrick et al. 1921). In fact, he
(Hedrick et al. 1921). In 178 ACN in Italy, Cato has commanded his orchardists to plant pears of
wrote the first book, written in Latin, on agri- distinct kinds for distinct purposes and has cited
culture, and described six pear cultivars (Hedrick the following three cultivars: ‘Dulciores’ for
et al. 1921). Cato describes almost every method fresh fruit, ‘Cocciore’ for cooking, and ‘Ser-
of propagating, grafting, caring for, and keeping otina’, a late maturing variety (Leroy 1867).
fruits known to twentieth-century fruit growers Following Charlemagne, there are no records on
(Hedrick et al. 1921). Following two centuries, agricultural activities for the next five centuries
Pliny described 41 pear cultivars in Historia (Hedrick et al. 1921). Undoubtedly, fruit tree
naturalis (Leroy 1867). From Pliny, we know farming must have been preserved in abbeys;
that the Romans valued pears for medicinal however, there are no records of names of the
purposes, as well as for food (Hedrick et al. pear cultivars cultivated in Western Europe dur-
1921). Subsequently and for a period of ing this period until the end of the fourteenth
1500 years, there are a few new facts that have century (Leroy 1867).
been offered regarding the evolution of the pear During the fifteenth century, the printing press
(Hedrick et al. 1921). Many Roman writers was by then developed, and books about horti-
mentioned pear, but they have all copied culture were written and printed (Leroy 1867).
Theophrastus, Cato, and Pliny (Hedrick et al. The Seminarium of Charles Estienne, printed in
1921). In Japan, the first evidence of pear culti- 1540, offered brief descriptions of 16 pear culti-
vation is found in the Chronicles of Japan (720 vars that are still known to this day (Leroy 1867).
ACN), which mention that cultivation of fruits From Le Théâtre d’Agriculture, written by De
and nuts has been promoted during the Jito Serres and published in 1608, we know that
Tenno era (686–696 ACN) to fight famine (Saito many pears of diverse shapes, colours, flavours,
2016). and perfumes existed in the year 1600 in France
1 Botany and Taxonomy of Pear 5

(Hedrick et al. 1921). Enthusiasm for pears and ‘Délice du Panisel’ (1760–62). ‘Beurré
rapidly increased due to the interest of a French d’Harpendont’ could still be found in tree nurs-
royal prosecutor, Le Lectier (Leroy 1867). Le eries worldwide, although it is now known as
Lectier collected all available fruits of his time ‘Glou Morceau’ in Anglo-Saxon countries and as
and in his country (Hedrick et al. 1921). In ‘Beurré d’Arenberg’ in France. Jean-Baptiste
Catalogue des arbres cultivés published in 1628, Van Mons has followed Hardenpont’s lead by
he classified 260 pear cultivars based on their developing about 500 new pear cultivars among
maturation. The French King Louis XIV (1638– thousands found in Belgium between 1758 and
1715) promoted pear cultivation, and during his 1900. Among these, ‘Beurré d’Anjou’ (syn. ‘Nec
reign, new cultivars were developed (Leroy plus Meuris’) has been exported to America
1867). Hitherto, the development of new culti- where it is still cultivated. It is important to point
vars was done through picking and transplanta- out that the designation of ‘Anjou’ or ‘d’Anjou’
tion of trees encountered in nature or in has been erroneously used for this variety when
cultivated gardens. Although it has been a com- first introduced to both America and England.
mon practice since ancient Rome, cultivar Nevertheless, almost 40 pear cultivars developed
selection of P. communis was mainly developed by Van Mons have remained under cultivation at
during the eighteenth century in Europe (Pesson the beginning of the twentieth century (Hedrick
and Louveaux 1984). In Japan, the concept of et al. 1921). In fact, it is Van Mons’ work that
cultivars and cultural techniques were developed has promoted fruit-growing in Europe and
during the middle of the Edo era (1603–1867). America, and pomologists are in general agree-
‘Shokokusanbutsuchou’ was the first recorded ment that until his time, no man has exerted such
Japanese pear cultivar in 1735, and it was men- profound influence on the field of pomology
tioned along with over 100 pear cultivars (Saito (Hedrick et al. 1921). Again, it is Belgian
2016). breeders from Pomone tournaisienne who have
During the eighteenth century in Europe, developed 160 pear cultivars, including ‘Beurré
knowledge and understanding of plant sexuality de Naghin’ (Wesel 1996). In the Belgian city of
have prompted the pursuit of plant breeding Mechelen, Pierre Joseph Esperen developed 70
(Leroy 1867). Growers have made crosses and cultivars, such as ‘Bergamotte Esperen’, while in
sowed seeds in order to develop new cultivars another Belgian city Jodoigne, 13 breeders
(Table 1.1) with improved pear fruit flavour, developed about 200 new pear cultivars (Wesel
texture, size, and colour (Hedrick et al. 1921). 1996). Among the latter group of cultivars, and
Most of these new cultivars have been developed of particular note, are ‘Triomphe de Jodoigne’,
in Belgium, and several of these cultivars are developed by the brothers Bouvier, ‘Alexan-
cultivated to this day (Leroy 1867). drina’, developed by Alexandre Bivort, and
Pear improvement efforts in Belgium within a ‘Madame Grégoire’, developed by Xavier Gré-
single century surpass all other previous efforts goire (Wesel 1996).
(Hedrick et al. 1921). Belgian pear growers and As new cultivars have been developed in
well-suited soil and climate conditions must be Belgium, similar efforts have been undertaken in
given credit for the development of the modern France, leading to such present-day cultivars as
pear (Hedrick et al. 1921). The first and most ‘Beurré-Hardy’, ‘Bonne Louise d’Avranches’,
famous Belgian to sow pear seeds in order to ‘Doyenné du Comice’, and ‘Triomphe de
obtain new cultivars was Abbot Nicolas Har- Vienne’, in the UK, resulting in ‘William’s (Bon
denpont (1705–1774), and a dozen or more new Chrétien)’, ‘William’s Duchess’, and ‘Confer-
pears have been credited to him (Hedrick et al. ence’, and in the USA, notably ‘Clapp’s
1921). Hardenpont’s best cultivars have been Favourite’. Although central and western Europe
known since 1758, including the popular have contributed some efforts for the develop-
‘Passe-Colmar’ (1758), ‘Beurré d’Hardenpont’ ment of pear cultivars, somewhat similar to those
(1759), ‘Délice d’Hardenpont’, ‘Beurré Rance’, efforts undertaken in Italy, France, Belgium, and
6 M. Quinet and J.-P. Wesel

Table 1.1 Major cultivars of European pear (Pyrus communis) identified during the eighteenth and nineteenth
centuries
Cultivar Synonyms Breeder(s) Year Country
Beurré d’Hardenpont Beurré d’Arenbert N. Hardenpont 1759 Belgium
Glou Morceau
William’s Bartlett Stair/William 1770 UK
Bon Chrétien Williams
Légipont Fondante de Charneux M. Légipont 1805 Belgium
Miel de Waterloo
Köstliche von Charneux
Durondeau Poire de Tongres Ch.-L. Durondeau 1811 Belgium
Beurré Durondeau
Beurré d’Anjou Nec plus Meuris J. B. Van Mons 1822 Belgium
Anjou
Joséphine de Malines J. Esperen 1830 Belgium
Beurré Hardy Ernest Bonnet 1830 France
Rocha P. A. Rocha 1836 Portugal
Doyenné du Comice Vereinsdechants birne Jardin du Comice 1849 France
Decana del Comicio
Beurré de Naghin N. de Naghin 1858 Belgium
Madame Grégoire X. Grégoire 1860 Belgium
Clapp’s favourite Clapps Liebling T. Clapp 1860 USA
Abbé Fetel Abate Fetel Abbé Fetel 1869 France
Triomphe de Vienne J. Colaud alias (Côte) 1870 France
Conference Firme Rivers 1890 UK
Packhams Triumph C. H. Packham 1896 Australia
Forelle >1670 Germany

England, it is Germany that is most noted for have originated in USA, until the middle of the
providing valuable literature in the field of nineteenth century, have come from imports due
pomology (Hedrick et al. 1921). to French, Dutch, and English settlements
In Japan, commercial pear production has (Hedrick et al. 1921). Moreover and of particular
substantially increased around the same period of impact on the US pear industry is the introduc-
time as in Europe due to successive discoveries tion of oriental (Asian) pears and their hybrids
of two chance pear seedlings, ‘Nijisseiki’ and (Hedrick et al. 1921). Asian pear cultivation has
‘Chojuro’, around the year of 1890 (Saito 2016). intensified in the USA around 1938 (Bretaudeau
During the Edo period in Japan (1603–1868), and Fauré 1991), and has since spread worldwide
over 150 cultivars have been documented (Silva (Bretaudeau and Fauré 1991). It is reported that
et al. 2014). Whereas cultivars of European pears the oriental, Chinese, or sand pear came into
have come to the New World almost entirely America from Asia by way of Europe through
from the countries of Belgium and France, along the Royal Horticultural Society of London
with three or four major cultivars of English (Hedrick et al. 1921). Hybridizations with the
origin that have been most commonly grown in European pear gave rise to ‘Le Conte’ (1846),
North America in the twentieth century (Hedrick ‘Kieffer’ (1873) or ‘Garber’ (1880) (Hedrick
et al. 1921). Most, if not all of the cultivars that et al. 1921). It is important to point out that
1 Botany and Taxonomy of Pear 7

cultivation of P. pyrifolia dates back to 693 ACN 1.3 Taxonomy and Phylogeny
in Japan (Bretaudeau and Fauré 1991). of Pears
During the twentieth century, private and
national research stations in Europe, North 1.3.1 The Pyrus Genus Within
America, and Asia established fruit breeding Rosaceae
programs to develop new commercial cultivars.
Overall, the number of newly developed and Both European and Asian pears belong to the
released cultivars of pear has been a lot less than genus Pyrus of the family Rosaceae within the
those for apple (Brewer and Palmer 2011). Order Rosales, belonging to the Rosids subclass,
Among the limited number of pear cultivar and within the Eudicot core (Chase et al. 2016).
releases developed from pear breeding programs The Rosaceae family is monophyletic with a
is ‘Concorde’, developed at East Malling (UK) in moderately large angiosperm lineage containing
1977 and derived from a cross between ‘Con- 90 genera and between 2500 and 2900 species
ference’ and ‘Doyenné du Comice’. However, (Stevens 2017). Rosaceae is a heterogeneous
efforts undertaken by Japanese and Chinese family that is divided into the following three
breeding programs during the twentieth- and subfamilies, according to APG IV, Dryadoideae,
twenty-first centuries resulted in the release of Rosoideae, and Amygdaloideae (Stevens 2017).
various new Asian pear cultivars (Jun and Previously, largely based on fruit and other
Hongsheng 2002; Teng 2011; Saito 2016). morphological characteristics, Rosaceae was
Overall, several pear breeding programs have divided into four subfamilies, including Rosoi-
focused their efforts on pest and disease resis- deae, Maloideae, Amygdaloideae, and Spi-
tance, fruit quality and appearance, duration of raeaoideae (Xiang et al. 2017). However, recent
harvest season, self-fertility, yield, and growth molecular analyses support the separation of the
habit (Jun and Hongsheng 2002; Brewer and former Rosoideae (s.l.) into Rosoideae (s.s.) and
Palmer 2011; Dondini and Sansavini 2012). It is Dryadoideae, and in combining the previous
only in the last 15–20 years that nearly 300 novel Maloideae, Amygdaloideae (s.s.), and Spi-
cultivars, including about 200 European pear and raeaoideae into the current Amygdaloideae (s.l.)
100 Asian pear cultivars, have been released (Stevens 2017; Xiang et al. 2017). The species
(Dondini and Sansavini 2012). Nowadays, there richness of Rosaceae could be partly related to
are several thousands of pear cultivars that are polyploidization and to species radiation in the
available worldwide. Among these, approxi- family’s history (Xiang et al. 2017). Relation-
mately ten cultivars account for 90% of the world ships among Rosaceae tribes and genera remain
production of pears (Pesson and Louveaux 1984; unclear, in part because of polyploidy events and
Miranda et al. 2010). However, due to cultivar rapid separation/diversification among some
history and propagation methods, some cultivars clades (Xiang et al. 2017). Phylogenetic studies
are known under different names in different of Xiang et al. (2017) suggest that Dryadoideae
regions or that different cultivars are grown/ is the basal clade of Rosaceae, and it is the sister
promoted as being the same; thus clearly indi- of the combined clade of Rosoideae and Amyg-
cating that pear cultivars are not as well charac- daloideae. The age of the crown Rosaceae is
terized as previously reported (Evans et al. about 101.6 Mya with the separation of Drya-
2015). Therefore, genetic molecular markers are doideae, followed by an immediate divergence of
currently being used to screen accessions of the two largest subfamilies Rosoideae and
different germplasm collections, and consider- Amygdaloideae at 100.7 Mya (Xiang et al.
able efforts are needed to verify and confirm 2017).
accurate identities of accessions in worldwide The subfamily Amygdaloideae contains about
national collections (Evans et al. 2015). 1000 species (Xiang et al. 2017), and it is divided
8 M. Quinet and J.-P. Wesel

into 11 tribes, including the Malinae (Stevens quinces (Chaenomeles), firethorns (Pyracantha),
2017). All, but two of the tribes of Amyg- and mountain ashes (Sorbus) (Campbell et al.
daloideae, must have diverged between 96 and 2007).
88 Mya., with no further activity for the next
20 Mya (Xiang et al. 2017). The Malinae may
represent a rapid but ancient radiation (Campbell 1.3.2 Phylogeny of Pyrus
et al. 2007; Stevens 2017; Xiang et al. 2017).
This is perhaps associated with whole genome The genus Pyrus is characterized by a high
duplication in the stem lineage, and accompanied genetic variability, and it consists of around 75
with climatic changes that must have occurred at species and interspecific hybrid species, along
the end of the Palaeocene and all through with thousands of cultivars (Ferradini et al. 2017;
towards the beginning of the Oligocene (Xiang Stevens 2017). Estimates of Pyrus diversity vary
et al. 2017). The stem group Malinae is dated between 50 and 80 species, according to various
back to the late Palaeocene, with subsequent publications (Table 1.2), and the numbers of
divergence in the Eocene and Oligocene epoques accepted species differ as a consequence of
(Lo and Donoghue 2012). poorly understood species limits (Korotkova
Despite efforts to elucidate relationships et al. 2014). Indeed, up to 900 Pyrus species
within the Malinae, relationships among the names have been recorded (Zheng et al. 2014).
major sublineages, generic limits, and divergence However, the number of primary (i.e., not of
times have remained uncertain (Campbell et al. hybrid origin) species has been relatively con-
2007; Lo and Donoghue 2012). Most probably, sistent, and approximately 20 putative primary
hybridization has played a part in the Malinae species are widely recognized (Zheng et al.
evolutionary history, as hybridization is unusu- 2014). Estimation of genetic diversity among
ally common among genera in this tribe Pyrus spp. has been difficult due to low mor-
(Campbell et al. 2007). Comparisons of genetic phological diversity, lack of differentiating
linkage maps within Malinae have suggested that characters among species, and widespread
all chromosomes of the genera in this tribe show cross-ability (Yao et al. 2010). Although they are
co-linearity despite considerable differences in interspecies compatible, Pyrus species are typi-
genome sizes (Yamamoto and Terakami 2016). cally self-incompatible (Yue et al. 2014).
The Malinae contains 1000 species organized The Pyrus origin dates back to the Oligocene
within 30 genera (Stevens 2017). However, epoque, about 33.35–25.23 Mya (Korotkova
Malinae is also known as Cydoniaceae, Mala- et al. 2018). It is a genus of deciduous trees and
ceae, Mespilaceae, Pyraceae, or Sorbaceae (Ste- shrubs occurring throughout temperate Eurasia,
vens 2017). Furthermore, Malinae is reaching the Atlas Mountains in North Africa,
characterized by a north temperate distribution, and extending to both Japan and South China
production of leaves with deciduous stipules, (Korotkova et al. 2018). Assessing species
flowers with a gynoecium that is at least half-way diversity in Pyrus is challenging due to high
inferior, and a fleshy hypanthium ‘pome’ fruit morphological plasticity and frequent hybridiza-
(Stevens 2017). Several important edible fruits tions within the genus (Korotkova et al. 2018).
are members of this tribe, such as apple (Malus), Thus, this genus is characterized by very low
pear (Pyrus), quince (Cydonia), loquat (Eri- genetic distances between taxa (Korotkova et al.
obotrya), chokeberry (Aronia), and serviceberry 2014). Currently, the genus is subdivided into the
(Amelanchier) (Campbell et al. 2007). In addi- following four sections: Pyrus sect. Pyrus, Pyrus
tion, the Malinae tribe includes valued orna- sect. Xeropyrenia Fed., Pyrus sect. Argyromalon
mentals, such as some cotoneasters Fed., and Pyrus sect. Pashia Koehne (Korotkova
(Cotoneaster), hawthorns (Crataegus), Japanese et al. 2018). However, phylogenetic analyses
1 Botany and Taxonomy of Pear 9

Table 1.2 List and origin of Pyrus species (Asanidze et al. 2011; Silva et al. 2014)
Species Country or region of origin
P. alnifolia (S. and Z.) Franch. and Sav. Russian Far East, China, Japan, Korea, Taiwan
P. americana DC Greenland, USA, Canada
P. angustifolia Aiton USA, Canada
P. arbutifolia (L.) L.f. USA
P. aria (L.) Ehrh. USA, Canary Islands, North Africa, All of Europe
P. armeniacifolia T.T. Yu China
P. aucuparia var. dulcis (K.) A. and G. All Europe
P. aucuparia var. randaiensis Hayata Taiwan
P. baccata L. Russia, Mongolia, China, Korea
P. baccata var. aurantiaca Regel Russia, Mongolia, China, Korea
P. baccata var. himalaica Maxim. China, Bhutan, India, Nepal
P. baccata var. mandshurica Maxim. Russia, China, Japan, Korea
P. betulifolia Bunge China, Laos
P. boissieriana Buhse Azerbaijan, Turkmenistan, Iran
P. bulgarica Kuth. and Sachokia (P.  nivalis Western Europe, Central Eastern and Southern
Jacq.)
P. calleryana Decne. China, Korea, Taiwan, Vietnam
P. calleryana var. dimorphophylla (Makino) Japan
Koidz.
P. calleryana var. fauriei (C. K. Schneid.) Rehder Korea
P. calleryana var. koehnei (C. K. Schneid.) T. China
T. Yu
P. cathayensis Hemsl. China
P. caucasica Fed. Eastern Europe and Central Greece
P. chamaemespilus (L.) Ehrh. Western Europe, Central Eastern and Southern
P. communis L. All Europe
P. communis subsp. gharbiana (T.) Maire Algeria, Morocco
P. communis subsp. P. marmorensis (Trab.) Morocco
Maire
P. communis subsp. P. pyraster (L.) Ehrh. Western Europe, Central Eastern, and Southern
P. communis var. cordata (Desv.) H.f. UK, Portugal, Spain, France
P. coronaria L. Canada, USA
P. coronaria var. ioensis Alph. Wood USA
P. cossonii Rehder Algeria
P. crataegifolia Savi Turkey, Albania, Serbia, Greece, Italy, Macedonia
P. cuneifolia Guss. Central Eastern Europe, South and Central
P. cydonia L. Iran, Armenia, Azerbaijan, Russia, Turkmenistan
P. decipiens Bechst. All Europe and North Africa
P. delavayi Franch. China
(continued)
10 M. Quinet and J.-P. Wesel

Table 1.2 (continued)

Species Country or region of origin
P. demetrii Kuth Georgia
P. discolor Maxim. China
P. diversifolia Bong. USA, Canada
P. domestica (L.) Sm. Algeria, Cyprus, Eastern Europe Central, West and Meridional
P. doumeri Bois Vietnam
P. elaeagrifolia Pall. Turkey, Ukraine, Albania, Bulgaria, Greece, Romania
P. elaeagrifolia subsp. kotschyana Turkey
P. floribunda Lindl. USA, Canada
P. folgner (C. K. Schneid.) Bean China
P. foliolosa Wall. Burma, Bhutan, India, Nepal, China
P. fusca (Raf.) C. K. Schneid. USA, Canada
P. georgica Kuth Georgia
P. germanica (L.) Hook. f. Middle East, Eastern Europe, Central, Southern and Northern
Asia
P. gharbiana Trab. Morocco
P. glabra Boiss. Iran
P. gracilis Siebold and Zucc. Japan
P. harrowiana Balf. f. and W. W. Sm. China, India, Nepal, Burma
P. heterophylla Regel and Schmalh. Kyrgyzstan, Tajikistan, China
P. hondoensis Nakai and Kikuchi Japan
P. hupehensis Pamp. China, Taiwan
P. indica Wall. South Asia and Far East Asia
P. intermedia Ehrh. All Europe
P. japonica Thunb. Japan
P. kansuensis Batalin China
P. keissleri (C. K. Schneid.) H. Lev. China, Myanmar
P. ketzkhovelii Kuth Georgia
P. korshinskyi Litv. Afghanistan, Tajikistan, Uzbekistan
P. korshinskyi Litv. subsp. bucharica (Litv.) B. K Former Soviet Union
P. kumaoni Decne. Middle East, Far East and South Asia
P. lanata D. Don Afghanistan, India, Nepal, Pakistan
P. malus subsp. paradisiaca (L.) Western, Eastern, and Central Europe and Greece
P. matsumurana Makino Japan
P. minima Ley UK
P. nebrodensis Guss. Italy - Sicily
P. nussia Buch.-Ham. ex D. Don Far East, South Asia
P. pinnatifida Ehrh. All Europe
P. pohuashanensis Hance Russia, China, Korea
P. praemorsa Guss South of Italy, France
P. prattii Hemsl. China
P. prunifolia Willd. China
(continued)
1 Botany and Taxonomy of Pear 11

Table 1.2 (continued)

Species Country or region of origin
P. pseudopashia T.T. Yu China
P. pyrifolia var. pyrifolia China, Laos, Vietnam
P. ringo var. kaido Wenz China
P. ringo Wenz. China, Korea
P. sachokiana Kuth. Georgia
P. salicifolia Pall. Iran, Armenia, Turkey, Azeebaijan
P. sanguinea Pursh Canada, USA
P. scabrifolia Franch. China
P. scalaris (Koehne) Bean China
P. sieboldii Regel China, Japan
P. sikkimensis Hook. f. China, Bhutan, India
P. sinensis var. maximowicziana H. Lev. Korea
P. spectabilis Aiton China
P. spinosa Forssk. Central Eastern Europe, South, and Central
P. sudetica Tausch Western Europe, Central Eastern, and Southern
P. syriaca Boiss. Caucasus and Middle East Region
P. taiwanensis Iketani and H. Ohashi Taiwan
P. torminalis (L.) Ehrh. North Africa, Middle East, South Caucasus, whole Europe
P. trilobata (Poir.) DC. Israel, Lebanon, Turkey, Bulgaria, Greece
P. trilobata (Poir.) DC. Turkey, Bulgaria, Greece, Israel, Lebanon
P. tschonoskii Maxim. Japan
P. turkestanica Franch. Kyrgyzstan, Tajikistan, Turkmenistan, Afghanistan
P. ussuriensis Maxim. Russia, China, Japan, Korea, Brazil
P. vestita Wall. ex G. Don China, Bhutan, India, Nepal, Myanmar
P. vilmorinii (C. K. Schneid.) Asch. and Graebn. China
P. xerophila T. T. Yu China
P. yunnanensis Franch. China, Myanmar
P. zahlbruckneri (C. K. Schneid.) Cardot China
P.  bretschneideri Rehder China
P.  complexa Rubtzov Former Soviet Union
P.  hopeiensis T. T. Yu China
P.  phaeocarpa Rehder China
P.  serrulata Rehder China
P.  sinkiangensis T. T. Yu China
P.  uyematsuana Makino Japan, Korea

have supported that Pyrus is a monophyletic et al. 2018; Wu et al. 2018). The first is an
group containing two major clades that diverged eastern Asian clade with a crown group age of
far prior to any possible human intervention 15.7 Mya, and the second is a western Eurasian
(Kim et al. 2015; Zheng et al. 2014; Korotkova clade that comprises species from Europe,
12 M. Quinet and J.-P. Wesel

Southwest Asia, and the Caucasus region, dis- P. communis and P.  bretschneideri are known
playing a crown group of 12.38 Mya (Korotkova to be polyploids (Ferradini et al. 2017).
et al. 2018). The separation of these two clades Currently, there are several studies aiming to
may be related to the recession of the Turgai estimate genetic distances among different pear
Strait, a Mesozoic epicontinental seaway that has cultivars/genotypes present in gene banks and in
separated Europe from Asia until the late Oli- various breeding programs (Bao et al. 2007;
gocene (Korotkova et al. 2018). However, Wu Bassil and Postman 2010; Silva et al. 2014;
et al. (2018) have estimated that both clades Chang et al. 2017; Ferradini et al. 2017; Wu et al.
diverged between 6.6 and 3.3 Mya. Their hypo- 2018). Pear cultivars can be subdivided into two
thetical common ancestor seems to have origi- major groups, the occidental (European) and the
nated in China before dissemination through oriental (Asian) pears, as confirmed by molecular
central Asia and then eventually on to western data (Bao et al. 2007; Bassil and Postman 2010;
Asia and Europe (Wu et al. 2018). Within the Yue et al. 2014; Ferradini et al. 2017). European
western Eurasian clade, a major period of cultivars belong to P. communis and are most
diversification has likely occurred in the Middle likely derived from one or two wild species,
to Late Miocene when Caucasian and Southwest P. pyraster (L.) Burgsd. and/or P. caucasica Fed.
Asian lineages have diversified (Korotkova et al. (Ferradini et al. 2017). Therefore, European pear
2018). Most of the extant diversity of Pyrus in cultivars have a narrow genetic base (Miranda
western Eurasia appears to have originated in the et al. 2010); whereas, cultivated pears native to
Pliocene and the Pleistocene (Korotkova et al. East Asia belong to the following five groups,
2018). Pyrus species diversity is concentrated in including the Ussurian pear (P. ussuriensis),
western Eurasia to eastern Asia, and particularly Chinese white pear (P.  bretschneideri), Chi-
in China (Silva et al. 2014). Speciation in Pyrus nese sand pear (P. pyrifolia), Xinjiang pear
is complex, and several currently accepted Pyrus (P. sinkiangensis), and the Japanese pear
species have not been recovered as mono- (P. pyrifolia) (Bao et al. 2007; Katayama et al.
phyletic, thus indicating that current species 2016). Phylogenetic studies of Pyrus cultivars
limits require re-evaluation (Zheng et al. 2014; native to East Asia have revealed contradictory
Korotkova et al. 2018). results; thus, additional studies are required to
Within the Pyrus genus, there are only a few resolve issues of origin and evolution of Asian
species that have been domesticated for com- pear cultivars (Bao et al. 2007; Bassil and Post-
mercial production (Bao et al. 2007; Wu et al. man 2010; Iketani et al. 2012; Chang et al. 2017;
2013). Most cultivated Pyrus species include Wu et al. 2018). However, Chang et al. (2017)
P. communis (European pear), and the Asian pear have explored the evolution routes of Pyrus in
species of P. ussuriensis Maxim., P. pyrifolia, China and highlighted the spread of pears from
P.  bretschneideri Rehd., and P. sinkiangensis the Shanxi province to other regions of northern
Yü (Wu et al. 2013; Ferradini et al. 2017). These China. From China, pears were then dissemi-
have been domesticated from the following wild nated throughout central Asia before they were
species, P. communis is derived from the wild spread over to western Asia and then on to
European species P. pyraster, while the culti- Europe (Wu et al. 2018).
vated P. ussuriensis is derived from the wild
P. ussuriensis, whereas P. pyrifolia and 1.3.2.1 Pyrus Species in Western
P.  bretschneideri are derived from the wild Eurasia
P. pyrifolia and finally P. sinkiangensis is In general, occidental pears are distributed in
derived from hybridization between the culti- Europe, northern Africa, Asia Minor, Iran, Cen-
vated P. communis and either the cultivated tral Asia, and Afghanistan (Zheng et al. 2014).
P. pyrifolia or P.  bretschneideri (Wu et al. They have been geographically divided into the
2018). Although the majority of cultivated pears following three subgroups: West Asian species,
are diploid (2n = 2x = 34), a few cultivars of European species, and North African species
1 Botany and Taxonomy of Pear 13

(Zheng et al. 2014; Zamani et al. 2017). It is display morphological adaptations such as nar-
reported that there are 12 primary species present row leaves (Korotkova et al. 2018). The other
in western Eurasia, including five European remaining species mainly inhabit mesophytic
species (P. communis, P. caucasica, P. pyraster, forests and display broad leaves (Korotkova et al.
P. nivalis Jacq., and P. cordata Desv.), five West 2018). Thus, wild pear species have diverged
Asian species (P. elaeagrifolia Pall, P. spinosa into numerous local ecogeographical races and
Forssk syn. P. amygdaliformis Vill., P. regelii species that are interfertile with the cultivated
Rehd., P. salicifolia Pall., and P. syriaca Boiss.), pear (Asanidze et al. 2011). It is important to
and three North African species (P. cossonii point out that the country of Iran is also rich in
Rehd. syn. P. longipes Balansa ex Coss. & Pyrus species, with about 23 taxa, and also has
Durieu, P. gharbiana Trab., and P. mamorensis both xerophytic and mesophytic species (Zamani
Trab.), while the remaining species are putative et al. 2017). These species occur throughout the
interspecific hybrids (Zheng et al. 2014). Further north-east region through northern hyrcanian
phylogeny studies have been conducted to char- forests to the north-west (Azerbaijan province)
acterize relationships among occidental primary and all the way to the southwest region in the
species (Zheng et al. 2014). It is revealed that Fars Province (Zamani et al. 2017).
European species may be the latest derived The cross-compatibility among various Pyrus
occidental species and displaying lower levels of species raises questions on the taxonomy of
genetic diversity compared to West Asian species Pyrus species (Zamani et al. 2017). For example,
(Zheng et al. 2014). Moreover, European pears P. caucasica, an endemic species of the Cauca-
are most likely independently derived from West sus, has been classified initially as a European
Asian species and North African species, as pear, P. communis, but has been subsequently
P. nivalis and P. cordata are more related to deemed as a separate species based on morpho-
West Asian species, primarily to P. spinosa; logical differences of leaf margins (Asanidze
whereas, P. caucasica, P. pyraster, and P. com- et al. 2011). Although earlier studies have
munis are more closely related to the North deemed P. caucasica as a completely indepen-
African species (Zheng et al. 2014). Among dent species because of its morphological dif-
West Asian species, P. regelii is an early ferences and its separate geographical
diverging and isolated species (Zheng et al. distribution, it is now considered as a wild sub-
2014), while the three African species are well species of P. communis (Asanidze et al. 2011).
differentiated with P. gharbiana and Furthermore, another wild ancestor of the culti-
P. mamorensis and are more related to European vated European pear, P. pyraster, native to
species (Zheng et al. 2014). Eastern and Central European countries, includ-
It has been reported that wild occidental pears ing the Balkan Peninsula and Turkey, has also
primarily inhabit two types of habitats, meso- been considered either as a species or a sub-
phytic forests and xerophytic open woodlands species of P. communis by different reports
(Zamani et al. 2017; Korotkova et al. 2018). (Asanidze et al. 2011; Korotkova et al. 2018).
Xerophytic woodlands constitute a Similar conflicting findings have been reported
vegetation-type characteristic for arid and semi- for other species, such as P. balansae Decne.,
arid regions of Southwest Asia, including the P. boissieriana Buhse, P. salicifolia, P. syriaca,
Caucasus ecoregion (Korotkova et al. 2018). P. georgica Kuth., P. demetrii Kuth., P. ket-
Xerophytic woodlands likely play an important zkhovelii S. Kuthath, and P. sachokiana Kuth.
role in the diversification of Pyrus as these (Asanidze et al. 2011). Recently, Aydin and
habitats comprise a considerable number of Dönmez (2015) have revised species taxonomy,
Pyrus species. The Caucasus ecoregion contains present in Turkey, and have proposed species
approximately 25 endemic species (Korotkova modifications. They have proposed that P. pseu-
et al. 2018). Moreover, the majority of Caucasian dosyriaca should be treated as a new botanical
pears inhabit xerophytic open woodlands and variety of P. syriaca, while P. serikensis and
14 M. Quinet and J.-P. Wesel

P. boissieriana are reduced to synonyms of The Ussurian pear is mainly cultivated in

P. cordata, and P. elaeagrifolia Pall., respec- North China, especially in Northeast China
tively. In addition, subsp. kotschyana (Boiss.) (Teng et al. 2015). The Chinese white pear is
Browicz is reassessed as P. kotschyana Boiss. ex cultivated in North China and occupies the most
Decne (Aydin and Dönmez 2015), while Zamani important position in commercial pear produc-
et al. (2017) have assessed the usefulness of tion (Teng et al. 2015). The Chinese sand pear is
biological markers to evaluate the taxonomic naturally distributed in south China and owns
significance of Iranian pear taxa. plentiful cultivar resources (Teng et al. 2015).
Pear improvement efforts undertaken in Eur- The Japanese pear refers to pears located in
ope have depended on P. communis and Japan, and has fruit traits similar to those of the
P. nivalis. Although P. communis is widely Chinese sand pear (Teng et al. 2015). Wild
cultivated worldwide, its origin is not well P. ussuriensis is widely distributed in
understood. It is likely that P. communis may north-eastern China, eastern Russia, the Korean
have other species in its genetic background, Peninsula, and central and northern Honshū in
including P. pyraster, P. caucasica, P. eleagri- Japan (Iketani 2016). In Japan, two botanical
folia, P. spinosa, P. nivalis, and P. syriaca (Silva varieties of P. ussuriensis, var. aromatica and
et al. 2014; Korotkova et al. 2018). On the other var. hondoensis, are native to the northern area
hand, P. nivalis is used in wine making and has and the central area of the main island, respec-
been of great importance in both Britain and tively (Iketani 2016; Katayama et al. 2016). At
France for over 400 years (Silva et al. 2014). least two native Japanese and one native Chinese
Pyrus species, namely P. ussuriensis,
1.3.2.2 Pyrus Species in East Asia P. calleryana, and P. pseudopashia T.T. Yu, are
Oriental pears are distributed from the Tian Shan included in the National Red List (Kell et al.
region and the Hindu Kush Mountains in Central 2015; Iketani 2016). Early on, the Japanese pear
Asia eastward to Japan (Zheng et al. 2014). is suspected to have originated from native plants
There are nine proposed primary Pyrus species in in Japan; however, it is subsequently reported
East Asia, five have originated from China that P. pyrifolia is most likely introduced to
(P. pyrifolia, P. ussuriensis, P. pashia D. Don, Japan during prehistoric times (Iketani 2016).
P. calleryana Dcne, and P. betulifolia Bge), two Phylogeny studies have revealed incidence of
from Japan (P. dimorphophylla Makino and close relationships among Asian Pyrus species.
P. hondoensis Yu), one from the Korean Penin- For example, Yue et al. (2014) have reported that
sula (P. fauriei Schneid.), and one from Taiwan the oriental pear cluster can be divided into two
Island (P. koehnei Schneid.) (Zheng et al. 2014). subgroups. One subgroup consists of three
The remaining species are most likely inter- P. betulifolia accessions, while the other sub-
specific hybrids although their parentages remain group consists of all other cultivars and species,
uncertain (Zheng et al. 2014). In China, pear namely P. pyrifolia, P. ussuriensis, P. pashia,
trees have originated in the mountainous regions P. dimorphophylla, P. fauriei, P. serrulata,
of Southwestern China, and have spread both P. hopeiensis, P. phaeocarpa, P. xerophila, and
westward and eastward (Chang et al. 2017). P. hondoensis. Likewise, Zheng et al. (2014)
A total of 69 Pyrus species are found in China. have supported the existence of subclades for
Of these, 13 have originated in China, including P. ussuriensis and P. pashia, but they have not
species with commercial cultivars, such as the resolve relationships among the remaining hap-
Chinese white pear (P.  bretschneideri), Chi- lotypes. According to Wu et al. (2018), Asian
nese sand pear and Japanese pear (P. pyrifolia), pear accessions are clustered into the following
Sinkiang pear (P. sinkiangensis), and the four groups: a first large group that includes
Ussurian pear (P. ussuriensis) (Kell et al. 2015; accessions of both P.  bretchneideri and
Chang et al. 2017). P. pyrifolia; a second group that includes wild
1 Botany and Taxonomy of Pear 15

accessions of China, Japan, and Korea; a third group, Pyrus Chinese white pear group, Pyrus
group that clusters wild and cultivated accessions Chinese sand pear group, and the Pyrus Japanese
of P. ussuriensis; and a fourth group that pear group (Iketani et al. 2012).
includes all cultivated accessions of
P. sinkiangensis.
Although genetic differentiation between 1.4 Botanical Description of Pear
groups of native populations and those of culti-
vars was usually high, cultivars were not well All Pyrus species are tree-like woody plants
differentiated from each other (Iketani et al. (Hedrick et al. 1921). They are medium-sized
2012). The classification of cultivated pears trees often with a tall, narrow crown, but with
could indeed be problematic due to only a few species that are shrubby. Leaves are
cross-compatibility and introgression between alternately arranged, simple, 2–12 cm in length,
species (Iketani 2016; Katayama et al. 2016). As glossy green in some species, or densely silvery
for cultivated Asian pears, Bao et al. (2007) hairy in some others (Hedrick et al. 1921). Most
demonstrated that Chinese sand pears and Chi- pears are deciduous, but one or two species in
nese white pears were clustered together, and that Southeast Asia are evergreen. Flowers are usu-
Japanese cultivars had sandy pears as parents, ally white, borne in corymbs on short spurs, or
while Ussurian pears clustered separately (Bao on lateral branchlets (Hedrick et al. 1921).
et al. 2007). However, Bassil and Postman Flowers are about 2–4 cm in diameter, and have
(2010) grouped Ussurian pear and Chinese white five sepals, five petals, numerous stamens, and
pear cultivars in the same clusters. According to five-locular ovary with usually free styles. The
Yao et al. (2010), some cultivars of Ussurian fruit is a pome, measuring 1–4 cm in diameter in
pear clustered with some Chinese white pears, wild species, and up to 18 cm in length and 8 cm
while other Chinese white pears generally clus- in width in some cultivated forms (Hedrick et al.
tered with Chinese sand pear and Japanese pears. 1921). The form of the fruit varies in most spe-
More recently, Chang et al. (2017) showed that cies from oblate, or globose, to pyriform
Japanese sand pear and Chinese sand pear cul- (Hedrick et al. 1921). The fruit is a pseudo-fruit
tivars shared similar genetic backgrounds and composed of the receptacle, or a calyx tube that
exhibited a high degree of kinship. Earlier, Ike- is greatly dilated and enclosing the true fruit,
tani et al. (2012) reported that Japanese pear which consists of five cartilaginous carpels,
cultivars had a simple genetic structure, while known as the core (Hedrick et al. 1921). The
Chinese and Korean pear cultivars were admix- flesh usually bears grit cells (sclereids) when
tures of Japanese pear and native P. ussuriensis. ripened on the tree (Hedrick et al. 1921). Leaf
Subsequently, Teng et al. (2015) showed that and fruit traits are commonly used to distinguish
there were no real genetic differences detected among Pyrus species (Asanidze et al. 2011;
among Chinese sand pear, Chinese white pear, Zamani et al. 2017). European pears are elon-
and Japanese pear. gated and have full-bodied textures, while Asian
Globally, Asian pear cultivars have been pears are round in shape and have sandy textures
deemed to be genetically continuous, and have a (Silva et al. 2014).
very narrow genetic diversity compared with that Pear trees are self-incompatible, exhibiting
of wild species (Iketani et al. 2012). In this typical gametophytic self-incompatibility, as
context, Iketani et al. (2012) have proposed that with other Rosaceous species (Sassa et al. 2009;
Asian pear cultivars should be regarded as a Franceschi et al. 2012). Gametophytic
single group, although this may not be accepted self-incompatibility is controlled by a single
by horticulturists. An alternative strategy is to multi-allelic locus, the so-called S-locus. In
divide Asian pears into four cultivar groups Pyrinae, the S-locus contains the single pistil-side
instead of species, namely Pyrus Ussurian pear S determinant, the S-RNase, which is expressed
16 M. Quinet and J.-P. Wesel

in the pistil, and multiple pollen-expressed S- good fruit development (Bretaudeau and Fauré
locus F-box genes, designated as SFBB (for 1991). Most pear cultivars are grafted onto clonal
S-locus F-box brothers), that are expressed in the quince rootstocks (Hessayon 1990). The most
pollen (Franceschi et al. 2012). Pyrus species are popular is quince A, producing trees which grow
pollinated by insects, and flowers produce nectar about 3–6 m in height (Hessayon 1990). Pears
to attract these insects (Pesson and Louveaux can be trained and grown as bushes, dwarf
1984; Mayer et al. 1990; Quinet et al. 2016). The pyramids, cordons, espaliers, or fans (Fig. 1.3)
sugar content of pear nectar is usually lower (Hessayon 1990). Pear trees favour sunny areas
(often <10–15%) compared to that detected in and do not tolerate shadowing (Hessayon 1990).
other fruit tree species (Farkas et al. 2002; Faoro Furthermore, pear trees have good tolerance to a
and Orth 2011; Quinet et al. 2016). Although wide variety of soil conditions, including those
intra-specific self-incompatibility is present, of soil texture and pH. However, they are exigent
interspecies hybridization is common in Pyrus for soil freshness, and are not well suited for
(Hedrick et al. 1921; Iketani 2016; Katayama either dry soils nor for flooded soils (Hedrick
et al. 2016; Zamani et al. 2017). et al. 1921; Hessayon 1990).
Due to self-incompatibility, pear cultivars are Although the wild European pear produces
vegetatively propagated by grafting. The Euro- fruits regularly, it rarely reproduces by seeds
pean and Asian pears readily intergraft with other (Hedrick et al. 1921). Suckering seems to be the
pears (Hedrick et al. 1921). The main rootstocks dominant form of proliferation of these wild
used for European pear are P. communis, forms, thereby allowing these wild types to
P. betaefolia, or quince (Cydonia oblonga), maintain their favourable biotopes.
while the main rootstocks used for Asian pear are European pear trees have an upright, oblong,
P. pyrifolia, P. communis, P. pashia, or pyramidal, and compact top (Hedrick et al.
P. calleryana, P. ussuriensis, or P. belulaefolia 1921). Branches are greyish brown or dark red-
(Bretaudeau and Fauré 1991). dish brown. Branchlets are glossy, smooth,
The next sections will focus on detailed glabrous, with more or less conspicuous lenticels
descriptions of the main cultivated pear species (Hedrick et al. 1921). Leaf buds are prominent,
P. communis for the European pear, and on plump, obtuse or pointed, mostly free, while
P. pyrifolia, P. ussuriensis, P.  bretschneideri, flower buds are larger and plumper than leaf buds
and P. sinkiangensis for the Asian pears. Distinct (Hedrick et al. 1921). Leaves are glossy dark
phenotypic traits have been selected during green, ovate to elliptic with crenate to serrate
domestication of European and Asian pears (Wu margins, and measure 7–9 cm (Rameau et al.
et al. 2018). 1989). The petiole is as long as the blade, and
when young, they are both pubescent (Coste and
Flahault 1903). Foliage turns into shades of red
1.4.1 European Pear and yellow in the fall season. Flowering occurs
in early spring and lasts between 6 and 20 days,
1.4.1.1 Description depending on the cultivar (Bretaudeau and Fauré
P. communis is a medium-sized tree, reaching 1991). Inflorescences are corymbs of 5–15
20 m tall and a diameter of 90 cm. Annual flowers, with centripetal flowering (Fig. 1.4a)
growth of wild P. communis is 0.5–1.5 m (Aas (Rameau et al. 1989; Bretaudeau and Fauré
1999). European pear trees bear fruit after 4– 1991). Flowers are hermaphroditic and creamy
8 years of growth, and their life spans could white (occasionally flushed with pale pink), and
reach up to 200 years, depending on the root- have a diameter of 2.5–3.5 cm (Fig. 1.4b) (Coste
stock used (Hedrick et al. 1921; Hessayon 1990). and Flahault 1903). They are composed of five
For cultivation, pear trees are pruned to facilitate triangular-lanceolate sepals of 5–9  3–4 mm,
harvest and to allow for light incidence into the five obovate (13–15  10–13 mm) white petals,
canopy of the tree to promote flowering and for about 20 stamens, with purple anthers and free
1 Botany and Taxonomy of Pear 17

Fig. 1.3 Description of

training systems. a Standard,
b spindle, c vertical cordon,
d vase, e–h pyramid, i–k
palmette, l U-shaped, m,n
palmette verrier, o double
U-shaped, p Cossonet system,
q oblique cordon, and r–u
horizontal cordon
18 M. Quinet and J.-P. Wesel

Fig. 1.4 Inflorescence (a),

flower (b), and fruit (c, d) of
Pyrus communis. c Whole
fruit and d longitudinal
section

filaments, and a single gynoecium composed of grit cells (Hedrick et al. 1921). Seeds are large,
usually five carpels (Bretaudeau and Fauré brown, or brownish, often tufted at the tips,
1991). The styles are free, and the ovary is sometimes abortive or wanting (Hedrick et al.
five-locular with two ovules per locule (Pesson 1921). Parthenocarpy is present in several
and Louveaux 1984; Bretaudeau and Fauré European pear cultivars, and it is characterized
1991). Anthers have a longitudinal dehiscence by the development of fruit without pollination
(Paris 1996). Pedicels are 2–3.5 cm long, pub- and fertilization of the egg, resulting in seedless
escent, or glabrate. fruit (Nyéki et al. 1998; Moriya et al. 2005;
Upon pollination and/or fertilization, flowers Quinet and Jacquemart 2015). However,
produce edible pear-shaped fruits that ripen from parthenocarpic fruits are generally smaller than
mid-summer to fall, depending on cultivar fertilized fruits (Moriya et al. 2005; Quinet and
(Hedrick et al. 1921; Bretaudeau and Fauré Jacquemart 2015).
1991). Pome fruits are green, yellowish or red-
dish green, globose, subglobose, ovoid, or pyri- 1.4.1.2 European Pear Cultivars
form, 30–160  15–120 mm in size (Fig. 1.4c, Over 3000 cultivars of the European pear are
d, Fig. 1.5). Sepals are persistent. Flesh is white, known (Table 1.3) (Hedrick et al. 1921). They
yellowish, sometimes pink or wine-red, rarely flower in early spring when temperatures reach
salmon-coloured; it is firm, melting, or buttery 10 °C (Bretaudeau and Fauré 1991). Different
and when ripening on the tree with few or many cultivars flower for periods lasting between 6 and
1 Botany and Taxonomy of Pear 19

Fig. 1.5 Fruits of European pear cultivars. a ‘Beurré d’Anjou’, b ‘Clapp’s Favourite’, c ‘Concorde’, d ‘Conference’,
e ‘Doyenné du Comice’, and f ‘Triomphe de Vienne’

20 days (Bretaudeau and Fauré 1991). Although Guyot’, and ‘Coscia’ (Dondini and Sansavini
the flowering period is usually short, fruit mat- 2012). In north America, ‘Beurré d’Anjou’,
uration takes place between the months of July, ‘Williams’, ‘Doyenné du Comice’, ‘Bosc’,
for early maturing cultivars, and January to ‘Concorde’, and ‘Forelle’ are largely grown
March, for winter maturing cultivars (Bretaudeau (USA Pears 2018). Below is a detailed descrip-
and Fauré 1991). European pears are usually tion of some of the most popular pear cultivars.
harvested at green stage, and are allowed to ripen ‘Abbé Fétel’ is a French cultivar, identified in
at room temperature. Some of the most popular 1866 (Dondini and Sansavini 2012). It produces
cultivated European pear cultivars include the large-sized and elongated fruits of medium
following listing. In Europe, eight cultivars rep- quality (Dondini and Sansavini 2012). The fruit
resent 80% of the production, and these include is pyriform, golden yellow, and at times it may
‘Conference’, ‘William’, ‘Abbé Fétel’, ‘Spa- develop a red blush (Dondini and Sansavini
dona’, ‘Doyenne du Comice’, ‘Kaiser’, ‘Dr. Jules 2012). This cultivar could also produce
20 M. Quinet and J.-P. Wesel

Table 1.3 List of some European pear and hybrid cultivars (Flores 1999; Drudze 2004; Jain and Priyadarshan 2009;
Bassil and Postman 2010; Dondini and Sansavini 2012)
Cultivar Synonyms Country Mode of selectiona
Abbé Fetel Abate Fetel France Chance seedling
Alexander Lucas Beurré Alexandre Lucas France
Ambrosia USA US 571  ‘Honeysweet’
Arganche Klementinka, Mustafabey, Zaharoasa de Yugoslavia
Vara
Ayers USA P. communis  P. pyrifolia
hybrid
Bambinella Malta
Bella di Giugno Italy
Belle Lucrative Belgium
Black Worcester UK
Blake’s Pride USA
Blanquilla pera de agua, blanquilla de Aranjuez Spain
Bosc Calebasse Bosc, Beurré Bosc, Carafon de Belgium
Bosc, Beurré d’Apremont
Beurré Clairgeau France O.P. Duchess D’Angouleme
Beurré Hardy Gellert’s butterbirne, French butter pear France Seedling
Beurré d’Anjou Nec plus Meuris Belgium Chance seedling
Anjou
Beurré Superfin France Chance seedling
Butirra Precoce Italy ‘Coscia’  ‘Bartlett’
Morettini
Butirra Rosata Italy ‘Coscia’  ‘Beurre Clairgeau’
Morettini
Carmen Italy ‘Guyot’  ‘Bella di Giugno’
Cascade Oregon ‘Max Red Bartlett’ 
‘Comice’
Catillac Cadillac, Gros monarque, Chartreuse France
Churchland Church USA Seedling
Clairgeau Beurré Clairgeau, Clairgeau de Nantes France
Clapp’s Favourite USA ‘Flemish Beauty’  ‘Bartlett’
Clara Frijs Denmark
Coloree de Juillet France
Concorde UK ‘Conference’  ‘Doyenné du
Comice’
Conference UK
Corella Australia
Coscia Italy
Delbard Premiere France ‘Akca’  ‘Dr. Jules Guyot’
Don Guindo Spain
Doyenne de Juillet Doyenne d’été Belgium
Doyenné du France O.P. seedling
Comice
(continued)
1 Botany and Taxonomy of Pear 21

Table 1.3 (continued)

Cultivar Synonyms Country Mode of selectiona
Dr. Jules Guyot Limonera France
Duchesse France Chance seedling
d’Angouleme
Earlibrite Clapp Favourite x Russet Bartlett Canada
Eletta Morettini Italy ‘Beurré Hardy’  ‘Passe
Crassane’
Farmingdale USA Chance seedling—O.
P. Anjou?
Flemish Beauty Fondante des Bois, Gros quessois d’été, Belgium Chance seedling
Gros Davy, Poire de Persil
Forelle Trout pear Germany
Gaspard USA
General Leclerc France
Gerburg Germany
Giffard Beurré Giffard France
Glou Morceau Beurré d’Hardenpont Belgium
Gorham USA ‘Bartlett’  ‘Josephine de
Malines’
Harobig Canada
Harovin Sundown Canada ‘Bartlett’  US56112-146
Harrow Crisp Canada ‘Bartlett’  US56112-146
Harrow Delight Canada
Harrow Gold Canada ‘Harvest Queen’  ‘Harrow
Delight’
Harrow Red Canada
Harrow Sweet Canada ‘Bartlett’  ‘Purdue 80-51’
Harvest Queen Canada
Highland USA ‘Bartlett’  ‘Comice’
Hortensia Germany ‘Nordhäuser Winterforelle’ 
‘Clapp’s Liebling’
Huntington USA seedling
Jeanne d’Arc France ‘Beurré Diel’  ‘Doyenne du
Comice’
Joséphine de Belgium
Malines
Jubileer D’Ar Bulgaria ‘Clapp’s Favourite’ 
‘Klementina’
Junsko Zlato Yugoslavia ‘Precoce de Trevoux’ 
‘Doyenne de Juillet’
Kieffer USA P. communis  P. pyrifolia
hybrid
Latgale Latvia ‘Kurzemes Sviesta’ 
‘Clapp’s Favourite’
Laxtons Superb UK ‘Marie Louise’  ‘Bartlett’
Le Conte USA Pyrus hybrid  P. lecontei
P. communis  P. pyrifolia
(continued)
22 M. Quinet and J.-P. Wesel

Table 1.3 (continued)

Cultivar Synonyms Country Mode of selectiona
Louise Bonne Bonne Louise d’Avranches, Louise Bonne France
de Jersey
Luscious USA
Merton Pride UK
(England, 1941)
Moonglow USA
Orcas USA Seedling
Orient USA P. communis  P. pyrifolia
hybrid
Packhams Packham Australia ‘Uvedale St. Germain’ 
Triumph ‘Bartlett’
Passe Crassane France Seedling selection
Pineapple USA P. communis  P. pyrifolia
hybrid
Rocha Portugal Chance seedling
Rosemarie South
Africa
Seckel Honey pear, Sugar pear USA
Starkrimson Red Clapps USA Mutation of ‘Clapp’s
Favourite’
Stinking Bishop Moorcroft, Malvern Hills, Malvern Pear, UK
Choke Pear, Choker
Summercrisp USA Unknown
Taylors Gold New A mutant clone of ‘Comice’
Zealand
Tosca Italy ‘Cossia’  ‘Williams’
Turandot Italy ‘Dr. J. Guyot’  ‘Bella di
Giugno’
Uta Germany ‘Madame Verte’  ‘Beurré
Bosc’
Vicar of de Curé, Belle de Berry, Belle Eloïse, Bon France
Winkfield Papa
Virgouleuse Virgoulette, Paradis d’Hiver, Chambrette, France
etc.
Williams Bartlett, Williams bon chrétien UK Chance seedling
Winter Nelis Bonne de Malines, Colmar Nélis Belgium
a
For those cultivars with blanks denotes unknown mode of selection/identification; O.P.: open pollination

parthenocarpic fruits (Dondini and Sansavini ‘Beurré d’Anjou’ is also known as ‘Anjou’,
2012). It has been brought back into commercial ‘Winter Meuris’, and ‘Nec Plus Meuris’ (Hedrick
orchards for its original elongated shape and et al. 1921). It is a Belgian cultivar
good fruit taste. In addition, it excels in southern developed/identified by Van Mons in 1823. It
European orchards due to its recent market produces medium-sized fruits of good quality
claims (Dondini and Sansavini 2012). that ripen in October–November (Bretaudeau and
1 Botany and Taxonomy of Pear 23

Fauré 1991). The fruit is doliform, yellow, blu- colour when ripe, along with a red blush on
shed heavily with red russet, and borne on a very light-exposed side (Dondini and Sansavini
short thick stems (Fig. 1.5a) (Hedrick et al. 1921; 2012). Flesh is cream-white, with a granular
Dondini and Sansavini 2012). Fruit flesh is yel- texture, slightly scented, juicy, and sugary. Its
lowish white in colour, luscious, buttery, slightly cropping is variable, and it is slightly susceptible
tart, and very sweet (Hedrick et al. 1921). This to internal breakdown (Dondini and Sansavini
cultivar is losing favour in Europe due to diffi- 2012).
culties in its management, size, or productivity, ‘Doyenné du Comice’, also known as
while it is widely grown in North America, South ‘Comice’ and ‘Fondante du Comice’, is a French
America, and South Africa (Dondini and Sansa- cultivar selected in 1849 (Dondini and Sansavini
vini 2012). 2012). It produces large-sized fruits of excellent
‘Concorde’, derived from a cross between quality that ripen in October (Bretaudeau and
‘Conference’ and ‘Doyenné du Comice’, and Fauré 1991). The quality is so good that the fruits
developed at the East Malling Research Station of this cultivar are called by many as the best of
in England (Hessayon 1990). It produces all pears (Hedrick et al. 1921). It is mainly cul-
medium-sized fruits of excellent quality that tivated as espalier trees. The fruit is turbinate and
ripen in October (Hessayon 1990). The fruit is has a pale green-brownish colour that turn lighter
golden green, oftentimes with a golden yellow in colour when approaching full ripeness
russetted spots, and has a vanilla sweet flavour (Fig. 1.5e), very sweet, creamy-coloured flesh,
and a firm texture (Fig. 1.5c) (Hessayon 1990). It along with a juicy and somewhat buttery texture
is known for its tall, elongated neck, along with (Hedrick et al. 1921; Hessayon 1990; Dondini
its firm and dense flesh. ‘Concorde’ is a late and Sansavini 2012). It is a late flowering culti-
flowering cultivar (Hessayon 1990). var (Hessayon 1990). ‘Doyenné du Comice’ is
‘Conference’ is an English cultivar and not very reliable under less than perfect growing
developed/selected at the end of the nineteenth conditions and requires warm temperatures, as
century (Dondini and Sansavini 2012). It pro- well as shelter from strong winds (Hessayon
duces medium-sized fruits of good quality that 1990). Unfortunately, it is losing favour in Eur-
ripen in October (Bretaudeau and Fauré 1991). ope due to difficulties in management of these
The fruit is long, pyriform, green, and prone to trees (Dondini and Sansavini 2012).
smooth russet on the skin, and it is sweet and ‘Forelle’ is a German cultivar, dating back to the
juicy when fully ripe (Fig. 1.5d) (Hessayon end of the seventeenth century, although its origin
1990). ‘Conference’ fruit has a long shelf life is unknown (Hedrick et al. 1921; Dondini and
(Dondini and Sansavini 2012). It is a mid-season Sansavini 2012). It produces small- to
flowering cultivar (Hessayon 1990). ‘Confer- medium-sized fruits of medium quality that ripen in
ence’ is reliable under less than perfect growing the winter (Dondini and Sansavini 2012). The fruit
conditions (Hessayon 1990). It develops is ovoid and has a greenish skin which turns bright
parthenocarpic fruits although pollination yellow, along with flecks of crimson-coloured
ensures a better crop (Hessayon 1990; Quinet spots when fully ripe. The flesh is crisp, firm yet
et al. 2014). It is the European pear par excel- juicy, with bright and candy sweet flavours.
lence and accounts for *32% of European pear ‘Forelle’ is distinguished among other pear fruits of
production (Dondini and Sansavini 2012). its kind by its trout-like specklings from which
‘Coscia’ is an Italian cultivar, developed/ comes the name Forelle, the German word for trout
identified in the 1800s, also known under the (Hedrick et al. 1921). This cultivar has recently
name of ‘Ercolini’ (Dondini and Sansavini found renewed interest as its fruit is pleasingly
2012). It produces medium-sized fruits of good different from other melting flesh types of tradi-
quality that ripen either in July or early August tional pear cultivars (Dondini and Sansavini 2012).
(Bretaudeau and Fauré 1991). The fruit is short, ‘Packham’s Triumph’ originated in Australia
pyriform, and light green turning yellow in at the end of the nineteenth century, and it is
24 M. Quinet and J.-P. Wesel

mainly cultivated in the southern hemisphere (Hessayon 1990). ‘Williams’ is a mid-season

(Bretaudeau and Fauré 1991; Dondini and San- flowering cultivar (Hessayon 1990). This cultivar
savini 2012). It produces medium to large fruits accounts for *13% of the European production
that ripen in October (Bretaudeau and Fauré (Dondini and Sansavini 2012). It is still unsur-
1991). The fruit is pyriform, has a bumpy green passed as the best summer pear cultivar in both
skin, and a sweet juicy flavour (Hessayon 1990; Europe and the Americas (Dondini and Sansavini
Bretaudeau and Fauré 1991; Dondini and San- 2012). It is also the only cultivar used by the
savini 2012). ‘Packham’s Triumph’ is an early canning industry for juice making and for
flowering cultivar. This cultivar is also losing fresh-cut slices, either alone or in fruit salads
favour in Europe due to difficulty in its man- (Dondini and Sansavini 2012).
agement, size, or productivity. Nevertheless, it is
widely grown in North America, South America,
and South Africa (Dondini and Sansavini 2012). 1.4.2 Asian Pear
‘Rocha’ is a Portuguese cultivar from 1840
and accounts for 90% of the production of pears Asian pears constitute a group quite distinct in
in Portugal (Dondini and Sansavini 2012). It aspects of tree and fruit as compared to European
produces very large fruits of excellent quality pear. However, not all characters absent in occi-
that ripen in the fall (Dondini and Sansavini dental species are found in all species of the ori-
2012). The fruit is turbinate and greenish yellow ental group (Hedrick et al. 1921). Among Asian
in colour with some russet (Dondini and Sansa- pears, most common differences, besides region of
vini 2012). It is sweet and fragrant with origin, are found in leaves and calyces (Hedrick
white-yellow flesh, and it can be eaten either et al. 1921). The leaves in most species are
while it is crisp or as it softens. This cultivar markedly acuminate, and their margins are
could also produce parthenocarpic fruits (Don- sharp-serrate or setose-serrate (Hedrick et al.
dini and Sansavini 2012). 1921). Persistent calyx is observed in P. ussurien-
‘Spadona’, also known as ‘Blanquilla’, is a sis, and few persistent calyces are present in
very old cultivar of unknown origin (Dondini P. pyrifolia and P.  bretschneideri (Iketani et al.
and Sansavini 2012). It produces small- to 2012). The main cultivated species are P. pyrifolia,
medium-sized fruits of medium quality that ripen P. ussuriensis, P.  bretschneideri, and
in August (Dondini and Sansavini 2012). The P. sinkiangensis. Flowering date depends on cul-
fruit is pyriform, with a smooth, pale green col- tivars, and fruit maturation ranges between July
our, and sometimes red-tinged when exposed to and October (Bretaudeau and Fauré 1991). Asian
sunlight. Its pulp is white, with a fine to pears reach optimum quality when allowed to
medium-fine texture, and a sugary taste. The fruit ripen on the trees, similar to apples and peaches,
does not have a good shelf life. It is mainly but not to European pears.
cultivated in Southern Europe (Dondini and
Sansavini 2012). 1.4.2.1 Description of the Cultivated
‘Williams’ is an English cultivar, first dis- Species
covered in 1765 by a schoolmaster, Mr. Stair
(Dondini and Sansavini 2012). It is also known Pyrus pyrifolia
as ‘William Bon Chrétien’ and ‘Bartlett’. It
produces large fruits of very good quality that P. pyrifolia is a vigorous, upright, and 7–
ripen towards the end of August or early 15-m-tall tree (Hedrick et al. 1921; eFloras
September (Bretaudeau and Fauré 1991). The 2008). Branchlets are slender, purplish brown or
fruit is pyriform to roundish, pale green to yellow dark brown when old, terete, tawny villous, or
in colour, shapely, along with a sweet and juicy tawny tomentose when young, soon glabrescent,
flesh (Hessayon 1990; Dondini and Sansavini glabrous when old, and sparsely lenticellate
2012). However, its storability is rather poor (Hedrick et al. 1921; eFloras 2008). Leaf buds
1 Botany and Taxonomy of Pear 25

are sharply pointed, plump, and thick at the base, P. pyrifolia hybridizes freely with P. communis,
with scales tomentose at margin and apex and several of these hybrids are important com-
(Hedrick et al. 1921; eFloras 2008). Stipules are mercial cultivars in North America (Hedrick
1–1.5 cm long, caducous, linear-lanceolate, et al. 1921). Hybrid pears are more pyriform, and
membranous with villous and entire margins, and are of much better flavour than those of their
an acuminate apex (eFloras 2008). Leaves mea- oriental parents, and their calyces are either
sure 7–12  4–6.5 cm, are ovate-oblong, some- persistent or deciduous (Hedrick et al. 1921).
times ovate, glabrous, or brown lanate when
young; the leaf base is rounded or subcordate, Pyrus ussuriensis
rarely broadly cuneate; the leaf apex is acute, and
the leaf margin spinulose-serrate (Bretaudeau Trees of P. ussuriensis are 15 m tall (eFloras
and Fauré 1991; eFloras 2008). Flower buds are 2008). Branchlets are yellowish grey to purplish
thick, short, conical, plump, free, and arranged brown when young, yellowish grey, or yellowish
singly on very short spurs (Hedrick et al. 1921). brown when old (Hedrick et al. 1921; eFloras
P. pyrifolia flowers in April (eFloras 2008). 2008). Branches are also glabrous or sparsely
Inflorescences are umbellate-racemose clusters of pubescent, and sparsely lenticellate (eFloras
6–9 white flowers with caduceus bracts (Hedrick 2008). Buds are ovoid with an obtuse apex, and
et al. 1921). Flowers measure 2.5–3.5 cm, are scales are sparsely pubescent or subglabrous at
composed of 5 sepals, 5 petals, 20 stamens, and margins (eFloras 2008). Stipules are caducous,
4–5 carpels, and are borne on slender pedicels of linear-lanceolate, 0.8–1.3 cm long, membranous
3–5 cm (Hedrick et al. 1921; Bretaudeau and with a glandular denticulate margin and acumi-
Fauré 1991). Hypanthium is cupular and abaxi- nate apex (eFloras 2008). Leaves are ovate to
ally glabrous (eFloras 2008). Sepals are 0.6– broadly ovate, glabrous or tomentose when
1.2 cm long, triangular-ovate, and young, soon glabrescent, with a rounded or
long-acuminate with an acuminate apex, and subcordate base, long spinulose-serrate margin,
glandular denticulate margins (Hedrick et al. and a shortly acuminate or caudate-acuminate
1921; eFloras 2008). The abaxial side of sepals is apex. The leaf blade measures 5–10  4–6 cm,
glabrous, and the adaxial side is brown tomen- and the petiole measures 2–5 cm (eFloras 2008).
tose (Hedrick et al. 1921; eFloras 2008). Petals P. ussuriensis flowers in May, and produces
measure about 2 cm, oval, and entire, with a white flowers with a diameter of 3–3.5 cm
short clawed base and a rounded apex (Hedrick (eFloras 2008). Flowers are grouped by 5–7 in
et al. 1921; eFloras 2008). Stamens are half as densely corymb with caducous, membranous,
long as petals (eFloras 2008). Gynoecium is and linear-lanceolate bracts of 1.2–1.8 cm
composed of a 4–5-loculed ovary, with two (Hedrick et al. 1921; eFloras 2008). Inflores-
ovules per locule, usually five glabrous styles cence peduncle and flower pedicel are tomentose
(rarely four), nearly as long as stamens (eFloras when young and soon glabrescent; flower pedicel
2008). In August, P. pyrifolia produce round, is 2–5 cm long (eFloras 2008). Flowers are
slightly pyriform fruits, with a diameter of 2– composed of 5 sepals, 5 petals, 20 stamens, and 5
2.5 cm and a brownish colour, with pale dots and carpels. The hypanthium is campanulate, abaxi-
caduceus sepals (eFloras 2008). Fruiting pedicel ally glabrous, or slightly tomentose (eFloras
is 3.5–5.5 cm long (Hedrick et al. 1921; Iketani 2008). Sepals are triangular-lanceolate, 5–8 mm
et al. 2012). Cultivated fruits are larger with a 5– long, abaxially glabrous, and adaxially tomen-
6 cm diameter (Hedrick et al. 1921). Sand pears tose with margins that are initially glandular
are commonly apple-shaped (Hedrick et al. denticulate and with an acuminate apex (eFloras
1921), and in China and Japan, there are a 2008). Petals are obovate or broadly ovate,
number of pomological cultivars, which, how- glabrous, and measure 1.8  1.2 cm (eFloras
ever, differ from each other, but less than culti- 2008). Stamens are shorter than petals, and are
vars of the European pear (Hedrick et al. 1921). nearly as long as styles (eFloras 2008). The
26 M. Quinet and J.-P. Wesel

gynoecium is composed of a five-loculed ovary Stamens are half as long as petals and as long as
with two ovules per locule and five styles that are styles (eFloras 2008). Gynoecium is composed
sparsely pubescent. Between August and Octo- of 4–5-loculed ovary, with two ovules per locule,
ber, P. ussiriensis produces yellow, subglobose and 4–5 glabrous styles (eFloras 2008). Between
fruits, of 2–6 cm in diameter, with persistent August and September, P.  bretschneideri
sepals, and a pedicel of 1–3 cm (Hedrick et al. produces ovoid or subglobose fruits that are
1921; eFloras 2008; Iketani et al. 2012). yellow with fine dots, and have a diameter of 2–
P. ussuriensis fruits require a ripening period in 2.5 cm (eFloras 2008). Sepals are caduceus, the
order to be edible (Teng et al. 2015). Cultivated fruiting pedicel is glabrous, and 1.5–3 cm long
fruits are much larger than wild fruits, although (eFloras 2008; Iketani et al. 2012). Fruits are
they are usually small and are not the tastiest of much larger under cultivation, are very juicy, and
pears to humans (Iketani et al. 2012; Teng et al. are shaped more like the European pear (Iketani
2015). et al. 2012).

Pyrus  bretschneideri Pyrus sinkiangensis

P.  bretschneideri is a small-sized tree, reach- Trees of P. sinkiangensis are up to 6–9 m tall

ing 5–8 m tall (eFloras 2008). Branchlets are (eFloras 2008). Branchlets are purplish brown or
purplish brown when old, terete, robust, densely greyish brown, terete, glabrous, and white lenti-
pubescent when young, glabrous when old, and cellate (eFloras 2008). Buds are ovoid, with an
sparsely lenticellate (eFloras 2008). Buds are acute apex and pubescent scales at margins
dark purple, ovoid with an obtuse apex, and (eFloras 2008). Stipules are 8–10 mm long,
pubescent scales at margin and apex (eFloras caduceus, linear-lanceolate, membranous, white
2008). Stipules are caducous, linear or tomentose, with an acuminate apex and sparsely
linear-lanceolate, 1–1.3 cm long, membranous, glandular and denticulate margins (eFloras
pubescent with glandular denticulate margin and 2008). Leaves are ovate, elliptic, or broadly
acuminate apex (eFloras 2008). Leaves are ovate ovate, either glabrous or white tomentose when
or elliptic-ovate, densely tomentose when young, young (eFloras 2008). Leaf petiole measures 3–
soon glabrescent with a broadly cuneate base, 5 cm, and leaf blade measures 6–8 cm  3.5–
spinulose-serrate margin, and acuminate apex 5 cm (eFloras 2008). Leaf base is rounded, leaf
(eFloras 2008). The leaf blade is 5–11  3.5– margin is crenate or subentire basally and ser-
6 cm, and the petiole is 2.5–7 cm (eFloras 2008). rulate apically, and leaf apex is shortly acuminate
P. bretschneideri flowers in April, and produce (eFloras 2008). P. sinkiangensis flowers during
umbel-like racemes with 7–10 white flowers, and the month of April, producing white flowers of
caduceus linear bracts of 1.5–3 cm (eFloras 1.5–2.5 cm in diameter, and these are organized
2008). Inflorescence peduncle is tomentose when in umbel-like racemes of 4–7 flowers (eFloras
young, soon glabrescent, and flower pedicel is 2008). The inflorescence peduncle and flower
pubescent and 1.5–3 cm long (eFloras 2008). pedicel are tomentose, when young, and
Flowers are 2–3.5 cm in diameter, and are glabrescent; the flower pedicel is 1.5–4 cm in
composed of 5 sepals, 5 petals, 20 stamens, and length. Bracts are 1–1.3 cm long, caducous,
4–5 carpels (eFloras 2008). Hypanthium is linear-lanceolate, membranous with long
cupular, slightly pubescent when young. Sepals tomentose margins, sparsely glandular, denticu-
are triangular, 3.5–5 mm long, abaxially glab- late, and with an acuminate apex (eFloras 2008).
rous, and adaxially brown tomentose with Flowers are composed of 5 sepals, 5 petals, 20
a glandular denticulate margin and acuminate stamens, and 5 carpels. The hypanthium is
apex (eFloras 2008). Petals are ovate with a cupular and abaxially glabrous (eFloras 2008).
shortly clawed base and rounded apex; sepals Sepals are triangular-ovate, 6–7 mm long, abax-
measure 1.2–1.4  1–1.2 cm (eFloras 2008). ially brown, tomentose, with an acuminate apex,
1 Botany and Taxonomy of Pear 27

and glandular denticulate margins (eFloras moderate eating quality, and a tough and gritty
2008). Petals measure 1.2–1.5  0.8–1 cm, texture. It has a high sugar content and a medium
obovate, shortly clawed at base, and obtusely low acid content. Fruits could be stored up to five
rounded at the apex (eFloras 2008). Stamens are, months (NSW 2017).
at a maximum, half as long as petals (eFloras ‘Hosui’ is a Japanese cultivar of P. pyrifolia
2008). The gynoecium contains a five-loculed that resulted from a cross between ‘Kosui’ and
ovary with two ovules per locule and five styles, ‘Hiratsuka 1’, although it has been previously
but not exceeding the number of stamens (eFlo- reported as a progeny of hybridization of ‘Ri-14’
ras 2008). P. sinkiangensis produces fruits in and ‘Yakumo’ (Saito 2016; NSW 2017). It is
August and September (eFloras 2008). Wild also known as ‘Housui’ (Saito 2016). This cul-
fruits are yellowish green, either ovoid or obo- tivar produces round, medium- to large-sized
void, with persistent sepals, and measure 2.5– fruits that ripen early to mid-September, 135–
5 cm in diameter (eFloras 2008). Fruiting pedicel 145 days after full bloom (Saito 2016; NSW
is 4–5 cm long, thickened distally, and glabres- 2017). The fruit is golden brown, russetted, along
cent (eFloras 2008). Cultivated fruits vary con- with conspicuous white lenticels. It has an
siderably, and combine characteristics of both excellent eating quality with high sugar and acid
P. communis and P. bretschneideri (Jun and contents and a fine-grained texture (NSW 2017).
Hongsheng 2002). Generally, the fruit shape is The flesh is crisp and juicy (Saito 2016). Fruit
more similar to P. communis, but with a long has a good keeping quality and can be stored for
pedicel. Some cultivars bear fruit with a persis- 3–4 months. ‘Hosui’ is a mid-season flowering
tent calyx, have a strong aroma, and require a pear (NSW 2017).
ripening period before they are edible, similar to ‘Huang Hua Li’ is a Chinese cultivar of
P. communis, while others are juicy and crisp, P. pyrifolia (Jun and Hongsheng 2002). It pro-
and do not require ripening as that for duces medium to large-sized round fruits that
P.  bretschneideri. ripen in mid-August (Jun and Hongsheng 2002).
Fruits have a smooth and yellow-brown skin
1.4.2.2 Asian Pear Cultivars colour.
There are several species and cultivars that are ‘Kikusui’ is a Japanese cultivar of P. pyrifolia,
cultivated as Asian pears (Table 1.4). The Japa- developed in 1927 from a cross between ‘Tai-
nese cultivars tend to be more round in shape, haku’ and ‘Nijisseiki’ (NSW 2017). It is also
while Chinese cultivars are more oval or pyri- known as the ‘twenty-first century’. It produces
form (pear-shaped). China accounts for most of oblate medium-sized fruits of good quality that
the world’s Asian pear production with tend to be lopsided. The fruit is yellowish green
P.  bretschneideri cultivars ‘Dong Shan Su in colour, tender, but cracks following a heavy
Li’, ‘Ya Li’, and ‘Huang Hua Li’ dominating rain (NSW 2017). It has a high sugar and acid
production (Bassil and Postman 2010). Pyrus contents. Fruits ripen mid-season, 135–145 days
pyrifolia cultivars ‘Kosui’ and ‘Hosui’ make up after full bloom, and can be stored up to five
to 65% of the production area in Japan, followed months (NSW 2017). ‘Kikusui’ flowers mid- to
by ‘Nijisseiki’ and ‘Niitaka’, while ‘Niitaka’ is late season, and it is partially self-fertile (NSW
the primary cultivar in Korea (Bassil and Post- 2017).
man 2010). Some of these cultivars are described ‘Kosui’, also known as ‘Kousui’, is a Japa-
below. nese cultivar of P. pyrifolia that has originated
‘Chojuro’ is a Japanese cultivar of P. pyrifolia from a cross between ‘Kikusui’ and ‘Wasekozo’
(NSW 2017). It has an early to mid-flowering (Saito 2016). It produces orbicular to oblate fruits
season, and it is partially self-fertile. It produces that ripen near middle to late August (Saito
oblate fruits of medium size that ripen 135– 2016). The fruit is orange in colour, over a
150 days after full bloom. The fruit is golden greenish yellow background, along with a par-
brown, fully russetted, and has a poor to tially russetted skin. Fruit flesh is soft, juicy, and
28 M. Quinet and J.-P. Wesel

Table 1.4 List of some Asian pear cultivars (Flores 1999; Bassil and Postman 2010; Yue et al. 2014; Saito 2016;
Chang et al. 2017; NSW 2017)
Cultivar Species Country Mode of selection*
Akizuki P. pyrifolia Japan Niitaka  ‘Hosui  Kosui
Arirang P. pyrifolia Korea
Atago P. pyrifolia Japan
Autumn Sweet
Ba Li Xiang [Ba Li P.  bretschneideri China
Hsiang]
Bong Ri P. pyrifolia, x Korea P. pyrifolia, Nijisseiki 
P.  bretschneideri P.  bretschneideri
Cheih Li P.  bretschneideri China
Chien Li P.  bretschneideri China
Chien Pa Li P. ussuriensis China
Chinfon Li P.  bretschneideri China
Choju P. pyrifolia Japan Asahi  Kimizukawase
Chojuro (Choujuurou) P. pyrifolia Japan Chance seedling
Cili P. pyrifolia China
Daisui Li
Dan Bae P. pyrifolia x P. ussuriensis Korea P. pyrifolia Chojuro  P. ussuriensis
Dangshan Suli P.  bretschneideri China
Dasui Li U.C. hybrids
Gold Nijisseiki P. pyrifolia Japan
Haeng Soo P. pyrifolia Korea P. pyrifolia, Kikuchi  Joseng Henjang
Hansen Siberian Pear P. ussuriensis China
Hakko P. pyrifolia Japan Yakumo  Kosui.
Harbin P. ussuriensis China
Hosui P. pyrifolia Japan Kosui  Hiratsuka 1
Huagai P. ussuriensis China
Hung Li P.  bretschneideri China
Huiyangqingli P. pyrifolia China
Huiyangsuanli P. pyrifolia China
Imamuraaki P. pyrifolia Japan
Jianbali P. ussuriensis China
Jinchuanxueli P. pyrifolia Japan
Kikusui P. pyrifolia Japan Taihaku  Nijisseiki
Kosui (Kousui) P. pyrifolia Japan Kikusui  Wasekozo
Manyuanxiang P. ussuriensis China
Meigetsu P. pyrifolia Japan Chance seedling
Nanguoli P. ussuriensis China
Nansui P. pyrifolia Japan
Niitaka P. pyrifolia Japan Amanogawa  Imamuraaki
(continued)
1 Botany and Taxonomy of Pear 29

Table 1.4 (continued)

Cultivar Species Country Mode of selection*
Nijisseiki (twentieth P. pyrifolia Japan Chance seedling
centyry)
Nijisseki (twentieth P. pyrifolia Japan Chance seedling
century)
Okusankichi P. pyrifolia Japan Old variety
Olympic
Pa Li P. ussuriensis China
Pai Li (Beijing white P.  bretschneideri China Old selection from Beijing region
pear)
Ping Guo Li (Pingo Li) P.  bretschneideri China Old selection from Jilin Province
Seigyoku P. pyrifolia Japan Nijiseiki  Chojuro
Seuri Li P. pyrifolia China
Shen Li China
Shin Go P. pyrifolia Korea Cheonjichon x Imamuraaki
Shin Li U.C. hybrids
Shinko P. pyrifolia Japan Nijisseiki  Amanogawa
Shin-Soo P. pyrifolia Korea Kikuchi  Kimizukawase
Shinseiki P. pyrifolia Japan Nijiesiki  Chojuro
Shinsei P. pyrifolia Japan Suisei x Shinko
Shinsui P. pyrifolia Japan Kikusui  Kimizukawase
Singo P. pyrifolia Korea
(Japan)
Tang Li P. ussuriensis China
Tse Li P.  bretschneideri China
Tsu Li P.  bretschneideri China Probably P. ussuriensis and
P.  bretschneideri
Xiangshui Li (Hsiang P.  bretschneideri China
Sui Li)
Xuehuali P.  bretschneideri China
Ya Li P.  bretschneideri China Old variety
Yakumo P. pyrifolia Japan Nijisseiki  Akaho
*For those cultivars with blanks denotes unknown mode of selection/identification

sweet, along with a very fine texture (Saito (Saito 2016). The off-white flesh is sweet and
2016). ‘Kosui’ is the principal cultivar in Japan juicy, but a bit coarser than other Asian pears
(Saito 2016). (Saito 2016).
‘Niitaka’ is a Japanese cultivar of P. pyrifolia, ‘Shinseiki’ is a Japanese cultivar of P. pyrifolia,
resulting from a cross between ‘Amanogawa’ developed from a cross between ‘Nijiseiki’ and
and ‘Chojuro’ (Saito 2016). It produces ‘Chojuro’ (NSW 2017). It produces flat-round
large-sized fruits of long shelf life (Saito 2016). fruits of medium size that ripen 125 days after full
The fruit is orbicular, orange-brown in colour, bloom. The fruit is yellow-green in colour, very
along with brown russeting, and it ripens begin- smooth, tender, and bruises rather easily (NSW
ning at the end of September to early October 2017). The flesh is juicy, mild-flavoured, along
30 M. Quinet and J.-P. Wesel

Fig. 1.6 Fruits of the Asian

pear cultivar ‘Ya-Li’

with a medium sugar and high acid contents (NSW species. Further investigations are required to
2017). Fruit storage is short, not exceeding two better understand the complex evolutionary his-
months. ‘Chojuro’ flowers late, and it is partially tories and relationships among species of Pyrus.
self-fertile (NSW 2017). Pear species could be divided into an eastern
‘Tsu-Li’ is an old Chinese cultivar that most Asian clade and a western Eurasian clade. In both
likely has resulted from a cross between clades, there are some species that are cultivated,
P. ussuriensis and P.  bretschneideri (NSW including the European pear P. communis and
2017). It produces ovate pyriform fruits of the Asian pears P. pyrifolia, P. ussuriensis,
medium to large size that ripen late, about 176– P.  bretschneideri, and P. sinkiangensis. There
189 days after full bloom (NSW 2017). The fruit are thousands of pear cultivars that are available
is light green to yellow-green in colour and may all over the world, with diverse fruit shape, taste,
have ugly lenticel spotting. It has a good eating and texture. However, only a few of these culti-
quality and contains some stone cells (NSW vars contribute to most of the world production
2017). It has a sweet taste with a trace of tartness of pears nowadays. Several pear breeding pro-
and has a high sugar content along with a mod- grams have been involved in developing new
erate acid content. Fruits can be stored for up to commercial cultivars. Undoubtedly, sequencing
six months at 0–1 °C (NSW 2017). and annotation of the pear genome, of both
‘Ya-Li’ is an old Chinese cultivar of European and Asian pears, will help scientists
P.  bretschneideri (NSW 2017). It produces and breeders in better understanding the genetics
turbinate to globular, acute, pyriform fruits of of pear and in making advances to develop
medium to large size and that ripen 175– improved genotypes with high fruit nutritional
190 days after full bloom. The fruit is pale yel- quality and tolerance to biotic and abiotic
lowish green, shiny, and has a good to excellent stresses.
eating quality with medium sugar and acid con-
tents and mildly sweet taste (Fig. 1.6) (NSW
2017). ‘Ya-Li’ flowers very early. In China, References
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 Pear Germplasm Needs
and Conservation 2
Joseph Postman

Abstract sequence repeat (SSR), markers, and

Pear (Pyrus) species are sources of food, chloroplast-derived markers are commonly
drink, landscape trees, and rootstocks. Differ- used to accomplish these tasks. Markers can
ent Pyrus species possess varied genetic traits also be used for pedigree analysis, which may
that render them useful for diverse purposes. either confirm or detect anomalies in pedi-
Pear genebanks preserve cultivars, or unique grees of genebank accessions. Advances in
genotypes, as grafted trees. They also store breeding, developing genetic markers, and
seedlots and seedling populations that may identifying major genes in pear cannot be
represent pear wild relative species. Seed and accomplished without access to diverse living
seedling collections usually represent species collections of Pyrus germplasm.
populations from distinct geographic locations
rather than unique genotypes. In the USA, the
USDA Agricultural Research Service’s
National Plant Germplasm System maintains 2.1 Commercial Uses of Pears
a genebank in Corvallis, Oregon, representing
world diversity for Pyrus that includes more Pears are produced commercially in mid-latitude
than 2500 unique clones or seedlots. Other temperate regions throughout the world, despite
pear genebanks around the world tend to be the fact that there are no Pyrus species native to
more specialized, focusing on accessions North America or from anywhere in the southern
native to the region or in support of focused hemisphere. Top pear producing countries, with
breeding programs. Molecular techniques and >400,000 metric tons harvested in 2016, are
genetic markers have become valuable tools China, Argentina, USA, Italy, Turkey, and South
for pear genebank management. Various types Africa. An additional 16 countries produced
of molecular markers can be used to assess >100,000 metric tons (Table 2.1; FAO 2018).
genetic diversity, identify gaps in germplasm Two distinct centers of origin or centers of
collections, and help detect redundancy and wild diversity are recognized for the genus
confirm synonymy. Microsatellite, or simple Pyrus, the Caucasus Mountains and China.
European pear species belong to section ‘Pyrus’
of the genus Pyrus (Table 2.2; USDA-ARS
2018a). These originated in regions around the
J. Postman (&)
USDA-ARS National Clonal Germplasm
Caucasus Mountains between the Black Sea and
Repository, 33447 Peoria Road, Corvallis, the Caspian Sea. The taxa Pyrus communis
OR 97333, USA ssp. caucasica (Fed.) Browicz, P. communis
e-mail: [email protected]

This is a U.S. government work and not under copyright protection in the U.S.; 35
foreign copyright protection may apply 2019
S. S. Korban (ed.), The Pear Genome, Compendium of Plant Genomes,
https://doi.org/10.1007/978-3-030-11048-2_2
36 J. Postman

Table 2.1 Pear yield in metric tons (MT) from 2010 to 2016 in countries producing >100,000 MT in 2016 (FAO
2018)
2010 2011 2012 2013 2014 2015 2016
China (mainland) 15,057,000 15,795,000 17,073,000 17,300,752 17,964,400 18,699,000 19,388,063
Argentina 670,000 812,633 825,115 890,000 840,000 869,000 905,605
USA 738,085 876,087 778,583 795,692 754,415 744,345 738,770
Italy 736,646 926,542 645,540 743,029 701,558 753,667 701,928
Turkey 380,003 386,382 442,646 461,826 462,336 463,623 472,250
South Africa 368,495 350,527 338,584 364,854 404,260 394,450 433,105
India 336,000 335,000 340,000 325,000 316,700 303,000 399,000
Netherlands 274,000 336,000 199,000 327,000 349,000 349,000 374,000
Spain 476,686 502,434 407,428 425,700 429,548 355,410 366,131
Belgium 307,270 284,827 236,400 305,000 374,300 374,630 331,550
Chile 181,387 196,743 199,247 226,189 240,399 280,870 299,432
Japan 284,900 312,800 299,000 294,400 295,100 276,500 278,100
Iran 121,012 126,115 129,317 140,090 279,580 285,000 254,599
Korea (South) 307,820 290,494 172,599 282,212 302,731 260,975 238,014
Algeria 234,274 233,147 211,191 240,709 228,114 255,344 211,943
Ukraine 141,700 153,100 157,500 169,400 157,690 170,610 156,000
Korea (North) 137,971 143,000 147,000 145,000 144,569 145,963 146,601
Portugal 176,764 230,447 116,287 202,483 210,009 141,186 137,805
France 146,552 162,905 117,262 142,923 132,588 140,833 129,627
Taiwan 174,858 150,013 137,911 109,105 134,549 127,016 111,424
Australia 95,111 123,267 119,274 109,206 98,035 105,243 104,928
Uzbekistan 72,700 68,796 74,000 80,000 87,000 95,000 100,948

ssp. pyraster (L.) Ehrh., and P. nivalis Jacq. overlap, ancient natural hybrids between P. com-
are the primary ancestors of large-fruited Euro- munis and P. pyrifolia, known as P.  sinkian-
pean pears (P. communis L.) (Fig. 2.1). gensis T.T. Yu, have been selected for their large
Asian pears belong to section ‘Pashia’ of the fruit. A commercial industry in the Xinjiang
genus. Asian pear species have a more ancient region produces fruit marketed as the ‘Chinese
center of diversity in the region around Zhejiang Fragrant Pear’ or the ‘Korla’ pear.
Province in China (Tenga et al. 2015). For a detailed review of the ancient geo-
Large-fruited Asian pears are primarily derived graphic origins and ancestral relationships of
from P. pyrifolia (Burm. f.) Nakai, Pyrus taxa, please refer to Chap. 4 of this volume
P.  bretschneideri Rehd., and P. ussuriensis by Volk and Cornille.
Maxim., as well as complex hybrids with other
species. In the Indian subcontinent, large-fruited
pears are derived from hybrids between P. pashia 2.1.1 Pears for Food
Buch.-Ham. ex D. Don and both European and
East Asian pears (Table 2.2; Fig. 2.1; Pear cultivars commercially grown for their fruit
USDA-ARS 2018a). In far-west China where the are valued for traits related to fruit quality and
range of European and Asian pear wild relatives tree architecture that are amenable to efficient
2 Pear Germplasm Needs and Conservation 37

Table 2.2 Pyrus species recognized by the USDA National Plant Germplasm System (USDA-ARS 2018a)
Taxon Genebank Pyrus Native origin
accessions section
Pyrus armeniacifolia T. T. Yu 0 Pashia
Pyrus betulifolia Bunge 65 Pashia China, Laos
Pyrus boissieriana Buhse 0 Pyrus Azerbaijan, Turkmenistan, Iran
Pyrus  bretschneideri Rehder (2) 24 China
Pyrus calleryana Decne. 111 Pashia China, Korea, Taiwan, Vietnam, naturalized in
North America
Pyrus  canescens Spach (3) 1 Pyrus
Pyrus communis L. 1011 (a) Pyrus Caucasus, Middle Asia, Western Asia, Europe,
widely naturalized
Pyrus communis subsp. caucasica 77 Pyrus Caucasus, Turkey, Ukraine
(Fed.) Browicz
Pyrus communis subsp. pyraster 83 Pyrus Turkey, Europe
(L.) Ehrh.
Pyrus  complexa Rubtzov 2 Pyrus Armenia, Azerbaijan
Pyrus cordata Desv. 22 Pyrus UK, France, Portugal, Spain
Pyrus cossonii Rehder 4 Pashia Algeria
Pyrus dimorphophylla Makino 19 Pashia Japan
Pyrus elaeagrifolia Pall. 31 Pyrus Turkey, Ukraine, Southeastern Europe
Pyrus fauriei C. K. Schneid. 34 Pashia South Korea
Pyrus gharbiana Trab. 8 Pyrus Algeria, Morocco
Pyrus glabra Boiss. 1 Pyrus Iran
Pyrus hondoensis Nakai and 41 Pashia Japan
Kikuchi
Pyrus  hopeiensis T. T. Yu 0 Pashia China
Pyrus hybrid 216
Pyrus koehnei C. K. Schneid. 16 Pashia China
Pyrus korshinskyi Litv. 5 Pyrus Kyrgyzstan, Tajikistan, Uzbekistan,
Afghanistan
Pyrus  lecontei Rehder 0
Pyrus mamorensis Trab. 15 Pyrus Morocco
Pyrus  michauxii Bosc ex Poir. 0
Pyrus  neoserrulata I. M. Turner 0 China
Pyrus  nivalis Jacq. (4) 20 Turkey, Europe
Pyrus pashia Buch.-Ham. ex D. 41 Pashia China, Afghanistan, Iran, Indian subcontinent,
Don Indo-China
Pyrus  phaeocarpa Rehder (5) 2 China
Pyrus pseudopashia T. T. Yu 2 Pashia China
Pyrus pyrifolia (Burm. f.) Nakai 147 (b) Pashia China, Laos, Vietnam, Naturalized in Japan
Pyrus regelii Rehder 12 Pyrus China, Kyrgyzstan, Tajikistan
Pyrus sachokiana Kuth. 2 Pyrus Georgia
(continued)
38 J. Postman

Table 2.2 (continued)

Taxon Genebank Pyrus Native origin
accessions section
Pyrus salicifolia Pall. 36 Pyrus Armenia, Azerbaijan, Iran, Turkey
Pyrus  sinkiangensis T. T. Yu (6) 7 China
Pyrus spp. [accessions unidentified 88
to species]
Pyrus spinosa Forssk. 57 Pyrus Turkey, Southeastern Europe, France, Spain
Pyrus syriaca Boiss. 14 Pyrus Armenia, Western Asia
Pyrus taiwanensis Iketani and H. 0 Pashia Taiwan
Ohashi
Pyrus trilocularis D. K. Zang and 0 Pashia China
P. C. Huang
Pyrus turcomanica Maleev 0 Pyrus Iran, Kyrgyzstan, Tajikistan, Turkmenistan
Pyrus ussuriensis Maxim. 94 (c) Pashia China, Japan, Russian Federation
Pyrus  uyematsuana Makino (7) 1 Japan, Korea
Pyrus xerophila T. T. Yu 2 Pashia China
Number of USDA genebank accessions, taxonomic section, and countries of native origin (USDA-ARS 2018a)
(1) Subtaxa not included, except for P. communis
(2) P.  bretschneideri = cultivated Chinese white pear is a complex hybrid, predominantly of P. pyrifolia
(3) P.  canescens = P.  nivalis  P. salicifolia
(4) P.  nivalis = P. communis  P. elaeagrifolia
(5) P.  phaeocarpa probably = P. betulifolia  P. ussuriensis
(6) P.  sinkiangensis is a complex hybrid involving P. communis, P. armeniacifolia, and P. pyrifolia
(7) P.  uyematsuana probably = P. dimorphophylla  P. ussuriensis
(a) Includes 985 European pear cultivars
(b) Includes 89 Asian pear cultivars
(c) Includes 49 Asian pear cultivars

Fig. 2.1 Diversity of Pyrus

germplasm
2 Pear Germplasm Needs and Conservation 39

production. Breeders seek genetic traits to for perry production. The presence of hard stone
increase fruit quality, size, and productivity, as cells in fruits of many pear species limits their
well as disease and insect resistance. Further- use in breeding fruit for eating, but has no impact
more, precocity, appropriate flowering and on fermented juice products.
fruiting seasons, and maintaining quality during
storage are also important. Resistance to the
insect pear psylla (Cacopsylla pyricola (Forster)) 2.1.3 Pears for Ornament
and to diseases fire blight (Erwinia amylovora
(Burrill) Winslow et al.), Fabraea (Entomospo- A third use of Pyrus germplasm is as ornamental
rium) leaf spot (Entomosporium mespili (DC.) trees. While fruit of pear species grown for food
Sacc.), and pear scab (Venturia pirina Aderh.) is must be large and flavorful, species with small,
particularly important for improving pear pro- obscure, and unpalatable fruit are valued in the
duction. Breeding for these traits has long been urban landscape. Numerous cultivars and selec-
major objectives of the USDA pear breeding tions of the Callery pear (P. calleryana Decne.)
program at Kearneysville, West Virginia, as well have been introduced to the nursery trade as
as at other pear improvement programs world- flowering street trees, many originating from
wide (Brewer and Palmer 2011; Pyrus CGC germplasm collected in China by USDA plant
2004). explorer Frank Meyer at the start of the twentieth
century (Meyer 1922). Although cultivars of
P. calleryana with profuse early spring displays
2.1.2 Pears for Drink of white flowers and stunning fall colors are
some of the most widely planted flowering trees
Fermented pear cider, perry, is rapidly increasing in North America, in recent years, profuse
in popularity in the USA and abroad. Hard cider reseeding of these cultivars has rendered them
was popular during colonial times in the USA, undesirable in some locations (Culley 2017).
and much earlier in Europe. In recent years, there Selections of the willow-leaf pear (P. salicifo-
has been a major revival in locally crafted beer, lia Pall.) are appreciated in the landscape for their
cider, and perry. Many new accessions of perry fine texture, gray, pubescent foliage, and some-
pear cultivars have been introduced into the USA times weeping growth habit (Dirr 1997). Other
from Europe in recent years, especially from pear species, including P. betulifolia Bunge,
England, to meet this increased demand. Genetic P. dimorphophylla Makino, P. elaeagrifolia Pall.,
traits required for perry pears are somewhat dif- P. regelii Rehder, and P. syriaca Boiss., have
ferent from those traits selected for fruit pear striking foliage, unusual flowers, or unique
consumption. Cultivars of both groups should environmental adaptations. These species should
have high fruit production, but, like a good wine be evaluated for landscape use. Although wood of
grape, the fruit of a perry pear must contain high various Pyrus species is used as material for
levels of acids and tannins, combined with good furniture, musical instruments, and kitchen
flavor that is retained throughout the fermenta- implements, there have been no deliberate efforts
tion process. In contrast, fruit with high tannin to select varieties for genetic traits desirable for
content is deemed undesirable for fresh con- these purposes.
sumption. Fruits produced for the fresh market
must also be attractive; whereas perry pears are
pulverized and pressed for their juice, thus fruit 2.1.4 Pears for Rootstocks
appearance is not critical. Most perry pears are
selections from the species P.  nivalis Jacq., In the USA, commercial pear production has
although many other pear wild relatives have declined in recent decades. Between 2011 and
fruit with high tannins along with a range of 2016, pear production dropped from 875,000 to
interesting flavors that have not yet been tapped 739,000 metric tons in the USA (FAO 2018;
40 J. Postman

Table 2.1). The US pear industry attributes lower (EMA), EMC, EMH, and Sydo (Elkins et al
production to declining consumption, higher 2012; Wertheim 2002). Unfortunately, there is
production cost relative to other tree fruits, and lack of suitable dwarfing rootstocks available in
competition from imported fruit. An important Asian pear production areas. Seedlings of
factor in higher production costs is lack of root- P. betulifolia, P. ussuriensis, and sometimes
stock options (Elkins et al. 2012). P. pyrifolia are used as rootstocks for Asian pears
As with most fruit trees, pear cultivars are in northern China. Seedlings of P. calleryana and
vegetatively propagated by grafting. The above P. pyrifolia are the primary rootstocks in south-
ground portion of a tree (fruiting cultivar) pro- ern China. The use of these rootstocks for
duces fruit, while the below ground portion high-density plantings results in excessive vigor
(rootstock) anchors the tree and takes up water and and contributes to high maintenance costs and
nutrients. Often, rootstocks have very different poor yield (Teng 2011). In Japan, where seed-
genetic traits than fruiting cultivars. Except in lings of P. betulifolia and P. calleryana are the
cases of a few naturally compact genotypes, the primary rootstocks, vigor control of Asian pears
overall size of a mature grafted pear tree is highly is also a challenge. Research is underway in
influenced by the rootstock. Unlike apples, which Japan to develop rootstocks that combine
have many choices of size-controlling rootstocks, dwarfing, ease of propagation, and adaptation to
ranging from very dwarf to very vigorous, pears local environmental conditions, but none are yet
have limited rootstock options. Currently, seed- available for commercial use (Tamura 2012).
lings of P. communis and occasionally P. betuli- One of the greatest needs of the US pear
folia are the most common pear rootstocks used in industry is a greater diversity of stress-resistant
the USA. Moreover, seedlings of P. calleryana are rootstocks that will promote dwarfing, precocity,
also used as rootstocks in warmer regions. Clonal and productivity of fruiting cultivars (Elkins et al.
rootstocks derived from crosses between fire 2012). Every pear species is potentially graft
blight-resistant pear cultivars Old Home (OH) and compatible with every other pear species, and
Farmingdale (F) are becoming popular in the some originate from regions with very diverse
USA, with selections OH  F 87 and OH  F 97 climates, soils, and biotic or abiotic stresses. The
being the most widely used. ‘Pyrodwarf’ and wide range of adaptation to various soil types,
‘Pyro 2-33’ from Germany, also derived from temperature, moisture, pH, and nutrients as well
crosses with ‘Old Home,’ are available in the USA as to soil-born insects, nematodes, and diseases
as well. These two selections are more dwarfing of Pyrus species suggests that there are many
than OH  F selections, but may have other unexplored opportunities to identify improved
shortcomings that will limit their use. pear rootstocks (Lombard and Westwood 1987).
Most pear production areas in Western Europe It is necessary to preserve pear genetic
depend on high-density plantings, thereby resources not only for their potential to develop
requiring dwarfing rootstocks, as various OH improved cultivars for fresh fruit and perry pro-
F rootstock clones are too vigorous. Quince duction, but also for unique uses in the landscape
(Cydonia oblonga) rootstock cultivars are the and for improved rootstocks.
only options for adequate vigor control in this
case. However, some pear cultivars are incom-
patible when grafted directly onto quince; 2.2 Pear Germplasm Conservation
therefore, a compatible interstem is required.
Research is ongoing in several European coun- Advances in basic taxonomy research, breeding
tries to develop better, productive, and dwarfing new cultivars, developing genetic markers, and
pear rootstocks from Pyrus species; however, identifying major genes in pear cannot be
except for ‘Pyrodwarf,’ none are in wide use. accomplished without access to diverse living
Currently, rootstocks in production in Europe collections of Pyrus germplasm. Accessing
include quince clones BA29, East Malling A germplasm for breeding or tissue for genetic
2 Pear Germplasm Needs and Conservation 41

analysis directly from wild populations or dis- along with smaller collections of local pear
persed production areas is very expensive and varieties maintained at Vladivostok, Volgograd,
time-consuming. Fortunately, ex situ germplasm and Pavlovsk (Maggioni et al 2004). Many
collections are available to provide ready access non-government organizations throughout the
to needed genetic diversity with ‘one-stop- world also maintain significant pear germplasm
shopping’ convenience. In North America, a collections.
large pear collection is maintained in the USA by
the USDA Agricultural Research Service
(ARS) as part of the National Plant Germplasm 2.2.1 USDA-NPGS ‘Clonal’
System (NPGS) and represents worldwide Pyrus Repositories
diversity (Postman et al. 2006). The Canadian
Clonal Genebank in Harrow, Ontario, maintains Prior to 1980, fruit and nut germplasm collections
about 100 pear accessions of interest to that in the USA were largely assembled and main-
country (AAFC 2018). tained by individual plant breeders at universities
Large pear germplasm collections in Western or research institutes and were often lost when a
Europe are located at the National Fruit Collec- faculty member or a scientist retired, changed
tion at Brogdale Farm in Kent, England; Centre their research focus, or encountered funding
Wallon de Recherches Agronomiques in Gem- shortfalls. In the 1970s, a national plan was pro-
bloux, Belgium; Le Centre INRA Angers-Nantes posed to establish a series of US germplasm
in France; Federal Research Centre for repositories with perpetual federal funding to
Cultivated Plants in Dresden-Pillnitz, Germany; provide security and stability for collections of
and University of Bologna in Bologna, Italy horticultural crops (Brooks and Barton 1977)
(Morgan 2015). which would augment the existing germplasm
In the Czech Republic, Pyrus genetic resour- collections maintained primarily as seeds. These
ces are maintained at the Research Breeding collections of fruit and nut crops have been tra-
Institute of Pomology, Holovousy; in Greece at ditionally maintained as ‘clonal’ collections, as
the NAGREF Pomology Institute, Naoussa; in cultivars are propagated by clonal techniques,
Hungary at the Research and Extension Centre such as grafting, runners, or cuttings, and main-
for Fruit Growing, Újfehértó; in Denmark at the tained as living trees, not as seed. The ‘clonal’
Royal Veterinary and Agricultural University, genebanks often maintain collections of seeds
Copenhagen; in Finland at Agrifood Research; in too, representing populations of wild relative
Norway at Planteforsk-Njos; in Sweden at SLU species. The first of what was to become a net-
Balsgård; in Poland at the Research Institute of work of eight National Clonal Repositories was
Pomology, Skierniewice; in Portugal at Chaves; established in Corvallis, Oregon, in 1980 to house
in Slovenia at Ljubljana; in Spain at Servicio de collections of 26 genera of specialty fruit and nut
Investigación Agroalimentaria, Saragossa; in crops, including Pyrus (Jahn and Westwood
Yugoslavia at the Center for Fruit Growing, 1982; Postman et al. 2006; Westwood 1982).
Čačak and Faculty of Agriculture, Belgrade. The National Clonal Germplasm Repository
In Asia, Pyrus genetic resources are main- (NCGR) in Corvallis is part of the National Plant
tained at the Zhengzhou Fruit Research Institute Germplasm System (NPGS). The mission of the
in Henan Province, China, and NARO Institute NPGS is to support agriculture by collecting,
of Fruit Tree Science in Tsukuba, Japan (Morgan conserving, characterizing, documenting, and
2015). In South Korea, the National Institute of distributing crop plant germplasm (USDA-ARS
Horticultural Science in Naju maintains large 2018b).
collections of mostly Asian pears (NIHHS 2016). When the NCGR was first established in the
In the Russian Federation, there are important 1980s, several large pear germplasm collections
Pyrus collections maintained at the Vavilov from around the USA were consolidated at the
Research Institutes in St. Petersburg and Maikop, Oregon site (Postman et al. 2010). Collections of
42 J. Postman

Pyrus species assembled in support of pear collection at the NCGR in Corvallis (Postman
rootstock research, collections of heirloom pear 2008). This location has an ideal climate for a
cultivars, along with sources of fire blight resis- living pear genebank with mild winters and dry
tance from the USDA pear breeding program summers resulting in low incidence of diseases,
served as the foundations of this collection including fire blight. The NCGR maintains
(Westwood 1982). approximately 2200 clonal accessions of pear, as
well as 400 seedlots representing 36 Pyrus taxa
(Table 2.2) originating from 55 countries
2.2.2 NPGS Pyrus Collection (Table 2.3). Pear wild relatives are more effi-
ciently and economically maintained either as
The most genetically diverse collection of world seed or as small populations of seedlings. About
pear germplasm is very likely the NPGS pear 20% of the clonal collection is backed-up onsite,

Table 2.3 USDA Pyrus germplasm accessions by country of origin (USDA-ARS 2018c)
Country Count Country Count
Afghanistan 2 Montenegro 3
Albania 32 Morocco 22
Armenia 45 Nepal 15
Australia 21 Netherlands 9
Azerbaijan 2 New Zealand 2
Belgium 51 Pakistan 49
Bulgaria 9 Poland 27
Canada 41 Portugal 4
China 127 Romania 31
Czech Republic 15 Russian Federation 70
Denmark 4 Serbia 19
Estonia 2 South Africa 9
France 180 Spain 2
Georgia 36 Sweden 7
Germany 20 Switzerland 5
Greece 2 Syria 4
Hungary 8 Taiwan 4
India 32 Tajikistan 1
Iran 3 Thailand 1
Israel 7 Tunisia 11
Italy 61 Turkey 44
Japan 85 Turkmenistan 15
Kazakhstan 15 Ukraine 10
Korea, South 37 UK 78
Kyrgyzstan 4 USA 786
Macedonia 38 Uzbekistan 16
Mexico 1 Vietnam 1
Moldova 3
2 Pear Germplasm Needs and Conservation 43

either as in vitro shoot cultures or as small potted materials from Greece, the Balkan region, several
greenhouse trees. Accessions at higher risk of loss countries in the Middle East, Central and South-
due to either lack of cold hardiness or suscepti- east Asia are under-represented (Table 2.3).
bility to disease are prioritized for backup. Field
collections are grown on 10 hectares of orchard
plots with a single tree per accession. Cultivars are 2.2.3 Documentation
grafted onto a standard clonal rootstock, and wild
species are grown from seeds on their own roots. Genebank accessions are only as valuable as the
The NCGR orchards include 850 wild relative information associated with them. Passport or
species trees and 1350 cultivars. This collection provenance data detailing a wild collection site
consists of representatives of over 1000 European can be associated with climate (e.g., high rainfall
cultivars, 185 Asian cultivars, and 125 hybrid and extreme temperatures) or soil data (e.g.,
cultivars. Fruiting cultivars or selections with tolerance to low pH soils) and suggest adaptive
desirable traits represent a unique arrangement of traits that these plants may possess. Field
genes and must be managed as living trees to observation data collected from permanent living
preserve unique named genotypes. collections provide information on important
Nearly all of the primary species of Pyrus are phenotypic traits such as flower and fruit phe-
represented in the NCGR collection, with much nology, resistance to locally prevalent diseases or
larger numbers of accessions representing spe- insects, or morphologic traits that have agro-
cies from which large-fruited European and nomic value. All germplasm housed at NPGS
Asian cultivars have developed from (Table 2.2; genebanks is documented in a public database,
Fig. 2.1). Exchanges of plant materials with the Germplasm Resources Information Network
foreign genebanks along with USDA supported or GRIN (Postman et al. 2010; USDA-ARS
expeditions to collect pear wild relatives near 2015). To search GRIN, please visit https://
centers of wild diversity around the Caucasus npgsweb.ars-grin.gov/gringlobal/search.aspx?
Mountains and in Asia have filled taxonomic and
geographic gaps in this collection and have
expanded the overall size of this holding (Post- 2.2.4 Distribution
man et al. 2012). The wild germplasm is main-
tained as seed, but sometimes, it is supplemented Propagation materials and tissues for germplasm
by a small population of seedlings. As limited characterization are freely available for research
field space, staff, and budget resources restrict the and education purposes from NPGS genebanks.
number of seedlings that can be established Each year, NCGR fills hundreds of orders for
long-term as living trees, a seedlot is often rep- pear germplasm, averaging about ten accessions
resented by three to five seedlings grown in the per order. Between 2010 and 2016, approxi-
orchard. A larger number of seedlings may be mately 1500 pear accessions have been dis-
grown for rare taxa, to represent germplasm tributed annually (USDA-NCGR 2017). Of all
likely possessing valuable genetic traits, or for distributed materials between the years 1980
taxa from an under-represented region. through 2018, 25 of the most requested pear
Taxonomic gaps in the NCGR collection accessions are listed in Table 2.4. Named culti-
include species native to North Africa (P. ghar- vars of P. communis tend to be the most
biana and P. mamorensis) and species native to requested, with perry (cider) pears being espe-
central and western Asia (P. armeniacifolia, cially popular, a good indication of the impor-
P. korshinskyi, P. syriaca, and P. xerophila) as tance of the rapidly expanding craft cider market.
indicated by the accession counts in Table 2.2. Red flesh pears, such as ‘Summer Blood Birne,’
There are also geographic gaps in the collection have also been in high demand, thereby
for species that may be represented elsewhere demonstrating an interest in developing pears
from their native range. For example, plant with this unique trait.
44 J. Postman

Table 2.4 Top 25 most requested USDA Pyrus accessions from 1980 to 2018; rank and number of samples shipped
(USDA-ARS 2018c)
Cultivar Accession Taxon Rank (shipped)
Seckel PI 541262 P. communis 1 (202)
Yellow Huffcap PI 541287 P. communis 2 (165)
Red pear PI 541317 P. communis 3 (160)
Bartlett PI 300693 P. communis 4 (159)
Thorn PI 541273 P. communis 5 (156)
Taynton Squash PI 541271 P. communis 6 (155)
Barland PI 541123 P. communis 7 (154)
Gin PI 541195 P. communis 8 (146)
Butt PI 541156 P. communis 9 (142)
Summer Blood Birne PI 312507 P. communis 10 (141)
Beurre Superfin PI 541150 P. communis 11 (136)
Blakeney Red PI 541151 P. communis 12 (131)
Joey’s Red Flesh Pear PI 617584 P. communis 13 (130)
Hendre Huffcap PI 541205 P. communis 14 (128)
Beurre Bosc PI 541387 P. communis 15 (125)
Warren PI 541448 P. communis 16 (123)
Winnals Longdon PI 541486 P. communis 17 (123)
Ya Li PI 506362 P.  bretschneideri 18 (121)
Aurora PI 541119 P. communis 19 (121)
Rousselet de Reims PI 541256 P. communis 20 (119)
Brandy PI 541305 P. communis 21 (118)
Abbe Fetel PI 260195 P. communis 22 (116)
Harrow Delight PI 541431 P. communis 23 (112)
Magness PI 541299 P. communis 24 (111)
Doyenne du Comice PI 271658 P. communis 25 (110)

2.2.5 Clonal Genebank Challenges weather conditions or necessitate maintaining a

backup tree in a greenhouse for security. It is also
Since a clonal genebank accession may be rep- critical, yet expensive, to ensure that collections
resented by a single tree without replication, are backed-up and secured, so that they are not
some observation data may be difficult to inter- lost in the event of physical, environmental, or
pret. Data collected over multiple years can biological disasters.
sometimes provide a measure of confidence in
these observations. Accessions may originate
from a distant country or a climate much different 2.3 Genetic Tools for Genebank
from that present at the genebank repository. It Management
can be a challenge to maintain living trees of
low-chill and non-hardy genotypes, or trees that Confirmation of fruiting cultivar identities in
are very susceptible to local diseases. Subtropical genebank collections requires detailed compar-
species such as P. koehnei or P. pashia may isons of tree and fruit characteristics to published
require additional protection against winter descriptions, old nursery catalogs, photographs,
2 Pear Germplasm Needs and Conservation 45

and other artwork. ‘The Pears of New York’ insuring security of germplasm collections
volume, published by the New York State through onsite backups. Likewise, maintaining
Agricultural Research Station (Hedrick 1921), is identical accessions at different genebanks or
one of the most important references for pear genebank locations also contributes to germ-
identification in the USA. This publication has plasm security.
80 full-page lithographs and multi-page descrip- The use of SSR markers has become a stan-
tions of the most promising pear cultivars of the dard tool for DNA fingerprinting to confirm
early 1900s, along with thousands of brief genetic identities of trees and whether or not any
descriptions of less important and obscure culti- two presumed duplicate trees are indeed a match.
vars. A more recent book details over 500 cul- A comparison of 61 pear accessions received
tivars and includes more modern cultivars from the Brogdale National Fruit Collection in
(Morgan 2015). Many other domestic and for- the UK to accessions of the same name at the
eign pomology references also document fruit NPGS pear collection has demonstrated that 44
cultivars of different periods. accessions have identical allele sizes at 12 SSR
Prior to the widespread use of color photog- loci (Evans et al. 2015); whereas, 12 accessions
raphy, USDA has employed professional artists have distinctly different SSR profiles at six or
to paint detailed, actual-size watercolor paintings more loci. Therefore, phenotypic observations or
of fruit cultivars entering the country, or growing additional SSR comparisons are required to
domestically. From 1886 to 1942, thousands of determine which of these accessions are true to
small watercolor paintings, lithographs, and line type (Evans et al. 2015). For example, the
drawings have been produced, and 7500 are Japanese cultivar Hosui in the Brogdale collec-
preserved at the USDA National Agriculture tion is not a match to a ‘Hosui’ accession found
Library (NAL) in Maryland. Many are available in the USDA collection. Following phenotypic
online, including almost 300 pear images comparisons and verification, it has been deter-
(Fig. 2.2; USDA-NAL 2018a). Collections of mined that the Brogdale tree has been incorrectly
historic nursery catalogs are also maintained at named. In another example, the cultivar Arabitka
NAL (USDA-NAL 2018b) and elsewhere. Con- has exhibited different SSR profiles in these two
ventional references including books, paintings, collections. Following comparisons with a large
catalogs, as well as other living collections are set of SSR markers, the tree at NCGR is found to
needed to verify identities of pear cultivars be a mislabeled ‘Vicar of Winkfield.’ Similar
before they can be used as standards for molec- efforts with apple accessions obtained from dif-
ular identification protocols. ferent European collections have revealed that
Genetic fingerprinting techniques facilitate incorrect labels and propagation errors are more
confirmation of collection materials with those common than collection curators would like to
from other, often distant, sources. Genetic sig- see (Evans et al. 2011).
natures are consistent across locations even In other instances, a misidentified accession
though phenotypes may vary across growth may arise when a graft union fails and the root-
environments. Identities of trees representing stock grows over, or it is inadvertently planted to
crop wild relatives must likewise be properly represent another genotype. The NCGR pear
identified to their correct species upon receipt collection is grafted onto a standard clonal root-
into a collection. stock, ‘OH  F 333,’ and the SSR fingerprint of
this rootstock has, on occasion, been detected
from a tree that should represent a different
2.3.1 Intentional and Unintentional genotype. Valuable information confirming syn-
Redundancy onymy of accessions having different common
names can also be gleaned from the use of SSR
Intentional redundancy, or maintaining duplicate markers. For example, the following cultivars
trees, is an important management strategy for have been found to be synonyms: ‘Bella di
46 J. Postman

Fig. 2.2 An ‘Onondaga’

pear fruit harvested by G.W.
Soudder in Rowayton,
Connecticut, on September
20, 1913, and painted by
USDA artist Amanda A.
Newton on October 7, 1913

Giugno’ = ‘Mirandino Rosso,’ ‘Forelle’ = another unique accession. When pear genebank
‘Helmershus Roda,’ ‘Jubileer D’ar’ = ‘Pautalia,’ collections have been fully genotyped and culti-
and ‘Flemish Beauty’ = ‘Lesnaia Krasavitza’ var identities validated, a database can be estab-
(Bassil and Postman 2009). Confirmation of lished to serve as a resource for identifying trees
synonymy can help a curator justify removal of a of historic significance or with unknown
redundant accession, thus freeing up space for identities.
2 Pear Germplasm Needs and Conservation 47

2.3.2 Assess Diversity and Identify parents in an effort to develop easy-to-propagate,

Collection Gaps blight-resistant, clonal pear rootstocks. Seeds
have been collected in 1952 from an isolated
Analysis of amplified fragment length polymor- ‘Old Home’ tree planted next to several ‘Farm-
phisms (AFLPs) has provided useful information ingdale’ pollenizers in British Columbia
about genetic relationships between different (Canada) and grown out at an Oregon nursery.
groups of pear cultivars and species (Bao et al. Over the next few decades, hundreds of OH  F
2008). AFLP results have been validated and seedlings have been evaluated for ease of root-
refined by more recent genetic analyses using ing, dwarfing potential, and resistance to impor-
SSRs, chlorophyll and genomic sequences, tant pear diseases including fire blight. A dozen
single-nucleotide polymorphisms (SNPs), and or so selections have been introduced to the
other novel techniques (Jiang et al. 2016; Kumar nursery trade, and more than 40 numbered OH
et al. 2017; Volk et al. 2006, 2019; Wuyun et al. F selections are deposited at the USDA pear
2015). The use of these tools to investigate spe- genebank for preservation. Some of these clonal
cies relationships and the history of Pyrus rootstocks have become widely used in propa-
domestication is reviewed in greater detail in gating fruiting cultivars by commercial nurseries
Chap. 4 of this volume. and grown worldwide for pear fruit production.
The development of molecular tools for In a recent study, cultivars ‘Old Home’ and
diversity analysis cannot be accomplished with- ‘Farmingdale,’ along with six OH  F clonal
out access to diverse living collections of Pyrus selections were included in an SSR fingerprinting
germplasm correctly identified to a species or a assessment. ‘Old Home’ was found to share an
cultivar. A common and sometimes unantici- allele with all of the OH  F selections at all 12
pated outcome of applying genetic analysis to loci, but there was no alignment between
diversity assessment is to sort out those geno- ‘Farmingdale’ and any of the OH  F selections
types that do not group with other samples of the at several loci. Pedigree analysis showed that
same species. Following closer examination of ‘Bartlett’ was actually the pollen parent for all six
the phenotypic profile of a tree that is an outlier OH  F selections evaluated (Fig. 2.3; Postman
on a genotypic dendrogram, an accession will et al. 2013). Thus, it was proposed that ‘Farm-
often be deemed either as misidentified or as a ingdale’ was not the pollen parent for any of the
hybrid species (Volk et al. 2006). These types of OH  F rootstocks.
assessments are particularly important for gene- It is not uncommon for marker analysis to
bank collections as scientists rely on these col- reveal anomalies in published cultivar pedigrees;
lections to provide true-to-type germplasm for however, in the case of OH  F rootstocks, new
use in their research and breeding programs. generations of rootstock candidates have been
developed using OH  F selections as parents,
with the intention of obtaining fire blight resis-
2.3.3 Identify or Confirm Pedigrees tance from ‘Farmingdale.’ Resistance is highly
heritable when ‘Farmingdale’ is used as either a
Some pear cultivars are the result of deliberate male or a female parent (Reimer 1950); however,
crosses, and others were chance seedlings of ‘Bartlett’ is not considered to be a good source of
unknown parentage identified as desirable trees. fire blight resistance. The case of OH  F high-
In the case of OH  F pear rootstocks, two fire lights the importance to breeders of having accu-
blight-resistant cultivars have been used as rate genetic identity and paternity information.
48 J. Postman

Fig. 2.3 Unweighted pair OHxF 230

group method with arithmetic OHxF 51
mean (UPGMA) cluster OHxF 333
analysis of 20 pear accessions Bartlet
based on 12 simple sequence OHxF 87
repeat (SSR) loci (Fig. 4 from OHxF 97
Postman et al. 2013) Old Home
OHxF 69.003
OHxF 69.001
OHxF 69.002
Harrow Sweet
Conference
Pyrodwarf
Comice
Rogue Red
Passe Crassane
Anjou
Farmingdale
Abbe Fetel
Hosui

0.1

References
2.4 Conclusions
AAFC (2018) Plant gene resources of Canada. Agricul-
Pear collections provide a diverse source of ture and Agri-Food Canada (AAFC). http://pgrc3.agr.
species and cultivars essential to the success of gc.ca/nodes-noeuds_e.html, 8 Aug 2018
research and breeding programs. Access to such Bao L, Chen K, Zhang D, Li X, Teng Y (2008) An
assessment of genetic variability and relationships
materials is necessary to develop improved cul-
within Asian pears based on AFLP markers. Scientia
tivars for fresh fruit production, for perry, and as Hort 116:374–380
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Acknowledgements The author would like to acknowl-
C-E, Fernández-Fernández F, Boudichevskaia A,
edge Jill Bushakra and Gayle Volk for reviewing and
Dunemann F, Stankiewicz-Kosyl M, Gianfranceschi L,
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Acta Hortic 124:57–66 0133686
 Genetic Diversity and Domestication
History in Pyrus 3
Gayle M. Volk and Amandine Cornille

Abstract 3.1 Introduction: Assessing Pyrus

The cultivated pear is a major fruit crop in Diversity
Eurasia that underpins many local economies.
However, its origin and domestication history, Cultivated pears are produced throughout tem-
as well as the diversity of wild pears in natural perate regions on both a commercial scale and
ecosystems, are at the early stages of explo- for local household use; however, their origin
ration. In this chapter, we provide an overview and domestication history are at the early stages
of the described diversity and genetic relation- of exploration. Over the past 4000 years, pear
ships among wild and cultivated Pyrus species. cultivation has led to the identification and/or
Non-discriminatory morphological characters, development of a vast number of landraces and
poor diagnostic genetic tools, and lack of recent cultivars through natural and artificial
access to samples scattered throughout world- hybridization. Vegetative propagation by graft-
wide genebank collections make it difficult to ing has allowed interesting and/or desirable
definitively elucidate relationships of pear phenotypes to be maintained and spread (Zohary
species and more generally Pyrus diversifica- and Spiegel-Roy 1975). As a result, cultivated
tion and domestication. High-throughput pears exhibit a wide range of desirable traits,
sequencing is providing advancements in our including fruit attractiveness, flavor, size, and
understanding of the domestication process of shape. Numerous molecular studies, primarily
the pear, and of biogeography, taxonomy, and based mostly on a few marker loci, have been
ecology of wild pears. This knowledge will be used to characterize the diversity of pear cultivars
crucial for future breeding programs focused on and the origin of this diversity in wild species.
improving quality and production traits. However, the genetics underlying key agronomic
traits are just beginning to be understood.
Assessments of pear species diversity and
distribution are usually determined using regio-
G. M. Volk (&)
USDA-ARS National Laboratory for Genetic
nal inventory and census counts. These records
Resources Preservation, 1111 S. Mason St., 80521 are often not collected using standardized tech-
Fort Collins, CO, USA niques and have gaps with respect to coverage. In
e-mail: [email protected] addition, recurrent hybridizations and resulting
A. Cornille introgressions among species have made it diffi-
Génétique Quantitative et Evolution—Le Moulon, cult to differentiate species. Consequently, it is
INRA, Univ. Paris-Sud, CNRS, AgroParisTech,
Université Paris-Saclay, 91190 Gif-Sur-Yvette,
difficult to identify the geographical range of
France wild Pyrus species.

This is a U.S. government work and not under copyright protection in the U.S.; 51
foreign copyright protection may apply 2019
S. S. Korban (ed.), The Pear Genome, Compendium of Plant Genomes,
https://doi.org/10.1007/978-3-030-11048-2_3
52 G. M. Volk and A. Cornille

Wild relatives of cultivated pears offer novel 3.2 Diversification of Wild Pears
allelic diversity and allelic combinations that can
provide sources of resistance and tolerance to The genus Pyrus is presumed to have originated
abiotic and biotic stresses for pear breeding during the Tertiary Period (65–55 million years
programs. Pyrus wild species such as P. com- ago [Mya]) (Silva et al. 2014), or in particular in
munis spp. pyraster, P. calleryana, P. ussurien- the Oligocene Epoque, 33–25 Mya (Korotkova
sis, P. pyrifolia, P. fauriei, P. dimorphoylla, et al. 2018) in the mountainous regions of Western
P. betulifolia, and P.  nivalis have desirable China or Asia Minor. Microsatellite or simple
levels of disease resistance to various pathogens, sequence repeat (SSR) markers, as well as geno-
including pear leaf spot (Entomosporium mespili mic studies, have revealed strong genetic differ-
(DC.) Sacc.), fire blight (Erwinia amylovora entiation between two main genetic groups, an
(Burr.) Winslow et al.), and pear psylla Occidental (European/Central Asian) and an
(Cacopsylla pyricola (Foerster)) (van der Zwet Asian (East Asia), which diverged between 6.6
et al. 1983; Bell 1992; Bell and Itai 2011). These and 3.3 Mya (Fig. 3.1) (Liu et al. 2015; Volk et al.
species can be used as parents in breeding pro- 2019; Wu et al. 2018). Two non-coding regions of
grams, as providers of specific alleles for intro- the cpDNA and one low copy nuclear gene have
gression, or as rootstocks. Many wild pear also demonstrated the differentiation between
species, including P. pashia, P. korshinskyi, wild Asian and Occidental pear groups (Zheng
P. syriaca, P.  hopiensis, P. gharbiana, et al. 2014). Altogether, this suggests spatial dis-
P. betulifolia, P. calleryana, P. cossonii. persal events to eastern and northern Eurasia,
P. dimorphophylla, P. fauriei, P. pyrifolia, whereby Asian wild species have diversified, and
P. ussuriensis, and P. xerophila, are recognized to western Eurasia, whereby Occidental wild
for their desirable rootstock traits, providing species have diversified (Figs. 3.2 and 3.3).
tolerance to extreme heat, humidity, and cold, as The use of classical microsatellite genetic
well as disease resistance (Ercisli 2004; Bao et al. markers has shed light on the genetic diversity of
2008; Zong et al. 2014b; U.S. Department of some wild pear species. Nuclear microsatellite
Agriculture 2017). data demonstrated that the genetic variation of
This chapter focuses on the measured diver- wild populations of P. calleryana, P. communis
sity of wild Pyrus species and described rela- subsp. pyraster, P. pashia, and P. ussuriensis is
tionships between wild species and cultivated higher within (ranging from 80 to 96%) than
forms. The life history traits of pears, with long among populations (ranging from 4 to 20%) (Liu
lifespans and high levels of gene flow among et al. 2012; Wolko et al. 2015; Zong et al. 2014a;
populations and species, combined with their Wuyun et al. 2015) (Table 3.1). This observed
ancient origin, render Pyrus as a valuable model wide range across wild Pyrus species may be in
for studying fruit tree species diversification. part due to physical sampling methods used; e.g.,
Expanded knowledge of pear genetic diversity distances between sites and familial relationships
and evolution will also assist in pinpointing among individuals. The heterozygosity of these
sources of allelic variation in the wild useful for populations ranges from 0.48 for P. ussuriensis
future breeding programs. Such studies are par- (Wuyun et al. 2015) to 0.76 for P. communis
ticularly timely, as wild gene pools may be subsp. caucasica and P. communis
sources of alleles for resistance to biotic and subsp. pyraster (Table 3.2; Asanidze et al. 2014;
abiotic stresses (van der Zwet et al. 1983; Bell Wolko et al. 2015). Hereafter, we review the
1992; Bell and Itai 2011), and these are currently literature on specific diversity and evolution of
under threat of fragmentation in their centers of Asian (pea pear and large-fruited) and Occidental
origin. pears.
3 Genetic Diversity and Domestication History in Pyrus 53

Occidental pears Asian pea pears Large-fruited

P. betulifolia Asian pears
P. communis
P. cordata P. calleryana P. pashia
West Asian Pyrus P. dimorphophylla P. pyrifolia
P. fauriei P. ussuriensis
North African Pyrus P. koehnei P. hondoensis
P. regelii

Fig. 3.1 Generalized diagram of network relationships and shared haplotypes of Pyrus species, from Volk et al.
(2019). North African Pyrus species include P. cossonii, P. gharbiana, and P. mamorensis, while West Asian Pyrus
species include P. elaegrifolia, P. glabra P. korshinskyi, P. sachokiana, P. salicifolia, P. spinosa, and P. syriaca

ussuriensis

sinkiangensis

dimorphophylla
fauriei
South hondoensis
China betulifolia Japan
Korea

pyrifolia
calleryana

pashia

Fig. 3.2 General overview of the geographic distribution of native East Asian wild Pyrus species
54 G. M. Volk and A. Cornille

cordata communis eleagrifolia

spinosa
sachokiana syriaca

salicifolia
regelii

boissieriana korshinskyi

gharbiana
cossonii=
longipes glabra

Fig. 3.3 General overview of the geographic distribution of native Occidental wild Pyrus species

Table 3.1 Microsatellite marker genetic diversities assessed within and among populations of Pyrus species
Taxon Site location Number of Total Among Within-population Citation
populations number of population genetic variation
(no.) individuals genetic variation (%)
(%)
P. calleryana Zhejiang 8 77 9 91 Liu et al.
Province, (2012)
China
P. communis Poland 6 379 4 96 Wolko
ssp. pyraster et al.
(2015)
P. pashia Yunnan 4 470 11 89 Zong
Province, et al.
China (2014a)
P. ussuriensis Heilongjiang, 13 153 20 80 Wuyun
Jilin, Inner et al.
Mongolia (2015)
Malus Kazakhstan 8 949 5 95 Richards
sieversii et al.
(2009)

3.2.1 Genetic Diversity of Asian Wild in diameter with two carpels (Jiang et al. 2016).
Pears In contrast, large-fruited Asian pear species
include, among others, P. pashia, P. pyrifolia,
Asian wild pears are often described as belong- P. ussuriensis, P. xerophyla, and P. hondoensis
ing to either the “pea pear” or the “large-fruited (Challice and Westwood 1973). It has been dif-
pear” groups. Pea pears, including P. betulifolia, ficult to genetically differentiate between “pea”
P. calleryana, P. dimorphophylla, P. fauriei, and and “large-fruited” pears (Jiang et al. 2016;
P. koehnei, produce fruits that are less than 1 cm Zheng et al. 2014). Genetic diversity assessments
3 Genetic Diversity and Domestication History in Pyrus 55

Table 3.2 Diversity assessments using microsatellite markers of wild populations of Pyrus and Malus species,
including number of individuals sampled (n), number of SSRs used to assess diversity (SSRs), number of effective
alleles per locus, expected heterozygosity (He), observed heterozygosity (Ho), and inbreeding coefficient (Fis)
Taxon Source n SSRs Effective He Ho Fis Citation
(no. alleles/locus
markers) (no.)
P. betulaefolia Northern China (Gansu, 326 13 4.11 0.70 0.69 0.009 Zong et al.
Shaanxi, Henan, Hebei, (2017)
Shandong)
P. calleryana Zhejiang Province, China 77 14 3.74 0.64 0.57 0.170 Liu et al.
(2012)
P. communis Georgia 112 11 17.00 0.76 0.135 Asandize
ssp. caucasica et al. (2014)
P. communis Poland 192 17 5.66 0.76 0.75 0.018 Wolko et al.
ssp. pyraster (2015)
P. ussuriensis Heilongjiang, Jilin, Inner 12 20 2.44 0.48 0.34 0.220 Wuyun
Mongolia et al. (2015)
P. ussuriensis China 12 20 2.63 0.56 0.39 0.233 Katayama
et al. (2016)
P. ussuriensis Japan 20 20 4.31 0.74 0.71 0.030 Katayama
et al. (2016)
P. ussuriensis Tibet 8 28 3.22 0.67 0.59 0.070 Xue et al.
(2017)
Malus Kazakhstan 949 7 14.70 0.75 0.69 0.052 Richards
sieversii et al. (2009)

of Asian wild pears have focused primarily on P. phaeocarpa (P. betulifolia  P. ussurien-
differences/relatedness of either within species or sis  P. pyrifolia), P. hondoensis (P. dimopho-
between wild species and cultivated forms. phylla  P. ussuriensis), P. neoserrulata and
Molecular genetic markers have facilitated P. serrulata (P. calleryana  P. pyrifolia), and
identification of basal species and hybrids in the P. hopeiensis (P. ussuriensis  [P.  phaeo-
Asian wild Pyrus group. Sequence-specific carpa or P. betulifolia]) (Jiang et al. 2016; U.S.
amplification polymorphism (SSAP) suggest Department of Agriculture 2017).
that P. betulifolia, P. pashia, P. pyrifolia, and
P. ussuriensis are primitive genepools of wild 3.2.1.1 Genetic Diversity Within
Asian species (Jiang et al. 2016). Other original the Asian Pea Pear
wild Asian species include P. koehnei and Species
P. fauriei (Zheng et al. 2014). Pyrus species of The following Pyrus pea pear taxa, P. betulifolia,
ambiguous identities or origins include P. calleryana, P. dimorphophylla, P. fauriei, and
P. dimorphophylla (sometimes classified as a P. koehnei are native to China, Japan, and the
variety of P. calleryana), P. calleryana (with leaf Korean peninsula (Fig. 3.2). Pyrus betulifolia is
shape similar to P. pashia and fruit similar to described as an ancient pear species that shares
P. betulifolia), and P.  bretschneideri (geneti- some traits with both Asian and Occidental pear
cally similar to P. ussuriensis). Asian wild pear types (Zong et al. 2014b, 2017). Diversity of this
species resulting from hybridizations between species, as measured using chloroplast intergenic
wild pear species include P. xerophila fragments and microsatellite genetic markers
(P. pashia,  P. ussuriensis  Occidental), (SSRs), has revealed that the Taihang Mountains
P. sinkiangensis (P. pyrifolia  Occidental), are natural genetic barriers, and that range
56 G. M. Volk and A. Cornille

expansion and contraction events must have (Wuyun et al. 2015). It is reported that Inner
occurred during and between glacial periods Mongolian populations may have experienced
(Zong et al. 2014b, 2017). Furthermore, popu- some bottleneck effects due to their demographic
lations within P. betulifolia are more easily dis- decline (Wuyun et al. 2015).
tinguishable using chloroplast markers rather As P. pashia is another ancient species, it may
than nuclear SSRs as pollen-mediated gene flow be intermediate between Asian and Occidental
has likely homogenized genetic diversity at the pear groups. Whereas, P. pashia is native to
nuclear level (Zong et al. 2017). Future work Southwest China and to the Himalayan region
using additional markers, such as single nucleo- (Fig. 3.2; Zong et al. 2014a). Due to high levels
tide polymorphisms (SNPs), will provide more of within-site diversity, based on SSR profiles,
insights into the population structure of Zong et al. (2014a) have proposed that some of
P. betulifolia. the sampled populations may have likely served
On the other hand, P. calleryana, native to as sources for range expansions during inter-
southern China, Japan, and the Korean Penin- glacial periods. Liu et al. (2013) have used
sula, is classified as a wild pea pear that shares chloroplast sequence data to assess the diversity
some similarities with both P. pashia and of individuals within 22 populations. As with
P. betulifolia (Jiang et al. 2016). In Southern other wild pear species, a high level of genetic
China, the range of native species of variation is detected within populations. Range
P. calleryana, P. pashia, and P. betulifolia is expansions may explain lack of correlations
found to overlap (Liu et al. 2012; Jiang et al. between genetic and geographic distances across
2016). Using both nuclear microsatellite and the range of P. pashia (Liu et al. 2013).
chloroplast sequence markers, two genepools are
identified in eight populations of P. calleryana in
the Zhejiang Province in China (Liu et al. 2012). 3.2.2 Genetic Diversity
These genepools correspond to two geographic in the Occidental Pear
regions, with one located in the northeast and the Species
other located in the southwest.
Occidental pear species are likely to have radi-
3.2.1.2 Genetic Diversity Within ated westward from China and currently occupy
the Large-Fruited Asian overlapping ranges (Fig. 3.3). Chloroplast and
Pear Species nuclear genes have been used to reconstruct the
The wild large-fruited Asian pear species include phylogeny of Occidental Pyrus species using 50
P. pashia, P. pyrifolia, P. ussuriensis, P. xero- accessions representing the following 11 species:
phyla, and P. hondoensis. Pyrus ussuriensis is P. communis, P. nivalis, P. cordata, P. eleagri-
native to northeastern and north-central Chinese folia, P. spinosa, P. regelii, P. salacifolia,
provinces, as well as to Japan (Fig. 3.2; P. syriaca, P. cossonii, P. gharbiana, and
Katayama et al. 2016). Each of chloroplast P. mamorensis (Zheng et al. 2014). It is found
sequences, SSAPs, and SSRs has been used to that all Occidental species, except for P. regelii
assess genetic variations among and within and P. gharbiana, have shared haplotypes.
P. ussuriensis populations throughout its native Moreover, it appears that P. regelii, the most
range. These genetic studies have revealed exis- easterly West Asian species, must have diversi-
tence of a spatial genetic structure across sam- fied early, becoming isolated, and it is the only
pling regions. Furthermore, within-population west Asian species P. regelii that is mono-
diversity is found to be high, likely due to phyletic (Fig. 3.1; Zheng et al. 2014; Volk et al.
self-incompatibility, while between-population 2019). In addition, P. regelii has an ancestral
differentiation is weak, except for those geneti- phenotype with dissected adult leaves and ovar-
cally distant populations from Inner Mongolia ies with few locules (Zheng et al. 2014).
3 Genetic Diversity and Domestication History in Pyrus 57

Based on a phylogenetic dendrogram, acces- 3.3 Domestication

sions of some Occidental species, including
P. spinosa, P. cossonii, P. regelii, P. gharbiana, Pyrus communis subsp. communis is a European
and P. mamorensis, are located on distinct bran- pear known for its soft and juicy flesh, and
ches (Zheng et al. 2014). In contrast, P. eleagri- includes cultivars such as ‘Bartlett’ and ‘Anjou’.
folia, P. nivalis, and P. salicifolia are spread In contrast, P. pyrifolia, the Asian pear, has a crisp
throughout the phylogenetic dendrogram (Zheng and juicy texture. Asian pears include a number of
et al. 2014). Recently, Volk and co-authors types of cultivated pears, including Chinese white
(2019) have observed lower levels of differenti- pear cultivars (such as ‘Ya Li’ and ‘Tse Li’) and
ation among Occidental species using chloroplast Japanese pears (such as ‘Kosui’, ‘Hosui’, and
sequence data (Fig. 3.1). Furthermore, ‘Nijisseki’). Genetic markers have been developed
P. spinosa, native to Turkey, Southeastern Eur- and used to reconstruct the domestication process
ope, France, and Spain, has primitive characters, that has resulted in the evolution of European,
suggesting that it may be yet another ancient Chinese white, and Japanese pear cultivars, as
species; whereas, P. salicifolia and P. nivalis well as various Asian landraces that include Chi-
have overlapping phenotypes with regard to leaf nese sand pears, Ussurian pears, and Xinjiang
shape (lanceolate or elliptical leaves) and level of pears. Recently, SNP data have elucidated this
hairiness (Zheng et al. 2014; Paganová 2003). dichotomy between Occidental and Asian culti-
Wild P. communis subsp. pyraster in Poland and vated pears (Kumar et al. 2017). These two pear
Germany have high levels of diversity within types, from Europe and Asia, respectively, origi-
populations, as well as weak correlations between nated from different wild pear relatives specific to
genetic and geographical distance (Wolko et al. their regions of origin (Fig. 3.4). This suggests
2015; Reim et al. 2017). Recent genomic two independent domestication events, one in
sequencing data reveal that many pear accessions Europe and one in Asia from distinct wild species,
assigned to Occidental species may be highly which was recently confirmed by fully sequenced
admixed (Wu et al. 2018). genomes of a large collection of wild and

Fig. 3.4 a Edible European

pear (P. communis); b Edible
Asian pear (P. pyrifollia) by
Mary Daisy Arnold, U.S.
Department of Agriculture
Pomological Watercolor
Collection. Rare and Special
Collections, National
Agricultural Library,
Beltsville, MD 20705
58 G. M. Volk and A. Cornille

cultivated pears (Wu et al. 2018). Specifically, The Japanese pear is the most commonly
P. communis subsp. communis is derived from grown commercial pear in Japan. Nishio and
P. pyraster, and P. pyrifolia is derived from the co-authors (2016) have used microsatellite
wild P. pyrifolia (Wu et al. 2018). markers to assess the genetic diversity and
ancestry of modern Japanese pear cultivars.
These cultivars are genetically similar to local
3.3.1 The Chinese White, Japanese, cultivars from the Kanto region of Japan. Iketani
and Chinese Sand Pear et al. (2010) have found that these local Japanese
Cultivar Groups cultivars are more similar to P. pyrifolia of China
than P. ussuriensis of Japan.
The cultivated Chinese white pears, Japanese Chinese sand pears are primarily local culti-
pears, and Chinese sand pears share a common vars grown in Sichuan Province, along the
ancestor, P. pyrifolia (Fig. 3.5a; Bao et al. 2007; Yangtze River, and in southern regions of China
Jiang et al. 2016). (Song et al. 2014). Chinese white and Japanese
The Chinese white pear is the most commonly pears have fewer numbers of haplotypes than
grown pear in northern China, and it is found at those of Chinese sand pears, suggesting that
the intersection of the native species ranges of Chinese sand pears have higher levels of diver-
P. ussuriensis and P. pyrifolia (Bao et al. 2007). sity, and are likely to be more basal than other
The Chinese white pears, grown in northern cultivars derived from P. pyrifolia (Teng et al.
China, may have originated from a gene pool 2015). Although Chinese sand pears may have
whereby P. ussuriensis has hybridized with been derived primarily from P. pyrifolia (Jiang
P. pyrifolia (Jiang et al. 2016). et al. 2009), there is some SSAP marker evidence
Pyrus  bretschneideri is a hybrid species suggesting that Chinese sand and Japanese pears
(sometimes considered to be P. pyrifolia) may have resulted from introgressive hybridiza-
between P. ussuriensis and P. pyrifolia. This tions between P. pyrifolia and P. pashia in
hybrid species, P.  bretschneideri, is consid- Southern China (Jiang et al. 2016).
ered as the source species for Chinese white Zangli pears are yet another Asian pear lan-
pears (Liu et al. 2015). drace, native to Eastern Tibet, Western Sichuan,
and Northwestern Yunnan provinces. Cultivars

Fig. 3.5 Relationships

between cultivated pears (a) P. pyrifolia (b)
(bold) and their progenitor
P. ussuriensis P. pyrifolia
species for a Chinese white
pear, Chinese sand pear, and
Japanese pear; b Ussurian Chinese Chinese Japanese
cultivated pear; c Xinjiang White Pear Sand Pear Pear
Ussurian Pear
pear; and d Occidental pears,
P. communis
subsp. communis P. ussuriensis P. pashia

(c) (d)
P. communis P. communis
Occidental ssp. caucasica ssp. caucasica
P. pyrifolia
Pear

Occidental Pear
Xinjiang Pear P. communis
ssp. communis
3 Genetic Diversity and Domestication History in Pyrus 59

of Zangli pears are resistant to bitter cold, dry air, 3.3.4 The Cultivated European Pear
and high winds (Xue et al. 2017). Microsatellite
markers have revealed that Zangli pears are The European pear, P. communis subsp. commu-
genetically similar to Chinese sand pears and that nis, is commercially grown, and is thought to have
they may have been introduced north from originated from smaller fruited P. communis
Yunnan and west from Sichuan (Xue et al. subsp. pyraster, a subspecies native to Eastern
2017). Europe, and P. communis subsp. caucasica, a
subspecies native to the Caucasus Mountains of
Russia, Crimea, Armenia, and Georgia (Fig. 3.5d;
3.3.2 The Ussurian Cultivated Pear Volk et al. 2006). Microsatellite markers have
successfully differentiated P. communis
Ussurian pear cultivars are native to the southern subsp. pyraster and P. communis subsp. cauca-
area of northeastern China, as well as to Hebei, sica, from P. communis cultivars (Volk et al.
Shanxi, and Gansu provinces (Fig. 3.2). 2006). In a later study, Asanidze and co-authors
Domesticated Ussurian pears are genetically and (2014) have compared local Georgian pear culti-
phenotypically distinct from wild P. ussuriensis vars to wild species of P. communis subsp. cau-
(Wuyun et al. 2015). Cultivated Ussurian pears casica, P. balansae, P. salicifolia, P demetrii,
are known for their strong cold resistance, and P. syriaca, P. ketzkhovelii, and P. sachokiana
they can endure up to −52 °C (Katayama et al. found in Georgia. Based on microsatellite marker
2016). The domesticated Ussurian pears have relationships and morphological similarities, it is
lineages from the following two species, likely that P. communis subsp. caucasica and
P. ussuriensis and P. pyrifolia (Fig. 3.5b; Jiang P. balansae (sometimes considered to be P. com-
et al. 2016; Yu et al. 2016). It is likely that munis; U.S. Department of Agriculture 2017) are
P. ussuriensis and P. pyrifolia have also hybri- progenitors of local Georgian pear cultivars
dized in the northern part of Japan, where the two (Asanidze et al. 2011, 2014).
species overlap (Katayama et al. 2016). Recently,
full-sequencing genome data have revealed that
cultivated P. ussuriensis is derived from the wild 3.4 Conclusions
P. ussuriensis (Wu et al. 2018). Various samples
selected for genomic and genetic analyses may Altogether, studies based on genetic data, mainly
have affected conclusions obtained from the of SSRs and chloroplast sequences, provide a
different studies. first glimpse of the genetic diversity and evolu-
tion of the Pyrus genus. Population genetic
studies have revealed that within-population
3.3.3 The Xinjiang Pear variation and gene flow among populations of
Pyrus species are high, as well as
Xinjiang pear cultivars are derived from between-species hybridizations recurrent. This
hybridizations between P. pyrifolia (possibly adds to the taxonomic complexity of differenti-
Chinese white pears) and Occidentals (Fig. 3.5c; ating Pyrus species, either based on morpholog-
Jiang et al. 2016). It is presumed that Occidental ical or genetic traits. Yet, many of the current
pears have been introduced from abroad in the findings are based on relatively few numbers of
Xinjiang Province in China (Chang et al. 2017). markers—nuclear or chloroplast microsatellite or
It has been reported that the ‘Korla’ pear, the sequence data. The use of genome-wide SNP
most famous Xinjiang pear cultivar, shares data using high-throughput sequencing tech-
chloroplast haplotypes with Chinese white pears, nologies holds promise in reducing costs per
as well as with other Xinjiang cultivated pear marker and per sample (see Kumar et al. 2017;
accessions (Chang et al. 2017). Wu et al. 2018). This research will be limited,
60 G. M. Volk and A. Cornille

however, based on the availability of true-to-type picture of the genomic diversity and evolution of
reference materials and access to wild popula- the Pyrus genus and, more generally, of pro-
tions of Pyrus species within the native range. cesses of adaptation in perennials.
Genebanks currently offer reference materials
and some collections of wild species material
with detailed passport information (collection References
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between the Caucasian species of wild pear (Pyrus
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 Genetics and Breeding of Pear
4
Lester Brewer and Richard Volz

Abstract Quince (Cydonia oblonga) rootstocks are

Although Pyrus consists of 22 primary species, preferred in Europe, as they offer vigor control,
nearly all scion breeding is focused on three precocity, and ease of propagation. To date,
species, including Pyrus communis (European utilization of quince rootstocks in North
pear), Pyrus pyrifolia (sand pear), and Pyrus  America has been restricted due to their lack
bretschneideri (white pear). Most scion breed- of cold tolerance. Identification and testing of
ing programs around the world are in one of cold hardy quince selections could change this.
two camps: those breeding for European Pyrus rootstocks are currently preferred in
(P. communis) soft- or firm-textured pears, North America and in Asia because of their
and those breeding for crisp-textured Asian cold hardiness; however, they are more vigor-
pears (P. pyrifolia and P.  bretschneideri). ous than quince, yet their yield efficiency is
Intercrossing among species is typically lim- lower. Thus, vigor control is among breeding
ited, except in New Zealand where it is a core targets for Pyrus rootstocks. Hybrids between
aspect of the breeding program. The lack of Pyrus species are now being used to overcome
effective control of pests and diseases in pear some of these deficiencies and to include
combined with increased consumer prefer- adaption to highly alkaline soils. In addition,
ences for fruits grown with low chemical other species, such as Amelanchier, are being
inputs and low environmental impacts is tested for their potentials to confer dwarfing,
driving breeding programs to incorporate plant excellent cold tolerance, potential non-host
resistance to major pests and diseases. On the resistance to pear decline, resistance to fire
other hand, the range of vigor-controlling blight, and good yield efficiency. Recent
rootstocks for pear production is limited. identification of genetic markers for scion
vigor control and precocity is a positive step
for future breeding of enhanced Pyrus root-
stocks. Overall, the development of cultivars
L. Brewer (&) and rootstocks with new or improved charac-
The New Zealand Institute for Plant and Food ters would be facilitated by the availability of
Research Limited, 55 Old Mill Road, RD3, Motueka
7198, New Zealand
molecular markers for traits of interest. How-
e-mail: [email protected] ever, pear breeding programs lag behind those
R. Volz
of apple in application of marker-assisted
The New Zealand Institute for Plant and Food selection and genomic selection to speed-up
Research Limited, Hawke’s Bay Research Centre, cultivar/rootstock development, and to ensure
Private Bag 1401, Havelock North 4157, New programs are more effective and efficient in
Zealand

© Crown 2019 63
S. S. Korban (ed.), The Pear Genome, Compendium of Plant Genomes,
https://doi.org/10.1007/978-3-030-11048-2_4
64 L. Brewer and R. Volz

their utilization of available resources. As objectives include high fruit quality, early
current genetic markers are validated in more ripening, long shelf-life, large fruit size, resis-
populations, and the pear reference genome tance to both scab (V. nashicola Tanaka et
sequence undergoes further refinement, these Yamamoto) and black spot (Alternaria alternata
technologies will play a larger role in pear (Fr.) Keissler pv. kikuchiana), and environmental
breeding programs. adaptation through the use of a range of species,
including P.  bretschneideri, P. pyrifolia,
P. ussuriensis Maxim., and P.  sinkiangensis
(Teng 2011). Furthermore, breeding programs in
Japan focus on genetic improvement of P. pyri-
4.1 Introduction folia cultivars, with breeding objectives targeting
superior fruit quality, early ripening,
Pear is assumed to be an ancient allopolyploid self-compatibility, and multiple disease resis-
that behaves as a diploid (2n = 2x = 34) (Crane tance for pear scab (V. nashicola) and black spot
and Lewis 1940). There are three important (A. alternata) (Saito 2016). As in Japan, Korean
centers of origin for the genus Pyrus. The first is breeding programs focus predominantly on
in the mountainous regions of Western China, enhancement of P. pyrifolia cultivars. Breeding
while the second is in Western Asia, comprising targets include season extension, storage ability,
Afghanistan, India, Tajikistan, Uzbekistan, and large fruit size, and high aroma, as well as pest
western Tian-Shan, and the third is in the Cau- and disease resistance, especially for leaf rot (A.
casus Mountains. Pyrus, belonging to the family kikuchiana) and pear scab (V. nashicola) (Shin
Rosaceae and subfamily Pomoideae, is a diverse et al. 2002); whereas, the New Zealand breeding
genus that includes 22 primary species ranging program utilizes interspecific hybrids with major
from the mostly soft-textured European pear, breeding objectives of producing convenient (not
Pyrus communis L., to the crisp-textured Asian messy to eat) fruit with high levels of flavor that
sand pear, Pyrus pyrifolia (Burm.) Nak., and the can be eaten either readily from the tree or after
Chinese white pear, Pyrus  bretschneideri storage, but with a minimum storage life of three
Rehd. (Bell 1991). months. Furthermore, additional important
In 2015, world production of pears has been breeding goals for the New Zealand program
estimated to be 26.6 million metric tonnes, with include increased fruit precocity and yield, high
approximately 20 million metric tonnes of those fruit quality free of internal disorders, variations
being crisp-textured Asian-style pears (Belrose in red skin colors, a range of fruit flavors and
2016). Breeding programs typically fall into one shapes, fruit skin that will not scuff, and disease
of two groups, those selecting new types of resistance, especially to both fire blight and pear
soft-textured European pears, mainly in Europe scab (V. pirina). The primary species used to
and North America, and those selecting generate interspecific pear hybrids in New Zeal-
crisp-textured pears, generally concentrated in and include P.  bretschneideri, P. pyrifolia, and
South Korea, Japan, China, and New Zealand. P. communis.
Breeders of European pears tend to target such It is important to point out that the North
fruit characters, as harvest season extension, red American and European pear markets are domi-
skin color, good fruit size, flavor, improved nated by a small number of old P. communis
textural attributes, storage ability, as well as cultivars, such as ‘Williams’ Bon Chrétien,’ also
growth habit, and resistance to various diseases known as ‘Bartlett’, ‘Conference’, ‘Abaté Fetel’,
and pests, especially against pear scab (Venturia and D’Anjou’ that have been selected before
pirina Aderh.), fire blight (Erwinia amylovora 1900. Pear fruit consumption rates in these
(Burrill) Winslow et al.), and pear psylla (Psyll- regions are generally either static or dropping
idae: Psyllinae: Cacopsylla spp.) (Dondini and (Belrose 2016). New cultivars have struggled to
Sansavini 2012). In China, breeding program get a foothold in these markets. This may be
4 Genetics and Breeding of Pear 65

attributed, in part, to the dominance of a small to come into fruiting, and juvenile trees have
number of supermarket chains, strong competi- many spines, rendering harvest and management
tion with other fruits in the marketplace, and difficult. Furthermore, interstocks are required
changing Western consumer food demands when quince rootstocks are used for seedling
(Brewer and Palmer 2011). Over the last few growth, which adds time and expense to the
decades, consumers desire more convenient fruit process. Availability of adapted, compatible, and
and snack foods that are ready to eat, and with dwarfing precocious Pyrus rootstocks would be
consistent quality. Developing products with of great benefit to pear breeding programs and to
these attributes would have a positive influence the pear industry as a whole.
on the economic returns for producers (Brewer New genomic technologies would offer
and Palmer 2011). Furthermore, new pear culti- opportunities for accelerating development and
vars incorporating improved resistances, espe- increasing efficiency and effectiveness of breed-
cially for pests and diseases that have the largest ing programs for developing new pear cultivars,
effects on profitable pear production, such as fire as well as new and improved pear rootstocks.
blight, pear psylla, and pear scab, are needed to This review focuses on modern pear breeding
achieve an additional goal of growing pears with approaches, as well as genetics of key selection
low chemical inputs. traits that are important for today’s pear breeders.
The minimal impact of new pear cultivars in It summarizes recent genomic-related research
European and North American markets contrasts aimed at improving efficiencies of pear breeding.
with the situation in China, wherein traditional
P. ussuriensis and P.  bretschneideri cultivars
maturing in mid- to late-season (i.e., mid-August 4.2 Breeding Systems
to September), such as ‘Dangshan Suli’, ‘Yali’,
‘Nanguoli’, and ‘Xuehauli’ comprise about 40% Pear has a gametophytic self-incompatibility
of all commercially grown cultivars (Cao et al. (GSI) system that ensures pollen fertilization of
2014). Over the past few decades, a substantial ovules in flowers and subsequent seed produc-
increase in Chinese pear production has been tion via outcrossing with other compatible pears.
attributed, in part, to nearly 100 new cultivars, As many of the important horticultural traits in
released to the pear industry over the last pear are likely controlled by multiple genes, this
50 years from government and university GSI system ensures that pear progenies are
breeding programs (Belrose 2016). Several of highly heterogeneous, with a wide diversity of
these new cultivars, such as ‘Cuiguan’ mature possible phenotypes. Nevertheless, the three
very early to early (July to early August), thus most important components of any pear breeding
extending the season for fresh-eating pear fruit. program are the following: (1) hybridization of
Interestingly, despite decline in total pear parents, carrying traits of interest, to generate
production in Japan over the past 40 years by seedling populations expressing new and
over 40%, there has been a reasonable uptake of improved characters, (2) identification of desir-
new cultivars (14% in 2012) (Saito 2016). Old able selections carrying those traits of interest
cultivars, such as ‘Nijisseiki’ and ‘Chojuro’ have among seedling populations, and (3) evaluation
been superseded by cultivars released from and testing of the best-performing selections.
breeding programs, including ‘Kosui’ ‘Hosui’,
and more recently ‘Akizuki’ and ‘Nansui.’ The
success of these new cultivars has been attributed 4.2.1 Hybridization
to traits, such as resistance to black spot and
improved eating quality. 4.2.1.1 Compatibility
In comparison with other perennial fruit GSI is a mechanism triggered by proteins coded
crops, traditional pear breeding is an expensive by a single locus on linkage group (LG) 17 with
and lengthy process as seedling trees take longer multiple S-alleles that determine inhibition of
66 L. Brewer and R. Volz

self-incompatible pollen tube growth without with chlorotic and necrotic leaf regions, then
damaging self-compatible tubes (Dondini and often dying within one month of germination
Sansavini 2012). Genotypes possessing one S- (‘Type 1’); and (ii) seedlings that initially
allele in common are partially compatible, and develop normally, followed by termination of
under certain conditions may produce progeny growth within three months after germination,
that exhibit reduced fruit set and seed production, with leaves beginning to cup downward and
while those possessing the same two S-alleles are progressively becoming chlorotic and necrotic
fully incompatible (Wang et al. 2017). To date, a (‘Type 2’). For those seedlings that grow nor-
large number of unique S-alleles have been mally, these have been classified as ‘Type 3.’
identified, 28 in P. communis (Gharehaghaji Interestingly, no significant differences in seed
et al. 2014; Goldway et al. 2009) and at least 48 weight or radicle length among these ‘Types’
across five Asian pear species (Wang et al. are observed in the above pear population at
2017). The repeated use of closely related parents planting (Montanari et al. 2016a). Furthermore,
in a breeding program may over time result in ‘Type 1’: ‘Type 2’ + ‘Type 3’ ratios are con-
deleterious concentration of a few S-alleles in sistent with a 3:13 segregation ratio, while Type
breeding material of potential value as parents. 2:Type 3 ratios fit a 1:1 segregation ratio, thus
Of 133 P. communis cultivars assessed for their indicating possible presence of major genes
S-haplotypes, 75 are found to carry the S101 controlling this interspecific (sub)/lethality trait.
allele, probably reflecting the extensive use of In addition, at least a single two-gene epistatic
‘Bartlett’ (S101/S102) as a parent (Goldway et al. interaction, between loci on LG1 and LG5,
2009). An understanding of compatible and originating from Asian and European species,
incompatible mating combinations is therefore respectively, is attributed to incidence of Type 1
critically important to a pear breeder, and this can HN, with at least one other locus on LG2
be derived either from knowledge of the S-hap- implicated in regulating this phenotype.
lotype(s) of parent candidates, or through past Molecular markers linked to both lethal phe-
knowledge of cross-performance. notypes have been identified for these loci, and
There are no major incompatibility barriers to these will be useful in selecting parents lacking
interspecific hybridization in Pyrus, and at least ‘sublethal’ alleles in order to maximize progeny
six naturally occurring hybrid taxa have been numbers from interspecific crosses (Montanari
reported (Bell 1991). Zielinski and Thompson et al. 2016a).
(1967) have found little evidence for hybrid
sterility from interspecific hybridizations. How- Self-compatibility
ever, post-zygotic gene flow barriers may exist Incompatibility has been overcome following
between different Pyrus species. In New Zeal- identification of a self-compatible natural mutant
and’s pear breeding program, some progeny from of ‘Nijisseiki’, referred to as ‘Osanijisseiki’
crosses between Asian- and European (Saito 2016). Crossing experiments have indi-
pear-derived parents have shed either little or no cated that this self-compatibility is due to a
pollen when anthers are dried (White and Brewer mutation in the pistil S locus, resulting in deletion
2002). Hybrid necrosis (HN) of young pear of the S-ribonuclease allele 4 (S4-RNase) in
seedlings has also been observed in some inter- styles rather than in pollen. ‘Osanijisseiki’ has
specific populations, but this has not been been used to develop a number of new self-
observed in intraspecific crosses. Two distinct compatible P. pyrifolia cultivars. In another
HN phenotypic classes have been identified in a approach, pollen from a heavily gamma-
genomic mapping study of an interspecific irradiated ‘Kosui’ tree has been used to polli-
(‘PremP003’  ‘Moonglow’) pear population. nate ‘Kosui’ flowers. This has resulted in iden-
These include the following: (i) seedlings that tifying a selection with a partial pollen mutation
cease growing soon after germination, initially causing loss of pollen incompatibility function,
4 Genetics and Breeding of Pear 67

but retaining its stylar self-incompatibility 4.2.1.3 Emasculation, Pollination,

(Sawamura et al. 2013; Saito 2016). and Seed Culture
Flowers are emasculated when the majority
4.2.1.2 Pollen Collection and Storage reaches balloon stage, at which point any open or
To ensure a full range of parents with different excess flowers are removed. A variety of meth-
flowering times are available for intercrossing, ods can be used for emasculation, including
pollen collection is best completed in advance of notched scissors, fine combs, finger nails, scal-
the crossing season (Bell et al. 1996). Pollen can pels, or tweezers (van der Zwet et al. 1977; Bell
be stored either from the previous year, or shoots and Janick 1990). Branches with emasculated
of up to 1.2 m long, with their base cut along a flowers can be bagged or whole trees covered
25° angle, can be collected at the tight-cluster with insect proof nets and plastic tents to prevent
flower stage before flowering begins, and kept in insect visitation. However, many breeders do not
a greenhouse at 20–25 °C until flowers are fully think that this is necessary, as long as the calyx,
open to collect anthers, and then extract pollen corolla, and stamens are removed before flowers
before dehiscence (van der Zwet et al. 1977). In are open (Bell and Janick 1990). Pollination is
addition, flowers at the balloon stage can also be ideally completed within 24–48 h following
collected from the orchard approximately 2 days emasculation.
before they are required (Visser and Oost 1981). Although many cultivars have a
Pollen can be extracted using a number of stigma-receptive period of up to 6 to 11 days,
methods, including rubbing anthers over a wire some have a shorter receptive period that can
mesh grid (1.5 mm2) onto paper sheets (Bell cause a significant reduction in fruit set after 48 h
et al. 1996), or combed from flowers using fine from the start of anthesis; e.g., ‘Doyenné du
combs onto foil trays to maximize pollen Comice’ (‘Comice’) (Sanzol et al. 2003). In such
recovery. Following extraction, pollen should be cases, pollen can be applied to stigmas using a
allowed to dry at approximately 23 °C for 24– variety of tools, including the stopper of a pollen
48 h, either on a laboratory bench or in an vial, glass rod, camel hair brush, strip cut eraser,
incubator. While pear pollen remains viable at and a fingertip (Bell et al. 1996; van der Zwet
room temperature for 2–3 weeks, it is best et al. 1977). In addition to the type of cultivar,
refrigerated at approximately 3–5 °C in plastic or temperature also strongly influences stigmatic
glass vials, and placed inside closed containers or receptivity, pollen tube growth, and/or ovule
stored in a desiccator with indicating silica gel development for successful pollination. For
over anhydrous CaSO4 to remove moisture and example, ‘Comice’ has a shortened stigma
maximize viability. receptivity period and reduced ovule longevity at
Pollen can be stored for 2 years at 2–4 °C and 17 compared to 13 °C (Tromp and Borsboom
10% relative humidity (Bell et al. 1996). Pollen 1994). Cool spring conditions decrease pollen
can also be frozen at −20 to −120 °C for 2– tube growth, delay ovule degeneration, and can
3 years (Bhat et al. 2012). When pollen is reduce the overall period for successful pollina-
required for use in the orchard, it is best that it is tion (Sanzol et al. 2003).
transported in a cooler bin or bag with frozen Pear seeds extracted from fruit produced in
pads or similar receptacles to keep it chilled. crosses require a chilling period or stratification
Prior to use, pollen viability can be checked while in a moistened state to break dormancy and
using acetocarmine or other stains following initiate germination (Bell et al. 1996). During
standard procedures (Bell et al. 1996). Pollen can stratification, seeds will absorb enough water to
also be germinated in a liquid medium containing increase their weight by between 100 and 150%
10% sucrose solution and 50 ppm boric acid, and (Brewer, unpublished). Species originating from
germination rate recorded after 2 h at 23 °C warm winter climates require a shorter stratifi-
(Visser and Oost 1981). cation period, and the optimum temperature for
68 L. Brewer and R. Volz

this process is higher (typically 7–10 °C) than for spines makes fruit thinning and harvest opera-
those from cold winter climates where the ideal tions difficult. In New Zealand, seedlings are
stratification temperature is 3–5 °C for 60– now managed by using rootstocks; wherein,
90 days. Sowing media used by breeders to seedlings are grown as fast as possible in a
germinate seeds include a seedbed with a greenhouse (or temporarily in the orchard, if
well-aerated medium, such as sand or vermi- required) before budding or grafting onto elite
culite, finely ground peat moss (Bell et al. 1996), Quince C rootstocks interstocked with elite
or dampened filter paper in petri plates or other ‘Beurre Hardy.’ The main benefit associated with
closed containers (Montanari et al. 2016a). When utilizing rootstocks is improved ease of man-
grown on filter paper, any fungal development agement, including crop regulation and harvest.
can be quickly identified and treated with a Also, the outcome is more representative of what
suitable fungicide before germinated seeds are might be expected in commercial production of
planted (Montanari et al. 2016a). Once seeds any future pear cultivar.
have begun to germinate, warm periods of either
one or more than 24 h at 20 °C can help stimu-
late consistent germination. 4.2.2 Polyploidy

4.2.1.4 Seedling Growing Methods Naturally occurring polyploidy has been identi-
Traditionally, pear seedlings grown on their own fied in both European and Asian cultivars,
roots have long juvenility periods. In fact, gen- including that of ‘Sha 01’, a tetraploid
eration cycles of up to 10 years have been (2n = 4x = 68) bud mutant of ‘Korla Pear’ (Cao
reported for European pears (Brewer and Palmer et al. 2002, 2014), a tetraploid ‘Bartlett’, and a
2011). Seedlings from Asian species are more triploid (2n = 3x = 51) ‘Beurré Diel’ (Moffett
precocious; i.e., they have significantly shorter 1933), and ‘Anli’, a P. ussuriensis cultivar
generation cycles (Brewer et al. 2008a). Reduc- (Cao et al. 2002). Triploids have been developed
tion of the generation cycle is a focus of many by crossing naturally occurring or induced tetra-
breeding programs, as this has the largest influ- ploids (following colchicine treatment) with
ence on the time taken for new products to reach diploid parents. Even though there is a range of
the market (Brewer and Palmer 2011). Breeding available polyploids, pear breeding programs
systems have been developed to reduce the time rarely use these as to develop new cultivars. In
taken for seedlings to come into bearing fruit. In crosses undertaken between Asian species, a
New Zealand, seedlings are grown in the green- range of tetraploid, triploid, and diploid combi-
house to accelerate growth rate and increase nations have been generated. For example,
internode numbers before planting them in an crosses between two tetraploids have yielded
orchard or a nursery. In the orchard, tree top progeny of which 97% are tetraploid and 3% are
bending is applied when seedlings have pro- aneuploid. Crosses of tetraploids with triploids,
duced at least 60 internodes. This bending has a and reciprocal crosses, have yielded progenies
number of benefits, such as reducing terminal with more or less equal numbers of triploids
growth while enhancing spur formation and (34%), aneuploids (33%), and diploids (26%),
flowering on mature wood. After bending the top while crosses between tetraploids and diploids
of the tree, a full trunk girdle is completed, have mostly produced diploids (61%) and
usually in the middle of summer (Brewer et al. triploids (36%) (Cao et al. 2002). Although there
2008a). Fruit on seedling trees with bent tops are is little documented information on fruit traits
generally harvested from the ground, meaning in such polyploids, the wide range of phenotypic
ladders or other harvest devices are not required variations observed in leaf traits suggest
for the first 3 years of fruiting. Seedlings grown there may be unexplored potential for variations
on their own roots are vigorous, and production in fruit traits among such polyploids (Sun et al.
of excess vegetative growth along with juvenile 2011).
4 Genetics and Breeding of Pear 69

4.2.3 Mutation Breeding estimates the extent to which a phenotype is

determined by parental genes that are largely
Mutagenic agents can be used to increase fre- additive in their effects. While general combining
quencies of mutations that would otherwise ability (GCA) measures the average performance
occur naturally at very low rates. Irradiation of a parent based on the performance of its pro-
(X-rays) is the most common method used to geny, specific combining ability (SCA) measures
modify well-adapted cultivars, typically to the additional genetic value due to interactions
improve them for either one or two traits. How- between particular parent genotypes.
ever, many of these mutations are unstable, and In this section, key desirable traits targeted for
only those that have proved to be stable have selection in pear programs will be discussed in
found a place in commercial production (Bell detail, including how the trait is measured and
et al. 1996). The Food and Agriculture Organi- what is currently known regarding its genetics.
zation of the United Nations (FAO)/International
Atomic Energy Agency (IAED) Database (2000)
records five European and two Japanese pear 4.3.1 Fruit Quality
cultivars registered as new mutant cultivars
(Ahloowalia et al. 2004). Most commercially Improved fruit quality is the cornerstone of every
available European pear cultivar mutations, pear breeding program. Fruit quality is a complex
whether naturally occurring or induced, involve trait, being a culmination of all external and
enhancement of red fruit skin color. Such stable internal characters of the fruit deemed of com-
red skin color sports have been used in various mercial importance. Contributing characters to
pear breeding programs for developing new fruit quality include the following: texture; fla-
red-colored fruit skin cultivars. vor; sweetness; sourness; skin scuffing; skin
Other mutations influencing disease resistance russet; physiological disorders; levels of bitter-
and responses to environment have been identi- ness; astringency; absence of grit cells within
fied. For example, the most important mutations flesh, skin, and around core tissues; skin color;
of Japanese pear include self-compatibility and general appearance; post-harvest performance;
resistance to black spot disease of ‘Nijisseiki’ and shelf-life. Breeders in different geographic
and ‘Shinsui’. These have now been used within regions place different emphasis on each of these
the Japanese breeding program (Ahloowalia et al. traits in selecting cultivars that perform best for
2004). Natural and induced mutations have also their specific breeding objectives under their
been identified for bloom time, blossom color, climatic conditions.
ripening period, and growth habit (Hancock and Breeders often rate overall fruit quality using
Lobos 2008). a composite score, determined from an amalga-
mation of phenotypic scores of many of the
individual traits listed above. This is predomi-
4.3 Target Traits for Selection nantly a hedonic score, and thus its narrow-sense
heritability is often very low (Bell et al. 1996). It
A good knowledge of the genetics controlling a has been suggested that eating quality in Euro-
target trait of interest is critical in optimizing pean pear is governed by non-additive gene
breeding strategies to maximize genetic gain and effects (i.e., through dominance and/or epistasis),
develop new cultivars carrying the desired trait. while narrow-sense heritability is completely
For those complex traits controlled by many absent for this trait (Bell et al. 1996). Further-
genes, estimates of heritability and combining more, specific combining ability (SCA) is much
ability provide information of the relative more important, thus suggesting that effective
importance of heredity compared with that of genetic gain for eating quality could be made by
environment in determining an individual’s selecting for individuals within families with
phenotype. Narrow-sense heritability (h2) high SCA (Bell et al. 1996). In other studies,
70 L. Brewer and R. Volz

heritability for overall fruit quality of either fruit types, firmness and initiation of starch
European pear or for mixed European and Asian hydrolysis (using a starch pattern index) may
pear families is low (h2 = 0.09–0.1) (White et al. serve as useful indicators to determine optimum
2000b). The heritability of a selection index for harvest time. Ideally, several samples should be
overall fruit quality weighted each trait in terms harvested from each seedling as fruit matures to
of importance before summing individual scores ensure that fruit from at least one of these fruit
is also low (h2 = 0.17) (Bell and Janick 1990). samples has been collected at optimum harvest
Environmental factors and developmental time.
(maturation and ripening) stages can have con-
siderable influences on many aspects of pear fruit 4.3.1.1 Texture
quality (Bell and Janick 1990). Although their Texture is a term used for the overall feel of food
interactions with genotypes have not been for- in the mouth and comprises properties that can be
mally documented, they must either be controlled evaluated by touch. It can include biochemical
or accounted for in order to accurately estimate components, such as particle size and shape,
genetic effects on fruit quality within a pear moisture content, lipid content, and cell wall
population. Pears of Asian parentages can be composition, as well as mechanical factors (Sams
harvested either near or at full eating ripeness 1999). Breeding programs often measure pear
when fruit starch has been converted into sugar. texture using a hedonic scale, which summarizes
In fact, tasting of the fruit may help determine influences of fruit firmness, hardness, juiciness,
stage of maturity. For those genotypes wherein flesh coarseness, grittiness, chewiness, crispness,
skin color changes during maturation, back- fruit fiber, skin chewiness, and oral sensory
ground color changes from green to response. This collective ‘eating experience’ has
yellowish-green which can signal optimum a very important influence on consumer accept-
maturity. Changes in flesh firmness (as measured ability of new products (Sams 1999). Although
hedonically or with a penetrometer) can also be a the genetics of pear texture is still poorly
useful measure of maturity. Furthermore, likely understood, seedling populations tend to reveal
commercial handling of fruit should also be taken continuous segregation for this trait, with a
into consideration; i.e., fruit harvested at an ear- general likelihood for polygenic control (Bell
lier stage of maturation for storage versus fruit and Janick 1990). Bell (1991) has suggested that
that will be consumed immediately after picking. moderate genetic gain could be achieved through
In contrast to Asian pears, fruit of most mass selection for texture as relatively large
European pears usually requires storage at cold ratios of GCA to SCA variance along with
temperatures to induce proper ripening (Sugar moderate narrow-sense heritability (h2 = 0.30)
et al. 2009). Lengths of chill induction periods have been observed.
required for European pear vary among different Firmness of ripe pear fruit varies considerably
cultivars. Summer maturing pears require a much among species. European pears are generally
shorter induction or no induction period (Bower eaten when soft, whereas Asian pear types are
et al. 2003) compared with later maturing pears, eaten firm. Most breeding programs concentrate
such as ‘Comice’ and ‘Beurré D’Anjou’ on one or the other, thereby attending to local
(‘Anjou’), which require 4 and 6 weeks of cold consumer demand for pear fruit that they are
storage, respectively; however, this is also accustomed to.
dependent upon harvest time (Sugar et al. 2009). In most European pear breeding programs,
If fruits are left on trees to ripen, internal soft, melting, or buttery, and juicy textures are
browning and other physiological disorders can most commonly selected for (Bell et al. 1996),
often develop during storage or during shelf-life. although occasionally either firm (Batlle et al.
Therefore, fruits are harvested well before 2008) or ‘almost’ crisp textures, similar to ‘Abaté
ripening when skin background color is still Fétel’, are also selected. In a study involving 10
green, and the flesh is hard and dry. For these European pear seedling populations, wherein
4 Genetics and Breeding of Pear 71

fruit are stored for 70 days at 0.5 °C followed by 4.3.1.2 Flesh Color
7 days at 20 °C, White et al. (2000a, b) have Although white and cream are the most common
found firmness heritability to be low (h2 = 0.06). flesh colors present in pear, green, yellow, pink,
This may reflect the low genetic variation and red colors are also known to naturally occur.
observed for fruit firmness among parents used in Segregation for white- and green-colored fruit
the study, and that ripening–inducing conditions flesh is controlled by a single gene, with white
have been adequate for this population. color being dominant, while green or cream
In contrast, heritability for fruit firmness esti- colors serving as alternative alleles (Bell et al.
mated for either Asian or interspecific hybrid 1996). Furthermore, segregation of progeny from
pear seedling populations tends to be moderate to crosses between the red-fleshed ‘Sanquinole’ and
high. For example, heritability estimates have the white-fleshed ‘Conference’ has revealed that
ranged from 0.14 to 0.56 for P. pyrifolia in a red flesh is dominant over white flesh (Bell et al.
Japanese breeding program (Saito 2016; Abe 1996).
et al. 1995), while estimates of 0.70 have been
reported in P. pyrifolia, P. ussuriensis, and P.  4.3.1.3 Flavor
bretschneideri seedling populations in a Korean Flavor is an important attribute of any pear cul-
breeding program (Shin et al. 2008). In New tivar. It encompasses a combination of sweet-
Zealand, heritability estimates for seedling pop- ness, sourness, bitterness, and astringency of oral
ulations with Asian, European, and interspecific sensory characters of pear fruits, along with
hybrid parentages (White et al. 2000b) or of pear volatile components sensed in the nose and throat
germplasm, including accessions of the same (Brewer et al. 2008b; Dondini and Sansavini
pear species, as well as those of interspecific 2012; Bell et al. 1996). An important aspect of
hybrids, are high (h2 = 0.62–0.67) (Kumar et al. flavor is the sugar–acid balance, which is
2017). Good genetic progress can be expected to enhanced by the presence of volatiles, particu-
be made in breeding for firm (or soft) textures larly in European pears (Eccher Zerbini 2002).
from such seedling populations where a wide As the presence of volatiles in Asian pear is less
range of fruit firmness is present. important, breeders have placed greater emphasis
Juiciness is an important component of fruit on high sugar levels when selecting genotypes
quality in both European and Asian pears. In for commercialization. Heritability estimates for
European pear, this trait is under both polygenic overall flavor, from subjective scores, vary from
and monogenic controls (Hancock and Lobos low (h2 = 0.06) in P. communis seedling popu-
2008; Zielinski et al. 1965). Using a hedonic lations to high (h2 = 0.54) in interspecific hybrid
method of evaluation for juiciness along a 0–9 seedling populations (Bell and Janick 1990).
scale, White et al. (2000b) have reported that
there is a low heritability for juiciness 4.3.1.4 Fruit Sweetness
(h2 = 0.04) in European seedling populations, High fruit sweetness is important for market
thereby indicating there is little variation present acceptance of any pear cultivar (Jaeger et al.
in parents used. Moreover, when Asian and 2003). Sweetness, scored subjectively on a
interspecific seedling populations are incorpo- hedonic scale or assessed as soluble solids con-
rated in the analysis, a slightly higher value centration, is a quantitative trait (Hancock and
(h2 = 0.21) is observed. Lobos 2008). In an early study by White et al.
Finally, for breeding programs of both Euro- (2000b), heritability of sweetness in European
pean and Asian pears, there is strong selection pear seedling populations and in hybrid Euro-
against presence of grit or stone cells in flesh pean–Asian pear seedling populations is found to
(h2 = 0.57), skin, and to a lesser extent around be low, h2 = 0.05 and h2 = 0.07, respectively,
the core, as well as toward fine (rather than and similar to that (h2 = 0.05) reported by Shin
coarse) texture (Bell and Janick 1990). et al. (1983). These seedling populations have
72 L. Brewer and R. Volz

been developed from crosses among parents commercial cultivars (Bell et al. 1996; Hancock
selected for ‘ideal’ levels of sugar. and Lobos 2008). Levels of total organic acid
In contrast, Abe et al. (1995) have reported vary within Pyrus taxa, wherein an average of
much higher heritability values (h2 = 0.37–0.5) 5.98 mg g−1 total organic acids has been repor-
using randomly selected combinations of hybrid ted for P. ussuriensis, 3.07 mg g−1 for P. 
seedlings from the Japanese pear breeding pro- bretschneideri, 2.66 mg g−1 for P. pyrifolia, and
gram at the National Agriculture and Food 2.42 mg g−1 for P. communis (Sha et al. 2011).
Research Organization (NIFTS). Progress in Moreover, relative and absolute acid levels can
breeding for higher sweetness in pear fruit could also be influenced by the environment (Hudina
be achieved by selecting for genotypes with high and Štampar 2004; Sha 2012; Sha et al. 2011).
flesh fructose concentrations. On a mole-to-mole Thus, levels of individual organic acids present
basis, fructose has a perceived sweetness that is in both European and Asian pears can also vary.
*1.4–2-fold higher than other storage sugars While malic and citric acids typically dominate,
present in pear fruit, including sucrose, sorbitol, quinic, oxalic, shikimic, fumaric, tartaric, suc-
and glucose (Harker et al. 2002; Saito 2016). cinic, acetic, and lactic acids are also present (Liu
Storage sugars in pear fruit consist of fructose, et al. 2016; Sha et al. 2011). Fruit of P. commu-
glucose, sorbitol, and sucrose (Saito 2016; Viera nis is found to have higher acetic acid levels,
et al. 2013). In a New Zealand study on seedling while fruit of P. ussuriensis has higher quinic
populations of interspecific hybrids with different acid levels than those of other Pyrus species (Sha
proportions of European, Japanese, and Chinese et al. 2011). Furthermore, malic and citric acids
(P.  bretschneideri) parentages, average sugar exhibit significant positive phenotypic correla-
levels are found to consist of 59% fructose, 13% tions with quinic acid; whereas, significant neg-
glucose, 20% sorbitol, and 8% sucrose (Viera ative correlations are observed between acetic
et al. 2013). In a Japanese study including 79 and lactic acid and between quinic and tartaric
Asian cultivars from Japan, Korea, and China, it acids (Sha et al. 2011).
is reported that average percentage concentrations In New Zealand, heritability of acidity eval-
of these sugars are found to consist of 36.7% uated on a hedonic scale was low in both Euro-
fructose, 15.2% glucose, 23.8% sorbitol, and pean seedling populations alone, and when Asian
24.4% sucrose. In the New Zealand study, indi- and interspecific seedling populations were
vidual sugar levels of glucose, fructose, and included, h2 = 0.07 and 0.09, respectively
sucrose contributed to higher genetic variance (White et al. 2000b). Low heritability (h2 = 0.17)
relative to total phenotypic variance (0.54–0.86) for titratable acid was also identified through a
compared with that for total sugars (0.31). Inter- genome-wide association study (GWAS) that
estingly, sorbitol levels have negative genetic included European, Asian, and interspecific
correlation (rG = −0.65) with fructose, a rela- hybrids (Kumar et al. 2017). However, Liu et al.
tionship that warrants further investigation. Thus (2016) reported high heritability of individual
far, genetic markers associated with soluble solids acids, including oxalic (h2 = 0.88, 0.57), quinic
concentration have been identified on LG10, LG5, (h2 = 0.71, 0.58), malic (h2 = 0.83, 0.77), shi-
and LG14, in an F1 population of ‘Bayuehong’  kimic (h2 = 0.82, 0.50), and citric (h2 = 0.75–
‘Dangshansuli’, but these have not been detected 0.80), when these were measured in consecutive
in all tested years (Wu et al. 2014). years in progeny of a reciprocal cross of
‘Dangshansuli’  ‘Hosui.’ It has been suggested
4.3.1.5 Fruit Acidity that there was a maternal influence for inheri-
Organic acids are yet another significant com- tance of these acids. Thus, when breeding for
ponent of pear fruit flavor serving to balance lower acid levels, a parent with the lowest levels
sweetness. For European pears, a range of acidity of oxalic, quinic, malic, and shikimic acids
between pH 2.4 and 5.4 can be acceptable in should be used as the female parent.
4 Genetics and Breeding of Pear 73

Single-nucleotide polymorphisms (SNPs) low heritabilities for isopentanol and hexanol

linked to titratable acidity have been identified on acetone (Li et al. 2004).
LG2 in a biparental cross between European and Breeding for flavors complemented by aro-
Asian species, and also in a genotyping-by- matic compounds is an important objective for
sequencing (GBS) study including European, the New Zealand Institute for Plant and Food
Asian, and interspecific hybrids (Liu et al. 2011). Research Ltd (PFR) pear breeding program.
A SNP associated with titratable acidity was also Crosses among P. communis, P. pyrifolia, and P.
identified on LG7 in a New Zealand GBS study  bretschneideri have generated interspecific
(Kumar et al. 2017). hybrids bearing fruit with a wide range of dif-
ferent flavors (Brewer et al. 2008b). Adverse
4.3.1.6 Fruit Volatiles flavors, such as alcoholic, grassy, and high acid,
Aromatic volatiles complement the sugar/acid are selected against. Interestingly, it has also
balance in fruit and provide a cultivar’s distinc- been possible to select for pears with novel fla-
tive flavor. This is important for European pear vors that can develop when fruit are either on the
cultivars, as they have a wide range of flavors, tree and/or at any time during storage. Some
from the subtle ‘Comice’ (Eccher Zerbini 2002) individual selections bear fruit that do not seem
to the strong distinctive flavor of ‘Bartlett’. to produce perceivable volatile flavors (Brewer
A total of 77 volatile compounds have been et al. 2008b), while others bear fruit requiring
identified in fruit of ‘Bartlett’ (Bell et al. 1996), chill induction before volatile flavors
with decadienoate esters contributing the most to develop. Clearly, there is much for pear breeders
its characteristic flavor (Eccher Zerbini 2002). to learn in developing cultivars carrying fruit
Fruits of other cultivars and selections, devel- with specific flavors (Xue et al. 2017b).
oped in breeding programs, with high levels of It is important to point out that those favorable
decadienoate esters are also deemed to possess a flavors detected in fruit flesh are rarely identified
‘Bartlett’ flavor. in the skin. This may indicate that flavor devel-
Fruits of Asian pear cultivars are not typically opment is differentially regulated in these tissues.
known for their strong aromas, particularly those Although bitterness, grassiness, and astringency
of Japanese pear, P. pyrifolia. However, fruits of can often be present in fruit skin, these are not
some cultivars of P. ussuriensis have strong perceived in fruit flesh (Brewer et al. 2008b).
aromas, and these differ in their volatile com-
pound compositions from those found in 4.3.1.7 Astringency and Bitterness
P. communis (Kang 2010). In addition, fruits of While all breeding programs for fresh con-
P. ussuriensis cultivars exhibit a very wide range sumption pears actively select against astrin-
of olefins, esters, alkanes, aldehydes, phenols, gency and bitterness in fruit flesh, often little
and ketones, and these cultivars serve as valuable attention is paid to fruit skin or areas around the
breeding material for these aromatic compounds. core. Breeding for cultivars destined for perry
Li et al. (2004) have identified variations in production is an exception, where both bitterness
complex levels of volatile compounds in fruits of and astringency are desired (Bell et al. 1996).
cultivars of P. ussuriensis, P. communis, P.  Low levels of astringency and bitterness can be
bretschneideri, and P. pyrifolia. Therefore, it is acceptable for fresh consumption when this
suggested that inheritance of these compounds is enhances the overall flavor perception. Bitterness
quantitative, and controlled by multiple genes. and astringency are associated with presence of
Analysis of 16 different volatile compounds from phenolic and polyphenolic compounds, including
two families of P.  bretschneideri  tannins and leucoanthocyanins (Bell et al. 1996).
P. ussuriensis has demonstrated high heritabili- High levels of fruit astringency can be present
ties for acetone, ethanol, propyl alcohol, and when wild germplasm is used as parents in
aldehyde, moderate heritabilities for ethylene, crosses for introgression of other desirable traits.
isopropanol, propionate ethyl, isovalerate, and In the New Zealand PFR breeding program,
74 L. Brewer and R. Volz

bitterness is often detected in the skin of fruit of at 16.3 cM from a QTL identified on LG17 of
seedlings, but not as much in flesh of this fruit. ‘Dangshansuli’. In the second year of this study,
Population-level improvements in decreasing marker Pyb13_250, associated with fruit size,
bitterness and astringency have been reported, as was identified at 99.3 cM on LG13 of ‘Bayue-
both traits have virtually disappeared by the third hong.’ Additional research should be conducted
generation (Brewer et al. 2008b), even though to validate these markers.
early research has indicated that there is a low
heritability (h2 = 0.01) for astringency (White 4.3.1.9 Functional Compounds
et al. 2000b). To date, breeding programs have put very little
effort into improving health attributes of pear
4.3.1.8 Fruit Size fruit by increasing levels of bioactive com-
Fruits of various pear species exhibit wide ranges pounds. However, consumer preferences are
for fruit size, as this is influenced by genetics, increasingly focused on health-promoting quali-
environment, and management factors, such as ties of fruits and vegetables, and consumers can
water availability, fruit set, fruit thinning, and make purchasing decisions based on phytonutri-
overall crop load. P. calleryana and P. betulae- ent levels present in these foods (Patil et al.
folia, commonly used as rootstocks, can bear 2016). Researchers have quantified some bioac-
fruit as small as 1 cm in diameter (Hancock and tive compounds present in pear cultivars and
Lobos 2008). These species would require sev- germplasm (Abaci et al. 2016; Kolniak-Ostek
eral generations of improvement for fruit to reach 2016; Galvis Sánchez et al. 2003; Tanrıöven and
a suitable commercial size and eating quality. Ekşi 2005; Yim and Nam 2015). Fortunately,
Cultivars of European, Japanese, and Chinese presence of significant differences in contents of
white pear, such as ‘Uvedales Saint Germaine’, these bioactive compounds among pear cultivars
‘Dongguanli’, and ‘Xuehuali’, respectively, can offers opportunities for improvement in future
produce very large fruit (Cao 2014). Pear fruit breeding efforts, as does higher concentrations of
size is under polygenic control, but a range of anthocyanins in red skin and flesh of pear (Abaci
heritability values, depending on the population et al. 2016; Yim and Nam 2015). Promotion of
used (Hancock and Lobos 2008). For example, in cultivars with research-supported health benefits
the NIFTS program in Japan, heritability values is already underway (Sarkar et al. 2015;
of h2 = 0.57–0.82 have been reported for Stephenson 2015; Barbosa et al. 2013).
P. pyrifolia (Saito 2016), and in the Korean
breeding program, heritability values ranging 4.3.1.10 Storage Period and Shelf-life
between h2 = 0.09 and h2 = 0.85 have been Maintaining fruit in good condition during cool
reported for interspecific hybrid populations storage and until the point of sale is an important
among P. pyrifolia, P. ussuriensis, and P.  attribute of any new cultivar, and it is an
bretschneideri (Shin et al. 2008). In this latter important goal in many breeding programs (Bell
study, heritability variations are dependent on the et al. 1996). The PFR interspecific pear breeding
parental cultivar used in these crosses. For program selection is strongly directed toward
example, ‘Whangkeumbae’ and ‘Gamcheonbae’ fruit that retains high-quality texture attributes
are found to have high heritabilities, h2 = 0.76– following a minimum cold storage period of two
0.85 and h2 = 0.47–0.84, respectively, for fruit months at 0.5–3 °C (Brewer et al. 2008b).
size, while ‘Niitaka’ has a low heritability Results from segregating seedling populations
(h2 = 0.11–0.29). indicate that fruit storage potential is under
Quantitative trait loci (QTL) were identified polygenic control (Bell et al. 1996). Thus, there
for fruit weight in progeny of ‘Bayuehong’  are several reasons why fruit may fail storage
‘Dangshansuli’ population, with a marker located testing. The most common of these are
4 Genetics and Breeding of Pear 75

post-harvest disorders, such as internal browning, 4.3.2 Fruit Attractiveness

chilling injuries, and flesh spot decay (Brewer
et al. 2008b). 4.3.2.1 Fruit Shape
Fruit ethylene production at harvest has been Pear fruit shape is under polygenic control with
negatively associated with storage life in round and ovate shapes observed more fre-
P. pyrifolia. Ethylene production in pear is con- quently than pyriform and turbinate shapes in
trolled by two 1-amino-cyclopropane-1-car- Asian, European, and interspecific hybrid seed-
boxylic acid (ACC) synthase genes, pPPACS1 ling populations (White and Alspach 1996).
and pPPACS2, with dominant alleles associated A high heritability (h2 = 0.55) for fruit length:-
with high and moderate ethylene levels, respec- maximum width ratio suggests a relatively rapid
tively. PPACS2 has been mapped along the top progress can be made in breeding for fruit shape
of LG15 in P. pyrifolia (Itai et al. 1999). Many (White et al. 2000a). For European pear,
older Japanese pear cultivars carry the dominant acceptable genetic advances could be made for
pPPACS1 allele, while newer cultivars tend to pyriform curvature (h2 * 0.5), whereas the
possess both recessive alleles. This finding location of the point of maximum curvature has a
reflects selection for material with longer low heritability (h2 = 0.01) (White et al. 2000a).
storage/shelf-life and lower ethylene production Therefore, identification of fruit shapes that are
in modern Japanese pear breeding programs (Itai different from the typical pyriform fruit can be
and Fujita 2008). Restriction fragment length made, especially when pyriform-fruited parents
polymorphism (RFLP) markers for these two are crossed with parents with either round- or
genes have been developed to predict low ethy- ovate-shaped fruit.
lene production in pear material in breeding
programs (Itai and Fujita 2008). Interestingly,
regulation of genes controlling ethylene produc- 4.3.3 Fruit Skin Ground Color
tion in P.  bretschneideri cultivars that are
either climacteric (‘Yali’) or non-climacteric Background color of pear fruit skin is dependent
‘(Hongli’) is suggested to be similar to that on the relative concentrations of green (chloro-
observed in P. pyrifolia (Yamane et al. 2007). phyll) and yellow (carotenoid) pigments present
However, P. communis cultivars do not carry in the skin epidermis. During the ripening pro-
these pPPACS haplotypes (Oraguzie et al. 2010), cess in most pear cultivars, background color
thus suggesting presence of a separate system of changes from green to either yellow-green or
ethylene control. yellow following increase of carotenoids and/or
A long shelf-life for fruit following cold breakdown of chlorophyll; however, the timing
storage is also important for any newly released of this color change can vary considerably (Bell
cultivar. Therefore, many breeding programs et al. 1996). In some cultivars, such as ‘Confer-
target a set shelf-life period following cold stor- ence’ the skin remains fully green, but only turns
age. At PFR, a period of seven days at 20 °C is a yellow when the fruit is fully ripe, while for other
minimum standard used to simulate a typical cultivars, this change occurs at the onset of
time period for purchase and consumption of ripening; e.g., ‘Packham’s Triumph’. Genetic
fruit (Brewer et al. 2008b). Taking advantage of studies in European pear indicate that back-
the extended shelf-life inherent in many old ground skin color is controlled by a major gene,
Chinese pear cultivars, the New Zealand program with yellow being dominant over green (Han-
maintains fruit from the best seedlings on a shelf cock and Lobos 2008). Inoue et al. (2006) have
at 20 °C until they either rot, turn internally used a bulk segregant analysis of two F1 Japa-
brown, or shrivel. This approach has allowed for nese pear progenies to identify a 425-bp random
identification of advanced selections for up to amplification of polymorphic DNA (RAPD)
30 days of shelf-life following cold storage marker associated with green skin color exhibit-
(Brewer and Palmer 2011). ing a recombination rate of 7.3%. This RAPD
76 L. Brewer and R. Volz

marker has been converted into a RAPD levels of five anthocyanin biosynthetic genes
sequence-tagged site (STS) marker to identify a involved in the anthocyanin biosynthesis path-
QTL at the top of LG8 at 2.2 cM (Yamamoto way, and thereby inducing red skin color devel-
et al. 2014; Inoue et al. 2006). opment (Ubi et al. 2006). On the other hand, high
temperatures reduce anthocyanin biosynthesis
through down-regulation of regulatory gene
4.3.4 Fruit Skin Over-Color transcription factors for anthocyanin production,
including those of MYB, bHLH, and WD40
Red fruit over-color is an important breeding (Steyn et al. 2005; Thomson et al. 2018) which
target for many programs around the world, as it can also reduce the stability of existing antho-
can greatly enhance attractiveness of fruit cyanins (Mori et al. 2007). Anthocyanin degra-
(Brewer and Palmer 2011). Currently, dation and color loss are reported to increase
red-skinned pears are sold at higher prices in linearly between 10 and 30 °C (Steyn et al.
international markets (Steyn et al. 2005). This is 2005), more so in ‘Rosemarie’ because of its
due to the low volume of these cultivars, but they lower capacity to synthesise anthocyanin (Steyn
also have high eating and storage qualities. Red et al. 2004). Higher concentrations of antho-
color pigmentation is the result of accumulation cyanin provide a buffer for color loss before high
of anthocyanins, specifically of cyanidin3- temperatures visibly affect red coloration of fruit
glactoside and cyanidin3-arabinoside, which are skin (Steyn et al. 2004). Conversely,
secondary metabolites synthesized, via the fla- high-colored cultivars, such as ‘Bon Rouge’ and
vonoid biosynthetic pathway, in hyperdermal ‘Flamingo’, do not respond to low temperatures
layers of the skin (Steyn et al. 2005; Thomson for anthocyanin synthesis, while ‘Rosemarie’
et al. 2018). Genetic expression of these antho- does.
cyanins is highly heritable, and hence can be It has been reported that in P. communis, high
readily exploited in breeding programs. How- red fruit skin color pigmentation is attributed to
ever, anthocyanin levels are not always consis- spontaneous bud mutations of green-skinned
tent, as these can change during fruit cultivars, including ‘Bartlett’, ‘Comice’, and
development, and may also vary under different ‘Beurré D’Anjou’, wherein not only the fruit skin
environmental conditions, although they can also is red, but also those of leaves, especially of new
be enhanced by various cultural production shoot growth (Booi et al. 2005). Often, these
practices (Thomson et al. 2018; Steyn et al. mutations are not stable, and some tissues of a
2005). tree, such as leaves and fruit, can revert back to
In most flowering plants, fruit red skin color the original phenotype (Booi et al. 2005). Nev-
levels tend to develop most strongly during ertheless, stable mutants of these cultivars have
ripening (Thomson et al. 2018). Some pear cul- been commercialized, such as ‘Max Red Bar-
tivars, such as ‘Bon Rouge’ (a mutant of ‘Bar- tlett’, ‘Bonne Rouge’, and ‘Sensation’, all sports
tlett’), ‘Flamingo’, and ‘Rosemarie’ appear to of ‘Bartlett’. However, many red mutants
deviate from this pattern as they attain their released commercially, including ‘Crimson
maximum anthocyanin levels midway between Gem’, a red ‘Comice’, have had limited success
anthesis and harvest. From then on, anthocyanin because of poor tree vigor and cropping (Dondini
synthesis decreases slowly until harvest time in and Sansavini 2012). Furthermore, mutagenesis
response to light, temperature, solar radiation, has also been used to develop commercial culti-
and competition for assimilates (Steyn et al. vars of ‘Bartlett’ with red skin pigmentation,
2005; Thomson et al. 2018). Color development such as ‘Homored’ (Dondini and Sansavini
in pears either requires or is enhanced by light 2012). The red tissue color induced by such
intensity, and wavelength (Thomson et al. 2018). mutations is controlled by a major dominant gene
Dramatic drops in temperature as well as low with a simple 1:1 segregation ratio for red:green
temperatures promote increases in transcript seedlings, for both leaf and fruit phenotypes, thus
4 Genetics and Breeding of Pear 77

indicating Mendelian inheritance for this trait leaves and fruit with varying intensities of red
(Booi et al. 2005). Subsequently, this red color color pigmentation (Volz et al. 2008). Some
has been mapped to LG4 using a simple mutants, such as ‘Starkrimson’, derived from
sequence repeat (SSR)-enriched map of an ‘Abbé ‘Clapp’s Favorite’, are not capable of transferring
Fétel’  ‘Max Red Bartlett’ seedling population red fruit skin coloration to their progeny as the
(Pierantoni et al. 2004; Dondini et al. 2008). mutation is only present in the epidermis, i.e., the
Pierantoni et al. (2010) have mapped germ layer does not carry the mutation (Bell et al.
PcMYB10, which encodes an R2R3-MYB tran- 1996).
scription factor involved in the control of the Some genetic sources for red fruit skin color
anthocyanin biosynthetic pathway, onto LG9 of in both Asian and European cultivars are totally
both ‘Abbé Fétel’ and ‘Max Red Bartlett’. This dependent on solar radiation and light to induce
corresponds to the same location as MdMYBa red blush development on fruit (Zhang 2012;
and MdMYB10 that control red color pigmenta- Steyn et al. 2005). Therefore, presence of a gene
tion in fruit skin of apple (Espley et al. 2007). (s) controlling red skin color from these sources
The pear transcription factor PyMYB10 gene, a cannot be inferred from red leaf color of seed-
likely ortholog of MdMYB10, has been positively lings. In a New Zealand study, segregation ratios
associated with anthocyanin biosynthesis in of 5(non-blush):3(red blush) for fruit blush,
ripening fruit of red-skinned pear, and its func- derived from P. pyrifolia cv. Huobali, are
tion has been confirmed (Feng et al. 2010; Yao observed in four seedling populations; whereas,
et al. 2017). Yet, another transcription factor, segregation ratios of 3(non-blush):1(red blush)
PyMYB114, has been identified on LG5 of Chi- are obtained in three other seedling populations.
nese pear (P.  bretschneideri), and its abun- Furthermore, when both parents are descendants
dance, correlated with PyMYB10 in enhancing of ‘Huobali’, segregation ratios of 3(non-blush):5
anthocyanin biosynthesis, is confirmed when (red blush) in four seedling populations and 7
co-transformed in both tobacco and strawberry (non-blush):9(red blush) in three other seedling
(Yao et al. 2017). Kumar et al. (2017) have also populations have been observed. These segrega-
identified a SNP associated with red skin phe- tion ratios indicate that a complementary
notype on LG9, but it is unclear whether or not it two-dominant gene control mechanism is pre-
is associated with PcMYB10. Recently, Ntladi sent, wherein both genes are required for color
et al. (2018) have mapped a major QTL near the development. A similar segregation pattern for
telomeric region on LG9 of ‘Abbé Fétel’ that is red blush color fruit may also be observed for
associated with genes MYB21 and MYB39, seedling populations involving P. communis cv.
which is found to be responsive to environmental Louis Bonne de Jersey, an old French cultivar
changes, and varies between years. with red blush fruit (Volz et al. 2008). However,
Breeding programs have used a range of different segregation ratios have been observed at
red-skinned bud sports, such as ‘Max Red Bar- the Zhengzhou Fruit Research Institute (ZFRI) in
tlett’, ‘Red Sensation’, and ‘Rosired’, as parents China in crosses wherein both parents, ‘Man-
to transfer the red color pigmentation to new tianhong’, derived from ‘Huobali’, and ‘Hongx-
cultivars (Dondini and Sansavini 2012). Earlier, iangsu’, derived from ‘Korla Pear’, have red skin
it has been reported that phenotypic selection for color fruit. Segregation ratios of 3(non-blush): 2
red leaf color is possible in segregating seedlings (red blush) and 9(non-blush):8(blush) in seedling
of young nursery plants (Booi et al. 2005), and populations of ‘Mantianhong’  ‘Hongxiangsu’
that it is easy for breeders to identify seedlings and ‘Yuluxiang’  ‘Mantianhong’, respectively,
carrying the dominant gene for red color without have suggested that the red skin color trait is
using marker-assisted selection (MAS). How- controlled by a single dominant gene that tends
ever, seedlings carrying a gene for red skin color, toward green-skinned segregation (Xue et al.
developed from red-skinned sports, develop 2017a).
78 L. Brewer and R. Volz

In the above Zhengzhou studies, red skin that both carry more than one source of red color
coloration mapped to a 111.9–177.1 cM QTL gene(s), as heritability is then found to be high
interval on LG5 (Xue et al. 2017a). This is a (h2 = 0.86) for red color fruit (Kumar et al.
different chromosomal location to the dominant 2017). Once crosses are made using parents
gene derived from the European pear ‘Bartlett’ carrying multiple sources of red skin fruit color,
which is mapped to LG4 (Dondini et al. 2008). MAS would be beneficial in identifying seed-
Recently, Ntladi et al. (2018) have also identified lings carrying specific sources of red color.
two SSR markers, NB101a and SamsCo865954,
that are closely associated with a major QTL for
skin blush on LG5 in ‘Flamingo’. These markers 4.3.5 Fruit Russet
are present in approximately 90% of seedlings
that scored a high blush level. Thereby, two Unlike many other fruits, the presence of russet
candidate genes, MYB86 and UDP- on fruit is acceptable for fresh market pears, as
glucosyltransferase, have been identified. Ear- long as russet is smooth, and ideally, fully cov-
lier, in an F1 population of 102 individuals from ering the skin (Bell et al. 1996). Russeting of the
a cross of ‘Bayuehong’ (‘Clapp’s Favourite’ (red fruit pericarp is attributed to accumulation of a
sport) and ‘Zaosu’)  ‘Dangshansuli’, QTLs for cork layer resulting from suppressed biosynthesis
control of red skin color have been mapped to of suberin, cutin, and wax, and this layer can be
LGs 4, 13, and 16 (Wu et al. 2014). Interestingly, either green or brown in color (Wang et al.
the QTL on LG4 is located at 4.8 cM (Wu et al. 2014). Inoue et al. (2006) have obtained a 3:1
2014), differing from that mapped for ‘Bartlett’ at segregation ratio for russet:non-russet and partial
64 cM (Dondini et al. 2008), while QTLs for red russet fruit in an F1 seedling population where
blush are located on LG13 or LG16, and are both parents have russeted fruit skin, and a 1:1
deemed to be novel. Collectively, these results ratio in an F1 seedling population derived from
suggest that additional research to elucidate these fully russeted and partially russeted parents.
different loci controlling red color in pear along White et al. (2000b) have calculated a low heri-
with their interactions must be conducted. tability (h2 = 0.16) for russet in ten European
It is critical to point out that breeding for pear seedling populations; however, when five
either full-red or blushed fruiting pear cultivars Asian and interspecific crosses are included, the
for hot climate regions is challenging, as fruit heritability is found to increase (h2 = 0.55). This
skin color loss, close to harvest time, can be high. finding is similar to heritability values reported
Therefore, it is important to choose cultivars with earlier (Bell and Janick 1990), as well as in a
the highest anthocyanin levels and fruit blush as GBS study of European, Asian, and interspecific
parents in breeding programs to minimize the germplasm (Kumar et al. 2017).
likelihood of anthocyanin degradation due to hot Early on, Kikuchi (1924, 1930) has proposed
temperatures and intense light exposures in these that pear fruit russet is controlled by two loci,
environments (Steyn et al. 2004). In the joint R and I. More recently, it is hypothesized that the
Spanish Institut de Recerca i Tecnologia Agral- R locus has a dominant effect on cork layer
imentàries (IRTA)/PFR breeding program, development, and the modifier locus I has a
selection of parents with high levels of red color dominant effect on russet suppression (Saito
and carrying more than one source of red color 2016). In this proposed model, RR genotypes are
genes have been successful in developing pear completely russeted, Rrii are partially russeted,
cultivars that retain high levels of red color at and RrI are partially russeted when environ-
harvest time under Spanish growing conditions mental conditions are ideal (Hancock and Lobos
(Batlle et al. 2008). 2008). A major QTL for russet has been identi-
It has been recently reported that very good fied on LG8 (Yamamoto et al. 2014; Kumar et al.
breeding progress can be made by using parents 2017; Inoue et al. 2006).
4 Genetics and Breeding of Pear 79

4.3.6 Fruit Skin Friction Discoloration 4.4 Tree Production

(Scuffing)
Cultivars that produce many branches; i.e.,
Marking of fruit skin (scuffing) during ‘feathering’, naturally facilitate clonal propaga-
post-harvest handling operations and in the tion of trees by nurserymen, especially for those
supermarket following cold storage is a serious trees that will be planted in traditional orchard
problem for many commercial pear cultivars, as systems, wherein within-row planting distances
this downgrades fruit quality and discourages are wider than those of closely planted systems.
purchase (Brewer et al. 2011; Saeed et al. European pear cultivars ‘Conference’ and ‘Abeté
2014). The mechanism causing scuffing Fetel’ produce high numbers of feathers in con-
involves a combination of physical stress and trast to ‘Passe Crassane’ (Dondini and Sansavini
biochemical reactions, in particular enzymatic 2012) and to Asian cultivars. Some Asian culti-
oxidation of polyphenols by polyphenol oxi- vars and interspecific hybrids develop few bran-
dase (PPO) (Saeed et al. 2014). Harvest matu- ches along with very upright-growing shoots.
rity can influence scuffing susceptibility, This suggests that heading of young trees planted
although this trait is genotype dependent (Saeed in a nursery or an orchard, along with use of
et al. 2014). plant growth regulator treatments may be
Analysis of interspecific seedling populations required to induce feathers. Currently, an
derived from European and Asian pedigrees has understanding of the genetic factors controlling
revealed that scuffing has a high narrow-sense feather/shoot production is lacking.
heritability of h2 = 0.72 with a high correlation
between years (Brewer et al. 2011). Using
germplasm accessions of similar, but wider 4.4.1 Precocity
genetic backgrounds, a subsequent GBS study
has confirmed this observed high heritability As perennial fruit trees have long juvenile peri-
(h2 = 0.61) and year-to-year repeatability ods, reducing this juvenility period is very
(Kumar et al. 2017). It has been reported that important for all these breeding programs
susceptibility to low-scuffing is derived from (Brewer and Palmer 2011). Pears grown com-
Asian pear (Brewer et al. 2011), and this is mercially in countries like New Zealand must be
supported by a finding that the largest effect SNP competitive with apples in terms of speed to
allele associated with scuffing is present in Asian production (Brewer and Palmer 2011). Progress
but absent in European pear accessions (Kumar can be made in breeding for a reduced juvenile
et al. 2017). Scuffing is a complex polygenic trait period in pears as this trait is under additive
as highlighted by the identification of 105 QTLs genetic control (Bell et al. 1996), and there is a
associated with 22 relevant fruit traits, including positive correlation between length of the juve-
those of average scuffing score, fruit firmness, nility period and precocity of selections propa-
polyphenoloxidase (PPO) activity, ascorbic acid gated onto rootstocks.
concentration, and production of 17 polyphenolic In general, seedlings of P. pyrifolia are more
compounds (Saeed et al. 2014). With this many precocious than those of P.  bretschneideri and
small-effect QTLs distributed over 11 chromo- P. communis (Bell et al. 1996). Selection of
somal regions (LGs 2, 3, 4, 7, 9, 10, 11, 13, 14, parents for reduced juvenile period and increased
15, and 16), it is suggested that genomic selec- precocity over several generations in the New
tion is better suited in identifying Zealand program has enabled development of
scuffing-resistant individuals early in the breed- seedlings that can come into fruiting within three
ing cycle. In a GBS study, Kumar et al. (2017) years following crossing in some interspecific
have identified a SNP for scuffing on LG15. hybrid progenies.
80 L. Brewer and R. Volz

4.4.2 Harvest Season Given the moderate to high heritability for

fruit ripening date reported above, choice of
Extending the harvest season will maximize use parents in breeding for early or late fruit ripening
of grower and packing house resources and will is important. If both parents are early season
support efforts in meeting market needs (Dondini cultivars, a greater proportion of their progeny
and Sansavini 2012; Bell et al. 1996; Brewer and will have this desired trait (Bell et al. 1996).
Palmer 2011; Saito et al. 2015). Although there is Similarly, if both parents are late-season culti-
a high demand for the first fruit of the new sea- vars, a larger proportion of their progeny will
son, many early season pear cultivars have poor mature later in the season, as compared with
fruit quality, small fruit size, uneven ripening, progeny from one early- and one late-season
and short storability due to internal breakdown parent (Bell et al. 1996). Newly improved early
(Bell et al. 1996; Dondini and Sansavini 2012; season European pear cultivars that have been
Saito 2016). In an Asian pear seedling popula- released from Italian breeding programs include
tion, Abe et al. (1993) have observed a high ‘Etrusca’, ‘Sabina’ (Bellini and Nin 2002),
positive correlation between mid-season ripening ‘Tosca’, ‘Norma’, and ‘Carmen’ (Rivalta et al.
parents and fruit weight. Furthermore, the pres- 2002), while in Japan, ‘Hatsumaru’ with fruit
ence of a strong link between high ethylene quality equivalent to ‘Kosui’ has recently been
production and early maturity in Japanese pear released (Saito 2016).
cultivars explains their observed poor storability
(Itai et al. 2003).
It has been reported that fruit harvest date is a 4.4.3 Parthenocarpy
polygenic trait, with low environmental influence
(Abe et al. 1993). A high heritability for ripening Parthenocarpy, development of fruit without
date, h2 values of 0.80–0.95, has been reported in fertilization of ovules and rendering fruit seed-
seedling populations of Asian heritage (Nishio less, is a useful commercial trait of European
et al. 2011; Abe et al. 1993). This has been fur- pear. This is especially important in some
ther confirmed in a recent study wherein heri- pear-growing regions in Europe whereby early
tability of h2 = 0.83 has been reported spring frosts and adverse conditions can prevent
(Hae-Sung et al. 2015). On the other hand, effective pollination. It is reported that in some
moderate heritability (h2 = 0.49) for ripening growing environments wherein pear cultivars are
date has been reported in seedlings of late capable of developing parthenocarpic fruit, pol-
ripening parents of European pear heritage (Bell linators are not deemed necessary (Bell et al.
et al. 1996). 1996; Nishitani et al. 2012).
QTLs controlling harvest date have been In a study investigating parthenocarpy in 31
identified at the bottom of LG3 (nearest marker: accessions of several pear species, including P.
BGA35) and at the top of LG15 (nearest marker:  bretschneideri, P. ussuriensis, P. pyrifolia,
PPACS2) of ‘Taihaku’ (Yamamoto et al. 2014). P. communis, and interspecific hybrids, it is
The PPACS2 probe for an ACC synthase coding found that five tested European pear cultivars
gene, identified in a DNA band of 0.8 kb in have consistently set fruit, and the fruit has
length, is found to be specific to P. pyrifolia enlarged size in the absence of pollination
cultivars producing moderate ethylene levels (Nishitani et al. 2012). Some Chinese and
during ripening and storage (Saito 2016; Itai European cultivars, such as ‘Mili’, ‘Wowoli’,
et al. 1999). Recently, Ntladi et al. (2018) have ‘Alexandrine Douillard’, ‘Bartlett’, and ‘La
detected a QTL on LG9 of ‘Flamingo’ explaining France’ are found to have partial compatibility
more than 30% of the phenotypic variance, with when self-pollinated. Moreover, it is observed
88% accuracy, for seedlings flowering earlier that Chinese and Japanese cultivars do not
than either parent in a progeny of ‘Flamingo’ demonstrate consistent and stable fruit set with-
‘Abate Fetel’. out fertilization when compared to European
4 Genetics and Breeding of Pear 81

cultivars. Among these cultivars, ‘La France’ is others, such as ‘Winter Bartlett’, ‘Red Bartlett’,
deemed the best-performing cultivar, as and ‘Max Red Bartlett’, approximately 1050 h of
non-fertilized fruit weighed only slightly less chilling is required (Kretzschmar et al. 2011).
than pollinated fruit. Furthermore, it is observed The majority of pear cultivars adapted to sub-
that fruit weight and size of non-fertilized fruit tropical growing conditions belong to P. pyrifo-
are inherited, thus it should be possible to lia. While most European pear cultivars are not
transfer this parthenocarpy trait from the Euro- well adapted to these growing conditions, there
pean pear cultivar La France to Japanese or are a few exceptions. These exceptions include
Chinese pears (Nishitani et al. 2012). ‘Hood’ and ‘Flordahome’ (requiring 250 chill
It has been reported that three phenyl- hours between 3–5°C), both are hybrids between
propanoid pathway-related genes are found to be P. communis and P. pyrifolia. ‘Flordahome’ has
either up- or down-regulated in highly been developed and released from the University
parthenocarpic pear cultivars (Nishitani et al. of Florida breeding program in 1982 (Sherman
2012). Therefore, breeding for parthenocarpy and Lyrene 2003).
may be accelerated by using molecular markers Interspecific hybridizations between P. pyrifo-
for these three genes once these markers are lia and P. communis have been used to develop
developed and validated across species (Nishitani low-chill European pears; however, fruit quality
et al. 2012). However, parthenocarpy is a low of low-chill P. communis cultivars, such as
priority in most Asian pear breeding programs ‘Kieffer’ (550 chill hours between 3 and 5 °C),
(Nishitani et al. 2012), as absence of seeds in ‘Le Conte’ (450 chill hours between 3 and 5 °C),
parthenocarpic fruits is associated with lower and ‘Garber’, is low (Hauagge and Cummins
fruit flavor and lower soluble solid concentra- 2013; Abd El-Zaher et al. 2015). Interestingly,
tions (Bell et al. 1996). F1 seedling populations in a Mexican pear
breeding program have resulted in seedlings with
chill requirements ranging from 0 to 500 chill
4.5 Adaptation to Abiotic hours (Rumayor et al. 2005). Moreover, ever-
and Biotic Stresses green types have been identified from an
open-pollinated seed population of ‘Hood’, as
4.5.1 Low-Chill Requirement these seedlings do not require low temperatures
to break dormancy (Rumayor et al. 2005).
Temperate zone cultivars are not well adapted for Finally, breeders in Egypt have used ‘Hood’,
regions with subtropical climates, wherein chill ‘LeConte’, and ‘Yali’ in crosses, and have
requirement, necessary to achieve adequate selected a range of seedlings requiring fewer than
flowering, is often unmet. Breeding for adapta- 200 chilling hours at 7 °C (Abd El-Zaher et al.
tion for low-chill requirement, i.e., flowering 2015; Stephenson 2015; Barbosa et al. 2013).
after fewer chilling hours, is one approach to
develop cultivars with satisfactory yields and
acceptable fruit quality in regions with warmer 4.5.2 Cold Hardiness
climates. As time of bud break is not a good
indicator of chilling requirement, it is preferable Pears are grown in many parts of the world
to screen seedling trees for number of buds where temperatures can drop low enough to
breaking (Rumayor et al. 2005). cause cold injury to shoots, spurs, trunks, and
Japanese pear cultivars (P. pyrifolia) require roots that may result in tree death. Plant cold
approximately 800 chill hours to break dormancy hardiness is a complex trait, as it is influenced by
(Yamamoto et al. 2010); whereas, the estimated temperature, day length, and plant physiological
minimum chill hour requirement at 3 ± 1 °C for status (Palonen and Buszard 1997). Thermal
some European pears such as ‘Rocha’, ‘Pack- analysis can be used for measuring cold hardi-
ham’s Triumph’, and ‘Forelle’ is 750 h, while for ness for some pear tissues (Quamme 1991).
82 L. Brewer and R. Volz

However, breeding for cold adaptation is best Screening for genetic resistance to a pest or
undertaken under actual growing environments, disease in a germplasm collection to develop new
which may include Northern regions of the USA, cultivars with either higher tolerance, or ideally,
Canada, Europe, Russia, and Mongolia. resistance is an attractive proposition for any pear
Although genetic progress has been made, and breeding program. The long-term efficacy of
pear cultivars have been developed that can resistance should be carefully considered, as
withstand winter temperatures as low as −30 to breakdown of resistance by different strains of
−40 °C, fruit quality is not deemed as satisfac- the pathogen or pest can occur (Bus et al. 2011).
tory as those commercial cultivars grown in Therefore, breeding for durable resistance using
major pear-growing regions (Bell 1991). multiple resistance genes should be a long-term
Low spring temperatures, particularly early goal for pear breeding programs, as it is already
spring frosts, often cause flower damage and crop the case for apple (Bus et al. 2011).
loss. As flower buds do not supercool, the earlier Fruit quality breeding objectives, mentioned
a cultivar flowers, the greater the risk of spring in earlier sections, should not be ignored while
frost damage (Bell et al. 1996; Palonen and breeding for disease/pest resistance as no matter
Buszard 1997). Breeding for late flowering to how strong and effective the resistance of a cul-
avoid frost or to promote parthenocarpy is an tivar, consumer’s interest is mainly focused on
option, as bloom date is highly heritable, but fruit attractiveness and eating quality. The
noting that late flowering is not linked to late genetic background conferring resistance/s
fruiting (Quamme 1991; Palonen and Buszard should also be taken into consideration. For
1997). pear, the breeding cycle is at least 5 years, and
Although inheritance of cold hardiness has evaluation before cultivar release can take in
not been investigated in pear, it has been reported excess of 15 years. Thus, introgression of resis-
that cold hardiness in apple is under polygenic tance genes carried by large-fruited eating culti-
control with additive effects, and with little evi- vars and land races of P. communis, P. pyrifolia,
dence for incidence of epistasis and dominance P.  bretschneideri, and P. ussuriensis into new
(Bell et al. 1996). A range of pear cultivars have cultivars would yield high fruit quality more
been classified for their vulnerabilities to winter readily than introgression of resistance genes
injury based on cold damage to xylem and frost from small-fruited Pyrus species of poor fruit
injury to buds. In general, it has been reported quality. More specifically, in breeding of Euro-
that pear xylem and flower bud hardiness are not pean pears, introduction of resistance genes from
highly correlated (Bell 1991; Bell and Itai 2011). other European pears is highly desirable, and
equally, introduction of resistance genes from
Asian species is more suitable in breeding for
4.5.3 Disease and Pest Resistance Asian pears.
This section of the review concentrates on
The genus Pyrus is susceptible to damage from current status of breeding for resistance to the
various numbers of diseases and pests (Bell et al. three major diseases of pear, including fire blight,
1996). The importance of a specific pest or dis- pear scab, and black spot, as well as for the
ease in any particular region will be dictated by important economic pest of pear psylla (Psylla).
the cost of control management as well as the
detrimental economic impact on the crop, par- 4.5.3.1 Fire Blight Resistance
ticularly whereby control is less than fully Fire blight, caused by the bacterium E. amylo-
effective. In some cases, susceptible cultivars are vora (Burrill) Winslow et al., is a serious disease
excluded from certain regions due to devastating of pear, and indeed of various other Rosaceae
effects of a pathogen or pest on tree productivity species (Van der Zwet et al. 2012). This disease
and fruit quality. originated in the USA, and has been first reported
in 1718 in the Hudson Valley, New York. Since
4 Genetics and Breeding of Pear 83

then, it has spread throughout every region of the clonal replicates of a genotype are screened,
USA, as well as throughout Europe, Middle East, frequency and severity of infection can be
Oceania (New Zealand), and has recently been determined and are combined to yield a calcu-
detected in Kurdistan and South Korea (Park lated index of fire blight susceptibility.
et al. 2017). The most common commercial Most strains of E. amylovora isolated from
cultivars grown today in North America and in apple are capable of infecting pear and vice versa
Europe are known to be either susceptible, such (Momol and Aldwinckle 2000). While this bac-
as ‘Bartlett’, ‘Abate Fetel’, ‘Beurré D’Anjou’, terium is a relatively genetically homogenous
‘Beurré Bosc’, ‘Comice’, or only moderately species (Khan et al. 2012), there is a diversity in
resistant such as ‘Conference,’ to fire blight. pathogenicity among different E. amylovora
These cultivars are grown in regions where cli- strains (Cabrefiga and Montesinos 2005; Wang
mates are not very conducive for fire blight dis- et al. 2010; Smits et al. 2017). However, unlike
ease development, so growers are able to manage in apple (Norelli et al. 1984), to date there is no
the disease somewhat satisfactorily. evidence that differential responses of the
Over the last 40 years, efforts have been pathogen to different resistant pear genotypes
undertaken to evaluate and assess fire blight exist. Pear genotypes with varying degrees of
resistance status of Pyrus germplasm (Bell et al. resistance to fire blight have been inoculated with
1996; Bell and Itai 2011; Peil et al. 2009; Van several different strains of the pathogen, includ-
der Zwet et al. 2012). While total immunity to ing some that have been previously shown to be
fire blight has not been observed, high levels of differentially virulent on apple. While differences
resistance have been identified in some pear in host resistance and strain virulence have been
species. The proportion of resistant material in confirmed, no interactions between host and
European, circum-Mediterranean, and Central strain have been observed (Quamme and Bonn
Asian species tends to be lower than that found 1981; Bell et al. 1990; Bell and Van der Zwet
in East Asian species. However, Van der Zwet 1996). This has led to the conclusion that dif-
et al. (2012) have scored 14 of 75 ‘popular ferentially virulent strains do not need to be
commercial’ European pear cultivars and 24 of considered in breeding for fire blight resistance in
76 Asian/Oriental pear as ‘most resistant.’ Since pear, at least in the USA (Bell and Van der Zwet
the year 2000, several new P. communis cultivars 1987). Nevertheless, given that differentially
have been released that are reported to have high virulent E. amylovora strains have developed
levels of fire blight resistance (Dondini and against fire blight-resistant apple cultivars, it
Sansavini 2012; Hunter and Layne 2004). seems advisable to aim for durable fire blight
Screening methods used to determine fire resistance in pear by incorporating multiple dis-
blight resistance of cultivars, breeding selections, ease resistance genes into pear breeding
and hybrid seedlings have been reviewed exten- programs.
sively (Bell et al. 1996; Peil et al. 2009). The genetics of fire blight resistance first
Long-term field assessments are required to received attention in the USA in the 1960s, when
confirm a genotype’s fire blight status, and a segregation for resistance in breeding progenies,
standardized scoring system for rating fire blight mainly of interspecific Asian  European
infection of trees has been developed. However, hybrids, derived from parents of known resis-
there can be substantial non-genetic variability in tance were observed. No immunity was detected
these assessments; hence, breeders have in any pear genotype, and segregation of seed-
endeavored to control the timing and entry point lings for necrotic lesions of shoots following
of fire blight inoculum to improve assessment of inoculation generally followed a continuous
inherent resistance. Artificial plant inoculations pattern. This suggested that inheritance for
and/or use of greenhouse/plastic tent facilities to resistance was quantitative with presence of
optimize environmental conditions are now several resistance genes, and there was no pattern
commonplace in breeding programs. Where of inheritance specific to a certain pear species
84 L. Brewer and R. Volz

(Layne et al. 1968; Van der Zwet et al. 1974). Venturia Nashicola
Further studies reinforced the hypothesis that Some wild species of Pyrus are fully resistant to
additive gene action was the main mechanism by V. nashicola (Ishii et al. 1992), but of more
which fire blight resistance was inherited in pear interest to breeders is the discovery that several
in the USA (Bell et al. 1977), Canada (Quamme commercial pear cultivars are immune to this
et al. 1990), Italy (Bagnara et al. 1996), and fungal pathogen, including the Japanese pear
France (Durel et al. 2004). At least 18 cultivar ‘Kinchaku’ and the Chinese pears
small-to-moderate-effect QTLs, some of which ‘Hongli’, ‘Mili’, and ‘Cangxili’. Furthermore, it
may be the same, have been identified for control has been demonstrated that progeny generated
of fire blight resistance in European and Asian from crosses between either ‘Kinchaku’ (Abe
pedigrees in three genetic mapping populations and Kotobuki 1998a) or genotypes derived from
(Bokscczanin et al. 2009; Bell 2018; Montanari ‘Kinchaku’ (Terakami et al. 2006) with suscep-
et al. 2016b; Dondini et al. 2004). This further tible cultivars segregate into seedlings either with
confirmed earlier findings that fire blight resis- no symptoms (resistant) or with abundant
tance is polygenically controlled. sporulation (susceptible) (Abe and Kotobuki
As considerable parent-to-parent variability in 1998a). The ‘Kinchaku’ resistance has been used
capacity to transmit resistance to progeny has extensively in Japanese breeding programs, and a
been observed, fire blight resistance cannot be scab-resistant cultivar, ‘Hoshiakari’, carrying the
entirely explained by the parent’s own phenotypic ‘Kinchaku’ resistance has been named and
resistance. This supports hypotheses proposing released (Saito 2016).
that non-additive genetic effects may also con- A dominant major gene (Vnk) controlling this
tribute to fire blight inheritance, although major scab resistance has been mapped to LG1, with
dominant resistance (Drain 1943; Thompson one SSR marker and five STS markers found to
et al. 1962) or sensitivity (susceptibility) genes in be tightly linked to this gene (Terakami et al.
P. communis (Thompson et al. 1975) are also 2006). Two flanking markers, used together,
likely involved. In genetic mapping studies, have accurately predicted resistant seedlings in
minor-effect QTLs controlling resistance have segregating progenies derived from ‘Kinchaku’
been detected in susceptible parents (Bokscczanin (Gonai et al. 2009). These markers are currently
et al. 2009; Montanari et al. 2016b). This may being used in MAS for scab resistance in Asian
explain recovery of resistant genotypes that are pear breeding programs in Japan (Yamamoto and
sometimes developed from susceptible parents Terakami 2016).
(Van der Zwet 1977; Bagnara et al. 1993). Immunity to V. nashicola in European pear
cultivars, including ‘Bartlett’ and ‘La France’,
4.5.3.2 Resistance to Pear Scab has been reported to be transmitted to their pro-
Pear can be infected by two species of Venturia, geny and purported to be controlled by single
inciting pear scab disease. V. pirina Aderh. dominant genes (Abe et al. 2000). Subsequent
infects P. communis, while V. nashicola (Tanaka genetic studies have indicated that a QTL for
and Yamamoto 1964) infects all cultivated spe- resistance from ‘La France’ (Yamamoto et al.
cies of Asian pear. Each fungal species is specific 2009) and a major dominant gene conferring
to its host pear species (Abe et al. 2008; Tanaka resistance from ‘Bartlett’ (Rvn2) (Cho et al.
and Yamamoto 1964), thus the economic sig- 2009; Bouvier et al. 2012) are likely to be the
nificance of each fungal species is tightly linked same, as both mapped to the bottom of LG2.
to the geographic distribution of the cultivated However, the scope of resistance to V. nashicola
host species. V. pirina occurs worldwide except may not be exactly the same for each cultivar, as
for East Asia, while V. nashicola is restricted to Yamamoto et al. (2009) have mapped a second
China, Japan, and Korea (González-Domínguez QTL for resistance, derived from ‘La France’, to
et al. 2017). LG14. Furthermore, two cleaved amplified
4 Genetics and Breeding of Pear 85

polymorphic sequence (CAPS) markers tightly independent major QTLs on LGs 3 and 7, and
linked to RVn2 have been developed for likely collectively explaining *88% of the observed
use in MAS (Cho et al. 2009). variation in susceptibility in progeny of ‘Abè
It has been reported that non-host resistance to Fétel’  ‘Max Red Bartlett’, a scab-susceptible
V. nashicola derived from European pears may cultivar (Pierantoni et al. 2007). A locus on LG1
provide broader spectrum resistance than host confers resistance derived from ‘Wilder’ with a
resistance derived from Asian pears, as they are major QTL (67%) co-localized with the major
effective against all races of the pathogen (Gill gene Vnk on the pear genome (Perchepied et al.
et al. 2015). Often, host resistance is 2015). Recently, a major resistance gene (Rvp1),
race-specific, involving gene-for-gene relation- derived from ‘Navara’, has been identified on
ships, and may be less durable. Indeed, five races LG2 (Bouvier et al. 2012), indicating that such
of V. nashicola, collected from various regions in genes are present in P. communis germplasm.
Asia, have shown differential reactions to dif- The SSR marker CH02b10 is mapped close to
ferent hosts (Zhao et al. 2012). However, use of this gene. As is the case for V. nashicola, V.
non-host resistance from P. communis in Asian pirina also shows strain heterogeneity in
pear breeding may be disadvantageous, as it may pathogenicity to different resistance reactions
incorporate less desirable alleles from European present in P. communis (Chevalier et al. 2004).
pear. Nevertheless, Kim et al. (2016) have The breeding strategy in P. communis should aim
introgressed resistance from ‘Bartlett’ into to bring together a number of resistance QTL and
P. pyrifolia ‘Whangkeumbae’ to develop a new major genes in order to achieve resistance dura-
Korean cultivar, ‘Greensis’. bility in new cultivars.
Partial resistance to V. nashicola has been Asian pear cultivars are generally resistant to
observed in several Asian pear cultivars and their V. pirina (Postman et al. 2005) and may serve as
progeny, as well as in progeny derived from useful sources of non-host resistance in European
European pear. Abe et al. (2000) speculated that pear breeding. However, these sources of resis-
this resistance reaction was under polygenic tance are less well understood. A major QTL is
control. Differences in incidence of necrotic leaf identified on LG4 from a breeding selection,
tissues have been observed among commercial likely derived from P. pyrifolia (Perchepied et al.
Korean cultivars in replicated field trials (Won 2015). Moreover, seven QTL controlling resis-
et al. 2011). Four major gene loci were involved tance (two each on LG7 and LG2, as well as one
in varying necrotic resistance reactions observed each on LG5, LG10, and LG17) have been
in leaf inoculation studies using a segregating identified in a complex interspecific hybrid
progeny, derived from two resistant seedlings of family derived from P. communis, P. pyrifolia,
‘Yali’ x ‘Jingbaili’ that have been backcrossed to and P. ussuriensis (Won et al. 2014). Further-
their parents, susceptible cultivars ‘Yali’ (P.  more, all of these QTLs have exhibited differ-
bretschneideri) and ‘Jingbaili’ (P. ussuriensis) ential responses to discrete V. pirina isolates,
(Zhang et al. 2012). except for the QTL on LG17 which is effective
against all strains. However, the host/non-host
Venturia pirina nature of the QTL has not been established in this
Most P. communis cultivars have demonstrated a study, as not all accessions in the pedigree have
range of susceptibility to V. pirina in the field, been available for marker analysis.
although results have not always been consistent While resistance to V. nashicola in leaf tissues
(Vondracek 1982; Postman et al. 2005). Hence, extends to the fruit (Abe et al. 2008), this is not
most scab resistance in P. communis is presumed always the case for resistance to V. pirina. Some
to be polygenic, and recent genetic mapping in Asian and European pear cultivars (Postman
several partially resistant cultivars has confirmed et al. 2005), as well as interspecific hybrids
this finding. For instance, resistance in ‘Abè derived from Asian and European pears (Brewer
Fétel’ is proposed to be controlled by two et al. 2009), have exhibited leaf resistance
86 L. Brewer and R. Volz

reactions to V. pirina, but have shown some scab inherent low narrow-sense heritability of this
on fruit, thus indicating presence of a differential resistance (Bell 2013b).
resistance reaction depending on tissue. Hence, Immunity to pear psylla within Pyrus has not
reliance on leaf resistance symptoms as an indi- been documented (Quarta and Puggioni 1985;
cator of total plant resistance may not always be Briolini et al. 1988). However, there is a wide
appropriate. Further studies are warranted to variation in resistance responses to C. pyricola
develop a better understanding of resistance among Pyrus species, first documented in North
response of pear fruit to V. pirina. America by Westigard et al. (1970) and Quamme
(1984), and well summarized by Bell and Itai
4.5.3.3 Pear psylla (2011). East Asian pear species are generally
Pyrus hosts several species of the pear psylla resistant, whereas mid-Asian, Mediterranean, and
(Psyllidae: Psyllinae: Cacopsylla spp.), but only European species exhibit a wide range of
three are of economic importance (Hodkinson response, from susceptible to resistant.
2009; Ouvrard 2017). Cacopsylla pyricola Introgression of psylla resistance from Asian
Foerster is the most widespread, and it is pre- pear species into high-quality P. communis cul-
sently found in Europe, the Middle East, North tivars was initiated in the USA, back in the
and South America, Argentina, Russia, South 1960s. It was reported that large-fruited
Korea, and Japan (Ouvrard 2017). Cacopsylla P. ussuriensis material crossed with P. commu-
pyri Linné dominates in Europe, but has also nis cv.Bartlett transferred its psylla resistance to a
been reported in the Middle East and Central majority of the progeny (Harris and Lamb 1973).
Asia, including China. Cacopsylla bidens Šulc is Subsequently, a backcrossing strategy to ‘Bar-
present in France, Italy, Greece, central Asia, tlett’, as well as to other P. communis cultivars
including India, as well as South America (Valle was followed in the USA (Harris and Lamb
et al. 2017). 1973), as well as in both Italy and France
The control of pear psylla in commercial pear (Lespinasse et al. 2008; Nin et al. 2012). Two
orchards is handled by using selective pesticides second-generation cousin hybrids, NY10353 and
along with a range of active natural predators NY10355, with improved fruit quality perfor-
(Trapman and Blommers 1992). However, the mance and resistance to Psylla, have been
psylla reproduces prolifically, with multiple extensively used in breeding programs in the
generations per year, and readily develops USA, Italy, and France (Pasqualini et al. 2006;
resistance to many pesticides (Civolani 2012). Nin et al. 2012; Dondini and Sansavini 2012).
All of the major commercial cultivars of One of the major hurdles in introgressing
P. communis are susceptible to pear psylla. psylla resistance into new pear cultivars has been
Therefore, incorporation of resistance to this pest the poor fruit quality of resistant progenitors and
into new cultivars has been an important objec- the seemingly difficult task of improving fruit
tive for several European pear breeding pro- quality in subsequent generations. Harris and
grams. Fortunately, partially resistant Lamb (1973) have suggested that the
P. communis cultivars, originating mainly in P. ussuriensis source of resistance avoided some
Eastern Europe, have been identified (Bell and undesirable fruit quality attributes, such as small
Stuart 1990; Sestras et al. 2009; Benedek et al. size and flesh grittiness. However,
2010; Bell 1992, 2013a), and used in some psylla-resistant selections originating from this
breeding programs (Branişte et al. 2008). How- source, as well as those derived from Eastern
ever, transmission of resistance to progenies has European-resistant P. communis cultivars, have
often been poor (Bell 2013b). For example, the not exhibited the quality required of a modern
old Italian cultivar ‘Spina Carpi’ is resistant, but new pear cultivar (Bell 2013b). Thus far, no
it does not transmit this resistance to its progeny cultivar has yet been released from these breed-
(Rivalta et al. 2002). This may reflect the ing efforts.
4 Genetics and Breeding of Pear 87

It has been reported that psylla resistance from USA has demonstrated similar responses to both
P. ussuriensis seems to be under polygenic susceptible and resistant pear germplasm from
control (Lespinasse et al. 2008). A major QTL different sources. Interestingly, the P. ussurien-
for control of pear psylla located on LG17 of a sis-derived resistance line developed in the USA
pear selection, NY10355 (Bouvier et al. 2011), for C. pyricola is also resistant to C. pyri in
has been confirmed along with two additional Europe (Robert and Raimbault 2005; Pasqualini
QTLs located on LG1 and LG4. A strong epi- et al. 2006), as well as to C. bidens in Israel
static interaction has been observed between the (Shaltiel-Harpaz et al. 2014). These reports sug-
latter QTLs and that on LG17 (Perchepied et al. gest presence of a relatively broad-spectrum
2016). Nearly all of the genetic variation in resistance for pear psylla. Nevertheless, given
psylla nymph infestation is explained by these the rapid development of pesticide-resistant
QTLs. The major resistance QTL on LG17 has strains of pear psylla over the last few decades
also been identified in segregating progeny of (Civolani 2012), breeding should aim for resis-
NY10353 (Dondini et al. 2015). The SSR tance that is durable through pyramiding of dif-
markers CH05G03 (Dondini et al. 2015) and ferent QTLs for resistance (Corwin and
NB126a-2 (Perchepied et al. 2016), closely Kliebenstein 2017).
linked to the QTL controlling resistance on The modes of host resistance to pear psylla
LG17, have been identified from NY10353 and have been studied extensively for several resis-
NY10355, respectively, and provide a first step tance sources (Bell and Puterka 2004). Both
in developing promising resources for MAS. nymphal feeding antixenosis (unpalatability) and
In other efforts, a P.  bretschneideri  nymph antibiosis (mortality) are deemed impor-
P. communis hybrid that is partially resistant to tant, but ovipositional antixenosis is less impor-
C. pyri is reported to transmit psylla resistance to tant for tested resistant selections derived from
its progeny when crossed with the P. communis both P. ussuriensis and P. communis. In contrast,
cultivar ‘Moonglow’ (Montanari et al. 2015). P.  bretschneideri resistance, derived from
This resistance, most likely to be derived from ‘Xuehauli’, is attributed to both antibiosis and
‘Xuehauli’, is different from those of other ovipositional antixenosis (Montanari et al. 2015).
P. ussuriensis lines as a QTL for resistance is To date, mapping studies have not yet conclu-
located on LG8, but not on LG17. This QTL sively revealed the presence of specific QTLs
explains up to 30 to 39% of the observed phe- associated with each of these different modes of
notypic variation in total numbers of psylla resistance (Montanari et al. 2015). Further
nymphs. Further, this QTL is found to be stable investigation is needed to better understand the
over two years of testing, along with an SSR genetic mechanism of these different components
marker, CH05a02, that is closely associated with of Pyrus resistance to pear psylla in order to
this QTL. Several other minor QTLs for resis- identify better resources for developing
tance, located on LG5, 11, and 15 (from psylla-resistant cultivars.
‘Moonglow’), have also been identified, but In summary, there is a reasonable under-
these are not stable over years of testing, and standing of the genetics of the major scab resis-
their significance is inconclusive. Some inter- tance gene Vnk for V. nashicola, and molecular
specific hybrids of susceptible P. communis  markers linked to this resistance are being used
resistant P. pyrifolia have also shown resistance in some Japanese pear breeding programs. Fur-
to psylla, but the genetic mechanisms of these thermore, numerous sources of resistance to V.
resistances are yet unknown (Robert and Raim- pirina, fire blight, and pear psylla have been
bault 2005; Pasqualini et al. 2006). identified, and these have been used in various
It is unknown if different biotypes of pear pear breeding programs. However, in contrast to
psylla exist that can overcome any of the above V. nashicola resistance, these sources of resis-
reported resistances. Puterka (1997) has found tance have more complex genetics that is not
that C. pyricola collected from five regions in the well documented. Efficient and effective
88 L. Brewer and R. Volz

incorporation of these various genes for resis- compared to that of P. betulaefolia seedling
tance to these different diseases and pest into rootstock) (Elkins et al. 2012), developed at the
future pear cultivars can only be enhanced fol- French National Institute of Agricultural
lowing thorough understanding of their genetics Research (INRA) and released in 1967 (Simard
involved in these traits, as well as subsequent et al. 2004), the dwarfing ‘Quince A’ (QA), and
development and application of their associated the dwarfing ‘Quince EMC’ (QC) rootstocks,
molecular markers. both released from East Malling Research Station
in the United Kingdom in the 1920s (Anon.).
Graft compatibility testing of pear cultivars on
4.6 Rootstock Breeding Quince rootstocks have suggested that ‘Beurré
D’Anjou’, ‘Comice’, ‘Old Home’, ‘Beurré
Pear growers have a limited number and range of Hardy’, ‘Flemish Beauty’, ‘Abbé Fetel’, ‘Passe
clonal rootstocks to choose from when designing Crassane’, and ‘Maxine’ are compatible, but
a new orchard, compared with their apple coun- ‘Bartlett’, ‘Beurré Bosc’, ‘Winter Nelis’,
terparts. This range is even more limited if a ‘Clapp’s Favourite’, and ‘Forelle’ are not
vigor-controlling rootstock is required, as (Lombard and Westwood 1987). Since the
dwarfing rootstocks equivalent to the precocious release of ‘BA29’, QA, and QC, the Quince
flowering and high-yielding apple rootstock Eline® rootstock has been released by Boomk-
‘Malling 9’ are lacking (Knäbel et al. 2015; wekerij Fleuren in Belgium. Quince Eline®,
Brewer and Palmer 2011). Rootstock options for originated from a Romanian breeding program,
pear growers include several Pyrus species and has been developed for increased frost resistance.
alternatives from other species, such as Cydonia This rootstock is comparable to QC for scion
oblonga (quince), Amelanchier alnifolia (ser- vigor and fruit size, and it is reported to have
viceberry), Actaea spicata (baneberry), Ame- good graft compatibility with most pear cultivars,
lanchier canadensis (juneberry), Amelanchier along with frost resistance to temperatures of
lamarckii (juneberry), Sorbus aucuparia about −25 °C (Anon.; Brewer and Palmer 2011).
(mountain ash), Sorbus alnifolia (alder-leafed In 2001, East Malling has released ‘QR193/16’
whitebeam), and Pyronia veitchii (C. oblonga  (EMH), originally claimed to control scion vigor
P. communis) (Elkins et al. 2012; Postman similar to that of QC; however, further research
1994). has indicated that vigor control ranges between
that of QC and QA (Webster et al. 2000).
Although EMH contributes to good fruit size
4.6.1 Quince—Cydonia oblonga development and has good stool bed perfor-
mance, it shows poor precocity relative to QC,
Quince rootstocks are preferred in Europe and it is susceptible to fire blight (Brewer and
because of their strong vigor control and pre- Palmer 2011). EMH has been selected from seed
cocity of the pear scion, as well as ease of presumed to have originated from Transcaucasia.
propagation (Brewer and Palmer 2011; Necas Research efforts at the University of Pisa in Italy
et al. 2016). However, these have several limi- on breeding rootstocks tolerant to calcareous
tations to more widespread use, including lack of soils have led to the release of the selection ‘Ct.S
cold hardiness, limited fire blight resistance, 212’; however, this is not resistant to fire blight,
scion incompatibility, and susceptibility to iron and more recently has demonstrated some
chlorosis (Elkins et al. 2012). There has been inconsistency in fruit production of grafted scion
limited breeding of quince rootstocks to address cultivars (Brewer and Palmer 2011).
these issues (Brewer and Palmer 2011). In a quest for developing more dwarfing
Scion vigor-controlling rootstocks include the quince rootstocks that have cold resistance, a
semi-dwarfing ‘BA29’ (60% of tree size large number of accessions have been selected
4 Genetics and Breeding of Pear 89

from the National Clonal Germplasm Repository 4.6.2.1 P. communis

at Corvallis (Oregon, USA) and have been P. communis is the species most widely used as a
screened for cold hardiness. A total of 22 quince rootstock in North America, with seedlings of
selections have been found to be as hardy, or ‘Winter Nelis’ and ‘Bartlett’ being the main
hardier, than standard commercial Pyrus root- rootstocks currently used commercially (Elkins
stocks, including ‘Old Home’  ‘Farmingdale et al. 2012). However, grafted pear trees are
87’ and ‘Old Home’  ‘Farmingdale 97’, sur- mostly vigorous, yet they are adapted to a range
viving temperatures as low as −30 °C. Among of climates and soil types (Hancock and Lobos
these, the ten best-performing selections are 2008). Although fire blight susceptibility is
currently being evaluated in research programs in common in P. communis, seedling populations
Wenatchee (Washington State) and Hood River have been established to develop rootstocks with
(Oregon) in the USA (Warner 2015). The fire blight resistance along with some tree size
best-performing rootstocks for cold tolerance reduction or dwarfing (Hancock and Lobos
have originated from Armenia, Turkmenistan, 2008). Globally, there are limited numbers of
Russia, Uzbekistan, the Russian Federation, P. communis rootstocks that offer significant
Georgia, and France, with the most cold resistant grafted tree size reduction. Research efforts in the
being C. oblonga-Arakseni, ‘Avia’ from Gebe- USA have demonstrated that size of a grafted
seud, and ‘Akhtubinskaya’, an open-pollinated pear tree on ‘Pyrodwarf®’ is similar to that
seedling 4 (Einhorn et al. 2017; Anon.). grafted on Quince ‘BA29’ (Brewer and Palmer
2011), and only 61–70% of that grafted on
P. betulaefolia seedling rootstocks (Elkins et al.
4.6.2 Pyrus 2012). However, tree performance has varied
depending on planting site, scion cultivar, and
Pyrus rootstocks are the preferred choice in management practices (Elkins et al. 2012). Fur-
North America, Asia, and Australia. A wide thermore, yield efficiency has been poor com-
range of species have been used in breeding pared to that obtained with QC, QA, and many
programs or in commercial orchards, including Amelanchier rootstocks (Einhorn et al. 2017). In
P. communis, P. betulaefolia Bge., P. calleryana 1996, the University of Bologna in Italy has
Dcne., P. pashia D. Don, P. xerophila Yu, released P. communis rootstocks ‘Fox 11’ and
P. ussuriensis Maxim, P. heterofolia, P. nivalis, ‘Fox 16’, and in 2008 has released ‘Fox 9’.
P. longipes, and P. pyrifolia Nakai (Brewer and However, all three rootstocks are more vigorous
Palmer 2011; Tamura 2012; Teng 2011; Simard than quince BA29 (Brewer and Palmer 2011).
et al. 2004). Pyrus rootstocks have good graft From a rootstock breeding perspective, it is
compatibility, a satisfactory range of cold adap- important to identify individuals carrying traits
tation, and can grow well in low to high pH soils. required as soon as possible, especially for the
However, they have limited vigor control and scion dwarfing trait. QTLs influencing expres-
precocity induction of the scion, varying levels of sion of scion vigor and precocity have been
tolerance to Candidatus Phytoplasma pyri located on LG5 and LG6 of ‘Old Home’ in an
(inciting pear decline), and are generally difficult ‘Old Home’  ‘Louise Bonne de Jersey’ seed-
to propagate (Brewer and Palmer 2011). A con- ling population. It is reported that the QTL on
tinuing challenge for pear rootstock breeders is to LG5 maps to a position that is syntenic to the
combine vigor control and precocity of the scion, apple ‘Malling 9’ Dw1 locus located at the top
that can be obtained from Quince rootstock end of LG5 (Knäbel et al. 2015). This QTL for
options, with other important traits required for a rootstock control of numbers of branches pro-
successful rootstock. This may require use of duced by a grafted scion cultivar is detected in
more than a single species to combine all of these three successive years, and it is co-located with
required traits. the flowering trait for total number of
90 L. Brewer and R. Volz

inflorescences on a tree. The microsatellite mar- compatibility, limited root suckering, adequate
ker Hi01c04, located within the QTL region on adaption to winter cold temperatures, and high
LG5, is heterozygous in both ‘Old Home’ and tolerance to pear decline, but only moderate yield
‘Louise Bonne de Jersey’, and its trait association precocity and performance, as well as moderate
is found to be consistent over a number of years. tolerance to bacterial canker (Lombard and
A small-effect QTL for root suckering is also Westwood 1987). The Brossier series, developed
detected on LG5 within the same genomic region in France in 1962, have utilized five
as that QTL for tree architecture (Knäbel et al. open-pollinated seedling populations of
2015). In the same population, QTLs have been P. nivalis to generate selections having a range of
identified on LG7 controlling development of rootstock vigor. Furthermore, seedlings have
adventitious roots on hardwood cuttings of both displayed good graft compatibility, low vigor,
‘Old Home’ and ‘Louise Bonne de Jersey’ and a range of tolerance to fire blight; however,
(Knabel et al. 2017). Both of these discoveries they have also displayed poor to very poor ability
will support efforts in developing genetic mark- for clonal propagation, ranging from 1 to 54%
ers useful in future breeding efforts of desirable for semi-hardwood cuttings. The best genotype
Pyrus rootstocks. selected in this series, G28-120, confers similar
tree vigor to that of ‘BA29’, it is graft compatible
4.6.2.2 P. longipes with ‘Bartlett,’ induces regular cropping and
Rootstocks of P. longipes offer very good tree good fruit size, but it is susceptible to fire blight,
root anchorage, graft compatibility, and high has low ability for clonal propagation (31% by
tolerance to the bacterial canker Pseudomonas hardwood cuttings), and does not transplant well
syringae, but provide only moderate precocity (Simard et al. 2004).
and yield efficiency, susceptibility to fire blight,
and limited tolerance to pear decline (Lombard 4.6.2.4 P. calleryana
and Westwood 1987). Breeding efforts at As a seedling rootstock, P. calleryana exhibits
Dresden-Pillnitz in Germany have used very good tree anchorage and graft compatibility,
P. longipes to target improved propagation abil- moderate yield efficiency and precocity, moder-
ity, dwarfing, resistance to biotic and abiotic ate susceptibility to fruit cork spot, and resistance
stress, superior tree anchorage, yield, and fruit to black end of fruit (a physiological disorder of
quality, as well as reduced suckering and burr fruit). Grafted trees on this rootstock display high
knot development (Fischer 2007). A wide range tolerance to various diseases and pests, including
of interspecific crosses have been made, and fire blight, Podosphaera leucotricha Salm.
seven new Pyrus rootstocks have been selected, (inciting powdery mildew), Agrobacterium
ranging from ‘very dwarfing’ to ‘medium tumefaciens Conn. (inciting crown gall), Phy-
strong’. One of these selections, ‘Pi-BU 3’, has tophthora cactorum Schroet (causing collar rot),
been reported to confer vigor that is 40–60% of Eriosoma Pyricola (woolly pear aphid), and
that of P. betulaefolia seedling rootstocks (Elkins Pratylenchus vulnus (root lesion nematode)
et al. 2012). Tree losses have been reported in (Lombard and Westwood 1987). Overall,
German trials which may indicate that some P. calleryana has a superior adaption to most
levels of graft incompatibility must have occur- environmental conditions compared with that of
red, and ‘Pi-Bu 3’ has not matched quince root- P. pyrifolia, but it is susceptible to lime-induced
stocks for yield or yield efficiency (Brewer and chlorosis, and it is only moderately tolerant to
Palmer 2011). pear decline (Tamura 2012; Teng 2011; Bell
1991).
4.6.2.3 P. nivalis Rootstocks of P. calleryana are commonly
Used as a rootstock, perry pear (P. nivalis) dis- grown as seedlings in Japan, and in both North
plays satisfactory tree anchorage, good graft and South China. Studies have been conducted to
4 Genetics and Breeding of Pear 91

identify and propagate superior strains using 4.6.2.6 P. heterofolia

clonal propagation (Teng 2011; Tamura 2012; At INRA, open-pollinated populations of
Banno et al. 1988). Some strains display good P. heterofolia (closely related to P. betulaefolia)
rooting ability as softwood cuttings, while others have been evaluated to select for agronomic
exhibit growth control of grafted scion cultivars traits, particularly for fire blight tolerance and
(Brewer and Palmer 2011) with marked dwarfing ability for clonal propagation. Seedlings have
when grafted with Japanese cultivars (Tamura also been screened for erect nursery habits,
2012). A particular clone, P. calleryana D6, is without branching, and for iron chlorosis toler-
considered to be superior in Australia, where it is ance (Simard et al. 2004). Scion growth grafted
the most commonly used pear rootstock. D6 is a onto selection ‘P2532’ is similar to that on
clonal stock selected from seed supplied by Quince ‘BA29’, but ‘P2532’ induces more vig-
Nanjing University (China) in 1929. The root- orous growth of scions, similar to that of ‘Old
stock is vigorous, producing a large tree when Home’  ‘Farmingdale 333’, and produces fruit
used for grafting scions, but it is compatible with of good size, but it is susceptible to fire blight
most cultivars (Anon. 2014). Currently, clonal (Simard et al. 2004).
reselection rather than breeding is being con-
ducted. Therefore, additional research efforts are 4.6.2.7 P. xerophila
required before a reliable dwarfing P. calleryana Rootstocks of P. xerophila may serve as good
rootstock is developed. options in semi-arid regions, as this species is
very drought tolerant. The cultivar ‘Mu-Li’ has
4.6.2.5 P. betulaefolia displayed superior root growth in highly alkaline
Rootstocks of P. betulaefolia have very good soil soils and can sustain growth in soils up to pH
anchorage and graft compatibility, produce vig- 8.0 (Tamura 2012).
orous trees with moderate precocity and yield
efficiency in scions, along with fruit that does not 4.6.2.8 P. pyrifolia Nakai
display black end, but with low tolerance to cork Although P. pyrifolia has been used as a root-
spot. P. betulaefolia has high tolerance to pear stock in southern areas of China, it is not the
decline, bacterial canker, leaf spot, powdery rootstock of choice in most countries. It is not
mildew, crown gall, collar rot, woolly aphid, and cold hardy, can be damaged under conditions of
root lesion nematode (Lombard and Westwood low temperatures (Yu-Lin 1996), displays poor
1987). Similar to P. calleryana, it exhibits tolerance to drought, but with flood and salt
superior adaption to various environmental con- tolerance, yet it grows poorly on alkaline soils, it
ditions, especially to hot humid conditions, and it is susceptible to pear decline, and adapts poorly
is used widely throughout Asia (Tamura 2012). to clay soils (Bell 1991; Tamura 2012; Elkins
In the USA, P. betulaefolia is used as a rootstock et al. 2012). It does not produce root suckers,
on heavy clay soils and used as a standard for exhibits good tree anchorage, graft compatibility,
high vigor (Elkins et al. 2012). Although high good yield efficiency, shows moderate precocity,
vigor is a disadvantage, P. betulaefolia root- and has moderate tolerance to fire blight, bacte-
stocks are very drought and salt tolerant, can rial canker, and powdery mildew, but can induce
withstand temperatures down to −45 °C if cold black end of in the scion (Lombard and West-
hardened, but have low tolerance to alkaline soils wood 1987).
(Tamura 2012). The use of P. betulaefolia root-
stocks is also effective for avoiding black end in 4.6.2.9 P. ussuriensis Maxim
European pears or ‘Yuzuhada’ in Japanese pears. Rootstocks of P. ussuriensis are the most cold
Similar to P. calleryana, some selections have hardy of the Pyrus species (down to −50 °C)
displayed good rooting, as well as size control of (Teng 2011) and deemed most suitable for North
scion cultivars (Tamura 2012). Eastern China (Yu-Lin 1996). Seedlings have a
92 L. Brewer and R. Volz

low tendency to produce root suckers, although either equivalent to or better than QA, and have
trees have good soil root anchorage, graft com- either equal or significantly higher levels of cold
patibility, good yield efficiency, but fruit of hardiness than commercial P. communis root-
scions is susceptible to black end. Furthermore, stocks. Some selections look very promising as
P. ussuriensis is susceptible to pear decline and dwarfing rootstock options for US growers
root lesion nematode, but it is highly tolerant to (Einhorn et al. 2017).
fire blight, powdery mildew, and woolly aphid
(Elkins et al. 2012; Lombard and Westwood
1987). 4.6.4 Sorbus Species

4.6.2.10 P. pashia Sorbus (mountain ash) is being assessed as a

Nepal pear (P. pashia) is commonly used as a potential pear rootstock that can provide scion
rootstock for Japanese pears in East Asia dwarfing for intensive production. Although
(Tamura 2012). It is also used as a rootstock in scion dwarfing of less than 40% of the size of
the Yunnan province of China (Yu-Lin 1996). In P. betulaefolia seedling rootstocks has been
China, there are wide variations in morphology reported, graft compatibility with Pyrus is con-
and vigor within seedling populations, thus pro- sidered poor to good (Elkins et al. 2012). The
viding opportunities for selecting dwarfing types dwarfing ability of Sorbus along with its high
(Teng 2011). P. pashia is not cold tolerant, and tolerance to several pests and diseases are its best
stems can be damaged at temperatures of −16 °C attributes as these trees have only moderate
and below. This species tolerates low pH soils, anchorage to the soil, and grafted scions exhibit
but not high pH, and can grow on either sandy or low precocity and yield efficiency (Lombard and
clay soils (Bell 1991). Trees have good root Westwood 1987).
anchorage and graft compatibility, but confer
only moderate precocity and yield efficiency.
P. Pashia has high tolerance to pear decline and 4.6.5 Interspecific and Intergeneric
bacterial canker, moderate tolerance to powdery Hybrids
mildew, collar rot, and woolly aphid, but low
tolerance to fire blight, leaf spot, and root lesion Researchers at INRA have used the best selec-
nematode (Lombard and Westwood 1987). tions from several different species to develop
rootstocks adapted to Northern European condi-
tions, and that are dwarfing, tolerant to fire blight,
4.6.3 Amelanchier Species exhibit good productivity, and are easily propa-
gated (Simard et al. 2004). Interspecific hybrids
Dwarfing rootstocks for pear have been selected have also been used in collaboration with IRTA
from Amelanchier seedlings at the Bavarian in Spain to develop rootstocks adapted to
Centre of Pomology and Fruit Breeding in Ger- Mediterranean conditions. Crosses between
many (Brewer and Palmer 2011). This species is ‘Pyriam’ (P. communis) and four Mediterranean
considered to possess moderate to high tolerance species have been used to combine additional
to fire blight, excellent cold hardiness, fair to necessary traits of iron tolerance, drought toler-
good graft compatibility with Pyrus (high for ance, and propagation ability (Simard et al.
‘Comice’ and ‘Beurré Hardy’), low production of 2004).
root suckers, and it is potentially a non-host for Materials of Pyronia (Pyrus  Cydonia) and
pear decline, but trees can have poor root Sorbopyrus (P. communis  Sorbus) are at
anchorage (Einhorn et al. 2017; Lombard and early stages of evaluation as potential pear
Westwood 1987). rootstocks. Pyronia is considered to have good
Most evaluated selections offer a higher yield graft compatibility with pear cultivars (Elkins
efficiency than ‘Pyrodwarf’®, and many are et al. 2012).
4 Genetics and Breeding of Pear 93

In summary, various Pyrus and non-Pyrus thereby reducing progeny size and cost of
germplasm are being used as pear rootstocks growing seedlings to maturity in the field (Luby
around the world. However, there have been little and Shaw 2001). However, for nearly all other
focused breeding efforts using this wide germ- selection traits that are important in pear, rela-
plasm over a sustained period to develop root- tionships between phenotype and genotype are
stocks that fulfill the requirements of a modern less clear. Knowledge is lacking as to how many
pear orchard. A better understanding is needed of loci, and which loci, are important in consistently
the genetics of important rootstock traits, explaining genetic variations observed in specific
including dwarfing, precocity, compatibility, and traits. Further linkage analyses using biparental
adaptation to a range of abiotic and biotic stres- genetic mapping families and GWAS across
ses. This is in stark contrast to our more less-related individuals in pear breeding germ-
sophisticated genetic knowledge of many of the plasm sets of interest will be required to deter-
fruit and tree characters of the scion itself. mine these large-effect marker–trait relationships,
and how MAS might be best implemented in
particular pear breeding programs.
4.7 Genomics-Assisted Breeding GS offers the potential of utilizing large
numbers of molecular markers distributed across
Compared with other rosaceous fruit crop spe- the genome, some of which may be linked to
cies, genomics-assisted breeding in pear is still in small-, as well as to large-effect loci to explain
its infancy. Over the last 20 years, new genomic and predict genetic variations in either one or
tools have been developed and applied to more traits simultaneously, and without neces-
improve the efficiency and effectiveness of sarily understanding the function(s) of causative
breeding in apple, peach, strawberry, and sweet loci involved (Kumar et al. 2012; Desta and Ortiz
cherry (Peace 2017; Laurens et al. 2018; van 2014). The advantage of this in fruit tree species,
Nocker and Gardiner 2014). Applications range such as that of pear with a 4- to 10-year juvenile
from a better understanding of trait genetics, period, is that selections can be evaluated as
through confirmation of parentage and pedigree, potential cultivars or as breeding parents well
calculation of relatedness among potential par- prior to fruiting. This can significantly reduce the
ents, to either single-locus (MAS) or time frame from crossing to commercial cultivar
whole-genome-wide marker-assisted (genomic release and increase the genetic gain per unit
selection [GS]) seedling and parental selection. time.
The development of genomic resources In Japanese pear, GS has been conducted
specific to pear is now progressing quickly and using only 162 genome-wide molecular markers
will enable genomic-assisted breeding to pro- in a set of 76 cultivars for nine traits having
ceed. The recently published draft genomes of reasonably high linkage disequilibrium (Iwata
the Chinese pear ‘Dangshansuli’ (Wu et al. 2013) et al. 2013a). These predictions have showed
and European pear ‘Bartlett’ (Chagne et al. 2014) mostly moderate correlations with observed val-
have facilitated development of new and lower ues (using leave-one-out cross-validations),
cost genotyping methods, such as GBS, to pro- indicating the potential of GS technology for use
duce high-density molecular markers on pear in this breeding germplasm, despite of the rela-
genetic maps (Kumar et al. 2017). tively low number of markers utilized. Further-
As we have described, the genetics of more, it has been demonstrated that GS can also
self-compatibility, scab resistance, and harvest predict segregation of traits in a Japanese pear
time have been reasonably well studied in Japa- progeny with reasonable accuracy, based on the
nese pear, with each controlled by either one or whole-genome molecular marker profile of the
two major genes, or by major-effect QTLs. two parents (Iwata et al. 2013b). Further studies
Markers linked to these traits are being used for exploring the potential uses of GS in pear
MAS in Japanese pear breeding (Saito 2016), breeding are warranted.
94 L. Brewer and R. Volz

In many parts of the world, the genetic makeup Abe K, Saito T, Terai O, Sato Y, Kotobuki K (2008)
of pear fruit available to consumers has not chan- Genotypic difference for the susceptibility of Japanese,
Chinese and European pears to Venturia nashicola,
ged over the last 100 years. Efforts to develop the cause of scab on Asian pears. Plant Breed 127
enhanced rootstocks for pear have advanced only (4):407–412
slightly, and pear production is often limited by the Ahloowalia BS, Maluszynski M, Nichterlein K (2004)
relatively poor performance of the rootstock of Global impact of mutation-derived varieties. Euphyt-
ica 135(2):187–204
choice, particularly when compared with the status Anon (2014) Rootstocks. Apple and Pear Australia,
for apple. This provides enormous market oppor- 23/5/2018
tunities for pear breeders to provide novel types of Anon About Q-Eline. http://www.q-elinenet/about-q-
pear fruit and new rootstocks, by taking advantage eline/, 25/5/2018
Anon Rootstock research at East Malling: a history. http://
of the wide and relatively untapped diversity www.emr.ac.uk/projects/rootstock-research-east-
among Pyrus, and across other genera for devel- malling-history/, 25/5/2018
oping new rootstocks. Anon Cydonia Catalog NCGR-Corvallis. https://www.
The biology of pear, as of many perennial tree arsusdagov/ARSUserFiles/20721500/catalogs/
cydcoldhtml, 25/5/2018
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relies solely on phenotype and pedigree to pro- (1993) Cross combinations for fire blight resistance in
duce new cultivars, will be a relatively slow and pear. Acta Hort 338:369–374
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Evaluation of fire blight resistance in pear: Combining
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 Linkage Mapping in Pear
5
Jun Wu and Mengfan Qin

Abstract quantitative trait loci (QTL) or key genes

The past three decades have witnessed the regulating a target trait of interest. Further-
development of genetic linkage maps and use more, they contribute to efforts to pursue
of DNA markers in mapping agronomic traits marker-assisted breeding (MAB) in pear.
in many crops. In comparison with other
plants, linkage mapping in pear has been
initiated rather late, not until the year 2001.
Pear is characterized by a typical self- 5.1 What Is a Linkage Map?
incompatibility, and it has a long generation
cycle. Therefore, genetic maps have been A linkage map, also known as a genetic map, is
constructed using F1 populations. This may an alignment of molecular markers or known
lead to the development of linkage maps of genes, and their positions relative to each other in
lower resolutions due to the lack of sufficient terms of recombination frequencies rather than
genetic variations. Fortunately, the develop- their specific positions along each of the chro-
ment of next-generation sequencing technol- mosomes of a genome. More specifically, a
ogy has allowed for detection of high-quality linkage map is based on the recombination fre-
genome-wide DNA markers, such as simple quency between markers during chromosomal
sequence repeats (SSRs) and single nucleotide crossovers that may occur during meiosis.
polymorphisms (SNPs), in larger size popula- Therefore, a higher frequency of recombination
tions, thus greatly improving the quality of suggests that there is a wider physical distance
genetic linkage maps. Overall, linkage maps between markers, while a lower frequency of
are highly useful for dissecting complex recombination suggests a narrower physical dis-
agronomic traits, and for identifying either tance between markers. The unit used to measure
distances among markers along a genetic map is
a centimorgan (cM), as this corresponds to a
recombination frequency of 1%. A linkage map
is a useful tool in pursuing research studies and
in breeding efforts as it serves to identify and/or
J. Wu (&)
M. Qin locate new markers, or genes, linked to known
State Key Laboratory of Crop Genetics and markers by testing for genetic linkages among
Germplasm Enhancement, Center of Pear them.
Engineering Technology Research, Nanjing
Agricultural University, Nanjing 210095, China
e-mail: [email protected]

© Springer Nature Switzerland AG 2019 103

S. S. Korban (ed.), The Pear Genome, Compendium of Plant Genomes,
https://doi.org/10.1007/978-3-030-11048-2_5
104 J. Wu and M. Qin

5.2 What Is a Mapping Population? (Pyrus communis L.) (Chagné et al. 2014),
thereby allowing for identification and develop-
To construct a genetic linkage map for a target ment of large numbers of genome-wide DNA
plant species, the first and most important effort markers. These robust markers have allowed for
is to establish a proper segregating population for the development of high-quality genetic maps.
this species. For cross-pollinated species, such as
pear, wherein self-incompatibility and inbreeding
depression render it impossible to develop map- 5.3.1 Initial Maps
ping populations, such as F2, backcross (BC), or
elite lines, such as recombinant inbred lines The first genetic linkage map for pear was con-
(RILs) or near-isogenic lines (NILs), whereby structed using random amplified polymorphic
self-breeding is required for several generations. DNA (RAPD) markers developed using an F1
Similar to other fruit tree species, the pear also mapping population of 82 individuals of the
has a long period of juvenility prior to reaching Japanese pear (P. pyrifolia Nakai) cultivars Kin-
the reproductive phase, whereby a trait is deemed chaku and Kosui, and consisted of two separate
stable. On average, it takes about 5–8 years to maps (Iketani et al. 2001). The linkage map for
establish a simple F1 pear population. Fortu- ‘Kinchaku’ had 120 markers, distributed over 18
nately, after thousands of years of distant linkage groups, and spanning 768 cM; whereas,
hybridizations, the genome of the pear is highly the map for ‘Kosui’ had 78 loci distributed across
heterozygous, and progenies of these hybrids 22 linkage groups, and spanned 508 cM. Both
have high levels of segregation for performance linkage maps had more than the expected 17
of agronomic characters (Wu et al. 2013). For linkage groups, the actual number of chromo-
these reasons, an F1 population is usually used in somes of the pear genome. In addition, the low
genetic linkage studies for pears. numbers of markers coupled with the disadvan-
tages of RAPD markers, such as poor repro-
ducibility and inability to distinguish between
5.3 Genetic Linkage Maps for Pear homozygote and heterozygous alleles, rendered
this genetic map of limited genetic information.
In pear, the history of genetic map construction Subsequently, Yamamoto et al. (2002) used
had gone through three stages. The first stage an F1 mapping population of 63 individuals
involved the construction of initial maps using derived from the hybridization of the European
low polymorphism DNA markers. Thereby, the pear (P. communis) cultivar Bartlett and the
number of linkage groups was not equal to the Japanese pear (P. pyrifolia Nakai) cultivar
number of chromosomes of the pear genome, and Housu. They constructed two parental maps
it was difficult to determine which linkage group using amplified fragment length polymorphism
corresponded to which chromosome. The second (AFLP) and SSR markers developed from pear,
stage involved the construction of reference (or apple, peach, and cherry. The map of ‘Bartlett’
frame) maps. Prior to the release of the reference consisted of 18 linkage groups with 226 markers,
genome of pear, most of these maps consisted of including 175 AFLPs and 47 SSRs, a single
17 linkage groups, which was also consistent isozyme, and a single S locus, spanning 949 cM
with that of the apple genome as pear and apple with an average interval of 4.2 cM. The map for
belong to the same subfamily Pomoideae, by ‘Housui’ consisted of 17 linkage groups with 154
using co-dominant simple sequence repeat loci, including 106 AFLPs, 42 SSRs, three iso-
(SSR) markers. The third stage involved com- zymes, and two phenotypic traits
plete sequencing of genomes of the Asian pear (self-incompatibility and leaf color), spanning
‘Dangshansuli’ (Pyrus  bretschneideri) (Wu 926 cM with an average distance of 6.0 cM. By
et al. 2013) and of the European pear ‘Bartlett’ identifying common SSR markers shared by the
5 Linkage Mapping in Pear 105

two parental maps, a group of ten linkage groups cultivars Bartlett and La France using two inde-
was then connected together. pendent F1 populations. The population of
During later efforts, Dondini et al. (2004), ‘Bartlett’ (P. communis)  ‘Housui’ (P. pyrifo-
Yamamoto et al. (2004), and Pierantoni et al. lia) was used to construct a map for ‘Bartlett,’
(2007) constructed yet another three sets of pear while the population of ‘Shinsei’ (P. pyrifo-
genetic maps using AFLPs, SSRs, and other lia)  ‘282-12’ (a Japanese pear selection
marker types, such microsatellite-anchored frag- derived from ‘Housui’  ‘La France’) was used
ment length polymorphisms (MFLPs) and resis- to construct a map for ‘La France’. These two
tance gene analogs (RGAs), among others. These maps relied on AFLPs and SSRs developed from
maps consisted of linkage groups that were higher both pear and apple. These two maps consisted
in number than the basic chromosome number of 17 linkage groups that were well aligned
(n = 17) for pear. Thus, these additional maps together, and corresponded to the basic chro-
could not represent the complete genome of pear. mosome number (n = 17) of the pear genome.
Incidentally, those SSR markers developed from
apple and used in constructing these pear maps
5.3.2 Frame/Reference Maps showed co-linearity with a saturated reference
map for apple. The map length for “Bartlett” was
Yamamoto et al. (2007) constructed two genetic 1016.1 cM, with an average distance of 2.3 cM
maps of the European pear (P. communis) between markers, while the map length for “La

Fig. 5.1 Distribution of SNP and SSR markers on 17 linkage groups of the first high-density genetic map of pear.
A black bar indicates a SNP marker, and a red bar indicates an SSR marker. Linkage group number is shown on the
x-axis, while the genetic disease is shown on the y-axis (cM)
106 J. Wu and M. Qin

France” was 1156.7 cM, with an average interval 2011), consisted of 5085 markers, including 1232
distance of 2.8 cM. Due to their high map SSRs and 3853 SNPs, spanning 3266.0 cM, with a
lengths and marker densities along with their mean interval distance between adjacent markers of
good co-linearities with an apple reference 0.64 cM.
genetic map, these were deemed as reliable ref- The above-mentioned high-density SNP-based
erence linkage maps for pear. linkage maps have greatly improved the quality
and resolution of genetic linkage maps for pear. In
turn, these maps are very helpful in pursuing
5.3.3 High-Density Linkage Maps fine-mapping of target genes, map-based cloning
of qualitative trait loci (QTL) for traits of interest,
With the development of next-generation and promoting the progress of pear-breeding
sequencing technologies and the release of whole efforts. The details of all published genetic link-
reference genome sequences for pear (Wu et al. age maps are listed (Table 5.1).
2013; Chagné et al. 2014), massive numbers of SSR
and single nucleotide polymorphism (SNP) mark-
ers can be identified directly from the pear itself 5.4 Applications of Genetic Linkage
with genome-width coverage. These genomic Maps
resources have promoted efforts for constructing
high-density genetic linkage maps for pear. Genetic linkage maps could be applied in many
The first SNP-based high-density genetic linkage fields. For example, gene mapping, QTL map-
map for pear has been constructed by Wu et al. ping, map-based cloning, marker-assisted selec-
(2014). This map was constructed using SNPs inte- tion (MAS) breeding, comparative mapping,
grated along with SSR markers developed by and auxiliary genome assembly, among others.
restriction-associated DNA sequencing (RAD-seq).
This map consisted of 3143 SNPs and 98 SSRs
(3241 markers in total), spanning 2243.4 cM, with 5.4.1 Gene Mapping
an average marker distance of 0.70 cM (Fig. 5.1,
Wu et al. 2014). These SSR markers were capable of Known genes of traits of interest could be used to
anchoring all 17 linkage groups to their corre- construct linkage maps by transferring them into
sponding chromosomes. Another high-density DNA or morphological markers to further
genetic linkage map for pear has been constructed investigate their inherited characters. For exam-
by Wang et al. (2017) using a hybrid population of ple, Iketani et al. (2001) mapped two alleles for
‘Red Clapp’s Favorite’ (P. communis)  ‘Mansoo’ resistance to pear scab and an allele for suscep-
(P. pyrifolia), containing 4797 SNP markers and tibility for black spot on a linkage map for the
spanning 2703.6 cM, with an average distance of Japanese pear cv. Kinchaku, and found that these
0.56 cM between adjacent markers. alleles were mapped onto different linkage
Genetic maps constructed by different hybrid groups. Yamamoto et al. (2002) mapped an
populations usually vary a lot from each other, and S locus (for self-incompatibility) on linkage
none of them could include the whole genetic groups Ba1 and Ho1 of the maps for the Euro-
information of pear. Yet, lack of common markers pean pear cv. Bartlett and the Japanese pear cv.
making it difficult to do comparison analysis among Housui, respectively, and corresponding to the
them. Thus, Li et al. (2017) collected nine published apple linkage group 17 (LG17). Later, Yama-
maps and merged them into a single integrated moto et al. (2004) used additional markers to
high-density consensus genetic map using common reconstruct linkage maps for ‘Bartlett’ and
SSR markers, of at least three common SSR ‘Housui’, and following comparative mapping
markers within the same group, presented in indi- with apple, they found that the S locus mapped
vidual maps as bridging markers. The integrated onto LG17 of both Japanese and European pears,
genetic map (I-PCG), using MergeMap (Wu et al. as well as that of apple. Following the
5 Linkage Mapping in Pear 107

Table 5.1 Summary of published genetic linkage maps for pear

Population Size Marker type No. markers Map No. LGs Interval
length (cM)
(cM)
M F M Reference(s)
F M F M F
‘Kinchaku’  ‘Kosui’ 82 RAPD 120 78 768 508 18 22 4.20 Iketani et al.
(2001)
‘Bartlett’  ‘Hosui’ 63 AFLP, SSR 226 54 949 926 18 17 4.90 Yamamoto
et al. (2002)
‘Passe Crassane’  ‘Harrow 99 SSR, AFLP, MFLP, 155 156 912 930 18 19 5.80 6.00 Dondini
Sweet’ AFLP-RGA, RGA et al. (2004)
‘Bartlett’  ‘Hosui’ 63 AFLP, SSR 256 180 1020 995 19 20 4.00 5.50 Yamamoto
et al. (2004)
‘Bartlett’  ‘Hosui’ 63 AFLP, SSR 447 1000 17 2.30 Yamamoto
et al. (2007)
‘Housui’  ‘La France’ 55 AFLP, SSR 414 1156 17 2.80 Yamamoto
et al. (2007)
‘Abbè Fétel’ (AF)  ‘Max Red 95 MFLP, SSR 123 110 908.1 879.8 18 19 7.40 8.00 Pierantoni
Bartlett’ (MRB) et al. (2007)
‘Bartlett’  ‘Hosui’ 63 AFLP, SSR 335 1174 17 3.50 Terakami
et al. (2009)
‘Yali’  ‘Jingbaili’ 145 AFLP, SSR 402 18 1395.9 3.80 Sun et al.
(2009)
‘Housui’  ‘La France’ SSR, SNP 370 415 1160 1177 17 20 3.14 2.84 Yamamoto
et al. (2009)
‘Niitaka’  ‘Suhyangri’ 94 RAPD, AFLP, SSR 106 122 1006 1168 19 19 9.49 9.57 Junkyu et al.
(2010)
‘Bayuehong’  ‘Dangshansuli’ 97 AFLP, SRAP, SSR 214 122 1352.7 1044.3 17 17 6.32 8.56 Zhang et al.
(2013)
‘Red Bartlett’  ‘Nanguo pear’ 74 SRAP 103 105 602.2 650 20 20 4.89 5.20 Zhao et al.,
(2013)
‘Bartlett’  ‘Hosui’ 63 SSR, SNP 485 965 17 1.99 Yamamoto
et al. (2013)
‘Bartlett’  ‘Hosui’ 63 SSR, SNP 951 1341.9 22 1.41 Terakami
et al. (2014)
‘Bayuehong’  ‘Dangshansuli’ 102 SSR, SNP 3241 2243.4 17 0.70 Wu et al.
(2014)
‘Akiakari’  ‘Taihaku’ 93 SSR, EST-SSR 208 275 799.1 1039.1 17 17 3.84 3.78 Yamamoto
et al. (2014)
‘Bayuehong’  ‘Dangshansuli’ 56 SSR 734 1661.4 17 2.26 Chen et al.
(2015)
‘Red Clapp’s 161 SSR, SLAF 4797 2703.6 17 0.56 Wang et al.
Favorite’  ‘Mansoo’ (2017)
Nine published maps – SSR, SNP 5085 3266 17 0.64 Li et al.
(2017)

development of reference linkage maps for both were determined (Yamamoto et al. 2009). Fur-
European and Japanese pears, the precise linkage thermore, the self-incompatibility locus S was
groups for pear scab resistance gene, located found to be located along the bottom of LG17
along the middle region of LG1, and black spot (Yamamoto et al. 2009).
response gene, located along the top of LG11,
108 J. Wu and M. Qin

As the red-fruit color for pears is a popular dwarfing trait of pear has been reported to be
trait for consumers, Dondini et al. (2008) have controlled by the PcDw gene in cv. Aihuali
used a morphological marker for “red color” and (Wang et al. 2011, 2016). Using bulked segre-
mapped it onto LG4 of ‘Max Red Bartlett’. gant analysis (BSA) with 500 RAPD and 51 SSR
Previously, the gene encoding this trait, markers from both pear and apple, four markers
MdMyb10, has been mapped on LG9 in apple. co-segregating with the dwarf character have
Subsequently, Pierantoni et al. (2010) have been identified (Wang et al. 2011, 2016). The
cloned PcMyb10, found to have 96% amino acid PcDw gene is mapped on LG16 of ‘Bartlett’, and
sequence identity with that of MdMyb10, from located within very close distances, of 0.4 and
both ‘Max Red Bartlett’ and ‘Williams’. The 0.8 cM, from markers CN993875 and
gene PcMyb10 is found to map on LG 9 of ‘Max QauSSR36 (Fig. 5.2, Wang et al. 2016). These
Red Bartlett’, thereby indicating that this gene is latter two molecular markers have been deemed
in fact directly responsible for the red skin color valuable in fine mapping and cloning of the
of ‘Max Red Bartlett’. PcDw gene.
The dwarfing trait is another important agro-
nomic character, as it highly impacts efforts for
pursuing high-density fruit tree production. In the 5.4.2 QTL Mapping
1930s, a pear mutant seedling with a significant
dwarfing characteristic has been identified in Marker-assisted QTL-map-based genetic linkage
France (Fideghelli et al. 2003). Studies have maps are powerful in dissecting the genetic basis
revealed that the dwarfing trait is controlled by a of traits in many plant species, including pear.
single dominant gene (Rivalta et al. 2002). The Thus far, about 20 QTLs have been detected in
pear. Most of these QTLs are related to fruit
traits, while some are related to disease resis-
BartlettLG16 AihualiLG16 tance, harvest time, as well as length and width
of leaves, among others (Table 5.2).
TsuENH022
0.2 CH05a09 The first attempt to pursue QTL mapping in
NH026a 0.0 PcDw pear focused on fire-blight disease resistance
CH05c07 0.9 TsuENH022 (Dondini et al. 2004). In an earlier study,
0.3 TsuENH079 5.9 S1212-SCAR318 fire-blight resistance in pear has been confirmed
CH02a03 8.2 KA14
2.0 TsuENH036 9.5 S1172-SCAR930 to be a quantitative trait (Dondini et al. 2002).
2.4 CH01f03a Subsequently, interval mapping was conducted
3.4 KA14
5.0 NH007b using a segregating F1 population (99 seedlings)
5.4 TsuENH054 of ‘Passe Crassane’  ‘Harrow Sweet’, and
5.7 CH05c06 identified four putative QTLs on LG2a, LG2b,
LG4, and LG9 in the ‘Harrow Sweet’ map
30.0 CH05a04 (Dondini et al. 2004). In another effort, QTLs for
33.8 NB133a pear scab disease resistance, reported to be a
34.5 TsuENH042
polygenic trait (Chevalier et al. 2004), have been
identified. Pierantoni et al. (2007) conducted
interval mapping using a segregating F1 popu-
lation of ‘Abbé Fétel’  ‘Max Red Bartlett’, and
52.5 NB116b detected two QTLs for pear scab resistance on
52.8 NB123a LG3 and LG7 that were different from those
55.3 GC/CAC-313
mapped on LG1 (Iketani et al. 2001; Yamamoto
Fig. 5.2 Mapping of a dwarfing trait gene, PcDw on et al. 2009). Later, Won et al. (2014) performed a
LG16, of pear cultivars Aihuali (Wang et al. 2016) and Kruskal–Willis analysis using an interspecific
Bartlett (Celton et al. 2009)
pear progeny, PEAR1  PEAR2, derived from
5 Linkage Mapping in Pear 109

Table 5.2 Summary of QTLs of agronomic traits in pear

Trait Linkage groups Population Reference(s)
Fire blight 2a, 2b, 4, 9 ‘Passe Crassane’  ‘Harrow Dondini et al. (2004)
Sweet’
Pear scab 3, 7 ‘Abbè Fétel’ (AF)  ‘Max Red Pierantoni et al. (2007)
Bartlett’ (MRB)
1 ‘Kinchaku’  ‘Kosui’ Iketani et al. (2001)
1 ‘Housui’  ‘La France’ Yamamoto et al. (2009)
2, 5, 7, 10, 17 PEAR1  PEAR2 Won et al. (2014)
1 ‘Housui’  ‘La France’ Yamamoto et al. (2009)
Black spot ‘Kinchaku’  ‘Kosui’ Iketani et al. (2001)
11 ‘Housui’  ‘La France’ Yamamoto et al. (2009)
Self-incompatibility 17 ‘Bartlett’  ‘Housui’ Yamamoto et al. (2002,
2004, 2009)
Skin color 4 ‘Abbè Fétel’ (AF)  ‘Max Red Dondini et al. (2008)
Bartlett’ (MRB)
9 ‘Abbè Fétel’ (AF)  ‘Max Red Pierantoni et al. (2010)
Bartlett’ (MRB)
8 (two year) ‘Akiakari’  ‘Taihaku’ Yamamoto et al. (2014)
4, 13, 16 (two year) ‘Bayuehong’  ‘Dangshansuli’ Wu et al. (2014)
Drawf 16 ‘Aihuali’  ‘Chili’ Wang et al. (2016)
Fruit weight 2, 7, 8, 10 ‘Bayuehong’  ‘Dangshansuli’ Zhang et al. (2013)
3, 11 ‘Akiakari’  ‘Taihaku’ Yamamoto et al. (2014)
13, 17 ‘Bayuehong’  ‘Dangshansuli’ Wu et al. (2014)
Fruit diameter 10, 15 ‘Bayuehong’  ‘Dangshansuli’ Zhang et al. (2013)
3, 11, 17 ‘Bayuehong’  ‘Dangshansuli’ Wu et al. (2014)
Fruit length 7 (two year), 8 ‘Bayuehong’  ‘Dangshansuli’ Zhang et al. (2013)
11, 17 (two year) ‘Bayuehong’  ‘Dangshansuli’ Wu et al. (2014)
Fruit shape index 1, 2 (two year), 7, 8 ‘Bayuehong’  ‘Dangshansuli’ Zhang et al. (2013)
SSC 2, 5, 6 ‘Bayuehong’  ‘Dangshansuli’ Zhang et al. (2013)
4, 8 ‘Akiakari’  ‘Taihaku’ Yamamoto et al. (2014)
5, 10, 14 ‘Bayuehong’  ‘Dangshansuli’ Wu et al. (2014)
Flesh color 9 (two year) ‘Bayuehong’  ‘Dangshansuli’ Wu et al. (2014)
Firmness 4 (two year) ‘Akiakari’  ‘Taihaku’ Yamamoto et al. (2014)
Skin smooth 2, 17 ‘Bayuehong’  ‘Dangshansuli’ Wu et al. (2014)
Length of pedicel 2, 14, 17 ‘Bayuehong’  ‘Dangshansuli’ Wu et al. (2014)
Calyx status 6 (two year) ‘Bayuehong’  ‘Dangshansuli’ Wu et al. (2014)
Juice content 1, 5 ‘Bayuehong’  ‘Dangshansuli’ Wu et al. (2014)
Number of seeds 5 (two year), 9, 14, 17 ‘Bayuehong’  ‘Dangshansuli’ Wu et al. (2014)
(two year)
Preharvest fruit 1, 15 (two year) ‘Akiakari’  ‘Taihaku’ Yamamoto et al. (2014)
drop
Harvest time 3 (two year), 15 (two ‘Akiakari’  ‘Taihaku’ Yamamoto et al. (2014)
year)
(continued)
110 J. Wu and M. Qin

Table 5.2 (continued)

Trait Linkage groups Population Reference(s)
8 (two year) ‘Bayuehong’  ‘Dangshansuli’ Zhang et al. (2013)
Leaf length 8, 15, 16 ‘Yali’  ‘Jingbaili’ Sun et al. (2009)
Leaf width 10, 15 ‘Yali’  ‘Jingbaili’ Sun et al. (2009)
Leaf length/width 5 ‘Yali’  ‘Jingbaili’ Sun et al. (2009)
Petiole length of 4, 15 ‘Yali’  ‘Jingbaili’ Sun et al. (2009)
leaf

European (P. communis) and Asian (P. pyrifolia development. Zhang et al. (2013) have identified
and P. ussuriensis) pears, and identified seven four QTLs for fruit weight using an F1 popula-
potential QTLs for pear scab resistance. Among tion of ‘Bayuehong’  ‘Dangshansuli’, and
these, two QTLs localized on LG2 of PEAR2, these are distributed along LG2, LG7, LG8, and
one QTL identified on LG5 of PEAR2, two LG10, but without repeatability between years.
QTLs detected on LG7, along with both PEAR1 However, when Wu et al. (2014) have used the
and PEAR2 maps, one QTL localized on LG10 same population to construct an SNP-based
of PEAR1, and one QTL detected on LG17 of genetic linkage map, they have identified two
PEAR1, were identified. QTLs for fruit weight on LG13 and LG17.
In another effort, 11 QTLs for four leaf traits, Interestingly, Yamamoto et al. (2014) have
including leaf length, leaf width, leaf detected yet another two QTLs for fruit weight in
length/width, and petiole length were identified Japanese pear cultivars Akiakari and Taihaku.
using interval mapping (Sun et al. 2009). Among
these, four QTLs were associated with leaf
length, and localized on LG8, LG15, and LG16. 5.5 Software Resources
In addition, two QTLs were associated with leaf
width, localized on LG10 and LG15, two QTLs Currently, there are limited numbers of software
were associated with leaf length/width, both available for constructing linkage maps using F1
localized on LG5, and three QTLs were associ- populations. The most widely used software is
ated with petiole length, localized on LG4 and JoinMap (Stam 1993). The JoinMap is a com-
LG15. The observed phenotypic variation mercial software that runs on an MS Windows
explained (% Expl) by those QTLs ranged from platform, providing a user-friendly interface.
7.9 to 48.5%. According to the “double pseudo-test cross”
Several studies have pursued QTL mapping of hypothesis (Hemmat et al. 1994), JoinMap relies
various pear fruit traits as most fruit-related traits on the “CP” model to construct genetic linkage
are polygenic and are controlled by quantitative maps for F1 populations. In general, markers for
loci. Some of these loci are localized either a heterozygous male parent and a homozygous
within the same or in adjacent regions of a female parent are used for paternal mapping;
genetic linkage map, depending on years of whereas, markers for a heterozygous female
testing or populations used. It is noteworthy to parent and a homozygous male parent are used
point out that QTLs of correlated fruit quality for maternal mapping. Furthermore, markers for
traits often tend to map to the same chromosomal two heterozygous parents can be used to identify
region (Zhang et al. 2013; Yamamoto et al. 2014; homozygous linkage groups in these parents.
Wu et al. 2014). For example, fruit weight (or As presented in the JoinMap manual, markers
fruit size), fruit diameter (transverse diameter), can be divided into the following five different
and fruit length (vertical diameter) are highly types: <abxcd>, <efxeg>, <lmxll>, <nnxnp>,
correlated during various stages of fruit and <hkxhk>. However, only <lmxll>, <nnxnp>,
5 Linkage Mapping in Pear 111

and <hkxhk> can be used for genetic linkage map regression mapping or maximum likelihood
construction. JoinMap offers several mapping mapping, as with JoinMap v4.1, which may help
parameters to choose from, depending on the improve map quality. The package of R/qtl is
user’s preference and requirements. However, a available at http://www.rqtl.org.
major limitation of the JoinMap software is its
computing capacity. With the availability of new
generation sequencing (NGS), millions of
high-quality markers can be identified, yet this is
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 Molecular Mapping of Major Genes
and QTLs in Pear 6
Paolo De Franceschi and Luca Dondini

Abstract all progenies and selected parental lines

Pear breeding programs are mainly focused on capable of conferring traits of interest to their
resistance to biotic stress and fruit quality progenies are described herein. The aim is to
traits. In the last two decades, major efforts provide breeders with tools to identify pear
have been undertaken toward identification of ideotypes in which several traits can be
major genes and quantitative trait loci (QTLs) combined into a single individual. Further-
linked to both biotic resistance and fruit more, knowledge of genes and their related
quality traits, along with their associated functions should serve as the basis for pursu-
molecular markers in order to enable ing new plant breeding technologies, such as
marker-assisted selection and breeding. This cisgenesis or DNA editing. These unprece-
chapter will cover most relevant results dented advances in genomics and breeding
reported so far pertaining to markers and strategies promise to enable dramatic
QTLs linked to resistance to pathogens and improvements in breeding efficiencies, even
pests (such as fire blight, scab, brown and for pears, that will also reduce time and costs
black spot, pear psylla, pear sludge, and blister incurred in today’s traditional genetic
mite), fruit quality (fruit size, firmness, skin improvement efforts.
overcolor, russeting, fruit sweetness, and fruit
acidity), and other traits (such as tree habit,
chilling requirement, and harvest time). Fur-
thermore, summaries of findings of studies 6.1 Introduction
conducted before and after the beginning of
the genomics era will be provided. In addition, Among the critical objectives of primary impor-
tance in pear breeding programs are resistance to
biotic stresses, ability to adapt to environmental
P. De Franceschi
L. Dondini (&) changes, and desirable fruit quality traits. In the
Department of Agricultural and Food Sciences,
University of Bologna, Viale Giuseppe Fanin 44,
past 20 years, major efforts have been under-
Bologna, Italy taken to identify disease resistance genes and to
e-mail: [email protected] develop molecular tools that will support breed-
Present Address: ing programs in overcoming these adversities. In
P. De Franceschi recent years, various studies have also aimed at
Consiglio per la Ricerca in Agricoltura e l’Analisi identifying genes responsible for fruit quality
dell’Economia Agraria, Centro di ricerca
Cerealicoltura e Colture industriali (CREA-CI), Via
traits whose activities result in high levels of
di Corticella 133, Bologna, Italy phenotypic variability observed in pears.

© Springer Nature Switzerland AG 2019 113

S. S. Korban (ed.), The Pear Genome, Compendium of Plant Genomes,
https://doi.org/10.1007/978-3-030-11048-2_6
114 P. De Franceschi and L. Dondini

Collectively, these studies have revealed that in those for Pyrus  bretschneideri, Chinese white
most cases disease resistance traits are rather pear (Wu et al. 2013b), and for P. communis,
complex; moreover, most fruit quality-related European pear (Chagné et al. 2014). In particular,
traits are also highly polygenic, in which many availability and utilization of next-generation
loci with minor phenotypic effects are involved sequencing (NGS) techniques, in most cases, for
rather than a few major genes with major effects. analysis of whole transcriptomes, have greatly
The synteny between the genomes of apple facilitated identification of those genes, and their
and pear, as well as transferability of molecular related allelic variants, underlying expression of
markers between these two species (Pierantoni agronomic traits, and in some cases, these have
et al. 2004), has aided in the development of the also allowed development of markers for use in
first genetic maps for pear, in which a number of marker-assisted selection/breeding (MAS/MAB).
qualitative trait loci (QTL) linked mostly to dis- Identifying major genes, their sequences, and
ease and pest resistance traits have been identi- functions has allowed efforts to pursue new plant
fied (see Chap. 5 on linkage maps, and literature breeding technologies (NPBT), such as the
cited in this chapter). development of cisgenic cultivars, as well as the
Earlier efforts in using molecular approaches introduction of specific mutations using
have proved to be very useful in studying CRISPR-Cas9 gene editing (Schaart et al. 2016).
monogenic and polygenic traits related not only Therefore, this chapter aims to provide a review
to resistance to various pathogens, inciting fire of genes and QTLs identified in Pyrus species
blight, scab, black and brown spot, and pests, that will support future breeding efforts.
such as pear psylla, but also to fruit quality traits,
such as fruit color and size, firmness, as well as
acid and sugar contents in pear. As most of these 6.2 Major Genes and QTLs
traits of pear are of polygenic nature, several for Resistance Against
QTLs have been identified. Pathogens and Pests
The first genetic maps for pear have been
mainly based on microsatellite or simple Often, plant breeders have very ambitious pro-
sequence repeat (SSR) and amplified fragment grams aimed at developing disease- and
length polymorphism (AFLP) markers (Yama- pest-resistant pear cultivars. Unfortunately, these
moto et al. 2002; Dondini et al. 2004; Pierantoni efforts have been limited in the past due to the
et al. 2004). However, nowadays the availability scarce knowledge of sources of genetic resistance
of a single nucleotide polymorphism (SNP) chip to various important diseases and pests. How-
for genotyping in pear (Montanari et al. 2013) ever, with recent advances in new genetic and
allows for the construction of new generations of genomic technologies along with the availability
high-density maps, using classical segregating of worldwide germplasm, collections of Pyrus
populations, thereby dramatically promoting have allowed for the accumulation of new
discovery of numbers of new loci, while reduc- knowledge of genetic and genomic resources for
ing time and effort involved. In turn, this has pear. Currently, a few monogenic sources, as
greatly facilitated efforts to identify and localize well as QTLs for disease and pest resistance,
QTLs for disease/pest resistance and those for have been identified. Furthermore, a number of
fruit quality, as well as identify genes responsible molecular markers have been developed that are
for these QTLs, and develop molecular markers potentially useful for MAS.
for assisted selection and breeding.
With the advent of the genomic revolution, in
particular the availability of whole genome 6.2.1 Resistance to Fire Blight
sequence approaches and technologies, complete
draft sequences for several genomes of various Few pathogens are as devastating as the bacterial
fruit tree species have been published, including pathogen Erwinia amylovora (Burrill) Winslow
6 Molecular Mapping of Major Genes and QTLs in Pear 115

et al. that incites fire blight disease in pears, as germplasm, such as ‘Old Home’, ‘Seckel’,
well as in apples. Despite the presence of quar- ‘US309’, and ‘Michigan 437’, P. ussuriensis,
antine measures in several countries, fire blight and P. pyrifolia, among others, and these have
disease continues to spread throughout the world been used to develop and release a number of
and contributing to severe yield losses. resistant cultivars, such as ‘Harrow Sweet’ and
The bacterium takes advantage of either nat- ‘Moonglow’ (Dondini and Sansavini 2012;
ural openings (flowers) or wounds (caused by Montanari et al. 2016). These plant materials
hail or pruning cuts, among others) to infect have been used to investigate the genetic basis of
plants; moreover, insects can also serve as car- resistance and to identify a number of QTLs
riers. When the bacterium infects plant tissues, it linked to resistance (Fig. 6.1).
spreads along young shoots producing a charac- Overall, three QTLs have been identified in
teristic symptom known as ‘shepherd’s crook’ linkage groups (LGs) 2, 4, and 9 of the European
(Dondini and Sansavini 2012). Lack of com- pear ‘Harrow Sweet’ (Dondini et al. 2004; Le
pletely effective control measures has accentu- Roux et al. 2012), while two additional QTLs were
ated the importance of the availability of fire identified on LGs 9 and 11 of a resistant accession
blight-resistant cultivars with durable resistance of P. ussuriensis (Bokszczanin et al. 2009, 2011),
as a promising tool for an effective management and a major QTL was found on LG 2 of ‘Moon-
strategy for this disease (Dondini and Sansavini glow’ (Montanari et al. 2016). Interestingly, some
2012; Montanari et al. 2016). Fire blight resis- QTLs have also been identified in susceptible
tance is known to be a polygenic trait (Le Lézec accessions, including those found on LGs 3 and 4
et al. 1997). Several sources of fire blight resis- of ‘Doyenne du Comice’ (Bokszczanin et al. 2009,
tance are known to be available in the pear 2011), as well as those located on LGs 7, 9, 10, 12,

Fig. 6.1 Schematic

representation of positions of
known QTLs for fire blight
resistance. Colors of QTL
bars correspond to supporting
literature
116 P. De Franceschi and L. Dondini

and 15 of PEAR3, an interspecific hybrid between 6.2.2 Resistance to Pear Scab

P.  bretschneideri and P. communis (Montanari
et al. 2016). The high numbers of QTLs identified Scab is one of the most serious fungal diseases
in this latter study were attributed to the use of a affecting the European pear, and it is incited by
high-density map for QTL analysis, wherein an the fungal pathogens Venturia pirina Aderh. and
apple and pear Infinium H II 9K SNP array was V. nashicola Tanaka et Yamamoto. Most com-
used for genotyping (Montanari et al. 2013), as monly grown European pear cultivars are sus-
well as for phenotyping conducted under different ceptible to scab, and unfortunately, there are no
environmental conditions, in both France and New commercial cultivars with high levels of resis-
Zealand. tance to scab. Furthermore, the severity of dis-
It is important to point out that the two major ease symptoms is also influenced by
QTLs identified in ‘Harrow Sweet’ and ‘Moon- environmental conditions, as well as by the
glow’ co-localize around SSR marker variability of V. pirina biotypes (Chevalier et al.
TsuENH017, in spite of the fact that the two 2004). On the other hand, European pear culti-
LOD curves in the two cultivars do not perfectly vars seem to serve as sources of resistance to V.
overlap. The same consideration can be taken nashicola (Abe et al. 2008; Cho et al. 2009;
into account for QTLs identified on LG 4 of Bouvier et al. 2012).
‘Harrow Sweet’ and ‘Doyenne du Comice’, In contrast to fire blight, there are a few
around SSR marker CH02C02, and those found monogenic sources for resistance to pear scab
on LG 9 of ‘Harrow Sweet’ and P. ussuriensis in that have been identified in both European and
a region around SSR marker CH05C07. Japanese pear cultivars (Fig. 6.2; Abe et al. 2008;
Unfortunately, monogenic sources for fire Cho et al. 2009; Bouvier et al. 2012). Using
blight resistance have not yet been identified. interspecific pear hybrids, a single dominant
However, there is a strong indication of the gene, designated as Vn, has been identified to
presence of several major resistance genes in confer resistance to V. nashicola and proposed to
specific regions of the pear genome that could be be present in European pears ‘La France’ and
transferred into new pear cultivars with durable ‘Bartlett’ (Abe et al. 2008). Subsequently, two
fire blight resistance. additional V. nashicola resistance genes have

Fig. 6.2 Schematic

representation of positions of
major genes and QTLs for
pear scab resistance
6 Molecular Mapping of Major Genes and QTLs in Pear 117

been identified, Vnk, mapped on LG 1 of ‘Kin- as qrvp-1, is mapped both as a major gene and as
chaku’(Terakami et al. 2006), and Rvn2, puta- a QTL on LG 1 (within the same region of the
tively derived from ‘Bartlett’ (Cho et al. 2009). Vnk gene for resistance against V. nashicola),
This latter gene has been mapped to LG 2; while the second locus, designated as qrvp-o4, is
however, it is proposed that Vn and Rvn2 could mapped as a QTL on LG 4. Using the cross
be indeed the same gene (Bouvier et al. 2012). ‘Euras’  P3480, it has been possible to pyra-
Furthermore, Bouvier et al. (2012) have reported mid these two sources of scab resistance into
on the presence of yet another monogenic source single genotypes (Perchepied et al. 2015). All
of resistance to V. pirina, the Rvp1 gene, located these findings are summarized in Fig. 6.2.
on LG 2 of the European pear ‘Navara’. Overall, the availability of several known
In addition to these monogenic sources of sources of pear scab resistance has enabled pur-
resistance, several QTLs for pear scab resistance suit of new breeding efforts aimed at selecting
have also been identified in recent years new pear genotypes with durable resistance to
(Fig. 6.2) (Pierantoni et al. 2007; Won et al. pear scab.
2014; Perchepied et al. 2015). Among these, two
QTLs have been identified on LG 3 and LG 7 of
‘Abbé Fétel’ following analysis of a progeny 6.2.3 Resistance and Susceptibility
derived from a cross of ‘Abbé Fétel’  ‘Max to Stemphylium
Red Bartlett’ (a ‘Bartlett’ red sport); however, no vesicarium
associations have been identified on LG 2 and to Alternaria
(Pierantoni et al. 2007), wherein the previously alternata
described Rvn2 gene derived from ‘Bartlett’ was
mapped (Cho et al. 2009). Among the various fungal threats to pears,
Progeny from the interspecific cross PEAR1 Alternaria alternata (Fries) Keissler and Stem-
PEAR2, derived from European (P. communis) phylium vesicarium (Wallr.) E. Simmons, causal
and Asian (P. pyrifolia and P. ussuriensis) pears, agents of black and brown spot, respectively, are
was inoculated with three single-spore isolates of among the most widespread diseases. Interest-
V. pirina and used to develop a high-density ingly, genetic resistance to black spot has been
linkage map (Won et al. 2014). Using this link- primarily investigated in Japanese pears, while
age map, QTLs were identified on LGs 7, 10, and that of brown spot has been investigated more so
17 of PEAR1 and on LGs 2, 5, and 7 of PEAR2. in European pears.
Furthermore, the QTL on LG 17 of PEAR1 was Early efforts have focused on inducing resis-
found to be effective against all V. pirina isolates, tance to A. alternata in black spot-susceptible
while the QTL on LG 7 of PEAR2 was effective cultivars of apple and pear using gamma-ray
against two isolates of V. pirina (Won et al. irradiation, and have suggested the presence of
2014). In addition, the QTLs on LG 7 of PEAR1 susceptibility genes that are inactivated by
and ‘Abbé Fétel’ seem to map in the same mutagenesis (Sanada et al. 1988; Saito et al.
position, while the QTLs of PEAR2 on LG 2 2001). Subsequently, these susceptibility genes,
seem to co-localize with Rvp1 and Rvn2 genes including Aki, Ana, and Ani, have been identified
(Cho et al. 2009; Bouvier et al. 2012). Interest- in different Japanese pear cultivars and then
ingly, this region has been deemed to be syntenic mapped to LG 11 of P. pyrifolia (Fig. 6.3).
to an apple scab resistance gene cluster on LG 2 These genes are proposed to be involved in
(Bouvier et al. 2012). and/or responsible for observed necrotic activi-
Using yet another high-density linkage map, ties of fungal toxins (Iketani et al. 2001; Ter-
Perchepied et al. (2015) have identified two new akami et al. 2007, 2016). The locus for black spot
QTLs for pear scab resistance against V. pirina in susceptibility on LG 11 of P. pyrifolia has also
P3480, a hybrid with resistance derived from been confirmed using a genome-wide association
‘Wilder’, and in ‘Euras’. One locus, designated study (GWAS) approach (Iwata et al. 2013b).
118 P. De Franceschi and L. Dondini

Fig. 6.3 Schematic

representation of positions of
major genes and QTLs for
black and brown spot
resistance

On the other hand, most pear cultivars are been conducted thus far (Bellini and Nin 2002).
highly susceptible to brown spot disease, with Nevertheless, resistance to psylla has been intro-
the important exception of ‘Bartlett’ and its duced from P. ussuriensis genotype ‘Illinois 65’
mutant sports, such as ‘Max Red Bartlett’ into a number of pear selections, including
(Llorente and Montesinos 2006). Susceptibility ‘NY10352’, ‘NY10353’, and ‘NY10355’ (Westi-
to S. vesicarium has been identified, wherein a gard et al. 1970; Harris 1973). The latter two
major QTL for susceptibility is located on LG 15 selections have been used to characterize resis-
of ‘Abbé Fétel’, and the putative position of a tance responses following pear psylla attack. For
susceptibility gene, designated as Sv, is estimated example, Pasqualini et al. (2006) have investigated
to be located at the lower end of the linkage behavior of psyllids on pear selections derived
group (Fig. 6.3; Cappai et al. 2018). from ‘NY10353’, while Salvianti et al. (2008)
Identification of genes controlling suscepti- have analyzed differential gene expression in
bility to black and brown spot diseases will aid in ‘NY10355’ following challenge with psyllids. In
pursuing new plant breeding technologies, such addition, Civolani et al. (2013) have monitored the
as CRISPR-Cas9 systems, to efficiently develop feeding activity of adults and nymph psyllids on
new pear genotypes with resistance to these ‘NY10353’, and have concluded that resistance
fungal pathogens using targeted gene inactiva- factors are located in the phloem sap of this
tion approaches (Cappai et al. 2018). selection.
A major QTL for psylla resistance is located on
LG 17 of pear selection ‘NY10353’ (Fig. 6.4;
6.2.4 Resistance to Pear Psylla Dondini et al. 2015). This QTL, linked to the nym-
and Other Pests phal vitality, is first identified using gene scanning,
and then subsequently validated following analysis
Pear psylla (Cacopsylla pyri L.) is a serious pest for of seedlings of a whole progeny derived from the
pear-growing areas due to the high susceptibility cross ‘NY10353’  ‘Doyenne du Comice’ (Don-
of almost all marketed pear cultivars. Therefore, dini et al. 2015). In addition, this QTL is also con-
breeding efforts have focused on identifying firmed to be present in ‘NY10355’ following
sources of tolerance or resistance to pear psylla. analysis of a progeny of ‘NY10355’ ‘Angelys’,
Pyrus fauriei, P. calleryana, and P. ussuriensis wherein ‘Angelys’ is used as a psylla-susceptible
have been identified as sources of psylla resistance parent (Fig. 6.4; Perchepied et al. 2016). Further-
(Dondini and Sansavini 2012). The genetic control more, Perchepied et al. (2016) have identified four
for resistance to pear psylla is reported to be QTLs on LG 1, wherein these QTLs on LG 1 have
polygenic; however, only limited studies have strong epistatic effects on the QTL on LG 17.
6 Molecular Mapping of Major Genes and QTLs in Pear 119

Fig. 6.4 Schematic

representation of the positions
of QTLs for psylla and other
pests resistance

Yet, another source of resistance to pear psylla aimed at selecting and developing new pear
has been identified, derived from the Chinese white cultivars with combined resistances to different
pear P.  bretschneideri. QTLs for resistance to pathogens and pests.
pear psylla have been identified on LGs 5 and 8 of
the hybrid ‘PEAR3’ [‘Xuehuali’ (P.  bretschnei-
deri)  ‘Max Red Bartlett’ (P. communis)], as well 6.3 Major Genes and QTLs for Fruit
as on LG 15 of ‘Moonglow’, the other parent of the Quality Traits
‘PEAR3’  ‘Moonglow’ progeny used in this
study (Fig. 6.4; Montanari et al. 2015). As most pear fruit quality traits are under highly
Very recently, QTLs for resistance to pear polygenic control, with rare exceptions such as
slug (the larvae of the sawfly Caliroa cerasi L.) the red skin fruit color in European pear, this has
and pear blister mite (Eriophyes pyri Pagen- hampered identification of major genes. How-
stecher) have been identified (Brewer et al. 2018) ever, with the advent of functional genomics,
using progeny derived from the cross transcriptomics, and proteomics, many candidate
‘PremP003’  ‘Moonglow’. Specifically, a genes or gene families controlling important
major QTL for resistance to pear blister mite was biosynthetic pathways involved in pear fruit
located on LG 13 of ‘PremP003’. For pear slug, quality have been and are currently under
three QTLs for oviposition were mapped on LG investigation (Lu et al. 2011; Nashima et al.
7 and LG 9 of ‘Moonglow’ and on LG 10 of 2013; Li et al. 2014a, 2014b, 2014c, 2014d; Wu
‘PremP003’, while another QTL for leaf damage et al. 2014b; Dai et al. 2015; Li et al. 2015; Xu
was located on LG 9 of ‘Moonglow’, just below et al. 2015; Reuscher et al. 2016; Song et al.
the oviposition QTL (Fig. 6.4; Brewer et al. 2016; Wei et al. 2016; Zhang et al. 2016; Shen
2018). et al. 2017). For further detailed review of
All the above findings are critical in setting up functional genomics studies, please refer to
molecular protocols and MAS breeding strategies Chap. 14.
120 P. De Franceschi and L. Dondini

6.3.1 Fruit Color ‘Williams’, and it is positively correlated with

anthocyanin accumulation during fruit develop-
Although most common pear cultivars have ment (Pierantoni et al. 2010). Furthermore,
either yellow or green fruit color, there is an methylation of the PcMYB10 promoter and its
increasing interest and appreciation for cultivars transcriptional silencing are associated with
with red skin fruit color. In addition to increased regression to the green color fruit skin phenotype
fruit appeal for consumers, red skin color is of the same cultivar (Wang et al. 2013). Interest-
deemed as a desirable nutritional trait due to the ingly, expression of PcMYB10 in the interspecific
antioxidant activity of anthocyanins, as these hybrid ‘Wujiuxiang’ (‘Ya Li’  ‘Bartlett’) is
flavonoid compounds determine red color positively correlated with anthocyanin accumula-
pigmentation. tion in response to both developmental and
Red skin fruit color in European pears is cold-temperature induction (Li et al. 2012). These
considered to be a monogenic dominant trait, as findings clearly point to the role of PcMYB10 in
confirmed following analysis of seven segregat- regulating the anthocyanin biosynthesis pathway
ing progenies having one of the following culti- during fruit development. Furthermore, it is pro-
vars, ‘Max Red Bartlett’, ‘Cascade’, or posed that PcMYB10 acts along with a complex
‘California’, as their red-skinned fruit parental containing two other proteins, bHLH (basic helix–
line (Dondini et al. 2008). Moreover, this trait is loop–helix 33) and WD40 (tryptophan-aspartic
mapped onto LG 4 in ‘Max Red Bartlett’, a acid repeat protein) transcription factors, that bind
spontaneous red mutant of ‘Williams’, syn. to promoters of genes for key enzymes of antho-
‘Bartlett’ (Fig. 6.5; Dondini et al. 2008). cyanin biosynthesis, among which is the gene
In Rosaceae, as in most other plant taxa, encoding for UDP-glucose: flavonoid-3-O-
anthocyanin accumulation is regulated mainly at glucosyltransferase, UFGT (Pierantoni et al.
the transcriptional level, with transcription factors 2010; Wang et al. 2013). This hypothesis is also
belonging to the Myb family playing a key role supported by expression analysis of other Euro-
(Lin-Wang et al. 2010). The pear transcription pean pear cultivars (Li et al. 2012; Wu et al. 2013c;
factor from European pear (P. communis) Yang et al. 2013; 2015). Nevertheless, PcMYB10
PcMYB10, an ortholog of the apple MdMYB10 is mapped on LG 9 of ‘Max Red Bartlett’ (Fig. 6.5;
(Espley et al. 2007), is reported to be expressed at Pierantoni et al. 2010). Therefore, it is independent
much higher levels in ‘Max Red Bartlett’ than in from the ‘Red’ locus, which maps on LG 4 of ‘Max

Fig. 6.5 Schematic

representation of positions of
major genes and QTLs for red
skin color and for fruit
russeting. QTLs refer mainly
to Asian pears, whereas
positions of the Red locus
(from ‘Max Red Bartlett’) and
of the PcMYB10 gene for
European pear are
reported herein
6 Molecular Mapping of Major Genes and QTLs in Pear 121

Red Bartlett’ (Dondini et al. 2008). However, the Xue et al. (2017) have adopted a modified
gene underlying this phenotypic change is yet to be QTL-seq method to compare two DNA pools of
identified, although it must indeed act somehow red-skinned and green-skinned pears derived
upstream of PcMYB10 in the regulation of gene from a cross between P. pyrifolia cultivars
expression. ‘Mantianhong’ and ‘Hongxiangsu’, both having
The red skin fruit color in Asian pears is less red fruits. This analysis has highlighted a
frequently observed, and its genetic basis is 582.5-kb region in chromosome 5 as the main
under investigation. In addition to overall lower responsible region for red/green fruit color
accumulation, patterns of anthocyanin synthesis development. This region is compatible with the
in P. pyrifolia, P. ussuriensis, and map position of PyMYB114 and confirms its
P.  bretschneideri are different from that position at the bottom of LG 5 as a region con-
observed in P. communis, albeit it still correlates trolling this trait in Asian pears. Moreover, unlike
with expression of common genes, mainly driven in European pear, this study has suggested that
by PcMYB10 orthologs (Feng et al. 2010; Zhang the green color is dominant over the red skin
et al. 2011b; Yu et al. 2012; Yang et al. 2014). color. Therefore, despite the presence of a com-
Expression analysis studies in Chinese pear fur- mon biosynthetic pathway for anthocyanin
ther support the presence of a common pathway biosynthesis along with a likely conserved role
for anthocyanin regulation, involving two Myb for Myb transcription factors, the genetic control
transcription factors, PbMYB10b and PbMYB9, of red skin fruit color appears to be different in
promoting expression of UFGT and of other Asian and European pears. However, recent
genes (Zhai et al. 2016). However, when the analysis of the Chinese pear cultivar ‘Red
genetic control of anthocyanin accumulation has Zaosu’, a bud mutant of ‘Zaosuli’, with red fruits
been investigated, discordant results have been and foliage, has revealed the dominance of red
obtained. In particular, three QTLs are detected over green phenotypes (Xue et al. 2018). Fur-
for fruit skin red color in a progeny having thermore, this trait is mapped to the corre-
‘Bayuehong’, a hybrid between the European sponding locus on LG 4 (Xue et al. 2018), at a
pear ‘Clapp’s Favorite’ and the Chinese pear position that matches with that of the ‘Red’ locus
‘Zaosuli’, as the red-skinned parent (Wu et al. of ‘Max Red Bartlett’ (Dondini et al. 2008). On
2014a). One of these QTLs is mapped onto LG 4, the other hand, a QTL for fruit skin blush is
but its position (4.8 cM) seems to be incompat- mapped on the bottom of LG 5 in a European
ible with that of the ‘Red’ locus (64 cM) found in pear progeny of ‘Flamingo’  ‘Abbé Fétel’
‘Max Red Bartlett’ (Dondini et al. 2008). The (Ntladi et al. 2018) and corresponding to the
other two QTLs have been located on LGs 13 main QTL previously characterized in Asian pear
and 16. However, subsequent analysis of the (Yao et al. 2017). These findings reinforce the
same population has led to the identification of a hypothesis that the same genes regulate antho-
new QTL located on the bottom of LG 5, and an cyanin biosynthesis and accumulation in Euro-
additional Myb transcription factor, PyMYB114, pean and Asian pears. However, the different
has been identified within this QTL region (Yao genomic positions to which this trait has been
et al. 2017). Expression of the PyMYB114 is associated with reflect its complex genetic con-
positively correlated with red skin coloration, as trol, with many loci playing a role and with the
genetic transformation experiments have con- red phenotype arising independently from
firmed ability of PyMYB114 to induce antho- mutations of various genes.
cyanin biosynthesis, confirming that there are It should also be noted that an important
transcription factors, other than the ortholog of component of the skin color depends upon
PcMYB10, that are also involved in expression of suberification of peridermal cells (russeting),
this trait. conferring a brown color, that is unrelated to the
122 P. De Franceschi and L. Dondini

presence of anthocyanins, which is more likely to 2013), unfortunately, the generated map was
occur in Asian rather than in European pears. In based mainly on AFLP and SRAP markers.
fact, a major QTL for this trait has been detected Thus, these QTLs could not be reliably anchored
near the top of LG 8 in Japanese pear ‘Akiakari’ to reference maps of pear and apple and render-
(Fig. 6.5; Yamamoto et al. 2014). ing it difficult to compare positions of these
QTLs with those detected in other studies. In yet
another study, QTLs for fruit size in Japanese
6.3.2 Fruit Size pears were found on LG 11 of ‘Akiakari’ and LG
3 of ‘Taihaku’ (Yamamoto et al. 2014), thus once
In pears, like in most cultivated fruit species, fruit again highlighting how segregation of this trait in
size is probably one of the traits that have different genetic backgrounds might depend on
changed most dramatically during the domesti- different loci.
cation process. Although the actual fruit size Given the complexity of this trait, it is not
always depends on the interaction between easy to identify candidate genes for pursuing
environmental and genetic factors, potential fruit gene expression studies. ‘Da Nanguoli’ is a
size is genetically determined and varies signifi- spontaneous large-fruited mutant cultivar of
cantly among different cultivars (Zhang et al. ‘Nanguoli’ (P. ussuriensis), and it has served as
2006). a useful tool for studying the genetic mechanism
Fruit size behaves as a typical quantitative of fruit size. A comparative study of transcript
trait, with many loci contributing to its expres- profiling between ‘Da Nanguoli’ and ‘Nanguoli’
sion. QTL analyses aimed at identifying genomic has revealed the presence of a large pool of genes
regions controlling fruit size have been per- whose expression is differentially modulated
formed mainly in Asian pears (Fig. 6.6). Using during the development of large-sized and
progeny of ‘Bayuehong’ and ‘Zaosuli’ small-sized fruits (Zhang et al. 2011a). While this
(P.  bretschneideri), two QTLs for fruit size finding suggests the importance of the role of
were identified on LGs 17 and 13, with the transcription factors in regulating cellular pro-
position of QTL 17 found to be compatible with cesses that determine fruit size, the causal
two additional QTLs for transverse and vertical mutation has yet to be identified.
fruit diameter (Wu et al. 2014a). Although this Analysis of cytological events involved in
progeny was previously analyzed, resulting in fruit development has revealed that fruit size is
the identification of several QTLs (Zhang et al. ultimately determined by the number and size of
mesocarp cells, and therefore may vary in
response to variations in both cell division and
expansion. Larger cell size is responsible for the
production of larger fruits in ‘Giant La France’, a
mutant of the European pear ‘La France’, and it
is found to be associated with variations in ploidy
of mesocarp cells rather than a result of a genetic
mutation (Isuzugawa et al. 2014). Interestingly,
polyploidization only impacts fruit flesh, leaving
other reproductive tissues diploid, thus suggest-
ing presence of factors determining occurrence
and persistence of DNA reduplication in recep-
tacles of ‘Giant La France’. Subsequently, two
candidate genes, PcWEE1, a cell cycle-
associated protein kinase, and PcCCS52A, an
Fig. 6.6 Schematic representation of positions of QTLs anaphase-promoting complex activator, have
for fruit size been isolated, based on homology with tomato
6 Molecular Mapping of Major Genes and QTLs in Pear 123

genes known to play similar roles, and are found surprisingly, QTLs for soluble solid content have
to be up-regulated in receptacles of ‘Giant La been detected in different genomic regions of
France’ (Hanada et al. 2015). This has suggested P. pyrifolia, LGs 4 and 8 (Yamamoto et al.
that differences in expression levels of these two 2014), P.  bretschneideri, LGs 5, 10, and 14
genes may induce DNA reduplication and con- (Wu et al. 2014a), and an interspecific hybrid
sequent increase in size of mesocarp cells population of Asian and European pear, LGs 9
(Hanada et al. 2015). and 10 (Saeed et al. 2014). Unfortunately, it is
When comparing common diploid pear culti- not possible to determine whether or not the two
vars, variations in fruit size are normally asso- QTLs for soluble solid content in LG 10 (Wu
ciated with variations in cell number rather than et al. 2014a; Saeed et al. 2014) overlap, although
in cell size (Zhang et al. 2006). Homologs of they seem to be located in the same chromosomic
fw2.2, a gene controlling fruit size by regulating region. A recent analysis conducted on a Japa-
cell division in tomato (Frary et al. 2000), are nese pear population derived from the cross
proposed to be involved in the same process in ‘Akizuki’  ‘373-55’, besides a QTL for total
different plant species including fruit trees. In sugar content on LG 11, has detected two QTLs
cherry trees, some of these fw2.2 homologs are associated with the conversion of sucrose to
co-localized with known QTLs for fruit size (De fructose and glucose on LGs 1 and 7 (Nishio
Franceschi et al. 2013). Two genes belonging to et al. 2018). Moreover, two acid invertase
this family, PbFWL1 and PbFWL2, have been (AIV) genes are found in close proximity of both
characterized in Chinese pear and are found to be QTLs, thus serving as interesting candidates for
expressed at higher levels in small-fruited culti- control of sugar conversion in pear fruits. On the
vars, consistent with the negative regulatory role other hand, a single QTL for fruit acidity, located
of fw2.2 in cell division (Tian et al. 2016). on LG 14, is reported (Yamamoto et al. 2014). It
Therefore, these two genes are good candidates is noteworthy to point out that the organic acid
for control of fruit size in pear. However, addi- content can also be significantly influenced by
tional studies are required to study functionality maternal inheritance, suggesting that non-nuclear
of these genes. genes may play important roles as well (Liu et al.
2016).
Fruit firmness is determined by cell wall
6.3.3 Fruit Sensory Qualities components, which are degraded by several
hydrolases during ripening and leading to fruit
Fruit taste is determined by many different bio- softening. QTLs for this trait have been identified
chemical factors, such as accumulation of sugars on LG 4 (Yamamoto et al. 2014) and LG 3
and acids, flesh firmness and texture, and emis- (Saeed et al. 2014). The latter linkage group, LG
sion of volatile compounds (aroma). However, 3, has effects on other ripening-related traits,
limited information is available regarding genetic such as fruit friction discoloration, polyphenol
regions controlling these traits in segregating oxidase (PPO) activity, and polyphenol content.
pear progenies, although QTLs for soluble solid Furthermore, QTLs associated with PPO activity
content, fruit acidity, and firmness have been have been identified on LGs 2 and 3, as well as a
identified (Fig. 6.7). number of QTLs associated with contents of 17
Soluble solid content of pear fruits is essen- polyphenolic compounds have also been identi-
tially determined by sugars and organic acids. fied (Saeed et al. 2014).
The amounts and ratios between these different In addition to the different enzymes that cat-
compounds are critical factors in determining alyze cell wall degradation, expansins are pro-
fruit taste and therefore deemed as key compo- posed to play a role in fruit softening as they
nents of fruit quality. As sugars and organic acids disrupt hydrogen bonds between cellulose
are primary metabolites, many factors can impact microfibrils and matrix polysaccharides, thereby
their synthesis and accumulation in fruits. Not rendering substrates available to hydrolases. An
124 P. De Franceschi and L. Dondini

Fig. 6.7 Schematic

representation of positions of
major genes and QTLs for
soluble solid content (SS),
firmness, and acidity

expansin gene, PcExp7, from P. communis, has SNP map, using a genotyping by sequencing
been mapped on LG 1 in a region in which a (GBS) approach, detecting three additional QTLs
firmness QTL has been detected in apple (Costa on LGs 5, 13, and 15 (Gabay et al. 2018), and
et al. 2008). The presence of a member of the confirming the presence of QTLs on LGs 8 and 9.
gene family coding for 1-aminocyclopropane- The latter was further confirmed in a different
1-carboxylate synthase, which plays a role in progeny of European pear (Ntladi et al. 2018). For
determining harvest time, may also be involved in further information on bud break, please look up
pear fruit softening (Iwata et al. 2013b; Yamamoto Chap. 12 of this volume.
et al. 2014). However, further studies are required Using a genome-wide association study
to ascertain whether or not such a candidate gene (GWAS) analysis of 76 cultivars of P. pyrifolia,
co-localizes with QTLs for firmness in pear. QTLs for harvest time have been mapped on LGs 3
(corresponding to SSR marker BGA35) and 15
(identified by the CAPS marker PPACS2)
6.4 Major Genes and QTLs for Other (Fig. 6.8; Iwata et al. 2013b). Incidentally, the
Traits marker PPACS2 identifies the position of a member
of the 1-aminocyclopropane-1-carboxylate syn-
Most efforts for developing molecular markers thase gene family (Iwata et al. 2013b; Yamamoto
for marker-assisted selection (MAS) have et al. 2014). In addition, both QTLs have been
focused on traits for resistance to pathogens and identified by analyzing a segregating progeny
pests, as well as on fruit quality traits. However, derived from the cross ‘Akiakari’  ‘Taihaku’
there are limited efforts in developing molecular (Yamamoto et al. 2014). Furthermore, both mark-
markers linked to other traits. ers BGA35 and PPACS2 have been validated by
Using a progeny derived from a cross between analyzing segregation data in six F1 progenies of
‘Spadona’ (with a low chilling requirement) and P. pyrifolia, demonstrating that alleles of 263 bp of
‘Harrow Sweet’ (with a high chilling require- PPACS2 and 136 bp of BGA35 are in linkage to the
ment) along with a comparative analogy to an early ripening fruit trait (Nishio et al. 2016).
apple linkage map, QTLs for bud break (follow- This QTL, together with another QTL found on LG
ing release from dormancy) have been found on 15, has been identified in the parent ‘Taihaku’.
LG 8, corresponding to SSR NAUpy98n, and LG Interestingly, results of findings on LG 3 of pear
9, between SSRs NH029 and CH01f03b (Gabay have also been confirmed in a subsequent GWAS in
et al. 2017). The same population was analyzed apple in which a major association for ripening time
more in depth by developing a high-resolution is found on chromosome 3 (Urrestarazu et al. 2017).
6 Molecular Mapping of Major Genes and QTLs in Pear 125

Fig. 6.8 Schematic flanking the Dw1 locus in apple (Hi01c04) also
representation of posi- segregates for dwarfing and precocity in pear
tions of major genes and
QTLs for harvest time with an allele of 116 bp in size associated with
these traits (Knäbel et al. 2015). The synteny
between the apple and pear genomes is very
important in identifying candidate genes for
controlling various traits, including these repor-
ted herein.
Using the same progeny described above,
QTLs controlling the development of adventi-
tious roots on hardwood cuttings have been
identified on LGs 7, 8, 10, and 11 of ‘Old Home’
and on LGs 7, 15, and 16 of ‘Louise Bonne de
Jersey’. In addition, a single QTL associated with
Although other traits such as plant vigor have
callus and root development has been found on
been phenotyped in 76 cultivars of P. pyrifolia,
LG 4 of ‘Louise Bonne de Jersey’ (Knäbel et al.
no associations could be found (Iwata et al.
2017). Furthermore, favorable alleles of markers
2013b); whereas, associations for plant vigor and
in QTL peaks of LG 7 (ss527788659 in ‘Old
early flowering have been detected in pear root-
Home’ and ss527789100 in ‘Louise Bonne de
stock breeding studies (Knäbel et al. 2015,
Jersey’) have demonstrated male and female
2017). By genotyping a very large progeny
additive and dominance effects for all years
derived from the cross ‘Old Home’  ‘Louise
(Knäbel et al. 2017). Therefore, the availability
Bonne de Jersey’, wherein all seedlings are used
of molecular markers will support breeding
for grafting the pear scion cultivar ‘Doyenne du
efforts aimed at selecting new pear rootstocks
Comice’, high-density linkage maps have been
that are easily propagated along with other
developed. Using these linkage maps, QTLs have
desirable traits such as vigor and early flowering
been identified on the top of LG 5 of ‘Old Home’
of known dwarfing rootstocks available for
for tree architecture, tree vigor, and various pre-
apples.
cocity traits, including number of branches per
Finally, an important trait for consideration
tree, tree height, number of inflorescences,
pertains to the S-RNase-based gametophytic
number of spurs per tree, trunk cross-sectional
self-incompatibility (GSI), previously reviewed
areas (TCA) of the rootstock and of the scion
by De Franceschi et al. (2012) and Wu et al.
around the graft zone, and root suckering (Knä-
(2013a). In addition to determining cross-
bel et al. 2015). Furthermore, except for a num-
compatibility of cultivars, GSI may also influ-
ber of inflorescences, additional QTLs have been
ence transmission of genes in proximity of the
identified for all other mentioned traits on the top
S locus. The S-RNase gene has been mapped on
of LG 6 of ‘Old Home’ and in the middle of LG
the bottom of LG 17 in both Japanese and
6 of ‘Louise Bonne de Jersey’ (Knäbel et al.
European pears (Yamamoto et al. 2002) and
2015). Other minor QTLs, for trunk
consistent with the position of the S locus in
cross-sectional areas of the scion and of the
apple (Maliepaard et al. 1998). Subsequently,
rootstock, are found on LGs 7 and 16 of ‘Louise
identification and mapping of S-locus F-box
Bonne de Jersey’, respectively (Fig. 6.9; Knäbel
brother genes, the male counterpart of S-RNase
et al. 2015). In a different study on apples, a
(Sassa et al. 2007), confirmed their linkage to S-
major QTL, controlling most of the dwarfing
RNase (De Franceschi et al. 2011). A detailed
effects conferred to a scion, has been identified
information and review of self-incompatibility of
on LG 5 of the apple rootstock ‘M9’ (Foster et al.
pear are provided in Chap. 10 of this volume.
2015). It is proposed that the SSR marker
126 P. De Franceschi and L. Dondini

Fig. 6.9 Schematic

representation of positions of
QTLs for vigor, flowering,
and root development in pear
rootstocks

made possible. For more information on func-

6.5 Conclusions tional genomics studies in pear, please read
Chap. 14.
Identification of major genes and QTLs linked to For those genes with strong effects on phe-
disease and pest resistance, fruit quality, and notypic variability, such as transcription factors,
other tree-related traits in Pyrus will certainly and for major QTLs, molecular marker selection
contribute to advances in MAS and in other offers serious advantages. Unfortunately, a
applications offered by the tools of genomics. In number of QTLs with minor effects on a phe-
particular, identification of QTLs will also assist notype have been presented in this current
in identification of additional genes, and possibly review. For these cases, the utility of linked
of related allelic variants, underlying observed markers for MAS is likely to be less effective in
phenotypic effects. These findings will in turn supporting pear breeding programs. This is par-
enable design of new additional markers for use ticularly true in instances wherein the cost for
in MAS. The release of the genome sequences genotyping seedlings must be justified when
for the Asian and European pears, along with the compared to conventional phenotypic selection
availability of high-throughput genotyping tech- methods. Nevertheless, novel approaches such as
niques, which allows for simultaneous analysis genomic selection are becoming more feasible
of thousands of markers, will offer opportunities and offer promise in making significant great
for more targeted and efficient selection of advances in this arena (Iwata et al. 2013a; Min-
desirable genotypes in a pear breeding amikawa et al. 2018).
population. Finally, it is important to conclude that once
The availability of tools for large-scale geno- genes and their related functions become known,
typing will also assist in pursuing GWAS a critical consideration must be taken into
approaches of pear germplasm collections, and account. Whether, we should choose to use new
enhance efforts in identifying genes and alleles plant breeding technologies, such as cisgenesis or
responsible for traits of interest. Unfortunately, DNA editing, in inserting mutations and altering
the time required for phenotyping remains the gene functions (Schaart et al. 2016), and how
greatest bottleneck in pursuing these approaches. best to exploit breeding advantages offered via
Nevertheless, genes controlling various traits can use of modified genes, either gene mutations or
be identified via transcriptomic approaches that gene editing, with significant reduction in time
next-generation sequencing technologies have and costs in developing and releasing improved
6 Molecular Mapping of Major Genes and QTLs in Pear 127

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 The Genome of Pear
7
Jun Wu, Shaoling Zhang and Xiaolong Li

Abstract 7.1 Genome of the Asian Pear

As the third most important temperate tree
fruit species, the pear occupies an indispens- The pear, belonging to the sub-family Maloideae
able position of high commercial importance in the Rosaceae family, is a diploid, shares a
and of beneficial nutritional value. Since the basic chromosome number of x = 17 (2n = 34),
release of genome sequences of the Asian pear and possesses a highly heterozygous genome.
and then of the European pear, comprehensive Studies have revealed that there is a high level of
‘big data’ explorations have been extensively heterozygosity in the pear genome and a 1–2%
carried out at the whole-genome level, includ- sequence divergence among alleles (Wu et al.
ing those of gene families, functional geno- 2013).
mics, and evolution analysis, among others. Using a combination of bacterial artificial
With the innovation of technology and chromosome (BAC)-by-BAC and next-generation
reduced costs of sequencing, increasing num- sequencing, a high-quality draft genome sequence
bers of genome resequencing and transcrip- of the Asian pear Pyrus  bretschneideri cv.
tome sequencing projects have also been Dangshansuli has been released (Wu et al. 2013)
undertaken based on the reference genome (Fig. 7.1). The assembled pear genome consists of
sequence of pear. These efforts will provide 2103 scaffolds with N50 at 540.8 Kb (Table 7.1).
credible data to support further functional This corresponds to 97.1% of the estimated gen-
analyses and valuable guidance in pursuing ome size (527.0 Mb), and a total of 512.0 Mb
germplasm improvement and breeding of sequence is assembled with 194 coverage.
pear. Herein, research advances in pear High-density genetic maps constructed using 2005
genome sequencing and its downstream anal- SNP markers have anchored 796 scaffolds, a total
yses are summarized and discussed, along of 386.7 Mb, and representing *75.5% of the
with future perspectives. assembled genome. In the pear genome, 42,812
protein-coding genes have been predicted with
transcript lengths of 2776 bp and coding lengths of
1172 bp (Wu et al. 2013). This indicates that, on
average, there are 4.7 exons per gene. In addition,
J. Wu (&)
S. Zhang
X. Li Illumina RNA-Seq sequences have provided
State Key Laboratory of Crop Genetics and strong support for these predictions. Moreover,
Germplasm Enhancement, Centre of Pear 297 miRNAs, 1148 tRNAs, 697 rRNAs, and 395
Engineering Technology Research, Nanjing
Agricultural University, Nanjing 210095, China
snRNAs have been identified. Furthermore, 53.1%
e-mail: [email protected] (271.9 Mb) of the length of the assembled genome

© Springer Nature Switzerland AG 2019 133

S. S. Korban (ed.), The Pear Genome, Compendium of Plant Genomes,
https://doi.org/10.1007/978-3-030-11048-2_7
134 J. Wu et al.

Fig. 7.1 A schematic diagram demonstrating the distri- ranges from 0 to 0.65 as illustrated by blue-colored lines.
bution of basic genomic elements in the Asian pear (D) Retrotransposon element (RT TE) density, wherein
genome. (A) Chromosome karyotype, wherein colored the rate of sites within the RT TE regions ranges from 0 to
segments are presented in accordance with the ancestor of 1, and this is illustrated by purple-colored lines. (E) SNP
Rosacea. (B) Gene density, wherein the rate of sites density, wherein the rate of SNPs per 100 kb ranges from
within a gene region per 100 kbp ranges from a minimum 0 to 0.03, and this is illustrated by green-colored lines.
of 0 to a maximum of 0.8 as illustrated by red-colored (F) GC content, wherein the rate of GC content ranges
lines. (C) DNA transposon element (TE) density, wherein from 0.25 to 0.45, and this is illustrated by black-colored.
the rate of sites within the DNA TE region per 100 kb This figure is taken from Wu et al. (2013)

is observed to consist of repetitive sequences. to genome size differences between the apple and
A high long terminal repeat (LTR) expansion rate the pear. Further, genes associated with disease
suggests that the pear genome is in continuous resistance, stone cells, sugar, and volatiles, as well
expansion. Compared to the apple genome, it is as self-incompatibility have been identified in the
proposed that the presence of large numbers of genome of the Asian pear.
repeat sequences in the pear is mainly contributing
7 The Genome of Pear 135

Table 7.1 Comparisons ‘Bartlett’ ‘Dangshansuli’

of the Asian pear genome
of ‘Dangshansuli’ and the Contigs
European pear genome of Number of contigs 182,196 25,312
‘Bartlett’
Total size of contigs (Mb) 507.7 501.3
N50 contig length (Kb) 6.6 35.7
Longest contig (Mb) 0.1 0.3
Scaffolds
Number of scaffolds 142,083 2103
Total size of scaffolds (bp) 577 512
N50 scaffold length (Kb) 88.1 540.8
Longest scaffold (Mb) 1.2 4.1
Anchored size to the chromosome (Mb) 171.3 386.7
Anchored rate to the chromosome (%) 29.7 75.5

density in the European pear genome is found to be

7.2 Genome of the European Pear similar to that detected in the Asian pear. Moreover,
a total of 60,820 and 51,425 SNPs have been iden-
The European pear, Pyrus communis, has a dif- tified and located within 1000 bases upstream and
ferent phenotype and fruit quality characteristics downstream, respectively, of a predicted gene.
than those of the Asian pear, including fruit Comparative proteome analysis of 13 different
shape, fruit taste, lignin content, and aroma, plant species has revealed that the European pear
among others. Therefore, sequencing and anno- has a close relationship with genomes of the
tating of the genome of the European pear are as Asian pear and apple and that higher numbers of
equally important as that of the Asian pear. protein clusters are shared between European and
Employing next-generation sequencing tech- Asian pears (Fig. 7.2). It is observed that the
nology (Roche 454), a draft genome sequence of the European pear genome has a total of 199.4 Mb
European pear cv. Bartlett has been recently of repeated elements; moreover, most common
assembled and released (Chagné et al. 2014). A total repeated elements are LTRs. These results are
of 142,083 scaffolds have been assembled, corre- consistent with those observed in the Asian pear
sponding to 577.3 MB with an average of 11.4 genome (Wu et al. 2013). In addition, a total of
genome coverage and representing 96.2% of the 41 predicted genes are identified as members of
expected 600 MB of the European pear genome the expansin gene family. Furthermore, it is
(Table 7.1). Furthermore, a genetic map consisting observed that there are more similarities between
of a total of 2279 single-nucleotide polymorphic apple and pear orthologs than that between
(SNP) markers, including 1391 and 888 apple and homologues of the same species, thereby con-
pear SNPs, respectively, is constructed to anchor firming that speciation must have occurred fol-
171.3 Mb of the assembled genome. Using a com- lowing the genome duplication event (Chagné
bined ab initio prediction and homology search et al. 2014).
approach, a total of 43,419 putative genes are iden- It is important to point out that current efforts
tified (Chagné et al. 2014). The number of predicted are underway in resequencing the European pear
genes is higher than those of most other plant spe- genome which will yield a higher-quality
cies, but it is similar to that identified in the Asian sequence draft of this genome. Thereby, it is
pear. This may be expected due to whole-genome expected that comparative findings between
duplication of members of Maloideae (Velasco et al. Asian and European pear genomes are yet to be
2010). In addition, the average predicted coding fully delineated and finalized.
region length (1209 bp), exon length, and gene
136 J. Wu et al.

Fig. 7.2 A phylogenetic tree of six rosids, four malvids, bottom of the figure corresponds to nucleotide substitu-
and three asterids constructed using 83 euKaryote tions per site. The high bootstrap values strongly support
Orthologous Genes (KOGs). Bootstrap values are listed that species in Rosaceae cluster together to the exclusion
along each of the branches. Nodes represent speciation of any other and that the separation event of the European
events, and branch lengths represent degrees of evolu- pear from the Chinese pear must have occurred following
tionary changes over time. The unit for the scale bar at the apple speciation (Chagné et al. 2014)

from the apple. Furthermore, investigation of the

7.3 Evolution of Pear Species: expansin gene family has also suggested that
Duplication Events divergence event of the European pear from the
and Chromosome Evolution Chinese pear must have taken place following
apple speciation (Chagné et al. 2014).
Similar to findings in apple, the pear genome has Substitutions per synonymous site (Ks) values
undergone two whole-genome duplication of 16,335 paralogous gene pairs suggest that the
(WGD) events, based on the estimation of four- recent WGD event in pear must have occurred at
fold degenerate site transversion (4dTv) values of 30–45 million years ago (Mya), and have also
13,372 pairs of paralogous genes (Fig. 7.3a). supported that a paleohexaploidization event
Meanwhile, its distribution supports the fact that must have also occurred in an ancient WGD that
this recent WGD event must have occurred first took place *140 Mya (Fawcett et al. 2009). As
and then followed by the divergence of the pear it is known, the pear and apple share the same
7 The Genome of Pear 137

Fig. 7.3 Summary of the duplication events and chro- pear, apple, and strawberry. b The evolutionary scenario
mosome evolution in pear. a The distribution of fourfold of nine chromosomes of the Rosaceae ancestor (Wu et al.
degenerate site (4dTv) distances of duplicate gene pairs in 2013)

numbers of chromosomes, as well as similar families must have diverged, as these must have
chromosome structures. It is proposed that both underwent multiple duplication events resulting
apple and pear must have been derived from a in expansion of the numbers of family members.
recent WGD of nine chromosomes of a Rosaceae Identification of a gene family can provide
ancestor, while triplication of seven ancestral abundant knowledge of gene functions, expan-
chromosomes of eudicots may have undergone sion ways, and expression patterns, thereby
additional rearrangements, thereby yielding nine providing a solid foundation for pursuing
ancestral chromosomes of the Rosaceae research studies related to gene function.
(Fig. 7.3b) (Wu et al. 2013). To date, with the release of pear genome
sequences, many gene families have been iden-
tified and explored at the whole-genome level.
7.4 Gene Families: Identification Particularly with large gene families, such tran-
and Functional Divergence scription factors as MYB, ERF, and MADS-box
families, among others, have been extensively
A gene family is a set of several similar genes explored, including identification, evolution, and
derived from a single ancestor. This family is function prediction (Li et al. 2016, 2018; Wang
formed following duplication of such a single et al. 2017). In addition, structural genes such as
original ancestral gene, and members of this SWEET transporters (Li et al. 2017), F-box genes
family generally have similar biochemical func- (Wang et al. 2016), B-box genes (Cao et al.
tions. Based on conserved domains of proteins, 2017), hexokinase encoding (HK) genes (Yu
predicted genes can be grouped into many dif- et al. 2017), and hydroxycinnamoyl transferase
ferent families of a genome, with each family encoding (HCT) genes (Ma et al. 2017) have also
possessing similar functions. In large gene fam- been globally identified in pear. Further func-
ilies, gene function among members of such tional verification has been carried out based on
138 J. Wu et al.

these gene function predictions. For example, Meyerozyma guilliermondii inhibits natural decay
genes MYB169 and MYB114, which were iden- of stored pear fruit and induces resistance to blue
tified from the MYB gene family and predicted mold decay caused by Penicillium expansum,
as regulators controlling lignin synthesis and expression of several defense-related genes, such
anthocyanin biosynthesis, respectively, have those coding for PAL, POD, and GLU have been
been verified in pear (Xue et al. 2019; Yao et al. found to be significantly modified following
2017). transcriptome analysis of fruit treated with M.
guilliermondii versus untreated pear fruit (Yan
et al. 2018). Recently, Li et al. (2019b) have per-
7.5 Multiple OMICS: Identifying formed a comparative transcriptomic analysis
Genes Related to Important revealing a distinctly different pattern of variation
Traits between expression and sequence diversity, and
identifying candidate selected genes associated
Transcriptomics, proteomics, and metabolomics with important fruit quality traits during domesti-
provide wide overviews of plant traits at the cation and improvement in pear (Li et al. 2019b).
mRNA, protein, and metabolite levels, respec- Integrating proteomics with transcriptomics is
tively (Palma et al. 2011). With the release of the a powerful approach for exploring functional
pear genome sequence, various OMICS studies, correlations between phenotype and genotype,
particularly for comparative OMICS analyses, and in establishing regulation networks (Palma
have been carried out to investigate biological et al. 2011). Studies have been completed using
phenomena in pear. Moreover, joint analysis of developing fruits of several fruit crops, including
multiple OMICS will aid in dissecting complex strawberry (Bianco et al. 2009), grape (Giribaldi
traits and thus will attract more attention with the et al. 2010), and papaya (Nogueira et al. 2012).
expansion of genomics data in the future. Using proteomics and transcriptomics, Li et al.
Transcriptome analysis is a powerful tool in (2015) have identified a total of 35 important
assessing expression levels of genes in specific differentially expressed proteins related to fruit
tissues and at various stages of development. Thus, quality in pear, including three genes related to
it is feasible to predict gene function(s) and sugar synthesis, a single gene related to aroma
investigate regulation mechanism(s). In general, a formation, and 16 genes related to stone cells’
transcriptome can be classified as either a reference content (Li et al. 2015).
or a non-reference (de novo assembly) transcrip- Metabolomics is yet another novel approach,
tome. For example, transcriptomes of endodor- building on genomics and proteomics, which
mant and ecodormant flower buds of Japanese pear performs quantitative analysis for all metabolites
have been analyzed to identify key genes involved in an organism, and traces correlations between
in regulation pathways during the release of metabolites and a phenotype. In pear, metabo-
endodormancy (Bai et al. 2013). In Chinese pear, lomics studies have been carried out to character-
comparative transcriptome analyses of pre- and ize complex phenotypes. Recently, comparative
post-ripening fruits have been carried out to iden- metabolomics analysis of flower buds during
tify candidate genes associated with fruit ripening endodormancy has identified and characterized
(Hao et al. 2018; Huang et al. 2014). As fruit aroma metabolic changes induced by chilling tempera-
is an important component of fruit quality in pears, tures, as well as simulated mild winter and/or cli-
candidate genes highly related to aroma biosyn- mate change scenarios during thermal fluctuation
thesis during fruit ripening have been identified in Japanese pear (Horikoshi et al. 2018). A total of
using unripe fruit of poor aroma and ripe fruit with 91 metabolites have been detected and classified
strong aroma of ‘Nanguoli’ (Pyrus ussuriensis) as into eight groups, including organic acids, fatty
tissues for transcriptome analysis (Wei et al. 2016). acids and sterols, amino acids, amino acid
Furthermore, as pear fruit, storability is an derivatives, phenol lipids, phenylpropanoids,
important postharvest trait, and as the yeast sugars and polyols, and other compounds. This
7 The Genome of Pear 139

study has contributed new knowledge on the bio- detected in Asian pears, while 5.35 Mb of the
logical mechanism of dormancy during tempera- genome sequence, containing 248 putative genes,
ture changes and has elucidated metabolic changes was identified as selective sweeps in European
during mild winters and future climate change pears. Notably, there was only 515 kbp of
scenarios (Horikoshi et al. 2018). overlap for regions with selective sweep signa-
tures between Asian and European pears, indi-
cating that different genome regions must have
7.6 Whole-Genome Resequencing undergone selection in Asian and European pear
genomes during domestication. Genes associated
Resequencing of a genome is an approach that with fruit size, sugar, organic acid, stone cell, and
aids in determining nucleotide order of a given volatile compounds were identified from these
DNA fragment for different individuals or pop- regions with selective sweep signatures.
ulations based on a reference genome and com- Therefore, it is possible to conduct additional
parative analysis. Following the alignment of analyses that will further reveal new knowledge
sequences of individuals or populations, large regarding other biological issues based on
numbers of SNPs, insertion/deletions (InDels), genomic data released from resequencing various
structure variations (SVs), and copy-number genotypes of pear.
variations (CNVs) could be identified from dif-
ferent individuals and populations and used to
perform downstream analyses. For example, 7.7 SNP Arrays
resequencing of wild plants and cultivated types
will allow for comparative analysis to reveal the A SNP array is a DNA microarray used to detect
origin of a species and its domestication during polymorphisms within a population. In plants,
evolution, as well as provide valuable genetic SNP arrays are useful tools for studying slight
resources and important references for plant variations among whole genomes, as well as for
breeding programs. conducting genome-wide association studies
Recently, genome resequencing of 113 pear (GWAS). Breeding efforts for a number of plant
accessions from worldwide collections, repre- species have been revolutionized following the
senting four different populations, including emergence of SNP arrays.
Asian wild, Asian cultivated, European wild, and With the release of the pear genome
European cultivated accessions, was performed sequence, along with the availability of large
(Wu et al. 2018). A total of 18.3 Mb SNPs were numbers of SNP data, designing high-density
identified in this study, and a weak domestication SNP arrays is now possible for pear. However,
has been observed based on analyses of popula- as of yet, the development of pear SNP array
tion structure, diversity, and linkage disequilib- has lag behind other plants, such as apple;
rium (LD) (Fig. 7.4). This comprehensive study therefore, efforts are underway to develop such
also clarified the process of divergence of Asian an array. Due to its efficiency, flexibility, high
from European pears, as well as dissemination throughput, and low cost, such a SNP array will
and independent domestication of Asian and likely be an important reference tool for GWAS,
European pears. The divergence time of Asian and highly useful in further germplasm
and European pears, 3.3–6.6 Mya, was first enhancement and breeding efforts in pear.
reported in this study (Fig. 7.5). Furthermore, Recently, an integrated 200 K SNP genotyping
evidence for rapid evolution and balance selec- array has been developed for pear by Dr. Jun
tion for S-RNase genes contributing to the Wu’s group at Nanjing Agricultural University,
maintenance of self-incompatibility in pear has and used for genetic mapping construction,
been found. Meanwhile, selective sweep signa- genome assembly improvement, and GWAS in
tures for a total of 9.29 Mb of the genome pear (Li et al. 2019a). Additionally, a 70 k
sequence, containing 857 putative genes, were Axiom® array has been developed by Dr. Sara
140 J. Wu et al.

Fig. 7.4 Phylogenetic tree, PCA, and LD analysis of 113 codes correspond to abbreviated names of pear species.
cultivated and wild pear accessions based on b PCA plots of the first two eigenvectors for all 113 pear
whole-genome SNPs. a Phylogenetic tree and population accessions. c LD decay determined by the correlation of
structure (K = 5) for all 113 pear accessions inferred from allele frequencies (r2) against distance (kbp) among
whole-genome SNPs, with apple (Malus  domestica) polymorphic SNP sites in different pear groups, including
used as an outgroup. Each color corresponds to a single cultivated Asian (red), cultivated European (light blue),
population as noted. In population structure, each acces- wild Asian (blue), and wild European (green) (Wu et al.
sion is represented by a bar. Pyw and Pyc correspond to 2018)
wild and cultivated accessions, respectively, while other
7 The Genome of Pear 141

Fig. 7.5 Genetic

relationships and divergence
times of pear species.
a Genetic relationships of
wild and cultivated pear
species. b Divergence time of
Asian and European pears. A.
Vitis vinifera; B.
Malus  domestica; C. Pyrus
communis; D.
Pyrus  bretschneideri; E.
Prunus persica; F. Fragaria
vesca; G. Populus
trichocarpa; H. Carica
papaya; and I. Arabidopsis
thaliana. This figure is taken
from Wu et al. (2018)

Montanari and co-workers, and has been determine whether or not any variant is associ-
presented at the RGC9, Nanjing, China, June ated with a particular trait. As mentioned above,
26–30, 2018 (http://rgc9.org/ep3_3.php). a SNP is the most popular variant used for
GWAS.
Thus far, GWAS has been extensively carried
7.8 A Genome-Wide Association out in apple. For example, Mcclure et al. (2018)
Study (GWAS) have conducted a GWAS using 172 apple
accessions, linking approximately 55,000 SNPs
A genome-wide association study, GWAS, is an with 10 phenotypes collected over two years, and
observational study of a genome-wide set of have identified loci associated with skin color,
genetic variants in different individuals to harvest date, firmness, and apple scab resistance.
142 J. Wu et al.

In another study, an Axiom® Apple 480 K SNP Horikoshi HM, Sekozawa Y, Kobayashi M, Saito K,
array along with a total of 1168 different apple Kusano M, Sugaya S (2018) Metabolomics analysis of
‘Housui’ Japanese pear flower buds during endodor-
genotypes has been used to investigate candidate mancy reveals metabolic suppression by thermal
genes responsible for flowering and ripening fluctuation. Plant Physiol Biochem 126:134–141
periods in apple (Urrestarazu et al. 2017). Huang G, Li T, Li X, Tan D, Jiang Z, Wei Y, Li J,
Recently, a limited GWAS has been conducted Wang A (2014) Comparative transcriptome analysis
of climacteric fruit of Chinese pear (Pyrus ussuriensis)
in pear wherein 214 pear accessions have been reveals new insights into fruit ripening. PLoS ONE 9:
used in marker-trait associations for several fruit e107562
color, fruit shape, and fruit quality traits using Kumar S, Kirk C, Deng C, Wiedow C, Knaebel M,
genotyping by sequencing (GBS) (Kumar et al. Brewer L (2017) Genotyping-by-sequencing of pear
(Pyrus spp.) accessions unravels novel patterns of
2017). However, with the release of pear genome genetic diversity and selection footprints. Hort Res
sequences along with resequencing data, a large 4:17015
amount of SNP data sets have become available; Li J, Qin M, Qiao X, Cheng Y, Li X, Zhang H, Wu J
thus, GWAS efforts in pear will be undertaken in (2017) A new insight into the evolution and functional
divergence of SWEET transporters in Chinese white
the near future. pear (Pyrus bretschneideri). Plant Cell Physiol
58:839–850
Li JM, Huang XS, Li LT, Zheng DM, Xue C, Zhang SL,
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 Repetitive Sequences in Pear
8
Shuang Jiang and Yuanwen Teng

Abstract been fully understood. In this chapter,

Repetitive sequences account for a large advances of transposable elements in Pyrus
proportion of the pear genome, suggesting are presented and discussed.
that they play critical roles in the evolution of
Pyrus. One form of repetitive sequences is
transposable elements, which have been pre-
dominantly investigated thus far, including 8.1 Introduction
DNA transposons and retrotransposons.
Approximately 22.5% of the ‘Bartlett’ gen- Repetitive sequences are highly diverse in their
ome (P. communis) and 42.4% of the ‘Suli’ organization, abundance, chromosome localiza-
genome (P. pyrifolia) are reported to be Long tion, and variations in sequences within and
Terminal Repeat (LTR)-retrotransposons between chromosomes, and account for a high
(LTR-RTs). Thus, investigation of transpos- percent of a plant genome. Among diverse
able elements will offer new insights of the groups of structural and functional repetitive
evolutionary history of Pyrus. LTR-RTs sequences, transposable elements have been
exhibit high heterogeneity and their copy widely identified and investigated (Kumar and
numbers vary with the Pyrus species. The Bennetzen 1999; Wicker et al. 2007) (Fig. 8.1).
dynamics of LTR-RTs are an important source Based on mode of transposition, there are two
of genetic variation in Pyrus species. As of groups of transposable elements. One group,
now, the function and development mecha- retrotransposons, transposes via RNA using a
nism of transposable elements have not yet ‘copy and paste’ mechanism; whereas, the sec-
ond group, transposons, transposes via DNA
using a ‘cut and paste’ mechanism (Wicker et al.
S. Jiang
Y. Teng (&) 2007).
The State Agricultural Ministry Key Laboratory of Long Terminal Repeat (LTR)-retrotransposon
Horticultural Plant Growth, Development and
Quality Improvement, Department of Horticulture,
(LTR-RT) is one form of retrotransposons
Zhejiang University, 310058 Hangzhou, Zhejiang, (Fig. 8.2), as LTR-RTs are flanked by two LTRs,
China and undergo replicative transposition. These
e-mail: [email protected] elements have been found in all plant species
S. Jiang investigated thus far (SanMiguel et al. 1996;
Shanghai Key Laboratory of Protected Horticultural Sabot and Schulman 2006; Wicker et al. 2007).
Technology, Forest and Fruit Tree Research
Institute, Shanghai Academy of Agricultural
In higher plants, the transposon of LTR-RTs may
Sciences, Shanghai 201403, China increase their copy numbers, and increase

© Springer Nature Switzerland AG 2019 145

S. S. Korban (ed.), The Pear Genome, Compendium of Plant Genomes,
https://doi.org/10.1007/978-3-030-11048-2_8
146 S. Jiang and Y. Teng

gene expression levels, or by driving genomic

rearrangements (Feschotte et al. 2002; Shapiro
2005). Kobayashi et al. (2004) have reported that
a retrotransposon inserted into a myb-related
gene is associated with pigmentation loss in
grape. While Butelli et al. (2012) have found that
insertion of a retrotransposon upstream of an
Fig. 8.1 Insertion of transposable elements
anthocyanin biosynthesis-related gene results in
cold-dependent fruit color development in blood
genome size (SanMiguel et al. 1998; Peterson orange. Furthermore, it is proposed that envi-
et al. 2002; Havecker et al. 2004). For example, ronmental stress and demethylation activate
more than 50% of the maize and wheat genomes retrotransposons and induce duplication events in
are made-up of retrotransposons (Meyers et al. a genome (Hirochika et al. 2000; De Felice et al.
2001; Daron et al. 2014). In a wild rice species, 2009; Tsukahara et al. 2009).
Oryza australiensis, it is reported that a rapid It has been reported that retrotransposons
twofold increase in genome size is likely attrib- display extreme sequence diversity, and more
uted to transposition of retrotransposons that than thousands of retrotransposon families in
must have occurred over the last 3 million years plants have been isolated (Havecker et al. 2004;
(Piegu et al. 2006). This has suggested that Wicker et al. 2007). An intact retrotransposon is
retrotransposons may play an important role in composed of two nearly LTR sequences flanked
expansion of a genome. Interestingly, retro- by target site duplications of usually 4–6 bp in
transposons isolated from plants appear to be length (Kumar and Bennetzen 1999). The inter-
rather young in age (El Baidouri and Panaud nal domain usually consists of two open reading
2013). Therefore, removal of retrotransposons frames (ORFs) required for transposition. In
must also occur in a plant genome. For example, particular, this internal domain contains a
the rice genome has been reported to have lost a primer-binding site (PBS), a polypurine tract
large number of retrotransposons, which, in turn, (PPT), and two functional genes, gag and pol.
has resulted in rapid reduction of genome size The pol gene encodes three enzymatic regions of
(Ma et al. 2004). a protease, reverse transcriptase, and integrase,
Retrotransposons act by inserting themselves while the gag gene encodes structural proteins
either within or near transcriptionally active involved in the maturation and packaging of
regions of a chromosome, thereby resulting retrotransposon RNA. Some other conserved
either in mutations by disrupting genes, altering sequence motifs of the primer-binding site and

Fig. 8.2 Classification of transposable elements

8 Repetitive Sequences in Pear 147

the PPT are also essential for retrotransposon retrotransposons is also found in other genomes of
replication. LTR-retrotransposons can be subdi- members of the Rosaceae family, such as that of
vided into the Ty1-copia and the Ty3-gypsy Malus (37.6%) and Prunus (18.6%) (Velasco
groups (Wicker et al. 2007). Within the pol gene, et al. 2010; Verde et al. 2013). Furthermore,
the order of reverse transcriptase in the Ty1- retrotransposons of the Pyrus genome have com-
copia group is in front of integrase, while that in plex structures (Yin et al. 2014), and some are
the Ty3-gypsy group, it is the integrase that is in reported to be inserted in many loci in genomes of
front. cultivated Pyrus species, but only in a few loci in
The pear, Pyrus species, is proposed to have genomes of wild Pyrus species (Jiang et al. 2015).
originated in the mountainous regions of western Frequent recombination events followed by
and southwestern China (Rubstov 1944). Pears transposition of retrotransposons may have played
are geographically classified into occidental and critical roles in the evolution of Pyrus genomes.
oriental pear groups (Bailey 1917). Major species More than one-thousand LTR-RTs have been
of oriental pears are native to China (Teng and isolated in the Pyrus genome, and it has been
Tanabe 2004). The oriental pear group consists found that retrotransposons are of high hetero-
of wild pea pears and cultivated species with geneity, thus contributing to difficulties in
large fruit, thus demonstrating their complex LTR-RT classification (Yin et al. 2014; Jiang
evolutionary history (Zheng et al. 2014). Retro- et al. 2016a). Two methods have been used to
transposons have been identified in pears and classify distinct families of LTR-RTs. The first
represent a large proportion (43%) of the Pyrus method is based on coverage and sequence
genome; therefore, they will provide new identities, wherein similar LTR-RTs made-up a
insights into the evolutionary history of pears single family (Du et al. 2010). Whereas, in the
(Wu et al. 2013). second method, families of LTR-RTs are classi-
fied based on mapping of these elements to an
existing database, such as Repbase. Using this
8.2 Retrotransposons approach, a total of 148 families have been
and Transposons in Two Pyrus identified in the assembled ‘Suli’ genome (Yin
Genomes et al. 2015). Recently, some new LTR-RT fam-
ilies, such as TGTT and AACA families, have
Recently, whole genomes of the Chinese white been found in the ‘Suli’ genome, containing the
pear ‘Dangshansuli’ or ‘Suli’ (Wu et al. 2013) and palindromic dinucleotide 5′-‘TG’-‘TT’-3′ and the
the European pear ‘Bartlett’ (Chagne et al. 2014) 5′-‘AA’-‘CA’-3′ motif at the start and at the end
were sequenced. The assembled ‘Suli’ genome of an LTR sequence (Yin et al. 2017).
consists of 2103 scaffolds with an N50 of Transposons move within a genome through
540.8 kb, totaling 512.0 Mb with 194 coverage. a ‘cut and paste’ strategy, and are characterized
The ‘Bartlett’ genome is not as well assembled, by their terminal inverted repeats (TIRs) of
but it consists of 142,083 scaffolds with an N50 of variable lengths. Currently, transposons are pre-
6569 bp, totaling 577.0 Mb with 11.4 coverage. dicted through a homology search. In Pyrus,
The assembled scaffolds have revealed that much 7.77% of the ‘Suli’ genome and 8.04% of the
of the Pyrus genome is retrotransposon-derived ‘Bartlett’ genome are reported to be transposons
(Wu et al. 2013). The LTR-RTs, long-interspersed (Wu et al. 2013; Chagne et al. 2014). The
elements (LINEs), and short-interspersed elements PIF-Harbinger is the largest family in these two
(SINEs) are classified into the retrotransposon pear genomes. This family carries terminal
group. However, LINEs and SINEs only account inverted repeats, and produces a 3 bp target site
for a little proportion of the Pyrus genome. A total duplication upon insertion. These TEs contain
of 42.4% of the ‘Suli’ genome and 22.5% of two ORFs, one encoding a DNA binding protein,
the ‘Bartlett’ genome are reported to be LTR-RTs while the other encoding a DDE/DDD trans-
(Table 8.1). This high-copy number of posase. The second largest transposon family in
148 S. Jiang and Y. Teng

Table 8.1 Distribution of % in ‘Suli’ genome % in ‘Bartlett’ genome

LTR-RTs in two Pyrus
genomes Repeated sequences 53.1 34.14
LTR/Ty1-copia 16.88 7.66
LTR/Ty3-gypsy 25.48 14.79
DNA transposons 12.12 7.28

Pyrus is hAT-Ac, which has been firstly reported problem may be resolved by increasing
from Zea mays as an activator or an Ac element. sequencing depth. Overall, based on
Common features of hAT transposons include whole-genome resequencing, a total of 14
sizes of 2.5–5 kb with short terminal inverted high-copy number LTR-RT families have been
repeats along with short flanking target site identified in Asian pears (Table 8.2) (Jiang et al.
duplications generated during the transposition unpublished data). Of these families, nine are
process. The PIF–Harbinger and hAT-Ac fami- copia-type and five are gypsy-type retrotrans-
lies account for half of the total size of trans- posons. Interestingly, some of these retrotrans-
posons in the Pyrus genome (Wu et al. 2013; posons have also been found in Malus and
Chagne et al. 2014). The functions of trans- Prunus genomes.
posons and their effects on trait performance are
not yet well-understood in Pyrus.
8.4 Insertion of Retrotransposons
and Marker Development
8.3 High-Copy Number
Retrotransposon Families LTR sequences of LTR-RT flank coding regions
at the 5′ and 3′ ends (Bergman and Quesneville
As it is difficult to analyze each of the LTR-RT 2007). Therefore, they are deemed well suited for
families, analysis of copy numbers of these developing new molecular markers (Fig. 8.3), as
families has been pursued instead. As it is they are ubiquitously distributed, with abundant
expected, high-copy numbers of LTR-RT fami- copy numbers, along with their insertion poly-
lies are more representative than low-copy morphisms (Flavell et al. 1992). Thus far, four
numbers of LTR-RT families. The BLASTN types of retrotransposon-based markers have
has been used to search assemble genome data been reported. For one type, retrotransposon-
based on numbers of LTR-RTs in order to based insertion polymorphism (RBIP) markers
identify which families yield high-copy numbers amplify the junction of LTR and flanking gen-
of LTR-RTs. Using this approach, a total of ten omes (Kalendar et al. 2011). For another type,
high-copy number of LTR-RT families have inter-retrotransposon amplified polymorphism
been identified in the ‘Suli’ genome (Jiang et al. (IRAP) markers of specific length have
2015). However, it is important to point out that been developed by amplifying the intermediate
this finding is influenced by numbers of incom- section of two nearby LTRs (Kalendar and Schul-
plete LTR-RTs, as well as by the method of man 2006). For a third type, retrotransposon-
genome assembly. Incomplete LTR-RTs are microsatellite amplified polymorphism (REMAP)
often lost in such a prediction; moreover, in the markers amplify specific lengths of LTRs to
current method of genome assembly, overlapping develop simple sequence repeats (SSRs) (Kalendar
reads are often ignored during the process of and Schulman 2006). Finally, sequence-specific
assembly. This would lead to recovery of some amplification polymorphism (SSAP) markers can
high-copy numbers of LTR-RTs, wherein highly be developed, as they are similar to amplified
similar members are assembled into either a fragment length polymorphisms (AFLPs).
single or a few sequences. Obviously, this Although both SSAP and AFLP makers
8 Repetitive Sequences in Pear 149

Table 8.2 High-copy ID Name in Repbase database

number of
LTR-retrotransposon 1 Gypsy-4_PX
families in Pyrus 2 Copia-100_Mad
3 Copia-24_PX
4 Copia-2_PX
5 Copia-90_Mad
6 Gypsy-3_PX
7 Copia-106_Mad
8 Copia-16_PX
9 Gypsy-46_Mad
10 Copia-13_PX
11 Gypsy-2_PX
12 Gypsy-5_PX
13 Copia-49_Mad
14 Copia-61_Mad

Fig. 8.3 Marker

development based on
retrotransposons

correspond to restriction size variations of a whole genome. Furthermore, sequence homology analysis
genome, SSAP markers also identify polymor- by BLASTN could not identify all retrotrans-
phisms produced by retrotransposon insertions posons, particularly those retrotransposons that are
(Waugh et al. 1997). These various types of specific to pear. Therefore, Jing et al. (2015) have
markers have already been developed in a variety developed 196 RBIP primers based on whole
of plant species, and are widely used in studies of genome sequence data of ‘Suli’. They have
genetic diversity, phylogeny, genetic mapping, and developed 24 pairs of primers, of the Ppcr1 sub-
cultivar identification. family of copia retrotransposons, Ppcr1, and have
Kim et al. (2012) have isolated retrotransposons used them to investigate genetic diversity among
from a bacterial artificial chromosome (BAC) li- 110 Pyrus accessions, including oriental and occi-
brary of Pyrus. Subsequently, these retrotrans- dental pears. The Ppcr1 is found to be inserted in
posons have been used to develop 22 RBIP many loci in genomes of cultivated Pyrus species,
makers, based on the copia-like retrotransposon but only in a few loci in genomes of wild Pyrus
Ppcrt4, and these markers have allowed for the species (Jiang et al. 2015). In another study, eight
differentiation of 61 of 64 Japanese pear cultivars polymorphic IRAP markers have been developed,
(Kim et al. 2012). However, a BAC library is too and a total of 76 alleles are amplified in 62 pear
limited in identifying retrotransposons in a pear cultivars (Sun et al. 2015). Overall, it is reported
150 S. Jiang and Y. Teng

that both RBIP and IRAP markers have provided of LTR-RTs should be quite informative.
only a few information sites. Therefore, SSAP (3) Construction of a mutant library of the Pyrus
markers have been developed, as they do overcome genome using LTR-RTs. As DNA transposons
this observed limitation. Jiang et al. (2016b) have are widely used in construction of mutant
developed 12 SSAP primers in Pyrus. Following a libraries; e.g., the Ac/Ds transposon tagging
population structure analysis, nearly all Asian pear method, use of LTR-RTs, which have transpo-
species and cultivar groups have been found to sition functions, could also serve as a valuable
undergo hybridization and must have originated tool in constructing mutant libraries of the Pyrus
from five primitive genepools (Jiang et al. 2016b). genome for pursuing functional gene analysis
Therefore, LTR-RT-based markers can serve as studies.
important tools for pursuing evolutionary analysis
studies of Pyrus.
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 Regulatory Sequences of Pear
9
Yongping Cai, Muhammad Abdullah and Xi Cheng

Abstract DNA sequences present in either coding or

Pear (Pyrus) is one of the leading and oldest non-coding regions of the genome. Cis-acting
cultivated fruit trees of temperate regions that is elements can also be covered by epigenetic
grown around the world. Compared to other information. On the other hand, trans-acting
Rosaceae species, pear research studies have factors are transcription factors (TFs) or other
lagged behind other members of the Rosaceae, DNA-binding proteins that bind to specific
such as strawberry, peach, and apple. However, sequences in cis-acting elements to either
the recent completion of whole-genome increase or suppress transcription of a given
sequencing projects for pear offers ideal oppor- gene. In this instance, chromatin remodeling
tunities for pursuing regulatory sequence involves dynamic modification of histones or
research studies. A regulatory sequence is a the DNA sequence itself to allow access of
segment of nucleic acids capable of either accessible regions within the DNA for trans-
increasing or decreasing; i.e., regulation of acting elements to regulate transcription. Fur-
expression of a structural gene. Furthermore, thermore, TFs may influence transcription of
the regulation of gene expression can be under- multiple genes, and they can function either in a
taken in different ways, such as during tran- complex manner or combinatorially to bind cis-
scription, mRNA processing, translation, and regulatory components at multiple transcription
via protein stability. It is commonly proposed factor binding sites to regulate a unique trait in a
that the regulation of gene expression occurs controlled pattern of gene expression.
primarily at the transcriptional level. A plant
transcriptional mechanism consists of two com-
plimentary regulatory modules, cis-acting and 9.1 Introduction
trans-acting elements. Cis-acting elements are
The genus Pyrus belongs to the family Rosaceae.
It is characterized by a wide genetic diversity
Y. Cai (&)
M. Abdullah
X. Cheng with several species and more than 4000 culti-
School of Life Science, Anhui Agricultural vars that can be divided into two major groups,
University, No. 130, Changjiang West Road, the occidental (European) and oriental (Asiatic)
Hefei 230036, China
e-mail: [email protected]; [email protected] pears. The pear is one of the oldest fruit crops
(over 3000 years) grown in the world, and has at
M. Abdullah
e-mail: [email protected] least 22 well-recognized species, including
P.  bretschneideri, P. ussuriensis, P. pyrifolia,
X. Cheng
e-mail: [email protected] P. sinkiangensis, and P. communis. The pear

© Springer Nature Switzerland AG 2019 153

S. S. Korban (ed.), The Pear Genome, Compendium of Plant Genomes,
https://doi.org/10.1007/978-3-030-11048-2_9
154 Y. Cai et al.

genome contains all of the coding and 2009). In general, conserved non-coding
non-coding DNA sequences controlling all sequences also have regulatory regions. Thus,
functions within all cell types of the pear. Pyrus these sequences are often the subject of analysis,
species are functionally diploid (x = 17, such as those of the CAAT box, CCAAT box,
2n = 34), and they are highly heterozygous due A-box, and Z-box, among others.
to self-incompatibility. Although genome sizes It is commonly known that expression of
of all Pyrus species are not yet available, the genes is a tightly regulated process. Specifically,
nuclear content of P. communis (European pear) expression must occur in the correct cell type to
is 1.03 pg/2C (Chagné et al. 2014). It is esti- an appropriate level and at the correct time during
mated that the P. communis genome is approxi- cell differentiation and development in response
mately 577 Mbp per haploid genome to internal and external signals. Failure of the
equivalents, while that of P.  bretschneideri regulation process of gene expression leads to
(Chinese pear) is 527 Mbp (Wu et al. 2013). In serious consequences in genetic disease (Barnes
addition, the total number of genes is estimated 2006). In the post-sequencing genomics era, with
to be approximately 43,000 (https://www. advances in both computational methods and
rosaceae.org/organism/Pyrus/all-species). genome-wide experimental approaches, it is
In general, DNA sequences consist of coding important to study how different regulatory
and non-coding regions. Coding regions consist of sequences and proteins interact to control gene
genes that encode proteins controlling various expression. Such control must occur not only at a
biological processes, as well as ribosomal RNAs single gene locus, but also globally across the
and proteins. Non-coding regions include mainte- genome within complex biological and tran-
nance elements, such as telomeres, centromeres, scriptional programs. Changes in gene transcrip-
and origins of replication controlling DNA repli- tion are mainly controlled by the transcription
cation. Furthermore, these non-coding regions factor protein that binds DNA to DNA and
consist of elements, such as promoters/repressors, modulates the transcriptional apparatus. TFs are
insulators, and regulatory RNAs that regulate the essential for regulating expression of many genes,
spatial and temporal expression of coding genes. and they may play important roles in plant
These latter regulatory sequences are capable of physiological processes, such as development,
either increasing or decreasing expression of biotic stress, abiotic stress, as well as structural
specific genes. Generally, regulation of gene and functional divergence. Many transcriptional
expression occurs at the level of RNA biosynthesis, factors have now been identified. In fact, there is a
and this is accomplished through sequence-specific paucity of data related to the regulation of tran-
binding of proteins or transcription factors. Inter- scriptional factors in the pear genome. TF pro-
estingly, transcriptional factors (TFs) may act as teins that bind to DNA-regulatory sequences,
either activators/repressors or both. Repressors usually localized in the 5-upstream region of
often act by preventing RNA polymerase from target genes, modulate the rate of gene tran-
forming a productive complex with the transcrip- scription. This may result in either increased or
tional initiation (promoter) region, while activators decreased gene transcription, protein synthesis,
facilitate the formation of a productive complex. and subsequent altered cellular function. Many
It is noteworthy to point out that DNA transcriptional factors have now been identified,
sequence motifs (or motifs) aid in predicting and a large proportion of the pear genome appears
epigenomic modifications, thus signifying that to code for these proteins. This is a review of the
TFs play a vital role in regulating the epigenome. regulatory sequences of pear, and will specifically
Regulatory sequences are commonly linked with focus on trans-acting factors and physiological
messenger RNA molecules that control mRNA function of TFs in normal cell development and in
biogenesis or translation (Adcock and Caramori plant physiological processes.
9 Regulatory Sequences of Pear 155

9.2 Transcription Factor Families elegans (3.6%), and D. melanogaster (4.7%)

in Pear genomes. Furthermore, this high ratio of TFs
detected in Arabidopsis is also accompanied by a
Transcription factors are classified into different high diversity of DNA-binding specification
families based on their DNA-binding domains when compared to those found in C. elegans and
(DBDs) (Riechmann et al. 2000). The general D. melanogaster. These collective findings sug-
characteristics of pear transcriptional factors are gest that plant transcriptional regulation may be
similar to those of other plants and eukaryotes. more complex and more diverse than that found
Pear TF families play important roles in tran- in mammalian systems. In fact, many specific
scriptional regulation of different processes, thus transcription factors identified in pear, Ara-
rendering the study of TFs essential for under- bidopsis, and in other plants possess
standing the functions of genes at the molecular DNA-binding domains found only in plants. For
level. example, WRKY, EIL, NAC, AP2-ERF, Dof,
Early on, it has been reported that the GARP, SBP, TCP, LFY, YABBY, TCP, and
Arabidopsis genome contains 1500 transcrip- AB13-VP1 (B3) are plant-specific transcription
tional factors (Riechmann et al. 2000); however, factor families (Cheng et al. 2018). Many plant
subsequent analyses reported that the Arabidop- transcriptional factors belonging to large families
sis genome has in fact 2000 TFs. Thus, the also share similar DNA-binding domain struc-
Arabidopsis TF database has been used as a tures. For example, each of NAC and AP2-ERF
model/basis for identifying and characterizing families contains >100 members. Furthermore,
TFs from pear, and from all other plant species. although MADS-box, bZIP, basic
Currently, the four descriptive databases for helix-loop-helix (bHLH), HB, and MYB are not
Arabidopsis TFs include the following: AGRIS plant-specific, they are also large families in plant
(http://agris-knowledgebase.org/AtTFDB/) (Davu- species. These TF families play critical roles in
luri et al. 2003), PTFDB (http://planttfdb.cbi.pku. plant growth and development, and against
edu.cn/) (Riaño-Pachón et al. 2007), RARTAF environmental changes.
(http://rarge.gsc.riken.jp/rartf/) (Iida et al. 2005),
and DATF (http://atrm.cbi.pku.edu.cn/) (Guo
et al. 2005). Each database has utilized different 9.2.1 MYB TF Family
algorithms, and offers different classification
criteria for TFs as the number of loci of each set MYB proteins correspond to a superfamily of
does not overlap fully (Table 9.1). Plant tran- transcription factors, known to be one of the
scriptional factors are characterized by a large largest transcription factor families in the plant
number of genes and by a variety of transcrip- kingdom. The genome of P.  bretschneideri
tional factor families when compared with those (Chinese white pear) contains 231 non-redundant
of either Caenorhabditis elegans or Drosophila MYB genes, including 35 R1-MYBs, 185 R2R3-
melanogaster. It is important to note that the MYBs, 10 R1R2R3-MYBs, and one 4R-like MYB
number of transcription factor genes is not protein (4R-MYB) (Li et al. 2016). MYB domain
dependent on genome size (Abdullah et al. repeats play a key role in pear and in other plants
2018a; Chen et al. 2018; Su et al. 2017). For regulatory networks controlling plant develop-
example, although the size of Arabidopsis is only ment, metabolism, cell differentiation, plant
*135 Mbp, it contains 2000 TFs, which is a defense mechanisms, and responses to multiple
significantly large number of TFs when com- stresses (Cao et al. 2016b). Members of the MYB
pared to other similar size genomes (Riechmann family are widely distributed in plants, animals,
et al. 2000). In addition, the ratio of number of as well as in other higher eukaryotes. This family
transcription factors to the total number of genes has been first identified in the avian myeloblas-
in the Arabidopsis genome is 5–10%, which is tosis virus as an oncogene, v-MYB, where its role
higher than ratios calculated in human (6.0%), C. is found to regulate the cell cycle (Ito et al.
156 Y. Cai et al.

Table 9.1 A listing of transcription factor families identified in Arabidopsis, their corresponding annotation, along
with the identified number of gene family members, as reported in different Arabidopsis TF databases
TF family InterPro or GenBank Riechmann RARTF AGRIS DATF PlnTFDB
SBP CAB56581 16 17 16 16 16
WRKY S72443 72 72 72 72 72
ARF AAC49751 23 71 22 23 23
AS2 0 0 0 42 0
ARID IPR001606 4 6 7 10 10
AB13/VP1* CAA48241 14 51 11 60 56
ALFIN-like AAA20093 7 47 7 7 7
AP2/EREBP IPR001471 144 93 136 146 146
TUB IPR000007 11 11 10 0 10
AUX/IAA AAC39440 26 21 0 29 27
Bhlh IPR001606 139 157 162 127 134
Bzip 81 56 73 72 71
AS2 0 0 0 42 0
C2C2(Zn)-BBX A56133 33 51 30 37 17
C2C2(Zn)-Dof CAA66600 37 33 36 36 36
C2C2(Zn)-GATA IPR000679 28 37 30 26 29
C2C2(Zn)-YABBY AAD30526 6 5 6 5 6
C2H2(Zn) IPR000822 105 177 211 134 96
C3H-TYPE(Zn) IPR000232 33 47 165 59 67
CCAAT A26771/P13434/Q02526 36 37 35 36 43
CPP(Zn) CAA09028 8 8 8 8 8
E2F/DP O00716/Q64163 8 8 8 8 7
EIL AAC49750 6 6 6 6 6
GARP AAD55941/BAA74528 56 51 55 53 52
GRAS AAB06318 32 32 31 33 33
HB IPR001356 89 97 91 87 91
HMG-box IPR000910 10 11 0 11 11
HSF IPR000232 26 27 21 23 23
JUMONJI T30254 9 13 5 17 17
LFY AAA32826 1 3 1 1 1
MADS IPR002100 82 106 109 104 102
MYB IPR001005/IPR000818 190 189 197 199 209
NAC BAB10725 109 106 94 107 101
Nin-like CAB61243 15 14 0 14 0
PCG 4 35 0 34 0
TCP AAC26786 25 24 26 23 24
Trihelix S39484 28 31 29 26 23
Others 20 215 127 231 375
Totals 1533 1965 1837 1922 1949
9 Regulatory Sequences of Pear 157

2001). Subsequently, the v-MYB gene is found to “MYB-related,” and it is divided into various
consist of three members, namely C-MYB, A- subclasses. The R3-type MYB-related genes
MYB, and B-MYB. Further studies have led to the have evolved from R2R3 to MYB, and they
identification of the first plant MYB gene, C1, control cell morphogenesis. The R1/R2-type
which is involved in anthocyanin biosynthesis in MYB-related genes encode proteins for core
kernels of Zea mays (Paz-Ares et al. 1987). Since components of the central circadian oscillator.
then, numerous members of the MYB gene family Those MYB proteins class with two repeats,
have been identified in genomes of Arabidopsis, R2R3, are widely found in plants, and these must
rice, maize, and soybean, among others, and are have evolved by loss of the R1 repeat from the
reported to be involved in regulating several R1R2R3-MYB gene ancestor, followed by sub-
cellular processes including cell cycle, cell mor- group expansion during plant evolution (Rosin-
phogenesis, and responses to both biotic and ski and Atchley 1998). While 3R encoded genes
abiotic stresses. of R1R2 MYB encoded genes have evolved by
The MYB protein family is comprised of three gaining R1 repeats through an ancient intragenic
domains, including the N-terminal conserved duplication (Jiang et al. 2004). It is the
DNA-binding domain, a central transcriptional R2R3-type MYB protein that has been catego-
activation domain, and a C-terminal domain that rized into 23 subgroups depending upon the
functions in transcriptional repression (Vargova conservation of the DNA-binding domain and
et al. 2011). The C-terminus is diverse, involved amino acid motifs present at the C-terminal
in modulating protein regulatory activity, and region (Dubos et al. 2010). Based on phyloge-
responsible for versatile regulatory roles of netic analysis, 185 PbMYB genes of
MYBs. The DNA-binding domain is highly P.  bretschneideri have been divided into 317
conserved and contains up to four imperfect subgroups, and these are well supported by
repeats, each of which consists of about 51–53 additional intron/exon structures and conserved
amino acids forming three a–helices. Depending motifs (Li et al. 2016).
on the number of repeats present in the MYB In a detailed study of this MYB class in
domain, the MYB family found in plants, it is revealed that this class of MYB
P.  bretschneideri is generally classified into proteins participates in plant tolerance to several
four subgroups, namely 1R (R1/2, R3-MYB), 2R biotic and abiotic stresses, hormone signaling,
(R2R3-MYB), 3R (R1R2R3-MYB), and 4R phenylpropanoid biosynthesis, secondary meta-
(harboring four R1/R2-like repeats) (Li et al. bolism, cell shape determination, and cell cycle
2016). Although four MYB classes are detected regulation (Table 9.2). Additionally, specific
in plants, it is R2R3-type MYB proteins that are clusters of orthologous and paralogous genes
the most common in plants, including those have also been identified that will facilitate the
found in P.  bretschneideri. In fact, a total of characterization of each subgroup in the R2R3-
185 R2R3-MYBs is detected in MYB gene family of pears (Table 9.2). Specifi-
P.  bretschneideri (Li et al. 2016; Cao et al. cally, the R2R3-MYB gene family is involved in
2016b), while the 4R-MYB class is the smallest both positive and negative regulation of many
and harbors four R1/R2-like repeats. In several stress-responsive pathways. So far, large num-
plant genomes, a single 4R-MYB gene is enco- bers of R2R3 MYB proteins have been reported
ded, but little is known about this MYB protein in various plant species, including 177 in sweet
group in plants. orange (Hou et al. 2014), 198 in Arabidopsis
The R1R2R3-type MYB proteins detected in (Yanhui et al. 2006), 183 in rice (Yanhui et al.
higher plants are usually encoded by five genes, 2006), and 209 in foxtail millet (Muthamilarasan
and they play significant roles in control of the et al. 2014). The R2R3-MYB-type subfamily
cell cycle. Yet, another heterogeneous class proteins have been reported to be involved in
includes proteins having either single or partial responses to environmental stresses in several
MYB repeats, collectively designated as plant species, including Arabidopsis, wheat, rice,
158 Y. Cai et al.

Table 9.2 A listing of R2R3-MYB transcription factors in pear, their counterparts in Arabidopsis and a few other
plant species, along with their likely functions
Pyrus  bretschneideri Corresponding counterparts Function(s) Reference(s)
in Arabidopsis thaliana
Pbr016839.1 AtMYB60 Response to Seo and Park (2010),
environmental stress Raffaele et al. (2008)
Pbr002014.1 AtMYB94
Pbr032528.1 AtMYB96
Pbr019262.1 AtMYB30
Pbr011441.1 AtMYB31
Pbr009823.1
Pbr033618.1 AtMYB10 Lignin biosynthesis Zhou et al. (2009), Zhong
et al. (2014)
Pbr041094.1 AtMYB58
AtMYB63
AtMYB72
Pbr028725.1 AtMYB3 Anthocyanin Vimolmangkang et al.
biosynthesis (2013)
Pbr013413.1 AtMYB7
Pbr014381.1 AtMYB32
Pbr038870.1 AtMYB4
Pbr020365.1 MdMYB3
Pbr038869.1
Pbr000876.1
Pbr020726.1
Pbr020733.1
Pbr011095.1 AtMYB15 Involved in cold stress Reyes and Chua (2007),
Agarwal et al. (2006)
Pbr025360.1 AtMYB13
Pbr030553.1 AtMYB14
Pbr031687.1
Pbr024975.1
Pbr031684.1
Pbr019908.1
Pbr028561.1 AtMYB113 Anthocyanin Li et al. (2012), Uematsu
biosynthesis et al. (2014)
Pbr016663.1 AtMYB114
Pbr016661.1 AtMYB75
Pbr042132.1 AtMYB90
PcMYB10
PpMYB10
MdMYB10
MdMYB1
(continued)
9 Regulatory Sequences of Pear 159

Table 9.2 (continued)

Pyrus  bretschneideri Corresponding counterparts Function(s) Reference(s)
in Arabidopsis thaliana
Pbr030848.1 AtMYB11 Flavonol biosynthesis Stracke et al. (2007)
Pbr008630.1 AtMYB12
Pbr011980.1 AtMYB111
Pbr001148.1
Pbr023487.1 AtMYB42 Lignin biosynthesis Zhao and Dixon (2011),
Patzlaff et al. (2003)
Pbr023482.1 AtMYB85
Pbr041889.1 AtMYB43
Pbr024975.1 AtMYB20
Pbr015763.1 AtMYB99
Pbr012750.1 AtMYB40
Pbr012624.1 PtMYB1
Pbr016625.1
Pbr014479.1
Pbr029909.1 AtMYB16 Epidermal cells Jakoby et al. (2008)
Pbr030136.1 AtMYB106
Pbr007283.1
Pbr030135.1
Pbr038434.1
Pbr019293.1
Pbr040860.1 AtMYB39 Trichome development Scoville et al. (2011)
Pbr031409.1 AtMYB9
Pbr013860.1 AtMYB107
Pbr021178.1 AtMYB74 Response to biotic Li et al. (2009), Cominelli
stress et al. (2008)
Pbr021193.1 AtMYB102
Pbr011268.1 AtMYB41
Pbr018024.1 AtMYB49
Pbr000268.1
Pbr001520.1
AtMYB28 Glucosinolate Gonzalez et al. (2009)
biosynthesis
AtMYB29
AtMYB76
AtMYB34
AtMYB51
AtMYB122
AtMYB47
AtMYB95
(continued)
160 Y. Cai et al.

Table 9.2 (continued)

Pyrus  bretschneideri Corresponding counterparts Function(s) Reference(s)
in Arabidopsis thaliana
Pbr026080.1 AtMYB6 Lignin deposition and Liang et al. (2005)
stomatal aperture
Pbr006685.1 AtMYB61
Pbr005982.2 AtMYB50
Pbr039864.1 AtMYB55
Pbr001888.1 AtMYB86
Pbr001950.1 AtMYB23 Epidermal cells Jakoby et al. (2008)
Pbr001952.1 AtMYB66
Pbr034884.1 AtMYB82
Pbr034885.1
Pbr003136.1
Pbr028978.1 AtMYB121 Root development Zhou et al. (2009)
Pbr033988.1 AtMYB71
Pbr020777.1 AtMYB79
Pbr035306.1 AtMYB48
Pbr023547.1 AtMYB59
Pbr016603.1 AtMYB27
Pbr033457.1
Pbr033089.1
Pbr018111.1 AtMYB101 Involved in Allen et al. (2007)
anther/pollen
development
Pbr033089.1 AtMYB97
AtMYB120
AtMYB65
AtMYB33
AtMYB104
AtMYB8
AtMYB81
Pbr017966.1 AtMYB24 Anther development Mandaokar and Browse
(2008)
Pbr027035.1 AtMYB21
Pbr024420.1 AtMYB57
Pbr028812.1 AtMYB62 Stress responses Devaiah et al. (2009)
Pbr013315.1 AtMYB116
Pbr038922.1
Pbr020295.1
Pbr001709.1 AtMYB78 Stress responses Devaiah et al. (2009)
Pbr008630.1 AtMYB108
Pbr014994.1 AtMYB112
(continued)
9 Regulatory Sequences of Pear 161

Table 9.2 (continued)

Pyrus  bretschneideri Corresponding counterparts Function(s) Reference(s)
in Arabidopsis thaliana
AtMYB2
Pbr028319.1 AtMYB52 Lignin, xylan, and Lee et al. (2009)
cellulose biosynthesis
Pbr010042.1 AtMYB54
Pbr039365.1 AtMYB56
Pbr016851.1 AtMYB69
Pbr002006.1 AtMYB117
Pbr035515.1 AtMYB105
Pbr039075.1 AtMYB89
AtMYB110
Pbr012310.1 AtMYB44 Abiotic stresses Jung et al. (2007)
Pbr008748.1 AtMYB77
Pbr025199.1 AtMYB70
Pbr015309.1 AtMYB73
Pbr022028.1
Pbr019687.1
Pbr035927.1 AtMYB1 Abiotic stresses Sun et al. (2014)
Pbr041921.1 AtMYB25
AtMYB109
Pbr028904.1 AtMYB53 Root development Gibbs et al. (2014)
Pbr001638.1 AtMYB92
AtMYB93
Pbr001932.1 AtMYB98 Embryogenesis Wang et al. (2009)
Pbr017972.1 AtMYB64
Pbr027028.1 AtMYB119
Pbr042296.1 AtMYB118
Pbr006264.1 AtMYB115
Pbr039284.1 AtMYB22
AtMYB100

soybean, maize, sorghum, and sugarcane. For transcriptional responses to water stress. In
example, MYB/MYC regulons, such as another study, wheat MYB TF genes including
AtMYC2 and AtMYB2 proteins, respond to TaMYB30-B, TaPIMP1, and TaMYB3R1 have
drought stress through abscisic acid (ABA)- been reported to regulate expression of drought
dependent signaling systems (Abe et al. 2003). stress-responsive genes (Zhang et al. 2012).
Moreover, AtMYB102 is found to assimilate Using microarray analysis, plants subjected to
signaling pathways following wounding and water deficit have revealed up-regulation of
osmotic stress signals in Arabidopsis (Denekamp several defenses and stress-related genes, TLP4,
and Smeekens 2003). Lippold et al. (2009) have RD22, and PR1a, by the TaPIMP1 MYB tran-
reported that AtMYB41 regulates short-term scription factor, and expression level of this
162 Y. Cai et al.

transcription factor is found to be positively suggesting a dynamic regulation of the

associated with drought tolerance (Zhang et al. miR159/GAMYB module during early stages of
2012). Subsequently, it is reported that enhanced fruit development. Specifically, SlGAMYB1/2
expression of dehydration-responsive genes is silencing in SlMIR159 overexpressing plants
observed in both ABA-dependent (ABF3, results in misregulation of pathways related to
RD29A, and RD29B) and ABA-independent ovule and female gametophyte development,
(ADH, CBF4, and COR15A) signaling path- leading to earlier fruit initiation and partheno-
ways in transgenic Arabidopsis plants overex- carpy (Silva et al. 2017). Orthologous genes
pressing TaMYB3R1 TF (Cai et al. 2015). commonly share similar functions and are clus-
Apparently, MYB proteins, such as AtMYB61, tered within the same clades and subclades;
AtMYB60, AtMYB96, and AtMYB44, control whereas, paralogous genes have generally dif-
stomatal aperture regulation in Arabidopsis under ferent functional roles. This suggests that closely
water deficit conditions (Jung et al. 2007; Liang related MYB transcription factors can recognize
et al. 2005). similar target genes and possess functional
Salinity impacts plants in numerous ways by redundancy (Ogata et al. 1999). Therefore, it is
causing metabolic imbalance, ion toxicity, critical that functional analysis studies, via
membrane disorganization, osmotic stress, and genetic transformation, should be conducted to
cellular dehydration, thus, in turn resulting in further delineate the functionality of PbMYBs
inhibition of plant growth and development. genes that have been thus far identified in pear.
Plants maintain salt tolerance through induction
of ABA and salt over sensitive (SOS) signaling
pathways. SOS is also an important signaling and 9.2.2 Heat Shock TF (HSFs)
regulatory pathway activated in response to salt
stress in an ABA-independent manner. A nega- Heat shock TFs (HSFs) play a central role in
tive regulator of SOS induction in Arabidopsis, controlling expression of heat responsive genes
AtMYB73, has been identified, and it is specifi- by mediating rapid accumulation of heat shock
cally activated under salt stress, thereby proteins in response to heat stress and to other
enhancing tolerance to high salt by modulating chemical stressors (Mehta et al. 2010; von
expression of SOS1 and SOS3 genes (Lee et al. Koskull-Döring et al. 2007). Thus far, a total of
2014). Similarly, OsMYB91 from rice has been 29 HSFs genes have been identified in Chinese
reported to increase salinity tolerance (Zhu et al. pear (P.  bretschneideri) (Qiao et al. 2015).
2015). Plant HSFs gene families contain various num-
Cold acclimation is an important process bers of genes, ranging between 20 and 52
through which plants increase their tolerance members, higher than in any given species
against low temperature with the help of several (Pirkkala et al. 2001). HSFs are not only
transcription factors. In this regard, MYB tran- involved in protection against stress damage, but
scription factors have played significant roles. they also play roles in degradation of proteins,
For example, MYB3 and MYB61 TFs have including intracellular distribution and folding
enhanced cold tolerance in Medicago truncatula (Wang et al. 2004). Furthermore, HSFs are also
by positively regulating expression of the cold involved in plant growth and development, as
acclimation TF gene MtCAS15 (Zhang et al. well as in responses to other abiotic stresses such
2016). In another study, a single R2R3-MYB as drought, cold, and salt (Shim et al. 2009). For
encoding gene, FtMYB12, from Tartary buck- example, HsfA9 is involved in seed maturation
wheat has been identified to mediate COR15A and embryogenesis in both Arabidopsis and
gene expression in order to improve cold toler- sunflower (Kotak et al. 2007), as well as in
ance (Zhou et al. 2015). Recently, it has been tomato (Solanum lycopersicum), while HsfA1a
reported that miR159-targeted SlGAMYB genes plays a central role in the regulation of heat stress
are essential for fruit ovule development, thus response in tomato (Mishra et al. 2002), and
9 Regulatory Sequences of Pear 163

HsfA4a acts in controlling tolerance to cadmium suggesting that these genes may be involved in
in rice (Oryza sativa) (Shim et al. 2009). some other signal transduction pathways in the
HSFs have a modular structure with an complex regulatory network of plant stress (Qiao
N-terminal DNA-binding domain (DBD) and an et al. 2015).
oligomerization domain (OD). The N-terminal
DNA-binding domain (DBD) is connected to
oligomerization (or HR-A/B region) by a flexible 9.2.3 WRKY TFs
linker of adaptable length (15–80 amino acid
residues). However, some HSFs also possess a The WRKY family is among the largest group of
well-defined domain consisting of a nuclear plant transcription factors and comprises of 103
localization signal (NLS) domain necessary for a members in the pear genome (Huang et al. 2015;
nuclear export signal (NES) domain and an acti- Rushton et al. 2010). The WRKY protein family
vator motif (AHA motif). In general, plant HSFs consists of either one or two conserved WRKY
can be divided into three classes, including A, B, domains containing a 60 amino acid sequence,
and C, based on structural characteristics of the comprising a short peptide, WRKYGQK, and
HR-A/B domain and their phylogenetic analysis. followed by either a C2H2 or a C2HC zinc finger
HSF encoding genes belonging to class A and C motif structure. These two motifs are essential for
have an HR-A/B region with insertions of either binding to the consensus cis-acting element, ter-
21 (class A) or 7 (class C) amino acid residues med the W-box (TTGACT/C). The WRKY
present within the A and B segments; whereas, family can be classified based on the number of
HSF encoding genes belonging to class B have no WRKY domains and the feature of the zinc finger
insertions, and are comparatively compact. motif. Furthermore, WRKY proteins can be
Plants have more than 20 HSF genes encoding divided into three subfamily types. Type I pro-
heat shock proteins (HSPs), more than other teins (the WRKY I subgroup) possess two
eukaryotes that contain only one to three such WRKY domains, while type II proteins contain a
genes. For example, Arabidopsis contains 21 WRKY domain and a C2H2 zinc finger motif, and
HSFs genes (15/A, 5/B, and 1/C), while verte- type III WRKY proteins have a WRKY domain
brates contain four HSF genes, and Drosophila (WRKYGQK) and a C2HC zinc finger motif.
contains only a single HSF gene (Guo et al. Since cloning of the first WRKY gene, SPF1
2008b; Scharf et al. 2012). HSF encoding genes from sweet potato (Ipomoea batatas), a large
have been widely studied in the model plant number of WRKY genes have been experimen-
Arabidopsis, as well as in other plants, such as tally identified in various plant species, such as
maize (Z. mays), rice (O. sativa), apple sweet kumquat (Fortunella crassifolia), rice (O.
(Malus  domestica), and poplar (Populus tri- sativa), soybean (G. max), poplar (P. tri-
chocarpa), among others (Table 9.3). Following chocarpa), and Arabidopsis (A. thaliana).
complete sequencing of the genome of the Chi- Additionally, large-scale systematic analyses of
nese pear (P.  bretschneideri), this has allowed the WRKY gene family have been undertaken for
for an opportunity to conduct an extensive study A. thaliana, O. sativa, P. trichocarpa,
of HSF encoding genes in pear. It is found that P.  bretschneideri, and Cucumis sativus as
the following HSF encoding genes in pear, WRKY TFs are important participants in plant
including PbHsfA6a, PbHsfA4b, PbHsfA3a, and signaling networks for various biotic stress
PbHsfA4d, are upregulated under high tempera- responses and abiotic stress responses (Chen
ture conditions, thus suggesting that these genes et al. 2012). WRKY TFs are involved in several
play critical roles in response to heat stress (Qiao developmental and physiological processes such
et al. 2015). However, it is important to add that as embryogenesis, seed coat development, tri-
unexpectedly some PbHsf genes are chome development, anthocyanin biosynthesis,
down-regulated under high temperatures, thus and hormone signaling. Transgenic Arabidopsis
164 Y. Cai et al.

Table 9.3 Classification of Hsf transcription factors along with numbers of gene families in six Rosaceae species,
including Pyrus  bretschnederi (Chinese white pear), Malus  domestica (apple), Prunus persica (peach), Fragaria
vesca (strawberry), Prunus mume (Chinese plum), and Pyrus communis (European pear) (Qiao et al. 2015)
HSFs Chinese pear Apple (25) Peach (17) Strawberry Chinese plum European pear
(29) (16) (17) (33)
HsfA1a Pbr025227.1 MdP0000517644 Ppa004782m gene13904 Pm023178 PcP005520.1
b Pbr041026.1 MdP0000156337 Ppa004559m gene10474 Pm011227 PcP027354.1
c Pbr031411.1 MdP0000232623 PCP027124.1
d MdP0000259645 PcP011761.1
HsfA2a Pbr019856.1 MdP0000489886 Ppa007300m gene02705 Pm005519 PcP044449.1
b MdP0000243895 PcP016141.1
c PcP034937.1
HsfA3a Pbr005496.1 MdP0000131346 Ppa015602m gene30146 Pm026236 PcP016675.1
b Pbr016805.1 MdP0000606400 PcP026047.1
c MdP0000174161
HsfA4a Pbr000538.1 MdP0000155849 Ppa006534m gene23802 Pm010169 PcP025026.1
b Pbr016090.1 Ppa015468m gene15872 Pm013905 PcP026169.1
c Pbr022463.1 PcP024177.1
d Pbr005379.1 PcP015400.1
HsfA5a Pbr016487.1 MdP0000301101 gene06570 Pm007815 PcP002437.1
b MdP0000613011
HsfA6a Pbr036788.1 Ppa1027143m gene29004 Pm009237 PcP030606.1
b Pbr014670.1 PCP018714.1
c Pbr018847.1
HsfA7a Pbr009953.1 Ppa010224m gene20347 Pm020253 PcP019575.1
b Pbr012908.1 PcP022776.1
HsfA8a Pbr012136.1 MdP0000191541 Ppa006514m Pm005887 PcP006787.1
b MdP0000172376 PcP031284.1
HsfA9a Pbr041474.1 MdP0000194672 Ppa016533m gene12667 Pm027197 PcP005035.1
b Pbr015630.1 MdP0000319456 PcP027517.1
HsfB1a Pbr025141.1 MdP0000527802 Ppa009274m gene24036 Pm026366 PcP024136.1
b Pbr030422.1 MdP0000578396 PcP030007.1
HsfB2a Pbr013953.1 MdP0000155667 Ppa009180m gene13301 Pm019357 PcP030684.1
b Ppa008441m gene32416 Pm023788 PcP033244.1
c PcP007662.1
HsfB3a Pbr002020.1 MdP0000622590 Ppa014675m gene02464 PcP029678.1
b Pbr030436.1 MdP0000202716 PcP024839.1
c Pbr002038.1
HsfB4a Pbr019653.1 MdP0000209135 Ppa026635m Pm005297
b MdP0000129357
HsfB5a Pbr016270.1 Ppa011804m gene02408 Pm010031 PcP044895.1
b PcP016888.1
HsfC1a Pbr014107.1 MdP0000230456 Ppa008830m gene30881 Pm027421 PcP000545.1
b Pbr016948.1 MdP0000320827 gene PcP022060.1
9 Regulatory Sequences of Pear 165

plants carrying AtWRKY52/RRS1, a type III Pan et al. 2017). Cardon et al. (1997) have
member containing WRKY and TIR-NBS-LRR identified the first SBP-box gene, SPL3, in Ara-
(TNL), have exhibited resistance to the bacterial bidopsis, and have observed its potential role in
pathogen Ralstonia solanacearum through nuclear regulating flowering under a duration of a long
interaction with the type III bacterial effector PopP2 photoperiod. Subsequently, the SBP gene family
(Deslandes et al. 2003). The AtWRKY52 TF also has been identified and thoroughly investigated
interacts with the RPS4 protein for dual resistance in many model plants including Betula pendula
against both fungal and bacterial pathogens. While, (silver birch) (Lännenpää et al. 2004), Gossyp-
WRKY proteins belonging to type II contain a ium hirsutum (cotton) (Zhang et al. 2014), Oryza
calmodulin (CaM)-binding domain, the C-motif sativa (Xie 2006), green algae (Kropat et al.
(DxxVxKFKxVISLxxxR), thus suggesting possi- 2005), Solanum lycopersicum (Salinas et al.
ble regulation by CaM and Ca2+ fluxes (Park et al. 2012), Cucumis melon (Ma et al. 2015), and the
2005). At this time, pear WRKY genes, showing moss Physcomitrella patens (Riese et al. 2007).
extensive autoregulation and cross-regulation, are In Arabidopsis, 16 SBP-box genes have been
yet to be investigated for their functional roles, yet identified, and their critical roles have been
it appears that these TFs facilitate transcriptional observed, and investigated in leaf development
reprogramming in a dynamic web with built-in (Guo et al. 2008a; Usami et al. 2009), leaf pri-
redundancy. mordia formation (Wang et al. 2008), early
flowering (Gandikota et al. 2007), gibberellic
acid (GA) responses (Zhang et al. 2007), copper
9.2.4 SQUAMOSA Promoter Binding homeostasis (Yamasaki et al. 2009), along with
Protein (SBP)-Box Genes nutritional changes and reproductive stage
development (Jung et al. 2011). Furthermore,
The SQUAMOSA promoter binding protein SPL8 mutants of Arabidopsis exhibit differences
(SBP)-box gene family is a group of in pollen sac development, contributing to
plant-specific TFs that play significant roles in reduced fertility, and regulating differential pat-
many biological processes, such as microsporo- terning of gynoecium development. Furthermore,
genesis, megasporogenesis, trichome develop- overexpression of SPL8 alters plant fertility
ment on sepals, ripening of fruit, stamen filament through crosstalk signaling of gibberellic acid
elongation, and homeostasis. SBP-box family (GA) (Zhang et al. 2007). Likewise, AtSPL11,
genes are present in all photosynthetic organ- AtSPL10, and AtSPL2 contribute to morpholog-
isms, from green algae to multicellular trees, ical changes in addition to reproductive phase
except for animals, prokaryotes, and fungi. and shoot maturation (Shikata et al. 2009). In
The SBP domain is comprised of *79 amino rice, overexpression of OsSPL14 regulates the
acids along with 10 conserved cysteine and his- reproductive stage of plant development, con-
tidine residues that are interrelated in nuclear tributing to significant increases in grain yield
localization and DNA-binding (Cardon et al. and in panicle branching (Miura et al. 2010).
1999; Zhang et al. 2015). SBP-box encod- Furthermore, SBP-box genes play potential roles
ing genes cover two zinc-binding sites in the modification of plant architecture and yield
(Cys-Cys-Cys-His and Cys-Cys-His-Cys), in traits through the initiation of lateral primordia.
which most have a three-stranded antiparallel In particular, SBP-box genes take on an inter-
beta-sheet (Yamasaki et al. 2004; Pan et al. 2017; phase role between phase change and home-
Abdullah et al. 2018b). ostasis. This role of the SBP-box gene family
Based on specific interactions with a promoter should be investigated further in diverse plant
sequence of the SQUAMOSA identity gene, systems. As of to date, studies are underway to
AmSBP1 and AmSBP2 are the first reported link SBP-box genes with identified flowering
genes identified in Antirrhinum majus (snap- pathways. These studies will investigate whether
dragon) (Klein et al. 1996; Cardon et al. 1999; or not homeostatic responses and transitions in
166 Y. Cai et al.

plant growth are common in different plant sys- roles in the development of gynoecia and anthers
tems, as well as determine whether or not (Lee et al. 2018). Furthermore, the GRF-GIF
SBP-box gene expression resembles the two complex is also critical for meristematicity
sides of a coin. A genome-wide investigation has (meristematic competence) and pluripotency of
been conducted in our laboratory wherein 32 carpel margin meristems (CMMs) and for arch-
SBP genes have been identified and isolated from esporial differentiation (Lee et al. 2018).
P.  bretschneideri (Abdullah et al. 2018b). The WRC domain plays a functional role in
Based on phylogenetic analysis, PbSBP proteins transcriptional control and DNA-binding, and it
have been classified into seven groups (Abdullah contains two distinctive features, a nuclear
et al. 2018b). This latter study on SBP-box genes localization signal (NLS) and a zinc finger motif
in pear will provide additional and detailed composed of three Cys and one His residues
information on the role of these genes in fruit (C3H motif) (Sauer et al. 2004). A barley tran-
crops. scriptional repressor (HRT) has a C3H motif, and
it is proposed to bind to a GA response element
(GARE), while the WRC domain is likely to act
9.2.5 GROWTH-REGULATING FACTOR as a DNA-binding domain (Noguero et al. 2013).
(GRF) TFs Most GRFs are strongly expressed in actively
growing and developing tissues, such as flower
The growth-regulating factor (GRF) family of buds, shoot tips, and growing leaves, as revealed
plant-specific transcription factors (TFs) serves by quantitative RT-PCR analysis and RNA
as positive regulators of growth and development gel-blots (Kim et al. 2003). Additionally, GRFs are
in flowering plants. Although GRFs have been more highly expressed in reproductive organs than
primarily studied in leaf tissues, GRFs play roles in vegetative organs. It has been reported that the
in both vegetative and reproductive shoot apical rice OsGRF10 and OsGRF3 interact and repress
meristems (SAMs), as well as during various the promoters of KNOTTED1-like homeobox
phases of reproductive growth in flowering (KNOX) family genes, which control meristem
plants. For example, in rice (O. sativa), OsGRFs development, thereby regulating meristem devel-
regulate stem growth induced by the phytohor- opment and restricting cell differentiation in apical
mone gibberellic acid (GA) (van der Knaap et al. meristems of shoots (Ma et al. 2017). Kim et al.
2000). (2003) have also reported that overexpressing GRF
GRF gene families have been investigated and genes in transgenic Arabidopsis plants result in
identified in various plant species, including A. larger leaves than those of wild-type plants;
thaliana, O. sativa, Z. mays, P.  bretschneideri, whereas, an Atgrf1/2/3 triple mutant has smaller
Brachypodium distachyon, and Brassica rapa leaves than wild-type Arabidopsis. Moreover,
(Cao et al. 2016a). It has been reported that overexpression of AtGRF5 results in early leaf
deduced protein products of GRF genes contain development, delay of the cell proliferation phase,
two conserved domains in the N-terminal regions, extensive division of chloroplasts, along with a
the QLC and WRC domains (Cao et al. 2016a). simultaneous suspension in the onset of the cell
Furthermore, SW12/SNF2 proteins containing expansion phase (Ma et al. 2017).
the QLC domain, a protein–protein interaction Various regulatory networks have been
domain that regulates interaction of these proteins involved in establishment and maintenance of
with homologs of SNF11 from Saccharomyces meristems and in promoting cell proliferation of
cerevisiae, form a complex that is involved in developing organ primordia, including the GRF
chromatin remodeling; whereas, the QLQ domain family of transcription factors (TFs). GRFs may
of GRFs facilitates interaction with also function in defense signaling and in stress
GRF-interacting factors (GIFs) (Choi et al. 2004). responses; for example, overexpression of
Moreover, GRF and GIF (GRF-interacting fac- DREB2A increases plant tolerance to heat stress,
tors) transcriptional complexes have biological osmotic stress, and other abiotic stresses, but it
9 Regulatory Sequences of Pear 167

also results in growth retardation and reduced 2015). It is important to point out that the zinc
reproduction (Jin et al. 2014). finger (C2H2, C2C2, and C3H) interacts with a
single zinc ion, but with new approaches, it has
been found that the plant RING finger and the
9.2.6 Zinc Finger Homeodomain TFs animal Lin-11/Is1-1/Mec-3 (LIM) domain inter-
act with two zinc ions (Yanagisawa 2004; Wang
Zinc finger homeodomain (ZHD) transcription et al. 2014).
factors are major regulators of specification of A cluster of novel zinc finger homeodomain
higher plants, and they are especially involved in (ZHD) proteins have been first isolated from
plant development (fiber development) and stress Flaveria as potential regulators of the gene
responses (Wang et al. 2015; Khatun et al. 2017). encoding C4 phosphoenolpyruvate carboxylase
Early on, homeobox genes have been first iden- (PEPCase), wherein the ZHD domain is capable
tified in the fruit fly, but subsequently, these of binding to DNA, predominantly to the regu-
genes have been found and isolated in several latory region of the C4 PEPCase encoding gene
organisms, including fungi, plants, nematodes, (Windhovel et al. 2001). Furthermore, it is
and humans (Bhattacharjee et al. 2015). As reported that the zinc finger domain is not
mentioned above, TFs can activate/repress target involved in DNA-binding, although it can boost
genes by direct binding to gene motifs or ele- the protein–DNA interaction facilitated by the
ments, and many TF families have evolved HD domain (Windhovel et al. 2001). ZHD pro-
through unique DNA-binding domains that teins have been identified and characterized in
advise their binding activity. One of the various plants, such as A. thaliana (Tan and Irish
well-characterized domains is the homeodomain 2006), G. max (Deng et al. 2002), O. sativa
(HD) which is encoded by 60 conserved amino (Jørgensen et al. 1999), and Triticum aestivum
acids (Wang et al. 2014; Mukherjee et al. 2009). (Bhattacharjee and Jain 2013).
In plant and animal genomes, homeobox Expression patterns of HD-Zip genes identi-
genes are characterized by large gene families, fied in pear have suggested that these genes are
and based on the number, nature, and spacing widely involved in salt stress, drought stress,
pattern, they can be also categorized into differ- and pathogen infection (Wang et al. 2015).
ent groups. Initially, the zinc finger has been Under conditions of drought stress, expression
classified into the following groups, KNOX, levels of 15 PbHB genes are found to be
ZM-HOX, BELL, AT-HB8, HAT, and GAL2 upregulated, while five other PbHB genes are
(Bharathan et al. 1997; Bhattacharjee et al. down-regulated (Wang et al. 2015). Specifically,
2015). Subsequently, homeobox genes of rice it is reported that PbHB2 is detected only at 6 h
have been classified into 10 subclasses, including following PEG6000 treatment, while PbHB1
HD-Zip I, HD-Zip II, HD-Zip III, HD-Zip IV, and PbHB20 are activated at 12 h, and PbHB25
KNOX I, KNOX II, BLH, WOX, PHD, and and PbHB4 are upregulated only at 24 h (Wang
ZF-HD (Bhattacharjee et al. 2015). In a com- et al. 2015).
prehensive study, homeobox genes have been Overall, several members of the ZHD class of
grouped into 14 subclasses, and by adding some proteins are critical components in regulating
new classes, such as DDT, NDX, PHD, blue light signaling, vascular development, outer
SAWADEE, LD, and PINTOX (Mukherjee et al. cell layer of a plant organ, response to stress, and
2009). control of anthocyanin processing (Khatun et al.
In pear, the HD-Zip has been extensively 2017). Early on, it has been reported that the
studied, and the zinc finger is categorized into 14 gene encoding for the ZHD protein is involved in
subgroups (Wang et al. 2015). Genome-wide the regulation of floral development, but subse-
analysis has identified 52 genes encoding quently it is found that the Arabidopsis protein
HD-Zip TFs within the pear genome (Wang et al. encoding gene, AtZHD1, binds to the promoter
168 Y. Cai et al.

of EARLY RESPONSE TO DEHYDRATION and MIKC* type, and furthermore, MIKCC can
STRESS 1 (ERD1) (Tran et al. 2007). Interest- be classified into 12 subfamilies (Becker and
ingly, the expression pattern of AtZHD1 is Theißen 2003).
inducible by salt stress, abscisic acid, and dehy- In the Chinese pear (P.  bretschneideri)
dration (Tran et al. 2007; Wang et al. 2014). In genome, a total of 95 MADS-box genes have
addition, as the ZHD protein can interact with been identified and categorized (Wang et al.
some NAC proteins, it has been found that 2017). Pear type I MADS-box genes have been
simultaneous overexpression of ZHD and NAC classified into three subfamilies, and type II
genes contributes to increased drought tolerance MADS-box genes are divided into 14 subfamilies
in Arabidopsis (Tran et al. 2007; Hu et al. 2008). (Wang et al. 2017). Remarkably, except for a
Thus far, 14 ZHD genes have been identified in highly conservative MADS (M) domain pos-
Arabidopsis, and their functions have been sessing about 60 amino acid sequences of the
characterized (Tan and Irish 2006). Recently, N-terminal regions, type II genes also contain an
ZHD coding genes have been identified in other Intervening (I), a C-terminal (C), and a Keratin
plant systems, and their functions have been (K) domain (Kaufmann et al. 2005). Compared
elucidated. For example, four rice ZHD genes with type II, type I genes are relatively simple
have been associated with gene regulation, while and lack the K domain, wherein a coding gene
two soybean proteins, GmZHD1 and GmZHD2, usually contains 1–2 exons (Parenicova 2003). It
have been identified to bind to the promoter of has been reported that MIKCC-group genes are
the gene encoding for calmodulin isoform 4 likely to be involved in developmental processes
(GmCaM4), thereby increasing expression levels of flowering plants. For example, Arabidopsis
of these proteins following pathogen induction flowering time genes, including AGAMOUS-
(Park et al. 2007; Hu et al. 2008; Wang et al. LIKE24 (AGL24) and SUPPRESSOR
2014). Furthermore, Hu et al. (2008) have OF OVEREXPRESSION OF CONSTANS 1
reported that the mini zinc finger (MIF) gene (SOC1), are involved in transition of vegetative
family, possessing the zinc finger, interacts with to reproductive stages of plant development
ZHD, and that their overexpression interferes (Ferrario et al. 2004; Khan et al. 2014). As
with the normal functions of ZHD proteins. If MADS-box genes play critical roles in develop-
this is indeed the case, then ZHD proteins may ment and in signal transduction of various
play important roles in regulating plant physiol- organs, such as development and maturation of
ogy and development. fruits (Ma and dePamphilis 2000), it has been
postulated earlier that the characteristics of plant
floral organ development can be explained by the
9.2.7 MADS-Box TFs ABC model (Weigel and Meyerowitz 1994).
Based on the ABC model for floral organ
MADS-domain transcription factors play impor- development, class A genes specifically regulate
tant roles in both development and evolutionary occurrence and development of the calyx, while
diversity, such as fruit development, floral organ classes A and B genes together control formation
conformation, and flowering time. MADS-box of petals, and classes B and C genes together
transcription factors are widespread in animals, determine the occurrence of stamens, while class
plants, and fungi, as they initiate target gene C genes regulate the development of carpels.
transcription by binding to the CArG-box Based on subsequent reverse genetics studies, it
domain in the cis-acting element of the target has been demonstrated that classes D and E
gene (Riechmann et al. 1996). Based on phylo- genes also play vital roles in regulating flower
genetic analysis, MADS-box genes have been morphogenesis. Among these, class D genes
classified into two large groups, type I (SRF) and mainly regulate the development of ovules
type II (MEF2). Type I is divided into Ma, Mb, (Colombo et al. 1995), while class E genes are
and Mr, while type II is divided into MIKCC type mainly involved in regulating the formation and
9 Regulatory Sequences of Pear 169

development of all floral organs (Pelaz et al. 9.2.8 B-Box TFs

2000). The MADS-box gene family has been
extensively studied in angiosperms, particularly The B-box (BBX) family of plant TFs is a
in the model plant A. thaliana (Ma and dePam- functionally diverse subclass of zinc finger TFs
philis 2000). In Arabidopsis, class A genes are containing an N-terminal B-box domain, either
represented by APETALA1 (AP1) and APE- alone or sometimes in combination with a CCT
TALA2 (AP2) (Mandel et al. 1992), class B genes [TIMING OF CAB EXPRESSION 1 (TOC1),
include APETALA3 (AP3) and PISTILLATA (PI) CONSTANS (CO), and CO-LIKE (COL)]
(Goto and Meyerwortiz 1994), class C genes are domain (Gangappa and Botto 2014). The BBX
represented by AGAMOUS (AG), class D genes domain is a short protein sharing a 40 amino acid
are represented by SEEDSTICK (STK/AGL11), residue in length. B-box proteins can be divided
SHATTERPROOF1 (SHP1/AGL1), and SHP2 into two types, B-box1 and B-box2, based on
(AGL5), and class E genes include SEPAL- their consensus sequence and the spacing of
LATA1,2,3,4 (SEP1/2/3/4 and AGL2/4/9/3) zinc-binding residues (Crocco and Botto 2013).
(Mandel and Yanofsky 1998). Among these BBX proteins play critical roles in regulatory
genes, besides AP2, all class A, B, C, D, and E networks controlling seedling photomorphogen-
homologous genes belong to the MIKCC-type esis, shade avoidance, photoperiodic regulation
MADS-box genes. These studies have demon- of flowering, and responses to biotic and abiotic
strated that type II MADS-box genes are mainly stresses. Conserved residues in CONSTANS
related to plant floral organ development. On the B-box motifs are known to be involved in
contrary, the function of type I MADS-box genes mediating protein-protein interactions (Datta
has seldom been reported. In limited studies, it et al. 2008). For example, the CONSTANS
has been reported that type I MADS-box genes B-box directly interacts with proteins containing
are mainly involved in the development of a coiled-coil domain for the SUPPRESSOR OF
female gametophytes, endosperms, or seeds PHYA1 (SPA1). The CCT domain, a basic motif
(Köhler et al. 2003). of 42–43 amino acids, performs a critical role in
In early studies in pear, it has been shown that nuclear protein transport and in transcriptional
MADS-box TFs play a vital role in fruit devel- regulation of BBX proteins. For example, the
opment and maturation (De Folter et al. 2004). CCT domain of CO plays a functional role in
More recently, it has been demonstrated that the mediating gene expression by binding the
PbMADS12 gene together with PbMYB10 and promoter of the FLOWERING LOCUS T
PbbHLH3, all from P.  bretschneideri, regu- (FT) (Griffiths et al. 2003). BBX proteins
late the anthocyanin pathway through activation sequence alignment has revealed that the CCT
of the promoters of PbUFGT1 and PbDFR1 domain also contains highly conserved sequen-
(Wang et al. 2017). Furthermore, PbMADS11 ces. In Arabidopsis, of a total of 32 BBX pro-
and PbMADS12 seem to serve as master regu- teins, 17 (BBX1-17) proteins contain a CCT
lators of anthocyanin biosynthesis in response to domain (Griffiths et al. 2003).
temperature and light (Wang et al. 2017). The The CONSTANS-LIKE 3 (COL3) is a critical
induction of the productive meristem identity protein-binding partner for B-BOX32 (BBX32)
MADS-box gene AP1 following repression of activity in Arabidopsis. The discovery of the
the pear TERMINAL FLOWER1, PpTFL1s, is interaction of B-BOX32 with COL3 can be used
proposed to play a primary role of the PpTFL1 in in combination with BBX32 for increased pro-
mediating floral induction in P. pyrifolia Nakai ductivity. This regulatory pathway could be
(Bai et al. 2017). applied as an efficient strategy for genetic
170 Y. Cai et al.

manipulation in crops for increased agricultural expressed at the P1 stage of pollen development
productivity (Tripathi et al. 2017). In general, (mature pollen grains), while two genes, PbBBX8
Arabidopsis BBX proteins are divided into five and PbBBX10, were expressed at the P2 stage of
subfamilies based on their type and number of pollen development (hydrated pollen grain) in
BBX motif and the presence/absence of a CCT pear (Cao et al. 2017). These findings suggested
domain (Gangappa and Botto 2014). Subfamily I that PbBBXs genes were important for signaling
(BBX1-6) and subfamily II (BBX7-13) possess processes during pollen growth in pear. Expres-
two B-boxes and a CCT domain, while sub- sion profiles of PbBBXs genes in different tissues
family III has a single B-box and a CCT domain. or organs were confirmed using qRT-PCR
Subfamily IV possesses two tandem repeats of revealing that PbBBX6, 8, 9, 11, and 19 were
B-box motifs, also referred to as the double not expressed in all tested tissues or organs, while
B-box (DBB); however, this subfamily lacks a PbBBX1, 2, 3, 4, 7, 10, 14, 16, 18, 20, 21, 22, and
CCT domain. Subfamily V (BBX27-32) contains 24 were expressed in leaves, and PbBBX13 and
only a short N-terminal B-box domain with 17 were highly expressed in roots (Cao et al.
either one or two B-box motifs (Gangappa and 2017).
Botto 2014).
A genome-wide survey of the B-box gene
family has been conducted in pear, 9.3 Concluding Remarks
P.  bretschneideri. Of 25 BBX encoded genes,
seven contained two B-BOX domains along with In general, TFs are a group of regulatory proteins
a conserved CCT domain, while four and five that control gene expression by binding to DNA,
PbBBXs were found to contain a single B-BOX and in doing so, they either activate or repress
and either a conserved CCT domain or only a mRNA transcription. Plant TF gene specificity is
single CCT domain, respectively, while the less obvious than that for animals. Thus far, there
remaining nine PbBBXs had two B-BOX are only a few cases that have been reported on
domains (Cao et al. 2017). The pear BBX enco- plant TF gene specificity, wherein NAC controls
ded genes showed wide variations in molecular development process and stress response, MADS
lengths, ranging from 142 to 859 amino acids. regulates flowering tissue differentiation, B3
Additionally, pear BBX genes showed highly controls auxin responses, and AP2/EREBP con-
similar structures within the same clade. For trol plant hormone responses, including those for
example, eight PbBBXs belonging to clades I and ethylene, jasmonic acid, auxin, brassinosteroids,
III had two exons, while PbBBXs belonging to and gibberellin, among others.
clade IV had three exons, and PbBBXs belonging Pear (Pyrus) TFs are characterized by a large
to clade V contained only a single exon, except number of genes, and by a diverse group of TF
for PbBBX24 and PbBBX25. These findings gene families. Furthermore, various cellular
suggested that exon gain or loss occurred during responses are regulated by highly divergent TFs,
evolution of the pear PbBBXs gene family, such as bHLH, MYB, homeodomain, and zinc
resulting in functional divergence among closely ring finger, among others. For each of the hor-
related PbBBXs (Cao et al. 2017). Moreover, 52% mones involved in plant and growth develop-
of PbBBXs, 13 genes, were not expressed during ment, it is likely that there are specific modes of
the different development stages of pear pollen signal transduction, from the receptor to the TFs
development, thereby suggesting that these genes involved in these processes. Studies of TFs in the
might be expressed in other tissues, such as stems, model plant A. thaliana have provided important
leaves, or roots, or under special conditions. Of insights on the roles of TFs in a variety of
the remaining 48% of PbBBXs, 12 genes, that plant-specific cellular responses, such as devel-
were expressed during development-dependent opment process, environmental stresses, such as
pattern of pollen development in pear, five genes, cold, responses to light, drought, high salinity,
including PbBBX6, 7, 9, 11, and 12, were and plant defense to pathogen infection. Genetic
9 Regulatory Sequences of Pear 171

and molecular studies of TFs in pear have elu- hormone-related genes and transcription factors.
cidated the roles of different families of TFs in J Exp Bot 68:4899–4914. https://doi.org/10.1093/
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 Self-incompatibility in Pear
10
Shaoling Zhang and Chao Gu

Abstract stylar-tissue mutations, including abnormal

Self-incompatibility (SI) has been widely expression and post-transcript modification,
investigated at both molecular and cellular are difficult to study, and are yet to be
levels in pear. This trait is controlled by a explained at the molecular level. Similarly,
single multi-allelic locus encoding at least two the mechanism of pollen tissue mutation and
components from the pollen and the pistil. The polyploidy require further investigations in
stylar-S determinant is an S-glycoprotein future studies.
(S-RNase) that can inhibit pollen tube growth
in a self-pistil, and induces a series of changes
in reactive oxygen species (ROS), calcium
(Ca2+), actin cytoskeleton, and phosphatidic 10.1 Introduction
acid, leading to programmed cell death in
incompatible pollen tubes. At present, a total Self-incompatibility (SI) is a common genetic
of 67 S-RNase genes have been identified and mechanism found in plants as it prevents
have served in selecting appropriate pollina- inbreeding by rejecting self-pollen, thereby pro-
tors in pear orchards. The pollen-S determinant moting outcrossing, and maintaining prior evo-
has also been investigated in pear. Although a lution of a species (De Nettancourt 2001). Many
group of F-box genes have been identified in flowering plants exhibit a wide range of SI, from
the S-locus, it remains unclear as to which 60 to 90 families (Brewbaker 1954), including
gene(s) are involved in self-incompatibility those of Cruciferae, Solanaceae, Rosaceae,
reactions. In pear, only a few cultivars have Papaveraceae, and Amaryllidaceae, among others
experienced loss of self-incompatibility, due (Lewis 1976). Self-incompatibility is controlled
to either stylar or pollen mutations, or due to by multiple alleles in a single locus, designated as
polyploidy. Except for the deletion of S4- the S-locus. Noteworthy, the genetic mechanism
RNase in cultivar Osa-Nijisseiki, other of SI is not identical in different plants. For
example, in Cruciferae, SI is determined by the
dominant S-allele in spores, and it is referred to as
sporophytic self-incompatibility (SSI); whereas,
in Solanaceae, Rosaceae, and Scrophulariaceae,
S. Zhang (&)
C. Gu
Centre of Pear Engineering Technology Research, SI is determined by a single S-allele in gametes,
State Key Laboratory of Crop Genetics and and it is referred to as gametophytic self-
Germplasm Enhancement, Nanjing Agricultural incompatibility (GSI).
University, Nanjing 210095, China
e-mail: [email protected]

© Springer Nature Switzerland AG 2019 179

S. S. Korban (ed.), The Pear Genome, Compendium of Plant Genomes,
https://doi.org/10.1007/978-3-030-11048-2_10
180 S. Zhang and C. Gu

The pear is a typical gametophytic shape with swollen tips prior to arrest of pollen
self-incompatible plant. In self-compatible pear tube growth in self-incompatible styles; whereas,
cultivars, the pollen can grow into the ovule no such morphological observations are noted in
through the self-stigma and the self-style. How- non-self-styles (Hiratsuka et al. 1985). These
ever, in self-incompatible cultivars, following findings suggest that S-RNase induces a series of
pollen germination on the self-stigma, pollen structural changes in pollen tubes during an SI
tubes could pass through the stigma and into the reaction. Using transmission electron micro-
self-style, and then simply stop during the scopy, similar structures are detected in incom-
expected period of pollen tube growth toward the patible and compatible pollen tubes during early
ovule. These inhibited pollen tubes have abnor- stages of pollen growth. However, after 24 h,
mal morphologies along with swelled tips. If the incompatible pollen tubes are filled with cyto-
style is cut from the top 1/3 or 1/2 section, the plasm and various organelles, while low amounts
ratio of pollen growth through the cut section is of cytoplasm are observed in tips of pollen tubes,
found to increase, but so is the inhibition of along with damaged organelles and thickened
pollen growth between 1/3 and 1/2 sections cell walls (Gao et al. 2015). These findings
(Zhang et al. 2000). These findings indicate that suggest that inhibition of pollen germination and
there is an inhibitor present in self-styles pre- pollen tube growth are influenced by levels of
venting pollen tube growth. S-RNase in pollen tubes.
Moreover, analysis of Ca2+ concentrations in
cytoplasm of pollen tubes treated by exogenous and
10.2 The Physiological Mechanism endogenous RNase reveals that the effects of stylar
of SI S-RNase treatments on Ca2+ concentrations are
different in self-compatible and self-incompatible
The inhibitor that prevents pollen tube growth pollen tubes. In fact, prior to germination, a
has been first unveiled by comparing expressed cytosolic Ca2+ gradient is detected around the ger-
proteins in styles of the self-incompatible Asian minal aperture in pollen tubes (Jiang et al. 2014; Qu
pear cultivar Nijisseiki and its mutant, the et al. 2007). Although the cause of this observed
self-compatible cultivar Osa-Nijisseiki (Sassa Ca2+ gradient is unclear, there is increasing evi-
et al. 1992). It is found that this inhibitor is an dence that the Ca2+ channel in the plasma mem-
S-glycoprotein, which is identical to an S-RNase, brane of pollen tubes plays an important role in this
and it is specifically expressed in styles, and not observed Ca2+ gradient detected in pear (Qu et al.
in leaves, pollen, or germinated pollen grains 2007). Interestingly, a similar phenotype is also
(Sassa et al. 1993). This S-glycoprotein can be observed in the flowering poppy weed plant
detected in styles approximately 8 days before Papaver rhoeas, wherein Ca2+ induces microfila-
flowering, and its levels continue to increase ment depolymerization and programmed cell death
during flower development (Hiratsuka et al. in self-incompatible pollen tubes (Wu et al. 2011).
1999). Interestingly, levels of expression of Coincidently, the stylar S-RNase in pear could
S-glycoprotein in styles vary in different pear interact directly with the actin protein PbrActin1 in
cultivars, but they are higher in self-incompatible an S-haplotype-independent manner, resulting in
cultivars than those in self-compatible cultivars depolymerization of the actin cytoskeleton, and in
(Zhang et al. 2000). turn promoting programmed cell death in
Based on both in vivo and in vitro studies, self-incompatible pollen tubes. The P156 of
lengths of pollen tubes are negatively correlated PbrS-RNase is essential for PbrS-RNase–PbrActin1
to levels of S-RNase (Hiratsuka et al. 1999, interactions, while the actin cytoskeleton-
2001). Furthermore, morphological differences depolymerizing function of PbrS-RNase does not
are also noted, wherein pollen tubes are curved in require an RNase activity (Liu et al, 2007; Chen
10 Self-incompatibility in Pear 181

et al. 2018). The induced actin cytoskeleton- 10.3 Self-incompatibility

depolymerization results in programmed cell Determinants
death in self-incompatible pollen tubes (Wang et al.
2009). The S-locus in pear should contain at least two
In general, overlapping phenotypes are genes that are, respectively, stylar-S and pollen-
observed in a GSI reaction. Recently, the reactive S determinants. If the single S-locus in pollen is
oxygen species (ROS) gradient has been inves- identical to one of the two S-loci in the style, the
tigated in tips of pollen tubes. It is found that this pollen presents an SI reaction. Thus, two pear
ROS gradient is disrupted by the stylar S-RNase cultivars with identical S-genotypes are deemed
in pears, which in turn leads to Ca2+ channel cross-incompatible (Fig. 10.1a), while two culti-
closure and microfilament depolymerization, vars with overlapping S-loci are semi-compatible
thereby stimulating degradation of nuclei. These (Fig. 10.1b), and two cultivars without any
findings suggest that ROS is an upstream regu- overlapping S-loci are deemed cross-compatible
lator for Ca2+ to mediate pollen tube growth in a (Fig. 10.1c).
GSI reaction (Wang et al. 2010). Moreover,
phosphatidic acid (PA) mitigates S-RNase sig-
naling in pollen by stabilizing the actin, as it has 10.3.1 Stylar-S Determinants
been recently observed. However, expression of
phospholipase D (PbrPLDd1) is enhanced by 10.3.1.1 Identification of Stylar-
PbrS-RNase cytotoxicity, resulting in increased S Determinants
PA levels in incompatible pollen tubes. Thus, S-RNases have been isolated from styles of pear
PbrPLDd1-derived PA initially prevents depoly- flowers using a two-dimensional gel elec-
merization of the actin cytoskeleton elicited by trophoresis (Sassa et al. 1993), and they are
PbrS-RNase, and delays SI signaling which leads found to belong to the T2/S ribonuclease super-
to pollen tube death (Chen et al. 2018). These family (Sassa et al. 1996). With the development
results provide further insights into the orches- of molecular technologies, additional numbers of
tration of the S-RNase-based SI response, in S-RNase alleles have been identified from pear
which increased PA levels initially play a pro- cultivars. Until now, 68 and 24 S-RNase alleles
tective role in incompatible pollen, until sus- have been individually isolated from Asian and
tained PbrS-RNase activity reaches the point of European pear cultivars, respectively, and S-
no return, and pollen tube growth ceases. genotypes have been determined in at least 462

Fig. 10.1 Schematic

diagram of
self-incompatibility reactions
182 S. Zhang and C. Gu

pear cultivars (Table 10.1). A detailed descrip- styles are rinsed with water, squashed on a
tion of how these various S-genotypes have been glass slide, and observed under an ultraviolet
identified is presented as follows: fluorescent microscope (Wang et al. 2009). If
either the majority, some, or none of the
(a) Cross-pollination test in the field: Based on pollen tubes could grow through to the bot-
the principle of GSI, it is expected that there tom section of a style, then this suggests that
are three different phenotypes that could be either two, one, or no S-loci in pollen tubes,
observed following cross-pollination. First, if respectively, overlap with the two S-loci of
the two cultivars are cross-incompatible, this the style.
indicates there are two identical S-loci shared (d) S-glycoprotein electrophoresis: As S-RNa-
between these two cultivars. Second, if two ses are specifically expressed in the style,
cultivars are cross-compatible, but half of the producing S-glycoproteins, then S-RNase
progeny is backcross-incompatible with the alleles could be determined by identifying
male parent, then this indicates that there is a S-glycoprotein products. In brief, soluble
single S-locus that is shared between these proteins are extracted from styles at
two cultivars. Thirdly, if two cultivars are pre-bloom stage, and then these are subjected
cross-compatible and all progeny are to isoelectric focusing-PAGE (Heng et al.
backcross-compatible with the male parent, 2015). Following silver staining, different
this indicates that there is no common S- S-glycoproteins will be readily identified,
locus present in these two cultivars. corresponding to the different S-RNase alle-
(b) In vitro culture of pollinated styles: As les present in the style.
pollen tubes are arrested in a style wherein (e) PCR amplification: Based on the polymor-
identical S-loci are present in these tissues, phism of the length of introns present in S-
an in vitro culture of pollinated styles, grown RNase alleles, allele-specific primer pairs are
on an agar medium, is used to discern the designed from conserved regions to identify
identity of S-loci present in each of the style different S-RNase alleles (Ishimizu et al.
and the pollen tube (Zhang et al. 2003). 1999). This PCR-based method has been
Following in vitro culture, if no pollen tubes widely used to identify S-RNase alleles and
could pass through the style, this indicates S-genotypes in pear cultivars. At present, a
that S-loci of the pollen tubes are identical to total of 92 S-RNase alleles have been isolated
those of the style. However, if few pollen from over 400 pear cultivars that have been
tubes are capable of passing through the successfully S-genotyped.
style, this indicates that there is a single S-
locus in the pollen tubes that is different from The S-RNase alleles isolated from Asian pear
that present in the style. While, if large cultivars are numbered with Arabic numerals,
numbers of pollen tubes are capable of while those isolated from European pear cultivars
passing through the style, then this indicates are initially numbered with lowercase letters and
that at least two S-loci present in pollen tubes then re-numbered with Arabic numerals (Gold-
are different from those present in the style. way et al. 2009). Unfortunately, the numbered S-
(c) Anatomical observations of pollen tube RNase alleles in Asian pears are out of order. For
growth in the style: At 96 h following pol- example, the two S-RNase alleles isolated from
lination, pollinated styles are fixed in an P.  bretschneideri, S20- and S29-RNases, share
FAA solution (formalin:acetic acid:70% identical sequences, while the S7-RNase in
ethanol at a ratio of 5:5:90 by volume) for P. pyrifolia shares identical sequences to S27-
about 24 h and then transferred to 100% RNase in P.  bretschneideri. Although similar
ethanol. Fixed styles are washed with water allele pairs have been recently merged and inte-
to remove ethanol, softened in NaOH, and grated (Table 10.2; Wang et al. 2017), identities
stained with an aniline blue dye. Stained of S-RNase alleles are still difficult to discern. In
 10
Table 10.1 Identified S-genotypes in cultivated and wild pears
Species Cultivar S-genotype Cultivar S-genotype Cultivar S-genotype Cultivar S-genotype
P.  brestschneideri Baipisu S34Sn Dongguoli S12S35 Jinhua no.4 S13S18 Wuzihuang S16S28
Banjinsu S5S21 Donghuang S20S34 Jinli S1S1 Xiangchi S1S21
Baoshansu S1S21 Dongningwu S1S17 Jinzhui S21S34 Xiangchun S3S19
Bayuesu S3S16 Eli S13S34 Jiuquanmaili S6S17 Xinli no.1 S8S22
Bingtang S16S19 Enli S1S19 Liuban S17S19 Xuehua S4S16
Boshanchi S19S27 Esu S15S38 Liuleng S16S19 Yali S1S21
Self-incompatibility in Pear

Chili S1S19 Gaopingdaihuang S1S2 Lixianxinbapan SeSx Yanguangli S1S17

Daaoao S12S12 Guanyangxueli S18S27 Mili S19S29 Yaoxianyinli S21Sx
Daaotu S11S22 Haitangsu S1S12S19 Pingguoli S1S17 Yingzhiqing S12S12
Dacili S19S27 Hongpisu S12S26 Qingli S19S19 Youli S16S19
Dahebai S16S19 Hongtaiyang S8S35 Qingpisu S34Sn Yunnanbaozhu S22SX
Dalijitui S17S19 Huangxiang S4S27 Shuidonggua S15S45 Zhaoxiandayali S1S1S21S21
Damianhuang S1S19 Jinanxiaohuangli S1S12S19 Shuihongxiao S16S19 Zhuzuisu S19S22
Dangshansuli S7S34 Jinbangtou SmS12 Taihuangli S2S14 Zisu S19S34
Daqingpi S19S34 Jinchuizi S16S19 Tianshengfu S12S29
Dashuihe S7S19 Jinfeng S17S19 Tianyali S1S21
Dayali S1S1S21S21 Jinhua S3S18 Wushantangli S8S19
P. pyrifolia Aikansui S4S5 Huagao S3S9 Mazili S1S29 Wandaxingao S3S9
Amanogawa S1S9 Huali No.1 S1S3 Meigetsu S8S9 Waseaka S4S5
Atago S2S5 Huali No.2 S3S4 Meirensu S4S12 Weiningdaihuangli S3S37
Baozhuli S22S42 Huanghua S1S2 Mianli S19S41 Wenshanhongxueli S31S36
Baxing S4S5 Whangkeumbae S3S4 Mimauraaki S1S6 Whasam S3S5
Bayun S1S4 Huangli S22S34 Minibae S3S31 Xianhuang S3S5
Cangxixueli S5S15 Huangmi S1S6 Niitaka S3S9 Xinhang S1S3
Chihuali S26S15 Huangpishui S16S42 Nijisseiki S2S4 Xinxue S5S6
(continued)
183
Table 10.1 (continued)
184

Species Cultivar S-genotype Cultivar S-genotype Cultivar S-genotype Cultivar S-genotype

Chisui S1S2 Huiyanghongli S46S47 Okusankichi S5S7 Xishui S4S5
Chixianfeng S5aS18 Huoba S26S36 Osa-Nijisseiki S2S4SM Xiuyu S4S5
Choju S1S5 Hangqing S1S4 Pre-Kisui S3S4 Xizilü S1S4
Chojuro S2S3 Huobali S26S36 Qingchangshilang S2S3 Yanbianmingyueli S3Se
Chubixiang S1S15 Jiangdao S5S8 Qingcheng S4S5 Yaoxianhong S1S21
Chulüxia S3S4 Jinchuanyeshengli S5S13 Qinggoushageda S36Sd Yewang S3S9
Cuiguan S3S5 Jincunxia S1S6 Qingkui S1S3 Yuanxiang S15S16
Cuilü S3S4 Jingyu S4S24 Qingxiang S4S7 Yunnanhuangpishui S16S19
Cuixing S1S4 Jinqiuli S3S9 Qingyu S3S4 Yunnanmali no.1 S9S42
Cuyu S3S4 Jinshui No.1 S3S29 Qiubai S19S34 Yunnanmali no.2 S42Sx
Daqingli S1S3 Jinshui No.1 S5S29 Sanhua S2S7 Yunnanwumingli S22S29
Deshengxiang S3S29 Jinzhuguoli S3S19 Shinkou S4S9 Yushui S3S4
Duyi S1S2 kikusui S2S4 Shinsei S8S9 Zaoli18 S4S28
Fuyuanhuang S16S33 Kimizukawase S1S5 Shinseiki S3S4 Zaomi S19S29
Guiguan S2S16 Kisui S4S5 Shinshui S4S5 Zaomixingao S3S9
Hakutasei S22S34 Kosui S4S5 Shisho S3S9 Zaoshengchangshilang S2S4
Hangqing S1S4 Lijiangbaili S22S42 Shounan S1S3 Zaoshenghuangjin S3S4
Hongli S4S36 Longquansu S3S22 shusui S1S5 Zaoyu S1S2
Hongsucui S4S12 Lüyun S3S29 Suisho S3S9 Zhaori S4S5
Hongxiao S16S19 Mandingxue S4S15 Suomei S36S37 Zhenghedaxueli S13S43
Housui S3S5 Mantianhong S4S12 Taiwanmili S11S22
Huafeng S3S9 Maogongli S12S13 Tianchengzi S7S12
(continued)
S. Zhang and C. Gu
 10
Table 10.1 (continued)
Species Cultivar S-genotype Cultivar S-genotype Cultivar S-genotype Cultivar S-genotype
P. ussuriensis Anshan No.1 S13S31 Jiaihuatubianti S1S17 Pitai S22S43 Tangli S27S30
Baibalixiang S19S31 Jianbali S12S30 Qinglong S2S3 Xiangshui S17S31
Balixiang S19S30 Jianbazi S27Sh Ruanbazi S16S36 Xiaoxiangshui S29S34
Dananguo S13S34 Jiazhibazi S5S19 Ruan’erli S17S31 Xiaoxiangshuiyabian SeSx
Fuanjianba S16S22 Jinbaili S16S30 Saozhoumiaozi S15S26 Xiehuatian S29S34
Ganguxiangshui SeSx Lanzhouruan’erli SdS12 Shanli S13S34 Xingchengxiehuatian S17S31
Self-incompatibility in Pear

Hanhong S27S34 Liaoyangdaxiangshui S16S12 Shanli no.2 S8S27 Yaguangli S19S30

Hanxiang S12S31 Longxiang S16S42 Shanli no.3 SbS41 Yanbiandaxiangshui S12S16
Honghuagai S19S32 Maili S31S40 Shanli no.4 ScS42 Yanbianxiehuatian S17S31
Huagai S34Sd Matihuang S16S19 Shanli no.5 S4S42 Yeshengleixingshanli1 S8S27
Huagaiwang S31S31S34S34 Nanguoli S11S17 Shanyali S30S36 Youhong S13S34
Huangjinduima S19S29 Neimenggushanli S29S41 Suandali S3S29 Zaobai S19S42
Hululi SaSb Pingxiangli S31Sd Suanliguozi S19S41
P. sinkiangensis Ganguheili S16S54 Huangmian S1S12 Lanzhouhuachangba S19S22 Qipanxiangli S22S28
Naixiteamuti S19S28 Jiuquanchangbali S6S22 Linxiadadiaodan S26Sx Seerkefu S22S28
Aolian SpS32 Jiuquanmaili S6S17 Linxiahuangma S3Se Sha-01 Xiangli S22S28
Ganguhongxia S16Sx Kanglebaiguo SbSi Linxiasala S16Se Sierkefuli S22S28
Guidechangba S19S22 Kangleganchangba S22Sd Linxiaxiangba S12S21 Wudoutianli S26Si
Hezhengganchangba S22Sd Kanglesumuli S1Sh Linyaomatianli S22Sd Xinjianghuangli S22S28
Hongnahe S22S28S40 Kuerle S22S34 Lǜjuju S22S28 Yilihongjuju S22S28
Huachangba S19S22 Kuerlexiangli S22S28 Manluwowoguo S12S21 Zaoshujuju S22S28
Huangjuju S22S34 Kuikejuju S22S28 Moli S26Sb Zhangyechangba S19S22
Huangma S31S40 Kunqieke S19S28 Qingmian S1S18
(continued)
185
Table 10.1 (continued)
186

Species Cultivar S-genotype Cultivar S-genotype Cultivar S-genotype Cultivar S-genotype

P. communis Abbé Fétel S104S105 Conference S108S119 Idaho S101S119 Rosired Bartlett S101S102
Akça S102S109 Coscia S103S104 Jeanne d’Arque S101S104 Rosmarie S101S116
Alexandrine Douillard S103S104 Covert S101S118 Joséphine de Malines S102S104 Royal Red S108S114
Angélys S105S119 Dagan S101S104 Kaiser S107S114 Saint Mathieu S114S116
Ankara S103S119 Dana’s Hovay S101S111 Kieffer S102S119 Santa Maria S102S103
Aurora S101S105 Delbard première S101S109 Kalle S101S108 Seckel S101S102
Ayers S101S102 Delfrap S101S109 Koonce S102S105 Seigneur d’Espéren S101S102
Ballad S101S119 Délices S101S102 Koshisayaka S102S119 Serenade S101S108
d’Hardenpont
Bartlett S101S102 Devoe S108S118 La France S101S119 Sierra S101S108
Bautomne S101S108 Docteur Jules Guyot S101S105 Lawson S115S117 Silver Bell S110S119
Besi de Saint-Waast S101S118 Doyenné d’hiver S101S119 Le Lectier S104S118 Red Jewell S101S102
Beurré Bosc S107S114 Doyenné du Comice S104S105 Limonera S101S105 Red Clapp’s S101S108
Beurré Clairgeau S105S118 Doyenné Gris S102S108 Louise Bonne S101S102 Red Hardy S108S114
d’Avranches
Beurré d’Anjou S101S114 Duchesse S101S105 Magness S101S105 Reimer Red S104S114
d’Angouleme
Beurré de S102S106 El Dorado S101S107 Marguerite Marillat S102S105 Sirrine S101S107
l’Assomption
Beurré Giffard S101S106 Eletta Morettini S105S114 Max Red Bartlett S101S102 Spadona S101S103
Beurré Hardy S108S114 Emile d’Heyst S102S119 Maxine S101S113 Spadona estiva S101S103
Beurré Jean Van Geert S102S104 Ercolini S103S104 Michaelmas Nelis S102S107 Spadoncina S102S103
Beurré Lubrum S101S104 Espadona S101S110 Moonglow S101S114 Star S101S108
(continued)
S. Zhang and C. Gu
 10
Table 10.1 (continued)
Species Cultivar S-genotype Cultivar S-genotype Cultivar S-genotype Cultivar S-genotype
Beurré Precoce S101S103 Ewart S102S114 Napoleon S101S102 Starking Delicious S101S113
Morettini
Beurré Superfin S101S110 Fertility S107S118 Norma S101S104 Starkrimson S101S108
Blickling S102S110 Flemish Beauty S101S108 Nouveau Poiteau S107S114 Summer Doyenne S101S106
Bon Rouge S101S102 Fondante Thirriot S101S103 Old Home S101S113 Sweet Blush S101S119
Blanquilla S101S103 Forelle S101S116 Olivier de Serres S101S110 Tosca S102S104
Self-incompatibility in Pear

Bon-Chrétien d’Hiver S101S118 French Bartlett S101S105 Onwards S101S104 Triomphede Vienne S105S110
Bristol Cross S102S119 Garbar S107S115 Orient S101S102 Turnbull Giant S104S113
California S101S104 General Leclerc S102S118 Ovid S102S118 Tyson S101S105
Canal Red S102S104 Gentile S101S106 Packham’s Triumph S101S103 Urbaniste S104S119
Cascade S101S104 Glou Morceau S104S110 Passe Crassane S110S119 Verdi S101S119
Chapin S102S115 Grand Champion S101S104 Pera d’Agua S101S102 William Precoce S101S105
Charles Ernest S105S110 Harrow Crisp S101S105 Pierre Cornelle S101S118 William’s S101S102
Clapp’s Favorite S101S108 Harrow Delight S101S105 Pierre Tourasse S102S105 William’s S101S102
Bon-Chrétien
Clapp’s Rouge S101S108 Harrow Sweet S102S105 Precoce di Fiorano S101S103 Washington S101S103
Colorée de Juillet S101S115 Hartman S101S104 Precoce du Trevoux S101S102 Wilder S101S111
Comte de Flandre S102S111 Harvest Queen S101S102 President Héron S110S118 Winter Cole S101S107
Comte de Lambertye S102S110 Highland S101S104 Rocha S101S105
Concorde S104S108 Honey Sweet S102S104 Red Anjou S101S114
Condo S104S119 Howell S101S104 Rogue Red S105S114
(continued)
187
Table 10.1 (continued)
188

Species Cultivar S-genotype Cultivar S-genotype Cultivar S-genotype Cultivar S-genotype

Interspecific 11-11 S2S26 Huasu S5Sd Qiyuesu S4Sd Yaqing S4S17
hybridization
2-15 S4S12 Jimi S1S16 Shenbuzhi S5Sd Zaoguan S4S17
Beifeng S4Sa Jinmili S21S28 Xingcheng2-23 S1S8 Zaomeisu S3S35
Bishan no.2 S4S16 Jinshuisu S4S21 Xinli S28Sd Zaosu S22S35
Chaoxianyangli SeS3 Jinxiang S34S37 Xinya S4S34 Zaosuwei S1Sd
Chikusui S3S4 Jinxiangshui S1Si Xuefang S4S16 Zaoxiangshui S26S42
Dongmi S1S42 Ningmenghuang S31S32 Xuefen S3Sx Zhongai No.1 S1S17
Hongxiu no.2 S1S12 Nongjiaxingao S3S9 Xuefeng S4S16 Zhongai No.2 S19S34
Huajin S4S4 Pingboxiang S1S8 Xueqing S3S16 Zhongli No.1 S4S35
Huangguan S4S16 Qinghua S1S4 Xueying S3S16 Zhongli no.2 S4S31
Landrace Danze S3S5 Hongjujuli S22S28 S7 S1S17 Wucang S2S3
Diaodan SdSe Kuitian SdSe Shageda S36Sd Xumo S2S5
Douli S30S31 Lingwuduli S27S36 Shunxiang SdSe Yucui S2S4
Heli S19S29 S5 S17S31 Xingyeli S22Sc
S. Zhang and C. Gu
10 Self-incompatibility in Pear 189

Table 10.2 Renumbering and integration of S-RNase alleles in Asian Pyrus species
New allelic Former allelic Pyrus species Genbank accession no.
designation designation
S6 S6 P. pyrifolia AB002142.1
S33 P. ussuriensis DQ138081.1
S7 S7 P. pyrifolia AB002143.1
S27 P.  bretschneideri EF643640.1
S8 S8 P. pyrifolia AB104908.1
S28 P.  bretschneideri EU375364.1, AY562394.1
S28 P. sinkiangensis EF566872.1
S34 P. pyrifolia DQ224345.1
S12 S12 P. pyrifolia EU117115.1, AB426604.1, AY249427.2,
HM047239.1
S12 P.  bretschneideri EU081889.1
S36 P. pyrifolia DQ417607.1
S13.1 S13 P.  bretschneideri DQ414812.1
S13.2 S13 P. pyrifolia AY249428.2, HM047240.1
S15 S15 P. pyrifolia EF643630.1, AY249430.2
S38 P. pyrifolia DQ666956.1
S16 S16 P. pyrifolia AY249431.2
S16 P.  bretschneideri DQ991388.1, EF643635.1
S31 P. pyrifolia DQ072113.1
S17 S17 P.  bretschneideri EU101466.1, AY249432.3
S34 P. pyrifolia DQ269500.1
S34 P.  bretschneideri DQ414813.1, DQ494676.1
S18 S18 P.  bretschneideri EF643636.1, AY249433.2
S39 P. pyrifolia DQ666957.1
S20 S20 P.  bretschneideri EU360894.1, AY250988.2
S29 P.  bretschneideri EU101462.1, AY601098.1
S31 S31 P.  bretschneideri DQ124366.1
S32.1 S32 P. pyrifolia DQ072114.1
S32.2 S32 P. ussuriensis EU336979.1, DQ124367.1
S38 S38 P.  bretschneideri EF643631.2, DQ839239.1
S39 S39 P.  bretschneideri EU336980.1, DQ995285.1
S42 S33 Inter-specific DQ082897.1
hybridization
S42 P. ussuriensis EF689006.1, EF088497.1, EF643637.1
S42 P.  bretschneideri EF689007.1

fact, re-identification of these alleles should be exceptionally high identities should also be tes-
performed in the same pear cultivars, as it is ted using cross-pollination of pear cultivars to
necessary. Moreover, several S-RNase alleles of determine functions of these alleles, such as
190 S. Zhang and C. Gu

those of S1-RNase in P. pyrifolia and S111-RNase 10.3.2 Pollen-S Determinants

in P. communis, which have yielded three dif-
ferent residues. Following Northern and Southern blot analyses
of pear styles, it has been observed that the S4-
10.3.1.2 Structural Features of S-RNase RNase in self-compatible pear cultivars is absent,
Alleles but this absence does not influence functionality
Pyrus S-RNase alleles have five conserved of pollen (Sassa et al. 1997). Thus, the likelihood
regions, including C1, C2, C3, RC4, and C5, that S-RNase controls pollen SI is dismissed.
along with a relative hypervariable (RHV) re- Therefore, what is the gene(s) controlling pollen
gion. Furthermore, amino acid sequences of SI? A series of propositions have been made.
these alleles contain cysteine and histidine resi- First, this gene(s) should be specifically expres-
dues that play important roles in S-RNase func- sed in the pollen, and not in other tissues,
tions (Fig. 10.2). The only RHV located between including the style. Second, this gene(s) should
the conserved C2 and C3 regions in Pyrus is be tightly linked with an S-RNase gene and will
different from the two hypervariable regions, not undergo recombination, as SI is well main-
HVa and HVb, found in Solanaceae and Plan- tained in gametophytic species. Third, this gene
taginaceae, although they have similar functions (s) must have high levels of sequence polymor-
in enriching indels and substitutes. Similar to phisms so that it could be specifically recognized
RNase T2 and RNase Rh, RHV sequences have by stylar S-RNases. Based on these propositions,
lower identities among different S-RNase alleles, flanking sequences around S-RNase alleles have
as well as catalytic histidine residues. Besides been analyzed using genome sequencing. An F-
RHVs, other variable regions have also been box gene is detected within the S-locus, and it has
detected between the conserved C1 and C2 been proposed as a good candidate gene con-
regions and the upstream region of the conserved trolling pollen SI in Prunus species (Entani et al.
C5 region, which are likely associated with S- 2003; Ushijima et al. 2003).
RNase allele-specificity. In pear, the pollen-S determinant has been
Pyrus S-RNase alleles contain only a single investigated using homologous cloning based on
intron, while Prunus S-RNase alleles have two conserved regions of F-box genes present in the
introns. This intron is inserted into the RHV S-locus of Malus (apple) and Prunus species.
region and exhibits strong length and sequence A series of two S-locus F-box genes have been
polymorphisms. Depending on the size of this detected that are specifically expressed in the
intron, ranging from 99 to 1,709 bp, a pollen and designated as S-locus F-box brother
PCR-based analysis could be used to distinguish (SFBB) genes (Sassa et al. 2007). Initially, three
different S-RNase alleles on either agarose or SFBB genes, SFBB4-a, SFBB4-b, and SFBB4-c,
polyacrylamide gels. This intron is subject to and an additional three genes, SFBB5-a, SFBB5-b,
mutations, thus allowing S-RNase alleles to and SFBB5-c, are found to co-segregate with S4-
effectively maintain their GSI functions. RNase and S5-RNase, respectively (Sassa et al.

Fig. 10.2 Structures of S-RNase alleles in Pyrus and Prunus

10 Self-incompatibility in Pear 191

2007). Of these six SFBB genes, SFBB4-b has SFBB2−u5, SFBB2−d1, SFBB2−d2, SFBB2−d3,
89.4% amino acid sequence identity to SFBB5-b, SFBB2−d4, and SFBB2−d5 (Okada et al. 2011).
while high amino acid identities are detected Furthermore, amino acid identities among these
between each of SFBB4-a and SFBB5-a (96.4%), SFBB genes range between 67.1 and 93.1%, thus
and of SFBB4-c and SFBB5-c (99.0%). This revealing high sequence polymorphisms. It is
finding suggests that SFBB-b is more likely to be noteworthy to point out that it is puzzling as to
a pollen-S determinant than either SFBB-a or which and how many SFBB genes are in fact
SFBB-c. involved in the SI reaction (Fig. 10.3).
To test for polymorphism of these SFBB genes, In Prunus, a gene controlling the pollen-S de-
SFBB-c genes have been isolated from different S- terminant has been identified, wherein an SFB
loci. Alignment of amino acid sequences showed allele is stably and tightly linked to the S-RNase
that these SFBB-c genes have lower sequence allele in any S-locus (Ushijima et al. 2003).
polymorphisms, with sequence identities ranging Therefore, the stability of SFBB genes in several
from 97.5 to 99.7% (Kakui et al. 2007). More- Pyrus S-loci has been investigated. Based on the
over, phylogenetic analysis of SFBB genes iso- classification of SFBB genes around S-RNase, a
lated from pear and apple has revealed that all series of primer sets have been designed for each
SFBB-c genes are clustered together, while SFBB-a class of SFBB genes (Kakui et al. 2011). As
and SFBB-b genes are distributed in different expected, a large number of newly SFBB genes
groups (Okada et al. 2011). Thus, the SFBB-c gene have been isolated from different S-loci. A phy-
is not deemed as the pollen-S determinant. logenetic analysis has revealed that genes SFBB2
−d1
In other efforts to identify the pollen-S deter- , SFBB2−d2, SFBB2−d4, and SFBB2−d5 are only
minant, two pear bacterial artificial chromosome present in the S2-locus, while SFBB4−d2 and
(BAC) libraries have been constructed containing SFBB4−u4 are present in the S4-locus, and no
S4-RNase and Ssm 4 -RNase alleles. It is found that orthologous genes have been found in other S-
the Ssm
4 -locus has a 236 kb deletion, along with loci (Fig. 10.3). Therefore, none of these genes is
34 open reading frames (ORFs), when compared the pollen-S determinant. Furthermore, additional
to that of the S4-locus (Okada et al. 2008). As the detected SFBB genes have been classified into
S4-haplotype of the pear cultivar Osa-Nijisseiki eight types, and designated as SFBB1 to SFBB8
lacks a pistil function, but retains a pollen func- (Kakui et al. 2011). Of these, SFBB8 is coded by
tion (Sato 1993), all 34 ORFs do not serve as an SFBB-c protein, while SFBB1 contains an
pollen-S determinants. Within the S4-locus, a S4F-box 0/SFBB4−d1 that is lacking in the Ssm 4 -
total of six SFBB genes have been detected haplotype, along with a truncated SFBB protein
within a 649 Kb region around the S4-RNase. found in the S5-haplotype. Due to these detected
Upstream SFBB genes are designated as SFBB4 S-haplotypes of the pollen, which have normal
−u1
, SFBB4−u2, SFBB4−u3, and SFBB4−u4, while functions in SI reactions, it is proposed that genes
downstream genes are designated as SFBB4−d1 coded by SFBB1, SFBB4, and SFBB8 are not
and SFBB4−d2 (Okada et al. 2011). In contrast, 10 likely to be involved in the pollen-S determinant.
SFBB genes are detected within a 378 Kb region Thus, the candidate gene(s) controlling the pollen
around the S2-RNase, and these are designated as SI is likely to be present in at least one of the
SFBB2−u1, SFBB2−u2, SFBB2−u3, SFBB2−u4, SFBB2, SFBB3, SFBB5, SFBB6, and SFBB7

Fig. 10.3 Pear SFBB genes around S-RNase within S2 and S4-loci
192 S. Zhang and C. Gu

types. With the development of gene editing and by gradual increases in these levels concomitant
genetic transformation in pear, it is anticipated with flower development. These findings indi-
that the gene(s) related to SI will be resolved in cated that S4-RNase had similar, but time-lagged
the near future, and that the pollen-S determinant expression patterns in cv. Osa-Nijisseiki com-
(s) will then be confirmed. pared to those observed in cv. Nijisseiki. More-
over, coded protein levels in cv. Osa-Nijisseiki at
2 DAA corresponded to those detected in cv.
10.4 The Breakdown Nijisseiki earlier than 4 DBA (Hiratsuka et al.
of Self-incompatibility 1999). Thus, it has been proposed that the
breakdown of SI in cv. Osa-Nijisseiki was likely
10.4.1 Stylar Mutants attributed to lower S4-RNase levels present in its
pistils.
10.4.1.1 Absence of the S4-RNase Allele Subsequently, nucleotide sequences of S2- and
in Cultivar Osa-Nijisseiki S4-RNase alleles were determined in stylar cDNAs
The pear cultivar Osa-Nijisseiki (P. pyrifolia) is a of cv. Nijisseiki (Norioka et al. 1995), but the S4-
bud mutant of the self-incompatible pear cultivar RNase allele could not be amplified from stylar
Nijisseiki. Reciprocal crosses have demonstrated cDNAs of cv. Osa-Nijisseiki. It was proposed that
that the pollen of cv. Osa-Nijissiki is the S4-RNase allele was unsuccessfully transcribed
self-compatible. Furthermore, it is found that in styles of cv. Osa-Nijisseiki (Norioka et al.
pollen of cv. Osa-Nijisseiki is cross-incompatible 1996). This finding was further supported by
with pistils of cv. Nijisseiki, while the reciprocal Northern blot analysis of S-RNases revealing that
cross has demonstrated that the pollen of cv. S2-RNase could be detected in both cvs.
Nijisseiki is compatible with pistils of cv. Osa-Nijisseiki and Nijisseiki; whereas, S4-RNase
Osa-Nijisseiki (Hirata 1989). This finding sug- was only detectable in cv. Nijisseiki (Sassa et al.
gests that the breakdown of SI in cv. 2007). To assess whether or not S4-RNase was
Osa-Nijisseiki has resulted from a stylar mutation. absent from the genome of cv. Osa-Nijisseiki,
To better understand the nature of the mutation probes for S2- and S4-RNase alleles were used in
in pistils of cv. Osa-Nijisseiki, an conducting Southern blot analyses. Surprisingly,
IEF/SDS-PAGE electrophoresis has been con- no hybridization signal was detected for the S4-
ducted to detect S-RNase expression. It was RNase probe in cv. Osa-Nijisseiki, while a signal
found that S2-RNase levels in cvs. Osa-Nijisseiki was detected for the S4-RNase probe in cv. Nijis-
and Nijisseiki were similar; whereas, S4-RNase seiki. In contrast, hybridization signals for the S2-
was weakly expressed in cv. Osa-Nijisseiki RNase probe were detected in both cvs.
compared to that in cv. Nijisseiki (Sassa et al. Osa-Nijisseiki and Nijisseiki (Sassa et al. 1997).
1993). These findings were further confirmed by Therefore, the S4-RNase was likely absent from the
later studies conducted by Wu et al. (2007) and genome of cv. Osa-Nijisseiki.
Zhang et al. (2000). Subsequently, expression of To further confirm the absence of S4-RNase in
S-proteins was analyzed in pear flowers at dif- cv. Osa-Nijisseiki, BAC libraries were con-
ferent stages of development (Zhang et al. 2000). structed for genomes of cvs. Osa-Nijisseiki and
It was found that S4-RNase was detectable in Nijisseiki. Following identification of BAC
pistils of cv. Nijisseiki at 8 days before anthesis contigs around the S4-RNase gene,
(DBA), and it was continuously synthesized until chromosome-walking was conducted to assem-
2 days after anthesis (DAA), with about 4.7-fold ble these overlapping BAC contigs and then used
increase in levels during these 10 days. In con- these for sequencing. Results of sequencing these
trast, S4-RNase was not detected in pistils of cv. BAC contigs revealed that a 236 kb region was
Osa-Nijisseiki earlier than 6 DBA, and only low deleted from the genome of the spontaneous
levels were detected at 4 DBA. This was followed mutant cv. Osa-Nijisseiki when compared to that
10 Self-incompatibility in Pear 193

Fig. 10.4 Nucleotide sequence analysis of a deletion junction and a deleted region in S4-haplotypes of cvs.
Osa-Nijisseiki and Nijisseiki

of its original self-incompatible cv. Nijisseiki  ‘Yali’ has yielded a 78.0% fruit set, thereby
(Okada et al. 2008). More importantly, this indicating that the pollen of ‘Yali’ is compatible
deleted region would have likely contained the S4- with pistils of ‘Yanzhuang’ (Li et al. 2009).
RNase allele (Fig. 10.4). Thus, it has been further Based on these findings, it has been determined
confirmed that S4-RNase was absent in cv. that the pistil and pollen of ‘Yanzhuang’ are
Osa-Nijisseiki, thereby resulting in an S4-haplo- functionally abnormal and normal, respectively.
type of a pistil that was functionally abnormal. The S-genotype of ‘Yali’ has been initially
identified as S21S34, as the nucleotide sequence of
10.4.1.2 A Likely Low Level the S34-RNase allele is identical to that of the S17-
of Expression of S21- RNase allele (Wang et al. 2017). Therefore, S-
RNase in Cultivar genotypes of ‘Yali’ and ‘Yanzhuang’ have been
Yanzhuang revised as S17S21. Genetic analysis has revealed
The pear cultivar Yanzhuang is a spontaneous that individuals in a self-pollinated progeny are
mutant of the self-incompatible cultivar Yali genotyped as S21S21 and S17S21 with a 1:1 ratio
(P.  bretschneideri). Almost 72.0% of ‘Yanz- (v2 = 0.02 < 0.05). This has indicated that the
huang’ fruit set is a result of self-fertilization, and S21-haplotype can be inherited in a self-pollinated
it displays a strong self-compatibility (SC). To progeny. Therefore, the S21-haplotype of the pistil
determine which of the reproductive tissues, in ‘Yanzhuang’ is deemed functionally abnormal.
either the pistil or pollen, have undergone To further explore the underlying reason(s)
mutation, reciprocal crosses have been made for these findings, expression levels of the S21-
between cvs. Yanzhuang and Yali (Li et al. RNase allele have been tested in pistils of both
2009). It is observed the cross of ‘Yali’  ‘ ‘Yali’ and ‘Yanzhuang’. Unfortunately, the S21-
Yanzhuang’ has only an 8.5% fruit set, indicating RNase allele is expressed at almost identical
that the pollen of ‘Yanzhuang’ is levels in pistils of ‘Yali’ and ‘Yanzhuang’, as
cross-incompatible with pistils of ‘Yali’. On the well as those of the S17-RNase allele. Thus, the
other hand, the reciprocal cross of ‘Yanzhuang’
194 S. Zhang and C. Gu

S21-RNase allele is normally expressed in pistils found to be compatible with ‘Zaoguan’ (Qi et al.
of ‘Yanzhuang’. 2011a, 2011b). Therefore, the pistil and the
SDS-PAGE and protein profiles have also been pollen of ‘Zaoguan’ are deemed functionally
used to assess levels of S21-RNase expression in abnormal and normal, respectively.
pistils of ‘Yali’ and ‘Yanzhuang’. It has been To determine which of the S-RNase alleles has
found that SDS-PAGE could not detect any dif- experienced a loss of function in an SI reaction,
ferences in protein profiles of S21-RNase between self- and cross-pollinated progenies of ‘Zaoguan’,
these two cultivars. However, protein profiles of ‘Xinya’, and ‘Yaqing’ are S-genotyped by poly-
styles of ‘Yanzhuang’ have revealed presence of a merase chain reaction (PCR) using allele-specific
single faint band, while those of pistils of ‘Yali’ primers. Genetic analysis has revealed that indi-
have revealed presence of two different bands. viduals in self-pollinated progenies of ‘Zaoguan’
These findings suggest that the S21-RNase protein are S-genotyped as S4S17 and S17S17, with a 1:1
is expressed at lower levels in ‘Yanzhuang’ than segregation ratio (v20.05,1 = 2.03 < 3.84). Likewise,
in ‘Yali’, thus contributing to the breakdown of SI individuals in two cross-pollinated progenies of
in ‘Yanzhuang’. ‘Zaoguan’  ‘Xinya’ and ‘Zaoguan’  ‘Yaqing’
This further begs the question as to what is the have also been genotyped as S4S17 and S17S17, and
reason for the observed low levels of expression yielding 1:1 segregation ratios of v20.05 = 0.41 and
of S21-RNase in pistils of ‘Yanzhuang’? Align- v20.01 = 0.87 < 3.84, respectively. These findings
ments of nucleotide and amino acid sequences of suggest that the S17-haplotype of pistils of ‘Zao-
S21-RNase allele in ‘Yanzhuang’ and ‘Yali’ have guan’ is functionally abnormal. However, the S17-
detected non-synonymous substitution(s) located RNase of ‘Zaoguan’ has identical amino acid
within the conserved C2 region (Wang et al. sequences to those of ‘Xinya’ and ‘Yaqing’, thus
2017). In this scenario, does this non-synonymous indicating that the S17-RNase of ‘Zaoguan’ has a
substitute(s) changes the function of the S21- complete gene structure. Therefore, what is the
RNase allele? These questions deserve further reason for the observed SI breakdown? Is it an
attention in future studies. issue of transcript levels? To address these ques-
tions, quantitative reverse transcription (qRT)-PCR
10.4.1.3 Post-Transcript Modification has been conducted in styles of ‘Zaoguan’, ‘Xinya’,
of S17-RNase (S34-RNase) and ‘Yaqing’. It is observed that each of S17-RNase
in Cultivar Zaoguan and S4-RNase have similar levels of expression
The pear cultivar Zaoguan (P.  bretschneideri), among these three pear cultivars. Thus, the S17-
derived from a cross between cvs. Yali and RNase is deemed to be normally transcribed in
Qingyu, has an 86.0% fruit set following these cultivars. Therefore, this begs the question as
self-pollination, thus demonstrating a strong to whether or not the function of the S17-RNase is
self-compatibility trait (Qi et al. 2011a, 2011b). blocked at the translational level? To address this
The current assigned S-genotype of cv. Zaoguan question, S-RNase proteins are extracted from
is S4S17, while the previous S-genotype desig- pistils of ‘Zaoguan’, ‘Xinya’, and ‘Yaqing’, and
nation has been S4S34. To determine which of the then subjected to two-dimensional gel elec-
reproductive tissues, either the pistil or pollen, trophoresis (2D-PAGE). It is found that both S4-
must have undergone a mutation, the two RNase and S17-RNase proteins are detected in both
self-incompatible cultivars Xinya and Yaqing ‘Xinya’ and ‘Yaqing’, while S17-RNase is not
have been selected from the progeny of ‘Yali’ detected in the pistils of ‘Zaoguan’ (Fig. 10.5).
‘Qingyu’. Both ‘Yali’ and ‘Qingyu’ have the Therefore, this indicates that S17-RNase (S34-
same S-genotypes as that of ‘Zaoguan’, and have RNase) is unsuccessfully translated to its corre-
been used in crosses with ‘Zaoguan’. Interest- sponding S-glycoprotein in ‘Zaoguan’. Taken
ingly, it is observed that the pollen of ‘Zaoguan’ altogether, it is proposed that the breakdown of SI
is cross-incompatible with the pistils of ‘Xinya’ in ‘Zaoguan’ is attributed to post-transcript modi-
and ‘Yaqing’, while the reciprocal crosses are fication of S17-RNase.
10 Self-incompatibility in Pear 195

Fig. 10.5 2D-PAGE profiles of style extracts of cvs. Zaoguan, Xinya, and Yaqing

10.4.1.4 Transcript Modification primers (Sanzol 2009). Genetic analyses have

of S21-RNase revealed that individuals in the cross-pollinated
in European Cultivars progeny of ‘Williams’  ‘Abugo’ are assigned
Abugo and Ceremeño S1S10, S1S21, S2S10, and S2S21 genotypes, thus
The following European pear cultivars, Abugo elucidating that S10- and S21-haplotypes are
and Ceremeño (P. communis), exhibit 74.1 and inherited in this progeny. Furthermore, individ-
65.4% fruit set due to self-pollination, respec- uals in the self-pollinated progeny of ‘Abugo’ are
tively, thus displaying strong self-compatibility genotyped as S10S21 and S21S21, with a 1:1
(Sanzol 2009). The assigned S-genotypes of observed segregation ratio (v2 = 0) (Sanzol
‘Abugo’ and ‘Ceremeño’ are S10S21 and S21S25, 2009); whereas, individuals in the
respectively. cross-pollinated progeny of ‘Abugo’  ‘Delbard
To determine which of the reproductive tissues, Esquise’ (S4S21) are genotyped as S4S10, S10S21,
either the pistil or pollen, must have undergone S4S21, and S21S21, with a 10:14:6:8 segregation
a mutation, the following two self-incompatible ratio (v20.05 = 2.4). These findings suggest that
cultivars, ‘Williams’ (S1S2) and ‘Passe Crassane’ the S21-haplotype of the pistil in ‘Abugo’ can
(S10S21), were used in cross-hybridizations (Sanzol accept pollen of the same S-genotype. Taken
2009). When ‘Abugo’ was used as a pollinator and together, it is proposed that the S21-haplotype of
crossed with ‘Williams’, a 29.1% fruit set was the pistil of ‘Abugo’ is functionally abnormal.
obtained, thus demonstrating cross-compatibility, However, does this imply that the S21-haplo-
and indicating that the pollen of ‘Abugo’ was type is also disordered in ‘Ceremeño’? To assess
normally functional for successful sexual fertil- this, several cross- and self-hybridizations have
ization. When ‘Passe Crassane’ was used as a been made (Sanzol 2009). Genetic analyses have
pollinator and crossed with ‘Abugo’, only 18.5% revealed that individuals in the self-pollinated
fruit set was obtained, thus demonstrating progeny of ‘Ceremeño’ are assigned S21S21 and
cross-incompatibility. These findings suggested S21S25 genotypes, thus indicating that the S21-
that pistils of ‘Abugo’ were functionally abnormal haplotype of the pollen is self-compatible.
and contributing to the observed GSI reaction. Genetic analyses of S-genotypes in
To assess the functional abnormality of the S- cross-pollinated progenies have revealed that
RNase allele, self- and cross-pollinated progenies individuals in the progeny of ‘Wil-
of ‘Williams’, ‘Abugo’, and ‘Delbard Esquise’ liams’  ‘Ceremeño’ are genotyped as S1S21,
are S-genotyped by PCR using allele-specific S1S25, S2S21, and S2S25, thus indicating that S21-
196 S. Zhang and C. Gu

and S25-haplotypes are inherited in this progeny whereas, in the reciprocal cross, 78.0% fruit set is
(Sanzol 2009). On the other hand, individuals in obtained, thus displaying strong
the cross-pollinated progeny of ‘Passe Cras- cross-compatibility (Li et al. 2009). This indi-
sane’  ‘Ceremeño’ are genotyped as S10S25 and cates that the pollen of ‘Jinzhui’ is functionally
S21S25, thus indicating that the S21-haplotype of abnormal.
the pollen in ‘Ceremeño’ has a normal function; To further study the functionally abnormal S-
whereas, individuals in the cross-pollinated pro- haplotype(s) in the pollen of ‘Jinzhui’, S-geno-
geny of ‘Ceremeño’  ‘Passe Crassane’ are types have been identified in self- and
genotyped as S10S21, S21S21, S10S25, and S21S25, cross-pollinated progenies (Li et al. 2009).
thus revealing that pistils of ‘Ceremeño’ are Genetic analyses have identified that 29 indi-
compatible with the S21-haplotype of pollen in viduals in a self-pollinated progeny of ‘Jinzhui’
the self-compatible ‘Passe Crassane’ (Sanzol are genotyped as S17S17 and S17S21, thus sug-
2009). Therefore, the S21-haplotype of pistils of gesting that it is only the S17-haplotype that is
‘Ceremeño’ is functionally abnormal. functionally abnormal (Li et al. 2009). However,
Expression of S21-RNase in pistils of ‘Abugo’, when the number of individuals is expanded to
‘Ceremeño’, and ‘Passe Crassane’ was deter- include a population of 94, these individuals are
mined using PCR analysis (Sanzol 2009). It was genotyped as S17S17, S17S21, and S21S21, which is
found that S21-RNase could not be detected in the similar to genotyping results obtained for indi-
pistils of both ‘Abugo’ and ‘Ceremeño’, but it viduals in the cross-pollinated progeny of
was detected in the pistils of ‘Passe Crassane’. ‘Yali’  ‘Jinzhui’. This outcome suggests that
This finding suggested that the breakdown of SI both S17- and S21-haplotypes are functionally
was attributed to abnormal expression of S21- abnormal (Wu et al. 2013).
RNase in the pistils of both ‘Abugo’ and ‘Cere- Subsequently, pollen grains and pollen tubes
meño’. Alignments of nucleotide sequences of ‘Yali’ and ‘Jinzhui’ have been grown in vitro
between normal and abnormal expressed S21- to further elucidate the viability of these tissues,
RNases identified three non-synonymous substi- and it is found that some pollen grains of ‘Jinz-
tutes in coding sequences, a retrotransposon hui’ are aborted (Wu et al. 2013). However, this
inserted within an intron, along with several observation is not sufficient to explain the
point mutations and indels found within the self-compatibility of ‘Jinzhui’. As it is highly
3’UTR (Sanzol 2009). Thus, it has been pro- unlikely that two S-haplotypes of pollen must
posed that the functionally abnormal So21-RNase have undergone simultaneous mutations, it is
was attributed to these observed mutations. proposed that it is more likely that a mutated
modifier, located outside of the S-locus, is the
one that takes part in such an SI reaction. In a
10.4.2 Pollen Mutants similar study involving apricot cultivars, it has
been found that the breakdown of SI may be
The pear cultivar Jinzhui is a spontaneous mutant caused by an M-locus, which is different from the
of the self-incompatible cultivar Yali, with a S-locus (Wu et al. 2011; Zuriaga et al. 2012).
72.0% fruit set, thus demonstrating strong
self-compatibility. Similar to ‘Yali’ and ‘Yanz-
huang’, the S-genotype of ‘Jinzhui’ is S17S21, 10.5 Polyploidy
although it has been previously assigned an
S21S34 genotype (Zhang et al. 2007). To assess The pear cultivar Sha01 is a spontaneous mutant
the mechanism of SI breakdown in ‘Jinzhui’, the of the self-incompatible cultivar Kuerlexiangli
pear cultivar Yali has been used in crosses with (P. sinkiangensis), genotyped as an S22S28, with
‘Jinzhui’. It is observed that the pollen of ‘Yali’ an 84.0% fruit set, and displaying a strong
is cross-incompatible with the pistils of ‘Jinzhui’; self-compatibility (Heng et al. 2011). To test for
10 Self-incompatibility in Pear 197

the breakdown of SI, ‘Kuerlexiangli’ is used in haplotypes in the pistils of ‘Sha01’ have the same
crosses with ‘Sha01’. When ‘Kuerlexiangli’ is allelic sequences and almost identical levels of
used as a pollinator, fruit set is 4.0%, thus expression to those of ‘Kuerlexiangli’ in a GSI
demonstrating cross-incompatibility; however, in reaction. Thus, this excludes the possibility that
the reciprocal cross, fruit set is 84.0%, thereby the observed cross-incompatibility between
demonstrating strong cross-compatibility (Qi ‘Kuerlexiangli’ and ‘Sha01’ results from low
et al. 2011a, 2011b). These findings reveal that levels of expression of S-RNase alleles in the
the pistil and the pollen of ‘Sha01’ are func- pistils of ‘Kuerlexiangli’.
tionally normal and abnormal, respectively. Therefore, it is important to identify the rea-
To assess whether or not S-RNases are dif- son(s) for the breakdown of SI in ‘Sha01’.
ferentially expressed in pistils of ‘Kuerlexiangli’ Allele-specific PCR has only identified two S-
and ‘Sha01’, qRT-PCR has been conducted. It is RNase alleles, S22-RNase and S28-RNase, in ‘Sha
found that expression levels of both S22-RNase 01’, as well as in each of the individuals in a
and S28-RNase in ‘Kuerlexiangli’ are almost self-pollinated progeny. Using genomic DNA as
identical to those detected in ‘Sha01’. Moreover, template, semi-quantitative PCR has revealed
alignments of nucleotide sequences of S22-RNase that densities of amplification products of S22-
and S28-RNase in ‘Sha01’ and ‘Kuerlexiangli’ RNase and S28-RNase alleles are different in the
have revealed no differences in these two alleles. self-pollinated progeny, with observed segrega-
These findings have suggested that the two S- tion ratios of 1:3, 2:2, and 3:1 in all tested

Fig. 10.6 A proposed scheme of how a hetero-diploid pollen is compatible with the S1S1S2S2 pistil because of
pollen grain leads to the breakdown of self-incompatibility competitive interactions. c During pollination of an S1S2
in tetraploid plants. a During pollination of the S1S2 pistil pistil with pollen from an S1S1S2S2 plant, the S1S1 pollen
with self-pollen, the S1 pollen and the S2 pollen will be and the S2S2 pollen will be rejected by the pistil. However,
rejected by the S1S2 pistil. b During pollination of an the S1S2 pollen is compatible with the S1S1S2S2 pistil
S1S1S2S2 pistil with self-pollen, both the S1S1 pollen and the because of competitive interactions
S2S2 pollen will be rejected by the pistil. However, the S1S2
198 S. Zhang and C. Gu

individuals. This indicates that the numbers of S22- hetero-diploid pollen (Fig. 10.6). Similar results
RNase and S28-RNase alleles are likely different in have been reported in the hetero-tetraploid Chi-
these individuals. Considering that these different nese cherry (Huang et al. 2008; Gu et al. 2010,
numbers have probably arose as a result of poly- 2013, 2014).
ploidy, the numbers of chromosomes and nuclear
DNA contents are measured in both pear cultivars,
‘Sha01’ and ‘Kuerlexiangli’, and in several indi- References
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As expected, it is determined that the number of Brewbaker JL (1954) Incompatibility in autotetraploid
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progeny are almost twofold than those determined in wild and cultivated plants. Springer, Berlin, p 322p
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that the observed ratio of individuals genotyped as Takayama S (2003) Comparative analysis of the
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approximately 1:4:1 (v20.05, 2 = 1.64 < 6.19) which allelic diversity. Genes Cells 8:203–213
is quite different from the expected ratio (v0.05, Goldway M, Takasaki-Yasuda T, Sanzol J, Mota M,
2 = 14.07 < 9.49). This indicates that ‘Sha01’ is a
2 Zisovich A, Stern RA, Sansavini S (2009) Renum-
bering the S-RNase alleles of European pears (Pyrus
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competitive interactions between the two S-haplo- cytoskeleton. Plant Cell 30:1023–1039
types in the pollen. Gao YB, Zhou HS, Chen JQ, Jiang XT, Tao ST, Wu JY,
Likewise, the pear cultivar Daguohuanghua is Zhang SL (2015) Mitochondrial dysfunction mediated
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result of self-fertilization is over 60.0% in cherry cultivars (Prunus pseudocerasus). Tree Genet
Genomes 6:579–590
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self-compatibility. Using ‘Huanghua’ as a polli- Zhang SL (2013) Inheritance of hetero-diploid pollen
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fruit set of the reciprocal cross is over 70.0%, Hetero-diploid pollen grains that represent
thus displaying cross-compatibility. These find- self-compatibility are incompatible with non-self
ings suggest that the pistil and the pollen of receptors in tetraploid Chinese cherry (Prunus pseu-
docerasus Lindl). Tree Genet Genomes 10:619–625
‘Daguohuanghua’ are functionally normal and Heng W, Wu J, Wu H, Cao Y, Nishio T, Zhang SL (2011)
abnormal, respectively. As sizes of leaves, fruits, Recognition specificity of self-incompatibility in
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 Stone Cell Development in Pear
11
Xi Cheng, Yongping Cai and Jinyun Zhang

Abstract and response to disease, as well as the

Pear (Pyrus spp.) is one of the most important mechanism of stone cell development in pear
deciduous fruit trees grown in the world. The fruits, including morphological characteristics,
genus Pyrus belongs to the subfamily Pomoi- distribution, development, components, for-
deae of the family Rosaceae. Stone cells mation, and regulation mechanism, will be
(sclereids), heavily lignified cells present in presented. Moreover, molecular mechanisms
fruit flesh, serve as a distinctive trait of pear of pear lignin metabolism, including pear
fruits. Stone cells are characterized by thick- lignin monomers type, biosynthesis pathway,
ening and lignified cell walls, and their and identification of key gene families will be
development is closely associated with lignin also summarized. Finally, we will share some
metabolism. The content and size of stone cell ideas relevant to future research directions
clusters are among the key factors in deter- pertaining to stone cells in pear.
mining the internal quality of pear fruits. Not
only are stone cells critically involved in fruit
texture, but they are also closely associated 11.1 Introduction
with the overall flavor of pear fruits. There-
fore, regulation of the size and content of Stone cells, also known as sclereids, are scle-
stone cell clusters is key for improving fruit renchyma cells that serve as a group of lignifi-
quality, and in promoting expansion of the cation cells found in plants that also include
pear industry. In this review, effects of stone tracheary elements, endodermal cells, seed coat
cells on fruit quality, including texture, flavor, cells, and siliques cells (Cai et al. 2010; Barros
et al. 2015). Based on their morphologies, scle-
reids can be divided into short sclereids,
X. Cheng
Y. Cai (&)
J. Zhang macrosclereids, osteosclereids, astrosclereids,
School of Life Science, Anhui Agricultural and trichosclereids, among others. Stone cells are
University, No. 130, Changjiang West Road, Hefei present in different plant tissues, including stems,
230036, China leaves, fruits, and seeds, as they play roles in
e-mail: [email protected]; [email protected]
structural support and protection functions, such
X. Cheng as in resistance against biotic and abiotic stresses
e-mail: [email protected]; [email protected]
(Zhao and Zhu 2014; Whitehill et al. 2016a, b).
J. Zhang In Pyrus spp., stone cells are quite abundant in
Horticultural Institute, Anhui Academy of Agricultural
Sciences, Hefei 230031, Anhui, China fruit flesh, thus serving a characteristic structural
e-mail: [email protected] feature of pear fruits (Wu et al. 2013).

© Springer Nature Switzerland AG 2019 201

S. S. Korban (ed.), The Pear Genome, Compendium of Plant Genomes,
https://doi.org/10.1007/978-3-030-11048-2_11
202 X. Cheng et al.

Stone cells in pear fruits are short sclereids, and cultivated pear species, including those of
their development is mainly determined by the P. communis, P. ussuriensis, P.  bretschnei-
pear genotype, but they are also regulated by deri, P. pyrifolia, and P. sinkiangensis.
external environment factors (Tao et al. 2009;
Brahem et al. 2017). Such stone cells present in
fruit flesh of pears tend to form clusters, and 11.2.1 SCC Contents in Different Pear
aggregation of multiple stone cells is referred to as Cultivars
stone cell clusters (SCCs) (Nii et al. 2008; Zhang
et al. 2017). Various studies have reported that Cao et al. (2010) analyzed and determined fruit
stone cells are key factors in determining pear fruit flesh SCC contents of 265 pear cultivars. These
quality (Li et al. 2019; Xue et al. 2019). Large size consisted of 117 cultivars of P.  bretschnei-
and content of SCCs in fruits will lead to decline deri, 89 cultivars of P. pyrifolia, 35 cultivars of
of internal quality characteristics of pear fruits, P. ussuriensis, eight cultivars of P. sinkiangensis,
which in turn, influence eating quality, as well as and 16 cultivars of P. communis. Mean values of
processing quality and overall economic value of SCC contents in fruit flesh (SCC content per
pear fruits (Choi et al. 2007; Wu et al. 2013; 100 g fresh weight) in these different cultivars,
Brahem et al. 2017; Cheng et al. 2017a; Zhang belonging to these various cultivated pear spe-
et al. 2017). Therefore, knowledge of the mech- cies, were as follows: P. ussuriensis
anism of development of stone cells, as well as (1.887 g) > P. sinkiangensis (0.939 g) > P. pyri-
that of regulatory controls of stone cell develop- folia (0.552 g) > P. communis (0.524 g) >
ment, will facilitate cultivation and development P.  bretschneideri (0.462 g). These findings
of pear cultivars with low content and small size were highly valuable in pursuing other studies to
SCCs. This will in turn enhance market compet- characterize genotypic differences among these
itiveness of pear cultivars and promote sustainable various cultivated species of pear.
development of the pear industry.
This chapter will cover current knowledge and
advances in our understanding of the develop- 11.2.2 Sizes of SCCs in Different Pear
ment mechanism of stone cells in pear fruits and Cultivars
will offer insights into future research studies in
this field. Sizes of SCCs (diameter of clusters) were sub-
sequently determined in 287 pear cultivars.
These included 118 cultivars of
11.2 Stone Cells and Cultivated P.  bretschneideri, 96 cultivars of P. pyrifolia,
Pear Species 44 cultivars of P. ussuriensis, 12 cultivars of
P. sinkiangensis, and 17 cultivars of P. commu-
Pears are mainly divided into two groups, nis. It was found that average proportions of
European and Asian pears (Wu et al. 2018). The SCCs with diameters larger than 300 lm were as
major cultivated species in Europe and America follows: P. ussuriensis (59.92%) > P. 
is Pyrus communis, European pears. Whereas the bretschneideri (58.27%) > P. pyrifolia
major cultivated species in East Asia include (50.07%) > P. sinkiangensis (47.76%) > P. com-
P. ussuriensis, P.  bretschneideri, P. pyrifolia, munis (41.02%). Furthermore, average propor-
and P. sinkiangensis (Xinjiang pear), all known tions of SCCs with diameters larger than 250 lm
as Asian pears (Lu et al. 2007; Wu et al. 2013, were as follows: P.  bretschneideri
2018). As content and size of SCCs vary in (81.78%) > P. ussuriensis (81.36%) > P. pyrifo-
different pear genotypes, this contributes to dif- lia (73.84%) > P. sinkiangensis
ferences in fruit quality among different culti- (73.68%) > P. communis (69.32%) (Table 11.1)
vated pear species. Therefore, it is critical to (Tian et al. 2011). Overall, it was reported that in
determine the content and size of SCCs in different pear cultivated species, the average
11 Stone Cell Development in Pear 203

Table 11.1 The average proportion (%) of SCCs, of different diameters, present in fruit flesh of five cultivated pear
species
Pyrus species Diameter of stone cell clusters (lm)
>500 (%) 300–500 (%) 250–300 (%) 200–250 (%) 150–200 (%) <150 (%)
P. ussuriensis 9.85 50.07 21.44 11.13 5.85 1.66
P. pyrifolia 9.78 40.29 23.77 14.68 8.84 2.64
P. communis 2.97 38.05 28.30 18.59 10.20 1.89
P. sinkiangensis 6.21 41.55 25.92 15.64 8.66 2.02
P.  bretschneideri 10.98 47.29 23.51 11.36 5.49 1.37

proportion of SCCs with diameters of 300– textural characteristics (Kim and Choi 2004a;
500 lm was the highest (Table 11.1). Yan et al. 2014). For example, although fruits of
P. ussuriensis cv. Beijing have a higher content
of SCCs than those of P.  bretschneideri cv.
11.3 Stone Cells and Pear Fruit Dangshan Su, they have a softer flesh texture
Quality Traits than those of ‘Dangshan Su.’ This is attributed to
a lower degree of polymerization of SCCs
Stone cells, including content, size, and degree of detected in ‘Beijing.’ In another example, fruits
polymerization, have a significant impact on the of P. pyrifolia cv. Wonhwang are found to have
internal quality of pear fruits (Choi et al. 2007; low SCC content and low degree of polymer-
Cao et al. 2010; Tian et al. 2011; Yan et al. ization, thus resulting in a highly soft fruit tex-
2014). In addition, stone cells are also associated ture. Therefore, a high degree of polymerization
with incidence of hard-end disorder, durability, of SCCs contributes to formation of
and juice composition of pear fruits (Konarska gritty-textured fruit flesh (Yan et al. 2014).
2013; Lu et al. 2015; Brahem et al. 2017). It is worth noting that although total amounts
Therefore, relationships between stone cells and of SCCs of fruits of some pear cultivars are
fruit quality traits will be discussed in further similar, flesh textures of fruits of these cultivars
detail. can be different. Some cultivars have higher SCC
contents, but if diameters of these SCCs are
smaller than others, then their flesh textures will
11.3.1 Flesh Texture be relatively soft. On the other hand, while some
cultivars have relatively low SCC contents,
Flesh texture is an important criterion for judging wherein diameters of their SCCs are larger than
the quality of pear fruits for fresh consumption. It those of other cultivars, then their fruit flesh
has been reported that the content of SCCs is textures are coarser (Cao et al. 2010; Tian et al.
positively correlated with adhesiveness and 2011). For example, the content of large diameter
chewiness of pear fruits, as well as with fruit SCCs (with a diameter between 80 and 260 lm)
firmness (Choi et al. 2007). Moreover, the high of fruits of ‘Dangshan Su’ are significantly higher
content of SCCs in flesh tissues will lead to a than those of P. pyrifolia, P. communis,
gritty texture and a coarse taste; however, it is not and hybrid P.  bretschneideri  P. communis,
significantly associated with fruit gumminess, and it is this high content of large diameter SCCs
springiness, and cohesiveness (Li et al. 2004; that leads to a coarse texture of flesh detected in
Choi et al. 2007; Konarska 2013). ‘Dangshan Su’ fruits (Li et al. 2017).
The content of SCCs not only influences fruit Studies have shown that if the diameter of
flesh texture and taste, but its degree of poly- SCCs is less than 150 lm, then the flesh texture
merization also has a significant impact on will be highly soft and with no grittiness;
204 X. Cheng et al.

whereas, if the diameter of SCCs is between 150 pear fruits (Nii et al. 2008; Lu et al. 2015; Wang
and 250 lm, then flesh texture will be soft, but et al. 2017a, b).
with a slight gritty taste. On the other hand, if the In comparison with normal healthy fruit, the
diameter of SCCs is more than 250 lm, then the content and diameter of SCCs of the calyx end of
flesh texture will be coarse and gritty. hard end fruit are significantly higher than those
Taking into account the influences of both of normal fruit at each of the stages of develop-
content and size of SCCs on flesh texture, it has ment (Lu et al. 2015; Wang et al. 2018). There-
been reported that SCC content, of diameters fore, massive accumulation of SCCs promotes
>250 lm in size, in fruit flesh per 100 g is an hardening of fruit flesh, which is one of the
important indicator of texture of flesh of pear factors triggering hard-end disorder.
fruits (Li et al. 2004, 2017; Tian et al. 2011).

11.4 Structural Components

11.3.2 Sugar Content of Stone Cells in Pear Fruits

Sugar is one of the main components of soluble The basic components of plant cell walls gener-
solids. In turn, soluble solid content is an ally include polysaccharides (cellulose, hemicel-
important indicator of the internal quality of pear lulose, and pectin polysaccharide), lignin,
fruits, as it impacts fruit flavor and consumer proteins, and mineral compounds, among others
appreciation (Choi et al. 2007; Gao et al. 2016; (Anderson et al. 2015; Zhong and Ye 2015). The
Han et al. 2017). proportion of cellulose in cell wall components is
It is reported that SCC content in pear fruits 40.6–51.2%, while hemicellulose is 28.5–37.2%,
has little effect on the contents of fructose, sor- and lignin is 13.6–28.1% (Pauly and Keegstra
bitol, and glucose; however, there is a significant 2008). Disruption of biosynthesis of these com-
negative correlation between SCC content and ponents will have an impact on cell wall devel-
sucrose content in pear fruit (Kim et al. 2004a; opment and even lead to cell wall deformity
Choi et al. 2007). Therefore, presence of high (Anderson et al. 2015; Zhong and Ye 2015).
contents of SCCs may lead to reduced levels of Therefore, it is important to have a good under-
sugar, thereby contributing to an inferior flavor in standing of structural components of stone cells
pear fruit. for future efforts in regulating development of
stone cells of pear fruits.
Stone cells of pear fruits are characterized by
11.3.3 Hard End Disorder thickened and heavily lignified secondary cell
walls (Tao et al. 2009; Jin et al. 2013). Plant
Hard end, or black end, is a physiological dis- secondary cell walls are mainly composed of
order of pear fruits (Wang et al. 2018). This lignin, cellulose, and hemicelluloses (xylan and
physiological disorder appears as a result of glucomannan). Among these three components,
delayed development of tissues, first resulting in cellulose microfibrils and hemicelluloses form
protrusion of the calyx and/or enlargement of the the skeleton structure of secondary cell walls,
calyx opening when the fruit is half-way through affording cell walls a degree of mechanical
its growth. At first, epidermal tissues of affected strength, while deposition of lignin enhances
areas turn shiny;however, as the disease pro- mechanical strength of these cell walls, and
gresses, tissues harden, and the surrounding contributing to their rigidity (Doblin et al. 2010;
mature calyx turns dry, prominent, and black in Keegstra 2010).
coloration. Hard end fruit loses its crisp and juicy Following analysis of cell wall components of
taste, and this will also have an adverse effect on pear stone cells, they are found to contain large
both internal and external qualities of affected amounts of lignin, cellulose, and hemicellulose
11 Stone Cell Development in Pear 205

(xylans). In addition, these stone cells contain 2013; Li et al. 2017). Furthermore, the content of
certain amounts of procyanidins. On the other pear stone cells exhibits an increase/drop pattern
hand, parenchyma cells of pear fruits contain during fruit development (Kim et al. 2004c; Cai
high levels of pectin (uronic acids and arabinose) et al. 2010; Tao et al. 2015). Moreover, the initial
and low levels of lignin. As a result, stone cells period of stone cell formation and the peak per-
are deemed harder and stiffer than parenchyma iod of stone cell content vary in different pear
cells (Brahem et al. 2017). genotypes and under different growing condi-
In other studies, it has been reported that tions (Li et al. 2017).
autofluorescence analysis indicated presence of The process of stone cell formation involves
high lignin content, as well as positive secondary thickening and lignification of cell
phloroglucinol-HCl (Wiesner) staining in stone walls of parenchyma cells of fruit flesh tissues, as
cells of pear fruits (Tao et al. 2009; Cheng et al. observed in microscopic anatomical studies
2017a). Although early studies have reported that (Figs. 11.1 and 11.2) (Nii et al. 2008; Nie et al.
the lignin content of stone cells is about 20–30% 2009; Jin et al. 2013; Zhao et al. 2013). Based on
(Lu et al. 2011; Yang et al. 2014), subsequent morphological characteristics of stone cells, the
analysis has determined that the lignin content in developmental process of stone cell formation
stone cells of different pear cultivars vary from can be divided into four stages. These stages are
34.25 to 39.46% (Tian et al. 2017). In addition, designated as follows: prophase, metaphase,
there is a significant positive correlation between anaphase, and telophase, corresponding to
SCC content and lignin content in flesh of pear Stage I (precursor occurrence), Stage II (cyto-
fruits. The lignin content of SCCs is also posi- plasm gathering), Stage III (protoplast shrinking
tively correlated with the lignin content in the and secondary wall thickening), and Stage IV
flesh (Tao et al. 2015; Tian et al. 2017; Zhang (formation of stone cells), respectively (Nie et al.
et al. 2017). Therefore, lignin is deemed as one 2009; Zhao et al. 2013). Other studies have
of the key components of stone cells. divided this process slightly differently into three
stages, based on the process of fruit enlargement
as follows: Stage I, extensive cell division;
11.5 Developmental Patterns Stage II, reduced rate of fruit enlargement; and
and Distribution of Stone Cells Stage III, rapid increase in fruit size until harvest
in Pear Fruit (Nii et al. 2008).
During bloom, the receptacle (later develop-
The content and size of stone cells in pear fruits ing into a fruit) is composed entirely of par-
are variable, as well as their distribution in dif- enchyma cells. At this time, cell walls are very
ferent tissues of the fruit. Thus, it is important to thin and cannot be stained with
understand the origin of these stone cells. In phloroglucinol-HCl (Fig. 11.2a), thus suggesting
particular, it is critical to address the following that lignin accumulation has not yet begun (Zhao
questions: Where do these stone cells come et al. 2013). Subsequently, it is found that cell
from? What is the process of their formation? walls of some parenchyma cells present in fruit
And how are they distributed within the fruit? flesh (mostly those adjacent to fruit vascular
bundles) begin to thicken unevenly, wherein
inclusions gradually disappear to form
11.5.1 Development thick-walled hollow cells. These cell walls of
and Morphology parenchyma cells will continue to thicken until
of Stone Cells entire cell walls are thickened, forming sclereid
primordium cells (Fig. 11.1a and 11.2b). Par-
Stone cells are initiated 7–15 days after flower- enchyma cells surrounding sclereid primordium
ing (DAF), and they form and develop between cells will also undergo secondary cell wall
23 and 67 DAF (Cai et al. 2010; Zhao et al. thickening and lignification (Fig. 11.1b). The
206 X. Cheng et al.

Fig. 11.1 Microscopic analysis of stone cell clusters Safranin. a–f The process of formation of SCCs in
(SCCs) development in pear fruit (the black pointer shows Pyrus  bretschneideri (15–67 days after flowering) and
a stone cell or a SCC). Stone cells were stained with 0.1% g The shape of SCCs of pear fruit of P.  bretschneideri

Fig. 11.2 Phloroglucinol-HCl staining and microscopic observation of parenchyma cells (a), sclereid primordium
cells (b), and stone cells (c) development in pear fruit

newly formed stone cells will gather around color (Fig. 11.2b), thus indicating that lignifica-
sclereid primordium cells, and stone cell aggre- tion has begun (Zhao et al. 2013).
gation will take place (Fig. 11.1c, d). When As the fruit continues to develop, the volume
stained with phloroglucinol-HCl, cell walls at of stone cells and the scope of aggregation con-
this stage of development are dyed light purple in tinue to expand. While cell walls continue to
11 Stone Cell Development in Pear 207

thicken, the cell lumen is continuously being filled

until it turns into a typical stone cell cluster made
up of a number of solid stone cells (Fig. 11.1e–g).
At this point, all stone cells could turn purplish red
in coloration following phloroglucinol-HCl
(Fig. 11.2c), thus indicating that stone cells are
fully lignified (Zhao et al. 2013).
Around 67 DAF, development of the majority
of stone cell clusters is stabilized, and parenchyma
cells will no longer differentiate into stone cells.
Following this period, parenchyma cells begin to Fig. 11.4 Scanning electron microscopic observation of
expand, and the fruit volume will rapidly increase. stone cell clusters (black arrows) and parenchyma cells
This will contribute to expanded spaces among (white arrow) within the flesh of a pear fruit
stone cell clusters, along with a drop in relative
contents of these clusters (Nii et al. 2008; Cai et al. small vacuoles gradually merge into a large cen-
2010; Choi and Lee 2013). By about 100 DAF, tral vacuole, after which the cytoplasm becomes
the content of stone cell clusters is completely dispersedly granular, and cellular contents grad-
stabilized (Nii et al. 2008; Li et al. 2017). ually shrink into the center of the cell. At the same
The process of stone cell formation is also a time, this process is accompanied by the appear-
plant programmed cell death (PCD) process ance of autophagic vacuoles (Cheng et al. 2019c).
(Zhao et al. 2013). During the period of par- Eventually, both the vacuole and cytoplasm dis-
enchyma cell differentiation into stone cells, appear, and the hollow cell lumen is filled entirely
autophagy is observed (Fig. 11.3). For those by thickened secondary cell walls (Jin et al. 2013;
parenchyma cells that do not differentiate into Zhao et al. 2013). Using electron microscopy, it
stone cells, they contain larger nuclei along with can be noted that the degree of polymerization of
small intracellular vacuoles within the dense stone cell clusters formed by multiple stone cells
cytoplasm. However, during the process of dif- is higher than that of parenchyma cells
ferentiation of parenchyma cells into stone cells, (Fig. 11.4). Indeed, stone cell clusters are sur-
rounded by parenchyma cells, and cell walls of
parenchyma cells are thinner than those of stone
cells (Yan et al. 2014; Brahem et al. 2017).

11.5.2 Secondary Cell Wall

Construction and Lignin
Deposition in Stone
Cells

Cell walls of stone cells are composed of a

middle lamella (ML), a primary cell wall (PCW),
and a secondary cell wall (SCW). Furthermore,
SCWs are generally subdivided into a secondary
wall outer layer (S1), a secondary wall middle
layer (S2), and a secondary wall inner layer (S3).
Interestingly, the ML between two stone cells
and PCW of stone cells are relatively thin, and
Fig. 11.3 Ultrastructural observation of autophagic vac- the combination of these two structures is tight.
uoles in stone cells Therefore, these structures form the composite
208 X. Cheng et al.

middle lamella (CML) which consists of ML and to cell walls (Figs. 11.5 and 11.6), thus indicat-
PCW. In addition, pits are also present along cell ing that the transport of intracellular material is
walls of mature stone cells (Figs. 11.5 and 11.6) active in these stone cells (Jin et al. 2013; Zhao
(Tao et al. 2009; Jin et al. 2013; Zhao et al. 2013; et al. 2013). In addition, lignin is generally
Cheng et al. 2019c). unevenly deposited, at first, along corner regions
During development of secondary cell walls of the primary cell wall, and then this expands to
of stone cells, large numbers of vesicles and other regions of CMLs, as well as to various
endoplasmic reticulum can be observed adjacent layers of SCWs. Lignin and cellulose microfibrils

Fig. 11.5 Microscopic and ultramicroscopic observation version of (d); f Cross-section of pits on the stone cell;
of pits (P) (white arrows) in pear stone cells. a–b Obser- g Microscopic observation of pits in pear stone cells; ER:
vations of the simple pit (longitudinal-section); c Obser- endoplasmic reticulum; M: mitochondria; SCW: sec-
vations of the pit pair (longitudinal-section); ondary cell wall; The red arrow indicates the pit pairs
d Ultrastructure of the pit cavity; e Shows a magnified formed between adjacent cells
11 Stone Cell Development in Pear 209

Fig. 11.6 Ultramicroscopy of lignin deposition during vesicles contain high electron-dense material (short black
stone cell development in pear fruit (Jin et al. 2013). arrows); e Lignin deposition (black stripes) within gaps
a Some cells within the pulp begin to exhibit uneven between cellulose microfibrils (white stripes); and f Some
thickening of the cell wall (short black arrow); b Depo- pits along stone cell walls, cross-section; CC: cell corner;
sition of lignin is initiated from the inner region of the S1 CML: composite middle lamella; S1: S1 layer of the
layer (short white arrows); c Lignin particles are deposited secondary wall; S2L: layer between S1 and S2; and S2: S2
unevenly along the inner regions of each microfibril in layer of the secondary wall
every S2 layer (short white arrows); d Many secretory

alternately arrange their depositions to build up changes dramatically over the growing season
the secondary cell wall (Jin et al. 2013) (Choi and Lee 2013). Specifically, it is observed
(Fig. 11.6). However, there are differences in the that from 15 to 55 DAF, the distribution of stone
degrees of lignification of different secondary cells in ‘Dangshan Su’ fruit gradually increases,
cell wall layers. In fact, the degree of lignification particularly during the period of 39–55 DAF,
of the S2L layer is the highest; moreover, the wherein the density of stone cells is high in the
degree of lignification of cell corner (CC) and tissue located between the core and the peel.
CML is higher than that of S1. It is worth noting Subsequently, during the period of 63 DAF to
that the highest degree of lignification of the S2L maturity, the density of stone cells within the
layer is also a characteristic of severe compres- fruit gradually drops, and it is primarily dis-
sion wood (Tao et al. 2009; Jin et al. 2013). tributed near the core. This may be attributed to a
faster rate of stone cell formation compared to
the rate of fruit enlargement prior to 63 DAF;
11.5.3 Distribution of Stone Cells thereby, a higher density of stone cells is
in Pear Fruits observed within the pulp at this stage (Cheng
et al. 2017a). As stone cells are completely
It is reported that distribution of stone cells developed after 63 DAF, the volume of par-
within flesh tissues of pear fruit is uneven, and it enchyma cells increases rapidly, and the rate of
210 X. Cheng et al.

Fig. 11.7 Stone cell staining of ‘Dangshan Su’ pear development were stained using the Wiesner method
fruits at different stages of development (Cheng et al. (phloroglucinol-HCl). DAF, days after flowering
2017a). Transverse sections of fruits at eight stages of

fruit enlargement also increases, thus contribut- caffeyl unit (C-unit), and a 5-hydroxy-guaiacyl
ing to reduction in distribution of stone cells unit (5H-unit) (Chen et al. 2013; Barros et al.
within the flesh (Fig. 11.7) (Nii et al. 2008; Cai 2015). These five structural units are formed by
et al. 2010; Choi and Lee 2013; Cheng et al. five monolignols, including p-coumaryl alcohol,
2017a; Li et al. 2017). coniferyl alcohol, sinapyl alcohol, caffeol alco-
During determination of stone cell content in hol, and 5-hydroxyconiferyl alcohol, respec-
different tissues of the fruit, including the pulp, tively. Various structural units are mainly
peel, and core, it is generally observed that there connected by ester bonds (C–O–C′) (including
is a higher stone cell content near the peel and the linkage bonds, such as b–O–4, a–O–4, 4–O–5,
core of the fruit (Kim et al. 2004c; Choi and Lee and a–O–c) and carbon–carbon bonds (C=C)
2013; Li et al. 2017). This high content of stone (including linkage bonds, such as 5–5, b–5, b–1,
cells present near the peel likely renders this and b–b) to form lignin polymers (Vanholme
tissue difficult for birds to feed on (Li et al. et al. 2010; Eudes et al. 2014).
2017). Depending on the pear cultivar, the mid- Synthesis of monolignol must have originated
dle pulp has a relatively lower stone cell content from synthesis of phenylalanine in plastids, and it
than that of other tissues (Li et al. 2017). In is subsequently converted into 4-hydroxyphenyl-
general, the size of stone cell clusters in the core propene alcohols by a series of enzymatic reac-
of the fruit is higher than that detected in other tions in the cytoplasm. There are various branches
tissues of the fruit (Tao et al. 2009; Li et al. of lignin metabolism in different plants; thus,
2017). lignin metabolism is a very complex metabolic
network (Liu 2012; Barros et al. 2015).
As mentioned above, stone cells are lignifi-
11.6 Stone Cells and Lignin cation cells, and that lignin is essential for their
Metabolism development. Thus, lignin monomer composition
and lignin synthesis pathway within the fruit are
Lignin is a polyphenolic polymer that is directly critical in defining the mechanism of formation of
deposited within plant cell walls. Lignin polymer stone cells. Therefore, we will next focus on pear
consists of either a single or five structural units, lignin structure, biosynthetic pathways, and
including a p-hydroxyphenyl unit (H-unit), a related structural genes.
guaiacyl unit (G-unit), a syringyl units (S-unit), a
11 Stone Cell Development in Pear 211

11.6.1 Composition and Structure development (Yan et al. 2014). Based on recent
of Lignin in Pear studies of lignin contents in pear fruits, levels of
G-units are generally higher than those of
Lignin monomer composition is known to vary in S-units, thereby resulting in a G/S ratio that is
different plant species, tissues, cell types, and cell greater than 1.0 (Cai et al. 2010; Jin et al. 2013).
wall layers. The lignin of gymnosperms is almost However, for some pear cultivars, such as
predominantly composed of G-units, while the ‘Dangshan Su,’ the level of G-units is more than
lignin of angiosperms is predominantly composed twofold higher than that of S-units (Yan et al.
of G–S-units (Campbell and Sederoff 1996). 2014). As an S-unit has methoxy groups at both
The lignin polymer in pear, an angiosperm, is C3 and C5 positions, while a G-unit has only a
primarily composed of G-units and S-units, with single methoxy group at the C3 position
no evidence of presence of either H-units, (Fig. 11.8), a G-unit can easily form stable, and
C-units, or 5H-units (Cai et al. 2010; Jin et al. not easily degraded, C=C bonds at the C5 posi-
2013). Certainly, the content and ratio of G-units tion. Therefore, the higher the G-unit content, the
and S-units (G/S) of lignin in different pear cul- more difficult it is to degrade lignin polymers. In
tivars are different. Moreover, even within the addition, presence of a higher G/S lignin ratio in
same pear cultivar, the content of lignin mono- a pear fruit, the more stable are those formed
mers is different at different stages of lignin polymers, the more difficult it is to degrade

Fig. 11.8 Metabolic pathways of lignin in plants. The shaded sections pertain to the main lignin biosynthesis pathway
in pear fruits
212 X. Cheng et al.

such polymers, thus leading to formation of high 11.6.2.1 The General Phenylpropanoid
density and of high degree of polymerization of Pathway
SSCs (Yan et al. 2014). The general phenylpropanoid pathway mainly
Organic elemental analytical results have converts L-phenylalanine into a hydroxycinnamic
demonstrated that the lignin of pear fruits mainly acid and an acyl-CoA ester. The enzymes respon-
contains carbon (C), hydrogen (H), and oxygen sible for this segment of the metabolic pathway are
(O) elements, but it also contains a small amount phenylalanine ammonia-lyase (PAL), cinnamate
of nitrogen (N) elements. Furthermore, the 4-hydroxylase (C4H), and 4-coumarate: coenzyme
structure of the lignin polymer of a pear fruit has A ligase (4CL) (Barros et al. 2015).
more side chains, along with more hydroxyl The metabolites of PAL and C4H, cinnamic
groups and less phenolic hydroxyl groups. The acid and p-coumaric acid, were detected at dif-
linkage bond of a lignin structural unit is gener- ferent stages of development of pear fruits. The
ally divided into the following four types, b–O– contents of cinnamic acid and p-coumaric acid
4, b–1, b–5, and b–b. were found to be higher during the vigorous
Of course, it is important to point out that period of stone cell formation and lignin
structural properties of lignin of different pear biosynthesis, thus suggesting that they are
cultivars will have some variations that may lignin-synthesizing key precursors (Cai et al.
influence stability of lignin polymers to a certain 2010; Wang et al. 2013). In addition, multiple
extent and ultimately influence formation of PALs, C4Hs, and 4CLs genes, along with their
stone cells. encoded enzymes, were detected by transcrip-
tomic and proteomic analyses, and expression
trends were found to be consistent with contents
11.6.2 Analysis of the Monolignols of stone cells and lignin in pear fruit (Li et al.
Metabolic Pathway 2015; Zhang et al. 2016, 2017). Thus, it has been
proposed that the general phenylpropanoid
The monolignols metabolic pathway can be sub- pathway was closely related to the synthesis of
divided into three components. An upstream monolignols in pear fruits.
pathway of monolignols metabolism is the gen-
eral phenylpropanoid pathway, which subse- 11.6.2.2 The Ester Intermediary
quently enters into an ester intermediary pathway, Pathway
and finally into synthesis of various monolignols The ester intermediary pathway mainly synthe-
via a monolignol-specific biosynthesis pathway sizes various hydroxycinnamic acids and coen-
(Fig. 11.8) (Barros et al. 2015; Pascual et al. zyme A thioesters (Pascual et al. 2016).
2016). Following transcriptomic, proteomic, Enzymes involved in this pathway include
metabolomic, gas chromatography–mass spec- hydroxycinnamoyl-CoA: shikimate/quinate
trometry (GC–MS), ultra-high-performance liq- hydroxycinnamoyltransferase (HCT), coumarate
uid chromatography–tandem mass spectrometry 3-hydroxylase (C3H), and caffeoyl-CoA
(UPLC–MS/MS), and high-performance liquid O-methyltransferase (CCoAOMT). In a study
chromatography (HPLC) analyses, the lignin of pear fruit transcriptomes and proteomes, genes
biosynthesis pathway in pear fruits has been well encoding these three enzymes have been detected
investigated. This has led to the unraveling of the (Wu et al. 2013; Li et al. 2015; Zhang et al. 2016,
subsets of pathways involved in the monolignols 2017). This has confirmed existence of this
metabolic pathway (Cai et al. 2010; Wu et al. metabolic pathway in pear fruits.
2013; Li et al. 2015, 2018a, b; Zhang et al. 2017).
11 Stone Cell Development in Pear 213

High levels of expression of HCT, C3H, and Furthermore, several microRNAs, such as
CCoAOMT have been detected in pear fruits at miR397a, are downregulated during the period of
early stages of development, therein promoting vigorous lignification of pear fruits, and expres-
conversion of p-coumaroyl-CoA into caffeoyl- sion patterns of miR397a during different stages
CoA, then to feruloyl-CoA, and ultimately lead- of fruit development are in contrast to those of
ing to accumulation of S-units and G-units. In multiple target genes such as LAC (Xue et al.
addition, the conversion reaction of caffeoyl-CoA 2018). It is proposed that miR397a can influence
to feruloyl-CoA is a rate-limiting step in lignin lignin biosynthesis by regulating expression of
biosynthesis in pear fruits (Wu et al. 2013; Zhang 27 LAC genes; thus, LAC is critical in lignin
et al. 2016). synthesis in pear fruits (Wu et al. 2014).
Xue et al. (2018) used 30 pear cultivars of
11.6.2.3 The Monolignol-Specific high-stone cell content (average stone cell con-
Biosynthesis Pathway tent ranged between 10.53 and 20.11%) and 30
Cinnamoyl-CoA reductase (CCR), cinnamyl pear cultivars of low-stone cell content (average
alcohol dehydrogenase (CAD), and sinapyl stone cell content ranged between 3.71 and
alcohol dehydrogenase (SAD) in the 6.78%). Single nucleotide polymorphism
monolignol-specific biosynthesis pathway are (SNP) mutations in a 3000 bp promoter region of
responsible for converting hydroxycinnamoyl- the PbrmiR397a precursor of these 60 cultivars
CoAs to monolignols. Ferulate 5-hydroxylas were compared using whole-genome resequenc-
(F5H) and caffeic acid 3-O-methyltransferase ing. It was observed that the TCA-element, a
(COMT) can then catalyze formation of precur- salicylic acid response element, in the
sors of S-units from precursors of G-units (Bar- PbrmiR397a precursor promoter had a single
ros et al. 2015). base mutation in high-stone cell content pears.
During the peak period of stone cell devel- This directly contributed to less effective salicylic
opment and lignin content accumulation in a pear acid induction of PbrmiR397a transcription,
fruit, transcription of multiple CCRs, CADs, thereby resulting in upregulation of expression of
SADs, and F5Hs has been detected by tran- the target gene, PbLACs, of PbrmiR397a in
scriptome and proteome analyses (Li et al. 2015; fruits. Thereby, this contributed to accumulation
Zhang et al. 2016, 2017). Interestingly, Wu et al. of lignin and development of stone cells
(2013) have not detected significant COMT (Fig. 11.9). In addition, dual-luciferase reporter
transcript levels. Therefore, this begs the fol- assays and genetic transformation also demon-
lowing question: Does COMT require very low strated that PbrmiR397a could affect plant lignin
levels of expression to meet the needs of catalytic content and cell wall development by regulating
reactions or is COMT responsible for catalytic LAC transcription (Xue et al. 2018).
steps that do not exist in pear fruits? This
important question is yet to be investigated.
11.6.3 A Low-Stone Cell Content Bud
11.6.2.4 Monolignols Polymerization Sport
Following synthesis of the lignin monomer, which is
catalyzed by peroxidase (POD, EC 1.11.1.7) and A mutation in meristematic cells of the growth
laccase (LAC, EC 1.10.3.2), it is coupled with point of a bud on a shoot of a fruit tree can lead
polymerize into a growing lignin polymer (Barros to the development of a bud sport mutant. When
et al. 2015). During the peak period of stone cells mutant buds grow into shoots and branches, they
development and lignin content accumulation, mul- can develop flowers and fruits that are likely to
tiple POD genes are upregulated, thus suggesting be different from the original cultivar in mor-
that these genes may playkey roles in monolignol phology, physiology, biochemistry, or even
polymerization (Cao et al. 2016a; Zhang et al. 2017). genetics. This trait can be either maintained
214 X. Cheng et al.

Fig. 11.9 The miR397a-LACs module regulates stone cell formation in pear fruit

through vegetative, or asexual, propagation, or it monomer synthesis and polymerization, they

can be even inherited by the offspring, if the have observed differences in transcription of
mutation occurs in germ cells (Foster and Aran- genes related to carbon metabolism and to some
zana 2018). hypothetical regulatory genes that are likely
Pyrus  bretschneideri cvs. Lianglizaosu and responsible for observed differences in both
Dangshanxinsu are new pear cultivars originating content and size of stone cell clusters in pear
as natural bud sports of ‘Dangshan Su’ (Wang fruits (Fig. 11.10).
et al. 2012; Xu et al. 2016; Zhang et al. 2017).
The contents of stone cells in fruits of these two
pear cultivars, ‘Lianglizaosu’ and ‘Dangshanx- 11.6.4 Gene Families Related
insu,’ are significantly lower than those of to Lignin Synthesis
‘Dangshan Su,’ and thus, these are designated as in Pear
low-stone cell content bud sports. Following
several years of observations and comparative Analysis of fruit transcriptome and proteome has
studies of these two bud sports with ‘Dangshan revealed that most of the lignin synthesis-related
Su,’ it is reported that the low-stone cell content genes have multiple members, and together,
trait is stable. Not only do these two bud sports these members play roles in fruit development.
have the original desirable traits of ‘Dangshan With completion of sequencing of the pear gen-
Su,’ they also set fruit containing significantly ome, screening and identification of members of
lower size and content of SCCs (Wang et al. the gene family related to lignin metabolism have
2012; Xu et al. 2016; Zhang et al. 2017; Cheng been successively conducted. The following
et al. 2019b). Therefore, discovery of these sections provide an overview of members of the
low-stone cell content bud sports provides ideal lignin gene family in pear.
materials for studying the molecular mechanism
of stone cell development. 11.6.4.1 The 4-Coumarate: Coenzyme
Using comparative transcriptome analyses A Ligase (4CL) Gene
between ‘Lianglizaosu’ and ‘Dangshan Su’, Family
Zhang et al. (2017) have reported that in addition The phenylpropanoid enzyme 4-coumarate:
to identifying structural genes related to lignin coenzyme A ligase (4CL) acts on the last step of
11 Stone Cell Development in Pear 215

Fig. 11.10 A model illustrating the putative mechanism of PPR: Pentatricopeptide repeat protein; PP2C25: Probable
pear stone cell development. PEP: Phosphoenolpyruvate; E4P: protein phosphatase 2C 25; PM: Plasma membrane; SCW:
Erythrose-4-phosphate; UGT: UDP-glucuronosyltransferase; Secondary cell wall; PCW: Primary cell wall; and ML: Middle
BGLU: b-Glucosidase; UDPG: Uridine diphosphate glucose; lamella
BAP: BON1-associated protein; NUDT: Nudix hydrolase;

the general phenylpropanoid pathway, with p- the branch point of the phenylpropane metabolic
coumaric acid, caffeic acid, ferulic acid, pathway, along with the synthesis of various
5-hydroxy ferulic acid, and sinapic acid serving secondary metabolites, are the precursors of lig-
as substrates in generating corresponding coen- nin, flavonoids, and chlorogenic acid (Barros
zyme A thioesters. These resultant thioesters, at et al. 2015; Cao et al. 2015, 2016b).
216 X. Cheng et al.

The 4CL gene family can be divided into two 11.6.4.3 The O-Methyltransferase
major subfamilies, Class I and Class II. In par- (OMT) Gene Family
ticular, members related to lignin synthesis are The O-methyltransferase (OMT) is a key enzyme
classified as either Class I in phylogenetic trees in the phenylpropanoid metabolic pathway,
or as Class II for members related to flavonoid which is responsible for the catalytic methylation
metabolism. A total of 29 members of the 4CL of lignin precursors, flavonoids, and a series of
gene family have been identified and screened in secondary metabolites. The OMT family can be
the pear genome, of which 16 members belong to subdivided into two types, Class I and Class II.
Class I and 13 members belong to Class II. Based Class I is caffeoyl-CoA O-methyltransferase
on analysis of expression patterns, it is proposed (CCoAOMT), which is mainly involved in
that Pb4CL1 plays a major role in lignin meta- monolignol biosynthesis, and Class II is caffeic
bolism, while Pb4CL2 and Pb4CL4 are likely to acid 3-O-methyltransferase (COMT), which not
participate in flavonoid metabolism in pear fruits only participates in lignin metabolism, but also
(Cao et al. 2015, 2016b). catalyzes the synthesis of flavonoids (Cheng et al.
2016).
11.6.4.2 The Hydroxycinnamoyl-CoA: There are 26 OMTs present in the pear genome,
Shikimate/Quinate including 19 COMTs and seven CCoAOMTs.
Hydroxycinnamoyl Based on phylogenetic tree clustering and expres-
transferase (HCT) Gene sion pattern analysis, PbCCoAOMT1 and
Family PbCCoAOMT3 are reported to play major roles in
The hydroxycinnamoyl-CoA: shikimate/quinate lignin metabolism and stone cell development in
hydroxycinnamoyltransferase (HCT) enzymes pear fruits (Cheng et al. 2016).
belong to the plant BAHD acyltransferase
superfamily, and have dual activities of shiki- 11.6.4.4 The Cinnamoyl-CoA Reductase
mate hydroxycinnamoyltransferase (CST) and (CCR) Gene Family
quinate hydroxycinnamoyltransferase (CQT). The cinnamoyl-CoA reductase (CCR) enzyme
HCT can catalyze formation of the coumar- belongs to the short-chain dehydrogenase/
oylquinate 3-monooxygenase (C3H) substrate reductase (SDR) family, which catalyzes the
coumaroyl shikimic acid/coumaroyl quinic acid, first reaction of lignin-specific synthetic path-
and at the same time, it can also catalyze C3H ways. The CCR substrates have been identified
production of the caffeoyl shikimic/quinic acid, as the following five hydroxycinnamoyl-CoAs,
which is further converted into caffeoyl-CoA. including p-coumaryl-CoA, caffeoyl-CoA,
A total of 82 PbHCTs have been identified in feruloyl-CoA, 5-hydroxyferuloyl-CoA, and
the pear genome of P.  bretschneideri, all of sinapoyl-CoA, and the product of which is the
which contain the conserved domains HXXXD corresponding hydroxyl cinnamaldehyde (Pan
and DFGWG. Approximately 25% of the mem- et al. 2014; Cheng et al. 2017a).
bers contain MYB transcription factor binding A total of 31 CCRs genes have been identified
sites. Transcriptome and qRT-PCR analysis have in the pear genome, and among these, there are
revealed that expression trends of PbHCT2, 28 CCR-like clade members and three PbCCR
PbHCT17, PbHCT18, PbHCT49, and PbHCT50, members belonging to a bona fide CCR clade.
at different stages of fruit development, are con- Furthermore, PbCCR1, 2, and 3 have high
sistent with changes of lignin contents in pear. genetic relationships with bona fide CCRs of
Furthermore, there is a high correlation between other species and share a characteristic conserved
expression levels of these five genes and contents motif, KNWYCYGK, whose spatial
of stone cells in pear fruits. Therefore, these five three-dimensional structure may be involved in
PbHCTs are proposed to play key roles in lignin the recognition of CoA. Following analysis of
synthesis and stone cell development in pear temporal-spatial expression patterns of these
fruits (Ma et al. 2017). three members, it is determined that PbCCR1 and
11 Stone Cell Development in Pear 217

PbCCR2 play major roles, while PbCCR3 may subdivided into three classes. Class I is present in
play a minor role in lignin synthesis in pear fruits bacteria, while Class II is present in fungi, and
(Cheng et al. 2017a). Class III is present in plants. Class III peroxi-
dases play important roles in plant lignin poly-
11.6.4.5 The Cinnamyl Alcohol merization, cell wall development, and in
Dehydrogenase resistance to stress (Barros et al. 2015; Cao et al.
(CAD) Gene Family 2016a).
The cinnamyl alcohol dehydrogenase (CAD) en- In pear, Class III peroxidase gene family
zyme belongs to a medium short-chain consists of 94 members belonging to 19 sub-
dehydrogenase/reductase (MDR) family, which families. Based on quantitative reverse tran-
is responsible for the reduction of hydroxycin- scriptase (qRT)—polymerase chain reaction
namaldehydes into hydroxycinnamic alcohols (PCR) analysis, it is revealed that five PbPODs,
(lignin monomers) (Pan et al. 2014). It has been including PbPRX2, PbPRX22, PbPRX34,
reported that within the CAD gene family, there PbPRX64, and PbPRX75, belonging to subgroup
is a class member, referred to as sinapyl alcohol C, may be involved in regulation of lignin syn-
dehydrogenase (SAD), which is responsible for thesis in pear fruits (Cao et al. 2016a).
the conversion of sinapaldehyde into sinapyl
alcohol (Li et al. 2001; Cheng et al. 2017a). 11.6.4.7 The Laccase (LAC) Gene Family
However, in recent years, other studies have Laccase (LAC) is the largest component of
suggested that the so-called SAD and its orthol- multi-copper oxidases (MCOs), and it is the key
ogous gene cannot be exclusively responsible for enzyme responsible for polymerization of
the synthesis of S-units, and their expression monolignols. Xue et al. (2018) have identified 38
cannot be changed by modifying their S-unit PbLACs in the pear genome, and have reported
contents (Barakate et al. 2011). Therefore, it is that PbLAC1, 2, 3, 15, 18, and 20 have higher
proposed that SADs may only act as alternative abundance of transcripts in early fruit develop-
or compensating CADs for bona fide CADs in a ment using transcriptome analysis. As stone cells
plant’s response to stress. are formed in large numbers during the early
A total of 26 members of the CAD gene stages of pear fruit development, it has been
family have been identified in the pear genome. proposed that six PbLACs are involved in poly-
These members are characterized by containing merization of lignin monomers during develop-
either an ADH_zinc_N domain or an ADH_N ment of stone cells. In particular, subcellular
domain (Cheng et al. 2017a). Following analysis localization analysis has indicated that PbLAC1,
of tissue specificity and temporal expression 2, and 18 are all localized in cell walls, and that
patterns in developing pear fruits, it has been simultaneous inhibition of expression of these
reported that PbCAD2 is upregulated, while three genes in pear fruit significantly reduces
PbCAD3 is downregulated during peak periods stone cell formation (Xue et al. 2018). Recently,
of fruit stone cell development and lignin content Cheng et al. (2019a) have also demonstrated that
accumulation. Combined with the tertiary struc- PbLAC (Pbr003857.1) is associated with lignin
ture of proteins, comparative key catalytic sites, synthesis and secondary cell wall development.
sequence similarities, and identity analysis, it has
been confirmed that PbCAD2, in a bona fide 11.6.4.8 The Dirigent (DIR) Gene
CAD clade (Class I), is related to lignin synthesis Family
in pear fruits (Cheng et al. 2017a). Dirigent proteins (DIRs) are closely related to
lignification of plant cells, and they play impor-
11.6.4.6 The Peroxidase (POD) Gene tant roles in secondary cell wall formation
Family (Burlat et al. 2001; Paniagua et al. 2017; Cheng
Based on sequence differences and catalytic et al. 2018). The dirigent protein model hypoth-
properties, peroxidases (PODs) can be esis suggests that formation of lignin oligomers
218 X. Cheng et al.

is carried out under the strict regulation of DIRs, contents of stone cells and lignin in the fruit
which controls formation of specific chemical (Cheng et al. 2017b; Li et al. 2018a, b). It is
bonds during the monolignol polymerization suggested that different male parental pollen can
process to form lignin polymers (Barros et al. influence expression of miRNA, such as
2015; Paniagua et al. 2017). pyr-miR1809 and pyr-novel-miR-144-3p, within
Cheng et al. (2018) have classified 35 mem- the fruit. In turn, this will regulate expression of
bers of the PbDIR family into the following four structural genes, such as laccase, in the lignin
subfamilies: DIR-a, DIR-b/d, DIR-e, and DIR-g synthesis pathway, eventually affecting devel-
subfamilies. Through systematic bioinformatics opment of stone cells in pear fruits (Cheng et al.
and qRT-PCR analysis, it is suggested that 2017b) (Fig. 11.11).
PbDIR4 belongs to a (+)pinoresinol-forming
DIR protein, and it is involved in formation of
lignin oligomers during development of stone 11.7.2 Bagging of Fruit
cells.
In pear production practices, bagging of fruits is a
common cultivation management measure. Pre-
11.7 Developmental Regulation harvest bagging can change the microenviron-
of Stone Cells ment around the fruit, thus affecting fruit quality.
Incidentally, effects of the type of bag used on
In addition to genetic factors influencing devel- fruit quality may also vary, and in some instances
opment of stone cells in pear fruits, the envi- may even contribute to negative outcomes
ronment plays a regulatory role in formation of (Wang et al. 2013, 2017a, b; Tao et al. 2015).
these stone cells, including light, water, mineral As an example, fruits of ‘Dangshan Su’ were
elements, and hormones. At present, there are bagged using double-layered paper bags, with
some available effective measures for controlling brown-colored outer layers and black-colored
the content of stone cells. inner layers, along with two gas-exchange holes
The roles of various factors involved in reg- present at bottom ends of these bags. It was
ulation of stone cells, as well as likely regulation observed that there were no significant differ-
mechanisms of these stone cells in pear fruits will ences in contents of stone cells between bagged
be herein presented. fruits and unbagged fruits. Moreover, patterns of
accumulation of stone cells and lignin contents in
bagged fruits and unbagged fruits were essen-
11.7.1 Pollination tially the same during fruit development. How-
ever, the activity of cinnamate-4-hydroxylase
As pear has a gametophytic self-incompatibility (C4H) in bagged fruits was lower than that in
(GSI) mechanism, the percentage of unbagged fruits during all stages of fruit devel-
self-pollinated fruit set in an orchard is rather opment (Tao et al. 2015).
low. Therefore, pear trees require In another study, effects of polyethylene (PE)-
cross-pollination to insure adequate fruit set. bagged and white non-woven polypropylene
Moreover, due to presence of the xenia phe- fabric bags on lignin content and metabolism in
nomenon in pear, fruit quality, including contents ‘Chili’ (P.  bretschneideri) fruits were investi-
of stone cells in fruits, is influenced to a great gated (Wang et al. 2017a, b). It was revealed that
extent by pollen from different pear cultivars. PE-bagged fruits had the highest lignin contents,
At present, the mechanism of pollination followed by unbagged fruits, and finally
affecting development of stone cells in pear is not non-woven fabric-bagged fruits, with the lowest
yet clearly understood. In cross-pollination of lignin contents. Moreover, white non-woven
‘Dangshan Su’ pear, it is observed that pollina- polypropylene fabric bags contributed to down-
tion of different pear cultivars can change the regulation of expression of Pb4CL, PbCAD, and
11 Stone Cell Development in Pear 219

Fig. 11.11 Pathway analysis of microRNA regulation of fruit quality traits in pear

PbPOD genes of the phenylpropanoid metabolic fruit development in P. pyrifolia cv. Niitaka.
pathway, and leading to lower levels of lignin They found that water stress at full-bloom and
synthesis; whereas, all these three genes were soon after full-bloom would lead to an increase
upregulated in PE-bagged fruits, and contributing in stone cell content until the fruit reaches
to higher levels of lignin synthesis (Wang et al. maturity, when compared to control
2017a, b). (non-stressed) fruits. However, water stress
It is critical to ask the question as to why does treatment prior to full-bloom did not have any
bagging affect lignin metabolism and stone cell significant effects on stone cell contents of
development of pear fruits? Thus far, it has been mature fruits. It has been proposed that water
speculated that expression of key genes in the stress at full-bloom and after full-bloom con-
lignin synthesis pathway may be modified due to tributed to lower levels of calcium (Ca) along
the effects of bagging on light intensity and light with higher POD activities in leaves and in fruit
quality on fruit development. pulp, but had no effects on contents of magne-
sium (Mg), N, phosphorous (P), and potassium
(K) in these tissues (Lee et al. 2006). However,
11.7.3 Water Stress none of these changes were observed in fruits of
trees subjected to water stress prior to full-bloom.
It has been reported that water stress has a sig- The above findings suggest that the mecha-
nificant effect on stone cell content in pear fruits nism of water stress leading to higher stone cell
(Kim et al. 2004b). Lee et al. (2006) investigated contents in pear fruits is as follows. Water stress
the influence of water stress on flowering and contributes to lower levels of calcium
220 X. Cheng et al.

accumulation in fruits, which leads to increased exogenous sprays of CaCl2 can increase calcium
POD activity, thereby resulting in accumulation content in pear leaves and fruits (peel and flesh),
of lignin in cell walls. In turn, this will ultimately thus influencing POD activity, which ultimately
promote formation of stone cells in fruit pulp regulates lignin synthesis and stone cell devel-
tissues. Therefore, water stress during early opment. It can be noted that POD activity is
stages of fruit development influences stone cell regulated by the content of calcium ions in pear
formation and development in pear fruits (Lee fruits. However, the effects of different treat-
et al. 2006). ments of CaCl2 and treatment times are different,
and the specific mechanism involved in these
responses is yet to be explored and elucidated
11.7.4 Exogenous Mineral Elements (Kim et al. 2004b; Lee et al. 2007; Lu et al.
2015).
In general, mineral elements are essential for Although there are various studies on the
plant growth and development. It is known that effects of boron and zinc ion sprays on control of
Ca2+ is the second signaling messenger in plants. stone cell contents in pear fruits undertaken by
As mentioned above (Sect. 11.5.3), calcium Chinese researchers, unfortunately none of these
accumulation in leaf and fruit tissues is related to have studies been published.
development of stone cells in pear fruits (Kim
et al. 2004b).
In recent studies, pear trees were treated with 11.7.5 Exogenous Hormones
different concentrations of CaCl2 (0.3, 0.5, and
1.0%), and it was found that CaCl2 treatments at Plant hormones can regulate multiple metabolic
0.5 and 1.0% reduced stone cell contents in fruits pathways in plants, in which gibberellin
compared to those of control (non-treated fruit). (GA) can promote growth and development of
However, findings were not as clear for trees crops, early maturity, improve quality, and
treated with 0.3% CaCl2. Nevertheless, CaCl2 increase production.
treatments at all three levels were found to reduce It has been reported that GA applications at
stone cell size, particularly that of the ratio of the the carpopodium stage of development can reg-
area greater than 200 lm2. In addition, 0.5% ulate metabolism of lignin in pear fruits (Yang
CaCl2 treatment could also reduce cell wall et al. 2014). During the rapid growth period of
bound and soluble peroxidase enzyme activities the fruit, the content of lignin in GA-treated fruit
(Kim et al. 2004b; Lee et al. 2007). is lower than that of the control (non-treated)
In another study, pear fruits, at 80 days after (Yang et al. 2014). Moreover, enzyme activities
flowering, were soaked with 0.5% CaCl2, and of CAD and POD in GA-treated fruit are lower
then fruits were harvested at maturity and stored than those in control fruit, while PAL activity at
(Lu et al. 2015). During storage, not only con- early stages of fruit development is significantly
tents of lignin in CaCl2-treated fruits were sig- lower than that in control fruit. In addition,
nificantly lower than those of control fruits, but expression levels of PpPAL1, PpPAL2, Pp4CL1,
also activities of PAL, 4CL, CAD, guaiacol Pp4CL2, and PpPOD1 in GA-treated fruits are
peroxidase (G-POD), and syringaldazine perox- lower than those in control fruits. Furthermore,
idase (S-POD) were significantly lower. In expression levels of PpCAD2 in GA-treated
addition, expression levels of CADs genes in fruits are significantly lower than those in con-
CaCl2-treated fruits were also significantly lower trol fruits during early stages of development.
compared to those of control (non-treated) fruits These findings suggest that GA may affect fruit
(Lu et al. 2015). lignin synthesis and stone cell development by
In summary, CaCl2 treatment can significantly regulating activities of key enzymes and
reduce lignin content, stone cell content, and expression of genes involved in lignin synthesis
stone cell size in pear fruits. It is suggested that (Yang et al. 2014).
11 Stone Cell Development in Pear 221

In addition to GA, salicylic acid (SA) has also development and lignin deposition do not stop at
been reported to regulate the development of these latter stages of fruit development. There-
stone cells in pear fruits (Zhang et al. 2002; Xue fore, this begs the question as to why does lignin
et al. 2018). Sprays of 0.02% SA on trees of accumulate following cell death?
P. sinkiangensis and P.  bretschneideri cv. In recent years, many experimental studies have
Yali found that these exogenous sprays can shown that lignin may accumulate from the
inhibit POD activity in the fruit, as well as reduce beginning of cell growth until cell death, and
content and size of stone cell clusters. As an accumulation of lignin continues following cell
important member of a plant’s disease resistance death (Voxeur et al. 2015). It is assumed that
signaling pathway, the role of SA has not yet neighboring cells may transport monolignol
been elucidated for its specific mechanism of polymerization-associated enzymes and monolig-
inhibiting the development of stone cells (Zhang nols to these dead lignified cells (Barros et al. 2015).
et al. 2002). Stone cell lignification may undergo a similar
process and pattern of development. Pits along
cell walls of stone cells may serve as material
11.8 Concluding Remarks transport channels to neighboring cells, thereby
and Future Prospects transporting active oxygen, polymerases, and
monolignols (Fig. 11.5) (Jin et al. 2013; Zhao
The effects of stone cells (or stone cell clusters), et al. 2013; Barros et al. 2015; Cheng et al.
both content and size, on fruit quality, as well as 2019c). Currently, plant cell lignification patterns
of components of stone cells (biosynthesis are mainly classified into three types, including
pathway and metabolic mechanism of lignin), cooperative lignification, partial cooperative lig-
development process, and distribution of stone nification, and autonomous lignification (Barros
cells, and regulation measures of stone cell et al. 2015). We speculate that formation of stone
development have been investigated. However, it cells may belong to either cooperative lignifica-
is still a long way to unravel the mystery of the tion or partial cooperative lignification. However,
mechanism of stone cell formation in pear fruits. at this time, there is a lack of relevant evidence,
Therefore, we would like to propose that the and therefore, future studies should be under-
following studies should be undertaken. taken to provide such evidence.

11.8.1 Lignification Patterns 11.8.2 The Branch Pathway

of Parenchyma Cells of Monolignol
in Pear Fruits Biosynthesis in Pear

As stone cells are lignified parenchyma cells in Although there is some level of understanding of
pear fruits, microscopic observations during early lignin biosynthetic pathways in pear fruits,
lignification have shown that there are large pathways of lignin metabolism are complex, with
numbers of Golgi organelles and transport vesi- several branches. Thus, these should be explored
cles present in secondary cell walls, thereby further. Numerous studies have demonstrated
indicating that there is an extensive material that either promotion or inhibition of a branch of
transport that is being undertaken during this lignin metabolism may yield different results,
period (Jin et al. 2013; Zhao et al. 2013). and perhaps lead to a novel lignin structure.
Although cellular contents are dissipated during Therefore, it is of great importance to further
latter periods of fruit growth and lose their abil- investigate and clarify branch pathways of lignin
ities to synthesize lignin, secondary cell wall metabolism, particularly those involved in
222 X. Cheng et al.

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 Genetic and Genomic Analyses
of Vegetative Budbreak in Response 12
to Chilling Units in European Pear
(Pyrus Communis L.)

Gilad Gabay and Moshe A. Flaishman

Abstract heritability, the potential for breeding a

Dormancy is critical for the normal yearly low-chilling requirement pear cultivar is high.
cycle of fruit trees in temperate zones due to However, chilling requirements are subject to
their requirements of exposure to certain a complex genetic mechanism which is prob-
numbers of chilling hours. Once the chilling ably determined by, or partially derived from,
requirement is fulfilled, vegetative budbreak multiple genes. Genetic factors affecting dor-
can occur when climatic conditions are favor- mancy have been identified for the first time in
able. Exposure to insufficient chilling units peach, wherein MADS-box genes associated
can lead to delayed vegetative budbreak. Bud with dormancy regulation have been reported.
dormancy has been studied in perennial fruit Six DORMANCY-ASSOCIATED MADS-BOX
trees within the context of the effects of (DAM) genes, and a genomic region, desig-
climate change. The recent rise in tempera- nated as the evergrowing (evg) locus, have been
tures worldwide has led to a reduction in identified. To date, three DAM genes, including
chilling units accumulation. Pear cultivars are PpDAM1, PpDAM2, and PpDAM3, have been
highly influenced by the number of chilling identified in Asian pear (Pyrus spp.). In previ-
units accumulated during the winter. How- ous genetic studies in apple, which has a high
ever, fruit of most low-chilling cultivars is level of synteny with pear, quantitative trait loci
considered to be of low quality. Study of the (QTLs) for chilling requirements have been
genetic mechanism underlying chilling identified. A QTL common to all families has
requirements would greatly accelerate adapta- been located on linkage group 9, suggesting
tion of new pear cultivars to warm climates. stability of this QTL over different families,
As vegetative budbreak date shows high climate regions, and years. However, in Euro-
pean pear, a major QTL has been detected on
linkage group 8, and an additional QTL on
linkage group 9 has also been confirmed.
G. Gabay
M. A. Flaishman (&) Differentially expressed genes in these regions
Volcani Research Center, Institute of Plant Sciences, include PcDAM1 and PcDAM2, putative
Derech Hamacabim 68, P.O. Box 15159,
Rishon Lezion, Israel
orthologs of PpDAM1 and PpDAM2. Due to a
e-mail: [email protected] significant genotype  environment (G  E)
G. Gabay
effect, QTLs associated with G  E vegetative
Faculty of Agriculture, Food and Environment, budbreak date have been detected. It has long
The Robert H. Smith Institute of Plant Sciences and been known that content levels of metabo-
Genetics in Agriculture, Hebrew University of lites are highly correlated with dormancy phase
Jerusalem, P.O. Box 12, 76100 Rehovot, Israel

© Springer Nature Switzerland AG 2019 227

S. S. Korban (ed.), The Pear Genome, Compendium of Plant Genomes,
https://doi.org/10.1007/978-3-030-11048-2_12
228 G. Gabay and M. A. Flaishman

transitions. Metabolites, such as phospholipids, dormant buds will break, and trees will resume
sugars, and fatty acids, including growth (Erez and Lavee 1971; Wigge 2013).
alpha-linolenic acid, play major roles in dor- The CR is generally characterized by the number
mancy regulation in pear. Several pear genes, of CUs needed for a given cultivar to achieve
such as 12-oxophytodienoate reductase 2-like 50% budbreak under favorable conditions. It is
(alpha-linolenic acid pathway), have been important to point out that CR and HR are syn-
found to be linked to dormancy regulation. chronized, and well-correlated. Hence, insuffi-
A proposed model for pear selection of traits cient CUs result in an extended HR period for
under a changing climate will be discussed. budbreak; whereas, overexposure to CUs that
extend CR results in a shorter period of favorable
conditions for budbreak induction (Ruiz et al.
2007). Therefore, when CR is not fulfilled, veg-
etative budbreak (VB) date is delayed. Hence,
VB date can indicate the CR of a particular
12.1 Introduction genotype.
European pear (P. communis) cultivars are
Perennial plants have developed a dormancy mostly bred in climate regions wherein winter
mechanism in temperate regions to overcome temperatures are low enough and sustained for
severe cold temperature conditions and frost. the duration of the winter season to satisfy CRs,
Dormancy is broadly characterized by three which in some cases differ from climates of other
states, including the following: (1) parador- growing regions, such as those of the Mediter-
mancy, when factors exterior to the bud regulate ranean region. Therefore, it is unlikely that pear
and maintain the dormancy state—this is usually tree performance for traits, such as flower
related to apical dominance; (2) endodormancy, development and fruit set, will be similar over
when factors related to chilling accumulation different environments (Labuschagné et al.
within buds regulate transition between different 2002).
dormancy states—this is the main state analyzed There are various models to estimate the
and discussed in this chapter due to its relevance, number of CUs. In the Mediterranean region,
which will be further explained; and (3) ecodor- characterized by low CU accumulation,
mancy, when environmental factors, which are endodormancy release is more strongly affected
mainly related to temperature, signal to the bud, by recent climate changes than in areas with
after sufficient chilling accumulation, to be higher numbers of CUs. In warm climate regions,
released from dormancy. a commonly used model to evaluate CU accu-
Most Rosaceae fruit tree species, including mulation is a dynamic model developed to
the European pear (Pyrus communis), enter a evaluate CRs in warm regions, such as those of
state of dormancy in response to decreasing the Mediterranean and California, as it accounts
temperatures. On the other hand, endodormancy for negative effects of high temperatures during
release depends on the number of chilling hours winter on CU accumulation (Erez et al. 1988).
(chilling units = CUs) that deciduous trees are Recent global warming conditions are expec-
exposed to during the winter season (Anderson ted to result in reductions of CU accumulation,
et al. 1986; Heide and Prestrud 2005). Chilling based on model climate predictions (Campoy
requirements (CRs) vary among cultivars and et al. 2011). As CU accumulation leads to
among plant species, and these correspond to the delayed budbreak, which is essential for normal
number of CUs required for budbreak during the flower and fruit development (Takemura et al.
spring. Once trees are exposed to favorable 2015), consequences of reduced CUs for warm
environmental conditions, such as rising tem- areas, such as the Mediterranean, may lead to
peratures, represented by heat requirement (HR), severe disorders in deciduous fruit tree growth
12 Genetic and Genomic Analyses of Vegetative Budbreak … 229

habits, such as reduced yield and abnormal fruit by, or partially controlled by multiple genes
set. Currently, a standard practice for inducing (Howe et al. 2000).
VB on required dates, for normal fruit growth
and for filling gaps between CR and actual CU
accumulation in warmer areas, is to spray 12.2.1 Main Effects of Heritability
chemical compounds. However, due to increas- and Variance
ing awareness of the environmental effects of this of Vegetative Budbreak
practice, there is a growing demand for fruit trees Date
with low CRs (Celton et al. 2011; Ubi et al.
2010; van Dyk et al. 2010). Therefore, a better It has been recently reported that VB date vari-
understanding of the genetics and of key factors ance is controlled by main effects in European
governing dormancy phase transitions is much pear (Gabay et al. 2017). VB date phenotyping,
needed. which indicates chilling requirements, has been
conducted over two consecutive years (2014–
2015) in two locations in Israel, Bet Dagan
12.2 Vegetative Budbreak Date (BD) and Tzuba (TZU), that highly differ in their
Variations and Chilling yearly average accumulated CUs (Fig. 12.1). The
Requirements in European pear material consists of an F1 population,
Pears derived from a cross between ‘Spadona’ (low
CR) and ‘Harrow Sweet’ (high CR), as well as
Cultivars of European pear (P. communis) are commercial pear cultivars and accessions differ-
highly influenced by the number of CUs accu- ing in their CRs (Fig. 12.1). Replications of these
mulated during the winter. Based on modeling genotypes have been exposed during the winter
and phenological data, the numbers of CUs to different CUs. Subsequently, trees have been
required for adequate budbreak can vary from transferred to the same region and exposed to
300 (cv. Spadona) to 1500 (cv. Bartlett). There- similar heat conditions to induce VB. This has
fore, most commercial pear cultivars are grown been conducted to determine the genetic com-
in temperate regions, classified as having inter- ponent of CR, and to distinguish it from the
mediate to high CUs, and are poorly adapted to genetic component for HR.
mild climates (Flaishman et al. 2001; Zohary As estimation of broad-sense heritability (H2)
1997). Recent global climate changes, along with is reliable for specific environmental conditions
increasing demands for growing pear trees in and populations (Souza et al. 1998), it is revealed
warm regions, have highlighted the importance that H2 estimations in this study are higher within
of developing low-CR fruit tree cultivars (Busov locations, and specific to location. Furthermore,
et al. 2016; Li et al. 2018). the determined overall mean broad-sense heri-
It has been reported that VB date shows high tability (H2 = 0.46) for pear is lower than those
heritably in apple, Malus  domestica (Labu- reported in other studies for apple, with esti-
schagné et al. 2002). Thus, there is a good mated values of 0.87 (Allard et al. 2016), 0.88–
potential for breeding low-CR pear cultivars 0.92 (Celton et al. 2011), and 0.62–0.92 (van
adapted to increasing temperatures (Allard et al. Dyk et al. 2010). However, specific H2 values
2016). However, CRs corresponding to VB dates per specific year and location for pear are similar
are subject to a complex genetic mechanism in to those obtained in apple, ranging between 0.84
deciduous trees, which is probably determined and 0.94.
230 G. Gabay and M. A. Flaishman

Fig. 12.1 a Average days for vegetative budbreak in X-axis corresponds to number of accumulated CUs. The
pear cultivars and accessions in two locations in Israel Y-axis corresponds to year and location. Tzuba = High--
over two consecutive years (2014–2015) (day 0 = 1st of chilling unit accumulation, in the Jerusalem mountains
January). b Accumulation of chilling units in two (720 m a.s.l); and Bet Dagan = Low-chilling unit accu-
locations over two consecutive years (2014–2015). The mulation, in a coastal area (50 m a.s.l)
12 Genetic and Genomic Analyses of Vegetative Budbreak … 231

12.2.2 Breeding Potential of Pears 12.3 Quantitative Trait Loci

for Low-Chilling (QTL) Mapping for Vegetative
Requirements Budbreak

A large genetic effect is observed for CR and its Genetic factors influencing CR were identified
heritability, thus indicating that there is a high for the first time in peach (Prunus), a member of
breeding potential for this trait (Celton et al. Rosaceae (Bielenberg et al. 2008). MADS-box
2011; Trainin et al. 2013; van Dyk et al. 2010). genes associated with dormancy regulation were
However, a significant genotype  environment identified, including six DORMANCY-ASSO-
(G  E) interaction is a major factor for VB date, CIATED MADS-BOX (DAM) genes along with a
and accounts for 35% of the observed phenotypic genomic region, designated as the evergrowing
variance in pear (Gabay et al. 2017; Labuschagné (evg) locus. These genes were proposed to play
et al. 2002). Furthermore, a significant effect of regulatory functions in bud set, vegetative
genotype on VB date has been observed and growth, and cessation of growth (Jiménez et al.
accounting for 35.8% of the phenotypic variance 2010). In a later study, quantitative trait loci
for VB date. Although genotypic effects influence (QTL) analysis was conducted using a large
the time of VB in pear, genotype  year and peach population, and a QTL associated with CR
genotype  location interactions should be also was identified in the same genomic region as that
taken into account when low-CR cultivars are of evg (Fan et al. 2010). QTLs associated with
being selected for. These interactions highlight the CR and dormancy regulation have already been
importance of selecting a particular genotype for a identified in other Rosaceae members. In Prunus,
targeted climatic region. Hence, genotypes have the same QTLs have been identified for both CR
different responses to number of CUs and to other and bloom date, thereby confirming presence of a
climatic components. This renders selection for strong correlation between these two traits (Dir-
such a trait complicated under instances of lewanger et al. 2012).
changing climates as genotypes may act differ- In previous genetic studies using full-sib
ently in upcoming years with predicted increases families in apple, which shares a high level of
in yearly average temperatures (Dirlewanger et al. synteny with pear (Celton et al. 2009), QTLs for
2012). CR have also been identified (Allard et al. 2016;
Cultivar selection in the targeted climate Celton et al. 2011; van Dyk et al. 2010). How-
region does not ensure the cultivar’s adaptation ever, the only QTL common to all families is
to that region, as CU accumulation can sharply located on LG9, thus confirming stability of this
decrease within a given climate region. Pheno- QTL over different families, climate regions, and
typic plasticity, a genotype’s ability to perform years (Allard et al. 2016; van Dyk et al. 2010).
stably across different years and climate regions, The first QTL analysis for a pear population
plays an important role in cultivar selection. This segregating for VB date (Gabay et al. 2017) has
is particularly critical for such traits as CR, confirmed QTL synteny between apple and pear
wherein CR is highly influenced by recent using data obtained from ‘selective genotyping’;
increases in worldwide temperatures. Therefore, i.e., tail analysis. This method can be used to
CR trait stability across environments should determine linkages between a genetic marker and a
serve as an important criterion during breeding. target trait at relatively low cost, and with a rela-
In addition, deciphering genetic and physiologi- tively small number of genotyped individuals
cal mechanisms of CR interactions with envi- (Darvasi and Soller 1992). Furthermore, two QTLs
ronment will further enhance pear breeding for have been detected within the same genomic
low CR. regions as those found in apple, LG9 and LG8, and
232 G. Gabay and M. A. Flaishman

these are determined to be stable over locations and as those found in apple, but at different levels of
years under diverse climatic conditions. However, significance and phenotypic variances explaining
recent advances in genotyping methods have these QTLs (Gabay et al. 2018). Furthermore,
enabled more accurate detection of such QTLs. QTL mapping using high-resolution genetic maps
has enabled accurate detection and confirmation
of QTL analysis results reported in both pear
12.3.1 Fine QTL Mapping Using (Gabay et al. 2017) and apple (Allard et al. 2016;
a High-Resolution Celton et al. 2011; van Dyk et al. 2010). The
Genetic Map above findings highlight the importance of con-
ducting independent genetic studies in pear, as
In an earlier study, QTLs associated with CRs well as in construction of high-resolution genetic
using a high-resolution genetic map in closely maps to accurately identify genomic regions
related species, including apple, have been iden- associated with complex target traits, as well as to
tified (Allard et al. 2016). Although, pear and accurately determine variance values explained
apple show high levels of synteny (Celton et al. by identified QTLs for these complex traits.
2009; Chagné et al. 2014), differences have been The reliability of a QTL for a trait of interest
observed between genomic regions of apple and its usefulness in pursuing efficient and
(LG9) and pear (LG8) associated with chilling effective marker-assisted selection strategies are
requirements. In apple, the most stable and sig- highly dependent on stability of this QTL under
nificant QTL has been detected on LG9 in various different environmental conditions/years, loca-
studies conducted in different locations and years tions, and genetic backgrounds (Allard et al.
(Allard et al. 2016; Celton et al. 2011; van Dyk 2016). Therefore, in a recent study, we have
et al. 2010). However, the most significant QTL identified 21 European pear cultivars with either
in pear is detected on LG8 (LOD score = 11.49), very low or high CR (i.e., ‘selective genotyp-
explaining 28% of the phenotypic variance of VB ing’). These cultivars have been subjected to
date (Gabay et al. 2018). This represents the first phenotyping and genotyping-by-sequencing
QTL detection in pear using a reliable genetic (GBS) analysis to evaluate those QTLs detected
map constructed using genotyping-by-sequencing in an F1 population of ‘Spadona’  ‘Harrow
(GBS) data for 162 F1 offsprings (Gabay et al. Sweet’ in diverse genetic backgrounds
2018). Additional QTLs for VB date have been (Fig. 12.1a). As numbers of accessions used to
detected on LGs 5 and 15 (Fig. 12.2), and these evaluate identified QTLs have not been sufficient
have also been previously identified in apple for pursuing genome-wide association stud-
(Allard et al. 2016). However, a new QTL asso- ies (GWAS), observed differences may result
ciated with VB date has been detected on LG13 in from other genetic variances that are not associ-
pear (Fig. 12.2). To the best of our knowledge, ated with CR (Zhu et al. 2008), and therefore our
this QTL has never been identified before in either results are deemed preliminary (Gabay et al.
pear or apple. Synteny within the subfamily 2018). It has been observed that significant
Amygdaloideae, which includes European pear molecular markers associated with VB date have
(P. communis) and apple (M.  domestica), has been detected in all LGs for which a QTL has
been reported for QTLs associated with traits such been detected in our F1 pear population. How-
as scab resistance (Bouvier et al. 2012), fire blight ever, not all of these markers are located within
resistance (Le Roux et al. 2012), and fruit soft- the highest peak of QTL intervals. Nevertheless,
ening (Costa et al. 2008). In addition, simple markers found significant in the highest peak of a
sequence repeat (SSR) markers have been found major QTL are detected on LGs 8 and 9 (Gabay
to be highly transferable between apple and pear et al. 2018). Therefore, it is assumed that these
(Celton et al. 2009; Yamamoto and Terakami regions control VB date in diverse genotypes of
2016). Interestingly, locations of QTLs found in European pear. In addition, genetic-relatedness
pear have been detected within the same regions analysis of pear cultivars included in this study
 12
Genetic and Genomic Analyses of Vegetative Budbreak …

Fig. 12.2 Positions of QTLs for VB date using a high-resolution consensus map of SPD  HS (Gabay et al. 2018). For
each QTL, one-LOD support intervals are shown. Black boxes correspond to QTLs for overall mean VB date (AVG). Blue
boxes correspond to QTLs for Tzuba (TZU) for high CU accumulation. Red boxes correspond to QTLs for Bet Dagan
(BD) for low CU accumulation
233
234 G. Gabay and M. A. Flaishman

has revealed presence of two groups corre- As low-CR cultivars may fulfill their CRs by
sponding to CR, thus suggesting that these culti- midwinter, unexpected warm temperatures dur-
vars share the same genetic mechanism governing ing this period may lead to early budbreak
the CR trait. However, a notable exception induction, but with likely subsequent drop in
detected among the low-CR group is pear cv. temperatures, this can result in frost damage.
Florida Home (Fig. 12.1a). Although this cultivar Therefore, it is essential to select high- and
is known to be an extremely low-CR cultivar, due low-CR pear cultivars with sufficient phenotypic
to its very early VB date, it is observed that it is plasticity and capable of withstanding such
not grouped close to other low-CR cultivars. This changes in weather conditions, thus demonstrat-
finding may be attributed to the fact that this ing stable performance within the same location,
cultivar, derived from a cross between European as climatic conditions may vary over the years.
and Asian pears (Villalta et al. 2005), has a sig-
nificantly different genetic background for CR
determination (Gabay et al. 2018). The major 12.4 Key Regulators During
QTL, on LG8, associated with VB date has been Dormancy Phase Transitions
confirmed across years and locations, having
experienced large differences in climatic condi- 12.4.1 Gene Expression, Gene
tions. Therefore, markers located within the Annotation,
identified QTL interval on LG8 for VB date can and Pathway
be used for future marker-assisted selection in Enrichment of Gene
pear breeding programs. Expression for Bud
Dormancy

12.3.2 G  E QTLs Associated Previous studies have reported on the importance

with Vegetative of DAM genes, along with other important genes,
Budbreak in regulating gene expression during bud dor-
mancy phase transitions. In apple, which has a
As there is a significant G  E interaction for high level of synteny with pear (Celton et al.
vegetative budbreak (Gabay et al. 2017), it has 2009), four DAM-like genes have been charac-
prompted efforts to identify QTL(s) associated terized, including MdDAMa, MdDAMb,
with CR, isolated from other environmental MdDAMc, and MdDAMd. Expression levels of
effects (Fig. 12.2). The most significant G  E MdDAMa (on LG16) and MdDAMc (on LG8)
QTLs are detected on LG9 and LG5, and sub- have been observed to differ over time, thus
sequently additional QTLs have been identified suggesting that these genes play roles in apple
on LG8 and LG17 (Gabay et al. 2018). All these tree dormancy (Mimida et al. 2015). To date,
QTLs suggest availability of pear genotypes, three DAM genes, including PpDAM1 (previ-
carrying useful genes/alleles, with differences in ously PpMADS13-1), PpDAM2 (previously
mean VB dates between two locations/different PpMADS13-2), and PpDAM3 (previously
climatic conditions. Hence, these QTLs could be PpMADS13-3), have been identified in pear
useful in predicting genotypic stability across (Pyrus spp.) (Tuan et al. 2017). Furthermore, it
diverse environments. This is important not only has been observed that expression levels of
to predict cultivar performance over climatic PpMADS13-1, a DAM homolog identified in
changes, but also for matching CRs of pear cul- P. pyrifolia (Japanese pear), are lower prior to
tivars to appropriate growing regions. Further- release of endodormancy (Saito et al. 2015). In
more, it is important to point out that adequate addition, expression levels of PpMADS13-2 and
VB date trait is relevant for both warm and cold PpMADS13-3 have been found to correlate with
regions for low-CR pear cultivars, due to frost different phases of dormancy (Saito et al. 2013).
susceptibility (Olukolu et al. 2009). In European pear, PcDAM1 and PcDAM2,
12 Genetic and Genomic Analyses of Vegetative Budbreak … 235

putative orthologs of PpDAM1 and PpDAM2, are 12.4.2 Major Changes in Metabolite
reportedly differentially expressed between dor- Content Levels During
mancy phases, thus confirming their roles in Dormancy and Their
dormancy phase transitions in European pear, Proposed Regulatory
P. communis (Gabay et al. 2019). Other notable Roles
genes involved in dormancy break and CR
determination include ParSOC1, an Arabidopsis Dormancy is often correlated with sharp changes
MADS-box gene homolog identified in apricot in metabolite content and composition (Del Cueto
(Trainin et al. 2013), and EARLY BUD-BREAK1 et al. 2017; Ionescu et al. 2017; Izadyar and Wang
(EBB1), first identified in poplar, and more 1999; Wang and Faust 1990). However, to date,
recently an apple homolog (MdEBB1) has also metabolite profiling of pear, and specifically of
been identified (Busov et al. 2016). dormancy in European pears, has not been well
Transcriptome profiles during different phases described. Along with genes associated with
of pear dormancy, as well as those during annual dormancy regulation, several metabolites and
growth cycles have been investigated (Bai et al. proteins, such as dehydrins, sugars, fatty acids,
2013; Gabay et al. 2019; Liu et al. 2012). polar lipids, and protein kinases have been
Annotation of gene profiles has been conducted reported to be involved in dormancy (Eremina
using GO terms, and KEGG pathway assignment et al. 2016; Maruyama et al. 2009). Lipids are
has been described. It has been observed that the proposed to play major roles in establishment of
‘Metabolic Pathways’ category is the most enri- dormancy by modifying the metabolite compo-
ched. This has suggested the importance of gene sition in plants for buds to deal with cold tem-
regulation of metabolic processes during transi- peratures. Changes in bud membrane metabolites,
tions between different phases of dormancy in dominated by fatty acids and lipids, during dor-
both Asian pear (Bai et al. 2013; Liu et al. 2012) mancy will offer optimal physiological conditions
and European pear (Gabay et al. 2019). Plant for budbreak response in the spring (Wang and
metabolic pathways are usually controlled by Faust 1990). Sugar accumulation (raffinose) has
diverse groups of genes and characterized by been detected during establishment of dormancy
complex gene regulation mechanisms (Xiao et al. in apple, suggesting that sugar accumulation may
2015). It has been observed that high numbers of protect dormant buds against draught during
differentially expressed (DE) genes, belonging to dormancy (Falavigna et al. 2018).
diverse gene families in the most enriched Previously, it has been reported that accumu-
KEGG pathway, are detected in all comparative lation of major groups of metabolites in various
dormancy phase transitions, thereby confirming groups of fruit crops is found to be correlated with
the proposal that metabolic processes are regu- chilling accumulation and dormancy break, such
lated by a complex genetic mechanism (Allard as those of unsaturated fatty acids in peach (Erez
et al. 2016; Celton et al. 2011; Heide and Pre- et al. 1997), sugars in apple (Falavigna et al.
strud 2005; Howe et al. 2000; Leida et al. 2010). 2018), and phospholipids in blackberry (Izadyar
Comparisons between pear cultivars differing in and Wang 1999). Recently, significant changes in
CRs have revealed that many of the biological more than 50 metabolites are detected between
processes of the GO analysis are detected earlier dormancy phase transitions in pear (Gabay et al.
in the low-CR cultivar Spadona compared to 2019). Specifically, three main groups of
those of the high-CR cultivar Harrow Sweet metabolites, including fatty acids, sugars, and
(Gabay et al. 2019). Hence, ‘Spadona’ must phospholipids, have been found to cluster toge-
respond earlier than ‘Harrow Sweet’ to drops in ther with similar patterns of changes during dor-
temperature. Biological processes, such as mancy, thereby suggesting their potential roles in
‘Metabolic Process’ and ‘Biosynthetic Process’ regulation of dormancy. Transcriptome analysis
must also be active earlier in ‘Spadona’ than in of ‘Spadona’ (low CR) and ‘Harrow Sweet’ (high
‘Harrow Sweet’ (Gabay et al. 2019). CR) has revealed that 22 DE genes related to the
236 G. Gabay and M. A. Flaishman

alpha-linolenic acid pathway, based on the sugars or some other factors that can sense suffi-
KEGG analysis, are detected. In particular, it has cient sugar accumulation. Moreover, when chil-
been observed that there is a significant and sharp ling is deemed sufficient, these sugars, or other
increase in alpha-linolenic acid content toward factors, alter the status of buds, from dormancy to
the end of dormancy in both pear cultivars. Fur- active growth, in the spring, as recently reported
thermore, fatty acid profiles in both cultivars are in grape (Khalil-Ur-Rehman et al. 2017).
found to be low during all phases of dormancy, In addition, increases in phospholipid content
but then this is followed by a sharp increase toward dormancy establishment have been
toward a break in dormancy. Previously, it has observed in pear (Gabay et al. 2019). This has been
been reported that fatty acid content is directly previously observed in peach bud dormancy, and
correlated with chilling accumulation (Erez et al. accompanied by chilling accumulation (Erez et al.
1997). In our recent study, although both pear 1997). However, this pattern is observed only in a
cultivars have been exposed to the same number high-CR pear cultivar. Therefore, additional
of CUs, they have exhibited different fatty acid studies should be conducted to confirm this finding
profiles during dormancy (Gabay et al. 2019). with additional groups of pear cultivars.
Moreover, in ‘Spadona’ (low CR), six additional It has been reported that large numbers of DE
unsaturated fatty acids have been detected, transcripts (n > 4000) are correlated to metabo-
including linoleic acid, which has significantly lites, along with significantly modified contents
changed during dormancy. Therefore, it is pro- at different sampling dates during dormancy, and
posed that changes in fatty acids, such as are likely controlled by multiple genes involved
alpha-linolenic acid, lauric acid, linoleic acid, in regulating metabolic processes (Xiao et al.
margaric acid, non-adecylic acid, palmitic acid, 2015). Genetic regulation of dormancy is com-
and stearic acid contribute to changes in mem- plex, and it is governed by multiple genes (Allard
brane metabolite composition that allow for et al. 2016; Celton et al. 2011; Heide and Pre-
budbreak. Moreover, it is also important to point strud 2005; Leida et al. 2010). This hypothesis is
out that accumulation of these fatty acids differs further confirmed by metabolite profiles of pear
between low- and high-CR pear cultivars. Hence, transcriptomes and their correlations to gene
fatty acid profile in low-CR pear changes earlier expression profiles during various phases of
than that in high-CR pear. dormancy (Gabay et al. 2019).
Furthermore, significant changes in contents
of 11 sugars are also observed in pear. In both
pear cultivars used in our study, changes in pat- 12.5 Integrated System Biology
terns of raffinose contents are found to be similar, Approaches to Decipher
thereby also confirming recent findings observed the Regulation Mechanism
during dormancy in apple (Falavigna et al. 2018). of Bud Dormancy
It has been suggested that raffinose protects apple
buds against drought (Falavigna et al. 2018). This 12.5.1 Co-localization
has been supported in our pear study, as raffinose of Differentially
accumulation is observed toward budbreak Expressed Genes, During
(Gabay et al. 2019). However, other sugars, such Dormancy Phase
as sucrose, undergo similar patterns of changes Transition, to QTLs
during dormancy. As it is reported, sugars are Associated with Chilling
necessary for regulation of bud regrowth regula- Requirements
tion (Roitsch and González 2004), and that bud- and Budbreak Date
break in the spring is highly influenced by
availability of sugar (Tixier et al. 2017). There- Although genes associated with pear dormancy
fore, it is assumed that the mechanism by which and VB may be located outside QTL intervals,
buds are signaled involves accumulation of QTL detection can lead to identification of
12 Genetic and Genomic Analyses of Vegetative Budbreak … 237

candidate genes underlying the QTL region, as may play roles in regulating metabolite synthesis,
previously described in tomato (Frary et al. 2000) which is essential for buds during dormancy and
and rice (Sallaud et al. 2003). Earlier, it has been then for budbreak in the spring (Gabay et al.
reported that DAM genes are located within the 2019). It is suggested that metabolites play
same genomic region of an identified QTL important roles in dormancy phase transitions
associated with CR in peach (Fan et al. 2010). In based on their profiles during dormancy
another study, wherein alleles of ParSOC1, an (Khalil-Ur-Rehman et al. 2017; Erez et al. 1997).
apricot MADS-box gene, are screened in 48 At the beginning of dormancy, phospholipids
apricot cultivars differing in CRs, a significant accumulate along with CUs and may be needed for
correlation is detected between allele segregation either sugar biosynthesis or to protect buds from
and CR (Trainin et al. 2013). In addition, a drops in temperature, and then followed by sugar
homolog of an AGAMOUS-LIKE24 (AGL24) accumulation. Sugars may play a role in signaling
gene in Arabidopsis thaliana, regulating flower- sufficient CU accumulation, allowing for budbreak
ing and is induced by vernalization, is located as soon as the temperature rises, as previously
close to the QTL on LG9, thus suggesting that reported in grape (Khalil-Ur-Rehman et al. 2017).
the same genetic factors determine CRs in both Increases in fatty acids during the last phase of
perennial and annual plants (Allard et al. 2016). dormancy, toward dormancy break, may lead to
Genes underlying five QTLs associated with membrane changes in buds, due to different
vegetative budbreak in pear, identified on LGs 5, metabolite composition, thus yielding optimal
8, 9, 13, and 15, have been identified and char- conditions for budbreak (Gabay et al. 2019).
acterized based on their levels of expression, as
well as their correlations to metabolites. These
genes, including PcDAM1, PcDAM2, and 12- 12.5.3 Putative Candidate Genes
oxophytodienoate reductase 2-like, involved in Associated
the alpha-linolenic acid pathway, have demon- with Regulation
strated significant changes in expression during of Dormancy
vegetative budbreak in pear (Gabay et al. 2019).
Among those genes demonstrating significant
differential expression and underlying QTLs
12.5.2 Key Regulators of Dormancy associated with VB date in pear include PcDAM1
and PcDAM2 (Gabay et al. 2019). Identification
Using an integrated systems biology approach, a of these genes represents a ‘proof of concept’ for
model for dormancy regulation involving those pursuing an integrated approach, as their roles in
most significant genomic regions associated with dormancy regulation have been previously
VB identified on LG8 (R2 = 28%) and LG9 described in both Japanese pear (Saito et al.
(R2 = 9.8%) in pear is proposed by Gabay et al. 2013, 2015) and apple (Mimida et al. 2015).
(2019). This model also takes into consideration Using this approach, other additional putative
metabolite contents during transition phases of genes, that may play roles in the genetic mech-
dormancy. Furthermore, as transcription factors, anism governing dormancy, have been detected.
such as DAM genes, are mostly expressed at the These include eight genes related to metabolic
beginning of and in mid-dormancy, these puta- pathways, and specifically to alpha-linolenic
tive candidate genes signal trees to enter into pathway (12-oxophytodienoate reductase 2-
dormancy when the temperature begins to drop like), and four genes encoded transcription fac-
(Table 12.1). Transcription factors can activate tors. In addition, using this integrated approach,
genes related to metabolic pathways, which are six new putative candidate genes, currently
mostly at their highest levels of expression dur- uncharacterized, are presumed to play major
ing later phases of dormancy. In turn, these genes roles in pear bud dormancy (Gabay et al. 2019).
238 G. Gabay and M. A. Flaishman

Table 12.1 Putative pear candidate genes associated with dormancy regulation
Gene Gene symbol Gene type1 Chr2
FT-interacting protein 1-like LOC103967842 TF 8
PcDAM1-MADS-box protein AGL24-like LOC103964948 TF 8
PcDAM2-MADS-box protein AGL24-like LOC103964950 TF 8
MADS-box protein AGL24-like LOC103964952 TF 8
3-hydroxyacyl-[acyl-carrier-protein] dehydratase FabZ-like LOC103967963 MP 8
Chlorophyll a-b binding protein CP24 10A, chloroplastic LOC103967973 MP 8
12-oxophytodienoate reductase 2-like LOC103967564 MP 8
Cytochrome b6-f complex iron-sulfur subunit, chloroplastic-like LOC103944475 MP 9
Palmitoyl-monogalactosyldiacylglycerol delta-7 desaturase, LOC103954983 MP 9
chloroplastic-like
Thymidine kinase a LOC103955051 MP 9
Chlorophyll a-b binding protein 151, chloroplastic-like LOC103955064 MP 9
Protein phosphatase 2C 56-like LOC103943902 MP 15
Uncharacterized LOC103964940 UF 8
Uncharacterized LOC103944526 UF 9
Uncharacterized LOC103944497 UF 9
Uncharacterized LOC103954139 UF 13
Uncharacterized LOC103943904 UF 15
Uncharacterized LOC103943918 UF 15
1
Gene type; TF = transcription factor, MP = metabolic pathways, UC = uncharacterized function
2
Chr = chromosome number of the pear genome

backgrounds. Furthermore, these have also been

12.6 Conclusions and Future previously identified in apple (Allard et al. 2016;
Research Directions Celton et al. 2011; van Dyk et al. 2010). These
identified VB QTLs associated with G  E
12.6.1 A Marker-assisted Selection interaction should also be taken into considera-
Strategy Taking tion when selecting pear genotypes under con-
into Consideration tinuous conditions of climate change.
G  E Effects A proposed selection strategy should take into
consideration such significant G  E effects
As pear breeding efforts are lengthy and (Fig. 12.3a). This model is developed based on
time-consuming, availability of tools that can data reported herein (Gabay et al. 2019)
facilitate and accelerate the breeding cycle is (Fig. 12.3b). Genotypes with G  E values
highly desired. In efforts to develop pear culti- equal to or near zero (i.e., category II; Fig. 12.3a)
vars with adaptability to changing climate con- are deemed to be more stable across different
ditions and warm growing regions, it is proposed environmental conditions. In addition, pheno-
that QTLs associated with VB date and identified typic values should also be taken into consider-
on LGs 8 and 9, for main G  E effects, can be ation based on the target location. Hence, a
used for marker-assisted selection (MAS). These low-CR pear cultivar should be selected for
QTLs have been identified in different pear cul- warm regions (i.e., Group I; Fig. 12.3a), while a
tivars of diverse genetic backgrounds, thus con- high-CR pear cultivar should be selected for cold
firming stability of these QTLs across these regions (i.e., Group III; Fig. 12.3a). For instance,
12 Genetic and Genomic Analyses of Vegetative Budbreak … 239

Fig. 12.3 Proposed model for pear selection in a breed- genotypes with large differences in performance (location
ing program under conditions of climate change. a 1. a < location b). b G  E values versus overall mean of an
Selection in a target location may result in an unsuitable F1 SPD  HS population. Genotypic differences under
cultivar in subsequent years due to climate change. 2. normalized scores for vegetative budbreak date between
Selection in multiple environments to evaluate phenotypic high-chilling unit location (TZ) and low-chill unit location
plasticity of selected genotypes. Group I—genotypes with (BD), and their means. The red star denotes cv. Spadona
low phenotypic performance. Group II—genotypes with (low-CR cultivar), and blue star denotes cv. Harrow
average phenotypic performance. Group III—genotypes Sweet (high-CR cultivar). Blue frames correspond to
with high phenotypic performance. Category I refers to genotypes with similar means for normalized vegetative
genotypes with large differences in performance (location budbreak date with high stability across environments
a > location b). Category II refers to genotypes with (genotype 309) and with low stability across environ-
phenotypic stability across different environmental con- ments (genotype 21) (Source: adapted from Gabay et al.
ditions (location a = location b). Category III refers to 2018)

low-chill cultivars should be selected from 12.6.2 Further Research

genotypes that demonstrate stability across
environments (genotype 309), while genotypes Further research should focus on detected QTL
with low stability across environments (genotype regions and putative candidate genes in European
21) should be discarded, although they have pear (Table 12.1), Asian pear (Saito et al. 2015,
similar means of normalized VB date (Fig. 12.3 2013), and apple (Allard et al. 2016; Mimida
b). Selection of new cultivars must be carried out et al. 2015). These QTLs and gene expression
in locations that can simulate climate conditions profiles should be further assessed in families
of the target location in which the cultivar will be and cultivars of different genetic backgrounds
grown. Hence, a breeder should consider using and under different climatic regions. As it is
the model of climate prediction to choose a assumed that CR has a great impact on flower
location that currently has the same climate development, fruit quality, and yield (Allard
conditions as the target location at the predicted et al. 2016; Bielenberg et al. 2008; Busov et al.
date for release. 2016; Khalil-Ur-Rehman et al. 2017; Lang et al.
240 G. Gabay and M. A. Flaishman

1987), associations between these traits with Campoy JA, Ruiz D, Egea J (2011) Dormancy in
chilling requirements and vegetative budbreak temperate fruit trees in a global warming context: a
review. Sci Hortic 130:357–372
date should be investigated. Celton J-M, Chagné D, Tustin SD, Terakami S, Nishi-
A low-chill apple cultivar, ‘Anna,’ has been tani C, Yamamoto T, Gardiner SE (2009) Update on
selected under warm temperature conditions and comparative genome mapping between Malus and
is considered a low-chilling cultivar. However, it Pyrus. BMC Res Notes 2:182. https://doi.org/10.1186/
1756-0500-2-182
has inferior fruit quality and poor storability Celton JM, Martinez S, Jammes MJ, Bechti A, Salvi S,
(Trainin et al. 2016). As most pear breeding Legave JM, Costes E (2011) Deciphering the genetic
efforts are conducted in cold regions (Zohary determinism of bud phenology in apple progenies: a
1997), it is difficult to determine whether or not new insight into chilling and heat requirement effects
on flowering dates and positional candidate genes.
fruit quality is associated with CR or that high New Phytol 192:378–392. https://doi.org/10.1111/j.
fruit quality cultivars are better adapted for cold 1469-8137.2011.03823.x
regions. Currently, we are pursuing pear breeding Chagné D, Crowhurst RN, Pindo M, Thrimawithana A,
efforts under warm climate conditions in Israel, Deng C, Ireland H, Fiers M, Dzierzon H, Cestaro A,
Fontana P, Bianco L, Lu A, Storey R, Knäbel M,
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009 Ubi BE, Sakamoto D, Ban Y, Shimada T, Ito A,
Ruiz D, Campoy JA, Egea J (2007) Chilling and heat Nakajima I, Takemura Y, Tamura F, Saito T,
requirements of apricot cultivars for flowering. Env Moriguchi T (2010) Molecular cloning of
Exp Bot 61:254–263. https://doi.org/10.1016/j. dormancy-associated MADS-box gene homologs and
envexpbot.2007.06.008 their characterization during seasonal endodormancy
Saito T, Bai S, Ito A, Sakamoto D, Saito T, Ubi BE, transitional phases of Japanese pear. J Am Soc Hortic
Imai T, Moriguchi T (2013) Expression and genomic Sci 135:174–182
structure of the dormancy-associated MADS box genes van Dyk MM, Soeker MK, Labuschagne IF, Rees DJG
MADS13 in Japanese pears (Pyrus pyrifolia Nakai) (2010) Identification of a major QTL for time of initial
that differ in their chilling requirement for endodor- vegetative budbreak in apple (Malus  domestica
mancy release. Tree Physiol 33:654–667. https://doi. Borkh.). Tree Genet Genomes 6:489–502. https://doi.
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(2015) Histone modification and signalling cascade of Richards SM, Liu SM (2005) Resistance to pear scab
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 Genetics, Genomics, and Breeding
for Fire Blight Resistance in Pear 13
Richard L. Bell

Abstract merged HS2a and HS2b, a single QTL is

Fire blight, caused by the bacterium Erwinia identified controlling disease incidence, sever-
amylovora (Burrill) Winslow et al., is the most ity, and the incidence severity (ISV) index. In
serious disease affecting the European pear, addition, three putative QTLs have been
Pyrus communis L., in North America, identified for disease incidence, severity, and
Europe, and the Middle East. Control of fire ISV on HS04. In a study of the progeny of
blight is difficult, thus rendering the develop- ‘Doyenné du Comice’  Pyrus ussuriensis
ment of resistant cultivars and rootstocks a No. 18, putative QTLs have been identified
high priority. The inheritance pattern of resis- on LG 11 of the P. ussuriensis parent.
tance is quantitative, and genetic control is Another QTL identified on LG 4 of ‘Doyenné
polygenic with additive effects, along with an du Comice’ has suggested that resistance
estimated narrow-sense heritability, from var- genes could be present in susceptible parents,
ious populations, of 0.40–0.50. There is some as observed in conventional segregation stud-
evidence for major gene inheritance for resis- ies. A follow-up study has identified a QTL on
tance. There have been five published studies LG 9 of the resistant parent, and additional
on presence of genetic markers linked to QTLs on LG 11, as well as on three other
quantitative trait loci (QTL). Microsatellite or linkage groups, have been also found. Fur-
simple sequence repeats (SSR) markers have thermore, four additional QTLs have been
been the most used marker type, but amplified identified in ‘Doyenné du Comice’. In an
fragment length polymorphisms (AFLPs) and interspecific seedling population of
single nucleotide polymorphisms (SNPs) have ‘PremP003’ (P.  bretschneideri  P. com-
also been used. In the first study of the progeny munis)  ‘Moonglow’ (P. communis), a
‘Passe Crassane’  ‘Harrow Sweet’, four major QTL is mapped to LG 2 of ‘Moonglow’,
putative QTLs have been identified, all which co-locates with a LG 2 QTL found in
detected in ‘Harrow Sweet’. A QTL is located ‘Harrow Sweet’. Three minor QTL have been
on linkage group (LG) HS2a, a second on identified on LGs 9, 10, and 15 of ‘PremP003’.
HS2b, a third on HS4, and a fourth on HS9. In The history of pear breeding for fire blight
a follow-up study with additional markers that resistance and notable cultivar releases will be
also discussed.

R. L. Bell (&)
94 Shady Meadows Court, Charles Town 25414,
WV, USA
e-mail: [email protected]

© Springer Nature Switzerland AG 2019 243

S. S. Korban (ed.), The Pear Genome, Compendium of Plant Genomes,
https://doi.org/10.1007/978-3-030-11048-2_13
244 R. L. Bell

13.1 Introduction and strategies along with evaluation/selection

techniques have also been previously reviewed
Fire blight, caused by the bacterium Erwinia by Bell et al. (1996a), Lespinasse and Ald-
amylovora (Burrill) Winslow et al., is the most winckle (2000), Hancock and Lobos (2008),
serious disease affecting European pear (Pyrus Fischer (2009), Lespinasse et al. (2011), Dondini
communis L.). Originating from North America, and Sansavini (2012), and Kellerhals et al.
fire blight has spread over to England, and in (2017); whereas, goals and progress have been
spite of quarantine and control measures, it has reviewed by Bellini and Nin (1997) and Brewer
continued to spread throughout Western, Central, and Palmer (2011).
and Eastern Europe, over to the Middle East, and
then to New Zealand (van der Zwet 2002). The
disease has influenced pear production more than 13.2 Breeding
any other single factor. Its prevalence has largely
limited large-scale production in North America 13.2.1 History of Breeding Scion
to mild and dry valleys of the Pacific coastal Cultivars
region of the province of British Columbia in
Canada, and to US states of Washington, Ore- The history of selection and breeding of pear for
gon, and California in the USA (Andersen 1956; fire blight resistance has been reviewed by Mag-
van der Zwet and Keil 1979). The disease is a ness (1937), van der Zwet and Keil (1979), Bell
constant threat, even in climatically favorable et al. (1996a), Bellini and Nin (1997, 2002),
production regions. Lespinasse and Aldwinckle (2000), Brewer and
The disease has been observed first in the state Palmer (2011), and Dondini and Sansavini
of New York, as early as 1780 (Denning 1794). (2012). The first fire blight-resistant pear cultivar
The pathogen infects nectarthodes of blossoms, grown in the USA is ‘Seckel’, which originated as
serving as the primary infection court, and a chance seedling in an area close to Philadelphia,
actively growing shoots and immature fruits, but Pennsylvania. Purposeful selection for fire blight
can also infect mature branches and trunks resistance in pears was initiated in the mid- to late
through wounds. Infection of shoots typically 1800s following the introduction of Chinese sand
produces a necrotic ‘shepherd’s crook’ symptom. pears [P. pyrifolia (Burm.) Nakai], probably via
Rootstocks can also become infected through Europe (Hedrick et al. 1921). The first fire
either infection of root suckers or transmission blight-resistant interspecific hybrids introduced to
from an infected trunk. the nursery trade have included ‘Le Conte’,
Control usually involves pre-bloom applica- ‘Kieffer’ (a chance seedling of ‘Bartlett’) and
tion of copper compounds, and subsequently ‘Garber’. These are all chance seedlings, not bred
with antibiotics or various biocontrols during cultivars, grown because of their fire blight
bloom. Despite these control measures, the dis- resistance; however, they are lacking in fruit
ease is often devastating. Once infection occurs, quality. The first large-scale evaluation and
even drastic pruning of infected tissues during selection effort involved the introduction of pear
the growing season cannot always stop disease species and species hybrids from Asia, and
progression. All of the major scion cultivars of evaluating these materials along with European
European pear currently in production and most pear cultivars for their resistance to fire blight
rootstocks are susceptible to fire blight, thus (Reimer 1925). A total of 85 European pear
rendering the development of fire blight-resistant (P. communis) cultivars or hybrids were artifi-
cultivars a high priority. cially inoculated, and data from natural field
Genetic resources for fire blight resistance and infection of an additional 500 cultivars/hybrids
other traits have been previously reviewed by have been recorded. The goals of these evalua-
Westwood (1982), Bell and Itai (2011), and Bell tions targeted the development of fire
and Leitão (2011). Moreover, breeding methods blight-resistant rootstocks and scion cultivars.
13 Genetics, Genomics, and Breeding for Fire Blight Resistance … 245

Seedlings of P. ussuriensis cv. Ba Li Hsiang introduced into the program as a source of fire
were found to be highly resistant, but have blight resistance, but it was found to be also a
proven unsatisfactory as rootstocks for P. com- source of resistance to the insect pest pear psylla,
munis scion cultivars. Hybridizations with major Cacopsylla pyricola Förster (Harris 1973; Harris
cultivars of P. communis have resulted in seed- and Lamb 1973).
lings bearing fruit of poor quality. The USDA pear breeding program was ini-
In 1915, Reimer discovered a fire tially operated from 1916 to 1919 at Michigan
blight-resistant cultivar in Illinois, ‘Farmingdale’, State University’s South Haven Horticultural
assumed to be a seedling of ‘Beurré d’Anjou’. Experiment Station (Magness 1937). The pro-
Other early breeding programs for fire blight gram developed a number of fire blight-resistant
resistance in the USA have been carried out at selections, including Michigan-US 437, which
the Georgia Experiment Station, releasing served as a progenitor of many of the selections
‘Pineapple’ (van der Zwet and Keil 1979). Fur- and cultivars produced by this program. The
thermore, the University of Tennessee has program was continued at a low level at the
released eight interspecific hybrids of P. commu- USDA’s Arlington Farm, and then beginning in
nis and P. pyrifolia, including ‘Ayers’, ‘Dabney’, 1960, a major expansion began at the Beltsville
‘Hoskins’, and ‘Mooers’ (Drain and Shuey Agricultural Experiment Station in Maryland
1954); ‘Carrick’ and ‘Morgan’ (Drain and Safley (Brooks et al. 1967), from which ‘Magness’,
1958); as well as ‘Orient’ and ‘Tenn’. While ‘Moonglow’, and ‘Dawn’ were released. Later
‘Orient’ is a seedling of an interspecific cross of on, it was found that ‘Dawn’ was moderately
P. pyrifolia  P. communis made by Walter Van susceptible to fire blight, while ‘Moonglow’ was
Fleet of Chico, California, who apparently pro- resistant and ‘Magness’ was highly resistant,
vided it to the Tennessee Agricultural Experi- except when infected via trunk wounds, such as
ment Station and to the United States Department those caused by limb spreaders used for tree
of Agriculture (USDA), ‘Tenn’ is a selection training. In 1979, the program was transferred to
from the Tennessee breeding program. None of the Appalachian Fruit Research Station in West
these cultivars have been widely planted com- Virginia, from which ‘Potomac’ (Bell et al.
mercially, as they are of mediocre fruit quality, 1996b), ‘Blake’s Pride’ (Bell et al. 2002),
but they are grown mainly by amateur backyard ‘Shenandoah’ (Bell and van der Zwet 2008),
orchardists. ‘Sunrise’ (Bell and van der Zwet 2011), and
The University of Maryland, in 1905, laun- ‘Gem’ (Bell et al. 2014) were released. Further-
ched a program mainly for developing hybrids of more, the USDA pear breeding program was also
‘Kieffer’ with common European cultivars; the likely source of the fire blight-resistant
however, no cultivars have been released. The ‘Warren’ pear, as most likely it was a
University of Minnesota began a limited breed- sister-seedling of ‘Magness’. The original seed-
ing program in 1908 to develop cold hardy, fire ling population of these two cultivars, ‘Warren’
blight-resistant cultivars, using mainly Man- and ‘Magness’, had been split and planted at two
churian P. ussuriensis germplasm hybridized to locations, the Arlington Farm and the USDA
European cultivars. Cornell University launched research station in Meridian, Mississippi.
a pear breeding program at their experiment The USDA station might have either shared
station in Geneva in 1892, and although the seedlings with or propagated selections at the
initial focus was on high fruit quality cultivars, Mississippi State University research station. The
the program was expanded to include fire blight identity of these two cultivars was confirmed
resistance as an objective using sources of fire using isozyme analysis, wherein isozyme profiles
blight resistance from P. communis including of these two cultivars were found to be almost
‘Seckel’ and ‘Worden Seckel’. A putative identical, as well as following morphological
P. ussuriensis  P. pyrifolia hybrid, Illinois 65 observations, wherein their fruits were also found
(syn. P. ussuriensis 65), was also initially to be almost identical.
246 R. L. Bell

Other breeding programs in the USA included (Thibault 1981), but eventually approximately
a short-lived program at the University of Illinois 60,000 seedlings were generated from 55 parents
(Hough 1944), producing several P. communis crossed in 200 combinations. This program’s
selections, as well as selections Illinois 65 and objectives pursued development of fire blight
Illinois 76, both were deemed as putative resistance by focusing on lack of secondary
P. ussuriensis  P. pyrifolia hybrids. These bloom, found to be a heritable trait (Thibault
selections were subsequently used as sources for et al. 1983), as well as reduction of shoot blight.
resistance to pear psylla, C. pyricola, as well as The program released a few cultivars, among
to fire blight by breeding programs at Cornell them ‘Angelys’. While only moderately suscep-
University, Rutgers University, and USDA. The tible to fire blight, it produced no secondary
Rutgers University program introduced ‘Mac’, bloom (Le Lézec et al. 2002). Another cultivar,
‘Star’, and ‘Lee’ (Hough and Bailey 1968), and ‘Cepuna’, matured in early September, was only
developed many selections derived from moderately resistant. Various aspects of this
hybridizations between P. communis cultivars program were reviewed by Le Lézec et al.
with selections of either P. ussuriensis or (1991).
P. pyrifolia. Purdue University released ‘Hon- Pear breeding at the Instituto Sperimentale per
eysweet’ (Janick 1977), ‘P448-2’ (‘Green la Frutticoltura in Forli, Italy commenced in 1968
Jade’™) (Janick 2004), and ‘H2-169’ (‘Ambro- (Rivalta et al. 2002). Breeding for fire blight
sia’™) (Janick 2006). The University of Cali- resistance, in cooperation with the INRA pear
fornia at Davis released ‘Elliot’, a seedling of breeding program, was carried out from 1980 to
‘Elliot 4’  ‘Vermont Beauty’, which gained 1995, during which time field inoculations were
favor in Europe, but marketed as ‘Selena’ carried out at the INRA station at Dax, whereby
(Ryugo 1982). fire blight was endemic. Resistant selections
The pear breeding program of Agriculture were propagated onto rootstocks, underwent
Canada began at Harrow, Ontario in 1962, and greenhouse bacterial inoculation tests at Angers,
was then transferred to Vineland, Ontario, from France, and pomological field evaluations were
1996 to 2000 (Hunter 2016). This program conducted in Italy. Susceptible cultivars, such as
released ‘Harvest Queen’, a cultivar with mod- ‘Max Red Bartlett’, ‘Bella di Guigno’, ‘Coscia’,
erate resistance to fire blight, as well as several and ‘Starking Delicious’, a cultivar with low to
fire blight-resistant cultivars, including ‘Harrow moderate resistance, were found to produce
Delight’ (Quamme and Spearman 1983), ‘Har- seedlings with suitable resistance to fire blight,
row Sweet’ (Hunter et al. 1992), ‘AC Harrow while some resistant cultivars, such as ‘Morgan’,
Gold’ (Hunter et al. 2002a), ‘AC Harrow Crisp’ ‘Dr. Molon’, ‘Sirrine’, and US309 produced
(Hunter et al. 2002b), ‘Harrow Sundown’ (‘Cold progenies of low resistance to fire blight
Snap’™) (Hunter et al. 2009), ‘AC Harrow (Lespinasse and Aldwinckle 2000). Selection of
Delicious’, and ‘Harrow Bliss’. The latter two parents from commercially acceptable germ-
cultivars have been marketed only in Europe. plasm was a more effective method of develop-
Additional fire blight-resistant selections, ing cultivars of commercial quality combined
including HW 602, HW 623, and HW 624, will with acceptable levels of fire blight resistance
also be named and commercialized. (Bagnara et al. 1996). Two fire blight tolerant
Following spread of fire blight disease to cultivars were developed and released. ‘Bohéme’
Europe, scion cultivar breeding programs added (ISF-FO 80-57-83), was selected from a seedling
fire blight resistance to their objectives in several population of ‘Conference’  ‘Dr. Jules Guyot’,
countries. The Institut National de la Recherche and ‘Aida’ (ISF-FO 80-104-72), was selected
Agronomique (INRA) pear breeding program, from a cross of ‘Coscia’  ‘Dr. Jules Guyot’. An
located at Angers, France, initially used a additional selection, ISF-FO 80-51-72, also a
half-diallel of four resistant selections crossed to seedling of ‘Coscia’  ‘Dr. Jules Guyot’, has
three susceptible European pear cultivars been undergoing evaluation.
13 Genetics, Genomics, and Breeding for Fire Blight Resistance … 247

The pear breeding program at the University with the more aromatic fruit flavor of P. com-
of Bologna in Italy, launched in 1978 (Musacchi munis cultivars. Transfer of disease (e.g., fire
et al. 2005), with resistance to fire blight blight) and insect (e.g., pear psylla) resistance
becoming one of its major goals. As of 2005, from Asian cultivars is yet another goal of this
three selections, either fire blight resistant or program. In 1999, ‘Crispie’ and ‘Maxie’, derived
tolerant, including DCA 92050701-14, DCA from hybridization of P. pyrifolia cv. ‘Nijisseiki’
91050701-41, and DCA 91050701-39, have with P. communis cv. Max Red Bartlett, have
been identified from a seedling population of US been released. A third-generation hybrid, ‘PIQA
309 (resistant)  ‘Abbé Fetel’ (susceptible). Boo’ (a numbered selection of PremP009), a
This program has also been investigating complex hybrid of P. communis, P. pyrifolia, and
molecular markers linked to resistance to fire P.  bretschneideri, has been recently released.
blight and to pear psylla (Musacchi et al. 2006). However, fire blight resistance ratings have not
Although fire blight resistance has not been a yet been published.
major goal of the German pear breeding program
at Dresden-Pillnitz, two released cultivars, ‘Iso-
lda’ and ‘Uta’, have some levels of tolerance to 13.2.2 History of Breeding
fire blight (Fischer and Mildenberger 2004). Rootstocks
Other resistant or tolerant released cultivars,
including ‘David’, ‘Hortensia’, and ‘Manon’, Fruiting-bearing, or scion, pear cultivars are
have also been released (Dondini and Sansavini clonally propagated, either by budding or by
2012). grafting onto either seedling or clonal rootstocks.
Using P. pyrifolia selections as sources of These rootstocks have been selected for, based
resistance, the Romanian pear breeding program on either availability of seed, as is the case with,
at the Fruit Tree Institute in Pitesti-Maracineni for example, ‘Bartlett’ and ‘Winter Nelis’ seed-
has released ‘Getica’ (Sestras et al. 2007; Bra- ling rootstocks, or their ability to positively
niste et al. 2008). The Fruit Research Station in influence production or various other traits, such
Voinesti has released ‘Corina’ and ‘Euras’, and as precocity of bearing, tree size control, adap-
the Fruit Tree Research Station in Cluj has tation to high pH soils, cold hardiness, ease of
released ‘Haydeea’. The Fruit Tree Institute has propagation from cuttings, and resistance to soil
also released ‘Monica’, a seedling of ‘Santa pathogens, woolly pear aphids, Armillaria root
Maria’  ‘Principessa Gonzaga’, both parents rot, pear decline phytoplasm, or fire blight
belonging to P. communis (Dondini and Sansa- (Lombard and Westwood 1987). Selection and
vini 2012). breeding for pear rootstocks have been previ-
The New Zealand pear scion breeding pro- ously reviewed (Lombard and Westwood 1987;
gram has used selections derived from several Bell et al. 1996a; Wertheim 2002; Webster 2003;
P. communis (‘Duchesse d’Angouleme’, Hancock and Lobos 2008; Fischer 2009; Lespi-
‘Moonglow’, ‘Harrow Crisp’, ‘Harrow Delight’, nasse 2009; Brewer and Palmer 2011; Dondini
‘Patrick Barry’, ‘Seckel’, and ‘Winter Cole’), and Sansavini 2012; Elkins et al. 2012). Elkins
P. pyrifolia (‘Nijisseiki’, ‘Okusankichi’, and et al. (2012) have also provided a listing of 36
NJ1), P. ussuriensis (‘Ping Guo Li’), rootstock breeding programs throughout the
P.  bretschneideri (‘Ya Li’ and ‘Xue Hua Li’), world.
and P. pyrifolia  P. communis hybrid cultivars Reimer (1925) has investigated fire blight
(‘Carrick’) as sources of resistance or high fruit resistance of Pyrus species and cultivars at Ore-
quality (Brewer and Palmer 2011; White and gon State University. These data are based on
Brewer 2002a, b). Selections from resultant observations of natural infections rather than
progenies are undergoing further evaluations. controlled inoculations. Lombard and Westwood
A major goal of this program is to combine the (1987) have also summarized general reactions
fine and crisp fruit flesh texture of Asian cultivars of clones or seedlings of 19 Pyrus species in this
248 R. L. Bell

collection for resistance to fire blight. Moreover, diverse (Simard et al. 2004). It has developed and
they have also summarized performance of var- released an open-pollinated ‘Old Home’ selec-
ious species deemed suitable as either seedling tion OH11 as ‘Pyriam’ in 1997 (Simard and
rootstocks or clonal rootstocks for pear cultivars. Michelesi 2002). This rootstock has been selec-
In addition, they have also included Cydonia ted for its ability to reduce scion vigor, and for
oblonga Mill., quince, as it is an important promoting production, fruit size,
source of dwarfing rootstocks, along with other graft-compatibility, nursery habit, and propaga-
genera within Rosaceae, for potential use as tion from softwood cuttings. Evaluations at sev-
rootstocks for pear. Unfortunately, all evaluated eral locations throughout France have shown
common clones of Cydonia have been found to good adaptability to calcareous soils, i.e., toler-
be susceptible to fire blight, whereas Asian pear ance to high pH-induced iron chlorosis.
species have been found to be more resistant to The Institute for Research and Technology in
fire blight, with some variabilities, but with Food and Agriculture (IRTA), an agricultural
P. ussuriensis and P. calleryana deemed the research organization of the government of Cat-
most consistently resistant. alonia, Spain, and the French INRA have initi-
Within P. communis, the first fire ated a joint pear rootstock breeding program in
blight-resistant rootstocks have been the ‘Old 1998 to develop pear rootstocks adapted to
Home’  ‘Farmingdale’ (OH  F) numbered Mediterranean growing conditions, specifically
series (Brooks 1984). However, the correct tolerance to high pH soils; i.e., iron chlorosis,
parentage of these rootstocks has been recently and to water scarcity (Asin et al. 2011). The
determined to be ‘Old Home’  ‘Bartlett’ using program involves crosses between the French
simple sequence repeat (SSR) molecular marker rootstock ‘Pyriam’ and four Mediterranean Pyrus
analysis (Postman et al. 2013). ‘OH  F 87’ is taxa, including P. amygdaliformis Vill.,
the most commonly used rootstock of this series P. amygdaliformis Vill. var. persica Bornm., a
in North America, as it is fire blight resistant, hybrid of P. communis var. cordata Desv. Hook
graft-compatible with all tested scion cultivars, f. (syn. P. cordata Desv.), and P. elaeagrifolia.
induces a semi-dwarf tree size, promotes preco- Pall. Seedlings resulting from these crosses were
cious fruit bearing, and produces better yield split into two sets, with one set being evaluated
efficiency than other rootstocks of this series. in France for rooting ability, upright growth
However, it is more difficult to propagate by habit, and graft-compatibility with ‘Bartlett’ (syn.
conventional cuttings and layering (Dondini and ‘Williams Bon Chretien’), used as the scion
Sansavini 2012). Another selection in this series, cultivar, while the second set being evaluated in
‘OH  F 40’, is also fire blight resistant, Spain for tolerance to iron chlorosis, vigor, and
graft-compatible, promotes good yield, and good graft-compatibility with ‘Conference’ used as the
fruit size. However, it induces higher vigor than scion cultivar. Open-pollinated ‘Bartlett’ seed-
Quince BA29, but it is less productive than lings have also been evaluated. Iron chlorosis is
Quince MC, and has lower yield efficiency. Yet measured using a visual rating scale (Sanz and
another selection, ‘OH  F 69’, has performed Montañes 1997), while nitrogen is determined
well in trials in California and in Europe (Elkins using a SPAD meter. It has been determined that
et al. 2008, 2011; Dondini and Sansavini 2012). seedlings of P. amygdaliformis, P. elaeagrifolia,
However, its yield efficiency is lower than those and the P. communis var. cordata hybrid are
of both quince and pear seedling rootstocks. In found to be more resistant to iron chlorosis.
most trials, ‘OH  F 69’ has demonstrated to be Moreover, open-pollinated seedlings of ‘Bartlett’
as vigor-inducing as that of seedling rootstocks, are reported to have lower vigor than other tested
but it is winter hardy and has resistance to both Pyrus materials. Furthermore, interspecific
fire blight and pear decline. hybrids and open-pollinated seedlings of ‘Bar-
The French INRA pear rootstock breeding tlett’ are reported to have similar percentages
program has been one of the largest and most (18%) of seedlings with no observed chlorosis
13 Genetics, Genomics, and Breeding for Fire Blight Resistance … 249

and with reduced vigor, less than 50% of that of potential as reliable and useful rootstocks for
the Cydonia rootstock BA29. Overall, seedlings pear scion cultivars.
of the hybrid P. communis var. cordata,
P. amygdaliformis var. persica clone, and of the
open-pollinated ‘Bartlett’ have yielded the high- 13.2.3 Disease Resistance Evaluation
est percentages of desirable selections. Methods
‘Pyrodwarf’ (Rhenus 1) and ‘BU 2/33’
(Rhenus 3) have been developed at the Geisen- As assessment of fire blight disease resistance is
heim Research Institute and Applied University rather difficult, several methods have been
in Germany from progeny of the resistant ‘Old developed to evaluate disease reactions in pear.
Home’  the susceptible ‘Bonne Louise d’Av- Methods of determining levels of host plant
ranches’ (Jacob 2002). ‘Pyrodwarf’ is reported to resistance/susceptibility have consisted of
induce low scion vigor, high fruit-bearing pre- short-term (disease severity in one year) and
cocity, high fruit yield efficiency, as well as long-term (cumulative disease severity over a
uniform and good fruit size. Furthermore, it has period of years) observations of infections caused
graft-compatibility with major scion cultivars, by natural epiphytotics, as well as short-term
good anchorage, winter cold hardiness, lacks data, collected based on artificial inoculations of
sucker development, and does not exhibit high actively growing shoots of established seedlings
soil pH-induced iron chlorosis. Unfortunately, and propagated trees under greenhouse and field
this rootstock has not performed as well in the conditions.
USA. On the other hand, ‘BU 2/33’ produces a A number of host, pathogen, and environ-
semi-dwarf to vigorous scion, but induces good mental factors influence expression and pheno-
production and yield efficiency. typic disease resistance. These include the
Although Cydonia is generally susceptible to following: (1) tree age, vigor, and infected tissue;
fire blight, studies at the Agricultural University (2) virulence of isolates; (3) inoculum concen-
in Plovdiv, Bulgaria have found that two edible tration; (4) inoculation method; and (5) tempera-
quince cultivars, ‘Hemus’ and ‘Triumph’, are ture and humidity conditions during pre- and
resistant to fire blight, while a third cultivar, ‘Du post-inoculation periods (Bell et al. 1996a). Thus,
Portugal’, is moderately resistant to fire blight fire blight disease resistance findings reported in
(Bobev and Deckers 1999). A number of selec- various studies are highly influenced by differ-
tions, such as IV-40, from subsequent breeding ences in any of these factors (van der Zwet and
progenies, generated by crossing these three Keil 1979). Young and vigorously growing
quince cultivars with susceptible cultivars shoots tend to be more susceptible to fire blight.
‘Asenitza’ and ‘Tzargradska’ and by Furthermore, blossoms are almost always more
open-pollination, are reported to be resistant to susceptible than either shoots, older branches, or
fire blight (Bobev et al. 2011). trunks, even when compared to shoots of fire
In other efforts, the edible quince breeding blight-resistant pear genotypes. In one study,
program at the Pomology Institute, NAGREF, in most genotypes resistant to shoot infection are
Naoussa, Greece, and the Technological Educa- reported to be moderately to highly susceptible to
tion Institute of Larissa, also in Greece, evaluated blossom infections (Le Lézec et al. 1985), but
49 genotypes, and found eight genotypes that with some exceptions, wherein both shoot and
were resistant to fire blight (Papachatzis et al. blossom resistance have been observed, such as
2011). those observed for pear genotypes HW 601 and
Additional cultivars and germplasm acces- ‘Potomac’, among other USDA selections.
sions have been found to be moderately resistant Interestingly, ‘Magness’ is essentially immune to
to fire blight (Bell, unpublished data). Although blossom infection, due to its underdeveloped
most of the genotypes discussed herein were of nectarthodes, an important infection court. Fur-
the edible types, these must be evaluated for their thermore, while screening of young seedlings in a
250 R. L. Bell

greenhouse, it has been observed that actively cultivars, it may be important to combine dif-
growing seedlings, with either 18–24 nodes or are ferent sources of resistance. If either individual
6–7 months old, are deemed best for distin- genes or quantitative traits loci (QTLs), along
guishing among levels of resistance/susceptibility with their markers, can be identified, then they
to fire blight (Carpenter and Shay 1953; should be combined or ‘pyramided’. Further-
Thompson et al. 1962; Layne et al. 1968). more, inoculum concentrations can influence
With the development of the tools of frequency and severity of infections, with lower
biotechnology, specifically of identifying concentrations resulting in less reliable results.
mutants derived from in vitro mutagenesis, Therefore, a concentration of at least 1  107
screening for somaclonal variants, and selection cfu
ml−1 is recommended, and should be used.
of genetic transformants, has led to the devel- For both outdoor and greenhouse inocula-
opment of in vitro methods for plant production. tions, sustained temperatures of less than 30 °C
Shoot proliferation can be used to produce plant (86 °F) should be prevalent. Moreover, high
materials that can be screened for disease resis- relative humidity (85–100% RH) conditions
tance at early stages of development, thus must be maintained before, during, and after
decreasing numbers of plants that must be rooted, inoculation, as it has been demonstrated that high
acclimated to greenhouse conditions, and then humidity increases the likelihood of success of
evaluated. Proliferating shoot cultures can be artificial inoculations (van der Zwet and Keil
inoculated in vitro, and those clones demon- 1979). Such high humidity conditions can be
strating low frequencies of necrosis are then maintained under greenhouse and nursery envi-
selected for propagation, and subjected to further ronments by constructing a plastic tent and
evaluations (Viseur and Tapia y Figeuroa 1987; placing a humidifier inside the tent for a period of
Hanke and Geider 2002; Paprstein et al. 2014). several days.
It is critical to point out that the strain of E. Various shoot inoculation methods have been
amylovora can affect the severity of infection, used, including the use of wounding with car-
due to differences in general virulence (Shaffer borundum, needles, hypodermic syringes,
and Goodman 1962) and differential virulence of pin-cushion equipped clamps (van der Zwet and
the bacterium; i.e., interactions between bacterial Keil 1979), and more recently and widely, scis-
strain and host genotype, as some bacterial sors. Using scissors involves dipping blades into
strains would infect otherwise disease resistant the inoculum, and then cutting the top two
host genotypes. There is at least one such case expanding leaves of an actively growing shoot
reported in apple (Norelli et al. 1984, 1986), but through the midrib. This method has been
most bacterial strains are not differentially viru- demonstrated to consistently yield high frequen-
lent, as it is apparently the case in pear (Quamme cies of infections.
and Bonn 1981). In this latter study, only nine On the other hand, blossom inoculation stud-
bacterial strains have been investigated and ies usually involve use of a uniform number of
assessed. It is noteworthy to point out that dif- newly opened blossoms per cluster. These blos-
ferences in general bacterial virulence may soms are inoculated individually with a small
influence ratings or measures of resistance, drop of inoculum using a repeating pipetter.
thereby potentially influencing fire Alternatively, whole clusters are inoculated using
blight-resistance findings. Moreover, it may be a sprayer, such as a DeVilbiss atomizer.
also important that while screening seedlings of For evaluation of epiphytotic infections,
breeding materials is to either use a mixture of Mowry (1964) has devised a rating index as
non-differential bacterial strains or screen indi- follows: (number of infected shoots  5) + (age
vidually against different bacterial strains, of infected wood  20). Yet another widely used
sometime during the breeding process. Addi- evaluation scheme, a 10-point scale based on a
tionally, to insure durable resistance of new modified Horsfall-Barratt scale (Horsfall and
13 Genetics, Genomics, and Breeding for Fire Blight Resistance … 251

Barratt 1945), has been later developed for trees may reflect differences in resistance response
that are at least 3 years old (van der Zwet et al. (Shaner and Finney 1977; Jeger and
1970). This latter scheme is based on a visual Viljanen-Rollinson 2001). This method involves
estimation of percentage of a tree that is infected, periodic measurements over a period of time, and
age of the oldest infected wood, and relative this has been used in fire blight disease evalua-
estimate of the proportion of shoots infected. tions (Momol et al. 1996). However, various
This scheme is intended for use in an orchard to modifications and improvements have been
rapidly assign disease severity scores. Citing made, including the development of yet another
various statistical deficiencies of the index, termed the area under the disease progress
Horsfall-Barratt disease evaluation system, Bock stairs (AUDPS) (Simko and Piepho 2012). This
et al. (2009, 2010) have recommended the use of index, along with its associated standardized
nearest percent estimates for citrus canker (sAUDPS) and relative (rAUDPS) variants, is
infections of leaves that may be also of use for recommended for use for quantification of fire
fire blight disease evaluations. Other systems for blight disease reactions.
fire blight disease categorical scales have relied Scoring of blossom infections following arti-
on use of regularly spaced intervals, usually five ficial inoculations involves determining fre-
intervals, of 20% per interval. quencies of infections of blossoms or clusters, or
In order to conduct artificial inoculations, of both frequency and severity, with the latter
actively growing shoots are often used. Mea- based on a scale of symptom progression through
surements of lesion lengths are converted to either an individual blossom and stalk into the
percentages of total shoot length (Lamb 1960). bourse and spur, or to some other woody tissue.
This type of data has been referred to as per- Again, various scales have been used to assess
centage lesion length (PLL). Sometimes, classes blossom disease severity (Bell et al. 2002; Bell
for disease severity, based on percent ranges, and van der Zwet 2008, 2011; Kellerhals et al.
have also been used (Thompson et al. 1962). In 2017).
some instances, data from unsuccessfully inocu- In vitro-cultured shoots have also been used to
lated shoots are excluded, as these are assumed evaluate fire blight resistance (Duron et al. 1987;
not to be representative of a true resistant reac- Brisset et al. 1988; Pinet-Leblay et al. 1996;
tion. In another approach, it is assumed that lack Abdollahi et al. 2004; Paprstein et al. 2014). In
of observed shoot infection represents a true general, shoot necrosis is correlated with known
resistant reaction. To account for these unin- susceptibility of a cultivar. Moreover, when
fected shoots, an Index of Varietal Susceptibility mesophyll protoplasts of the fire blight-resistant
(IVS), based on both frequency of successful ‘Old Home’, the susceptible ‘Williams Bon
inoculations (F, 0–1) and severity (S, 0–100), has Chretien’ (syn. ‘Bartlett’), and the highly sus-
been devised (Thibault et al. 1987). This has ceptible ‘Passe Crassane’ are co-cultured with E.
been extensively used by the INRA program in amylovora, protoplast viability, time to division,
France (Le Lézec et al. 1997), among many other and time to 10-cell colony stage development
breeding programs. The foregoing indices of have correlated with known resistance/
resistance are based on either maximum lesion susceptibility of these cultivars (Brisset et al.
length or lesion length after a set period of time 1990). Therefore, these alternative systems for
following inoculation. fire blight disease evaluations have been deemed
As shoot lesions may develop at different useful for instances wherein use of the pathogen
rates, depending on the host genotype or even in a greenhouse or outdoors is prohibited due to
individual replicate shoots, the area under the quarantine regulations, or for purposes of
disease progress curve (AUDPC) index has been studying various aspects of host-pathogen
developed to account for these differences and interactions.
252 R. L. Bell

13.2.4 Germplasm Zwet and Keil 1979). Although some moderately

resistant and resistant cultivars of P. communis
There are at least 29 Pyrus taxa that are widely have been identified, overall this species has been
accepted as species, and nine naturally occurring generally deemed as susceptible (Zeller 1978,
interspecific hybrids (Bell et al.1996a; USDA, 1990; van der Zwet and Keil 1979; Thibault et al.
ARS 2018). One of the major species cultivated 1989). Moreover, although most of P. pyrifolia
for edible fruit is the West European pear, cultivars are susceptible, some moderately resis-
P. communis, which is the primary focus of this tant germplasm has been identified (Zeller 1978;
chapter. Other edible Pyrus species of use in van der Zwet and Keil 1979; Lespinasse and
breeding of fruit cultivars include P. pyrifolia Aldwinckle 2000). Furthermore, although
(Burm. f.) Nakai, P. ussuriensis Maxim., and the P. betulifolia Bunge is generally deemed as sus-
naturally occurring interspecific hybrid, ceptible, a few resistant clones have been identified
P.  bretschneideri Rehd. The latter species is at (van der Zwet et al. 1974a), such as Reimer’s
times classified as a subspecies, P. pyrifolia resistant selection, which has been used as a
spp. sinensis T.T. Yu. In South Asia, P. pseu- seedling rootstock.
dopashia T.T. Yu is also cultivated. Large On the other hand, the ornamental species
numbers of cultivars, breeding selections, and P. calleryana Decne. has been observed to have
wild germplasm of several species have been a high proportion of fire blight-resistant clones,
evaluated for their resistance/susceptibility to fire including ‘Bradford’, ‘Capital’, and ‘White-
blight. General ratings of resistance/susceptibility house’; whereas, ‘Aristocrat’, ‘Autumn Blaze’,
reactions at the species level have been presented and to some degree ‘Redspire’ have been deemed
by Westwood (1982), Lombard and Westwood more susceptible (van der Zwet et al. 1974b; Fare
(1987), Bell (1991), and Bell and Leitão (2011). et al. 1991). Nevertheless, ratings for ‘Bradford’
Commonly used ancestral sources of fire blight have been variable (Bell et al. 2004). Generally,
resistance have included the resistant selections clones of the native species P. ussuriensis are
US309 and Michigan-US 437, and the moderately quite resistant, with 64% being moderately
resistant cultivars of ‘Seckel’ and ‘Roi Charles de resistant to resistant (Hartman 1957; van der
Wurtemberg’. In a 2-year study of epiphytotic fire Zwet et al. 1974b; van der Zwet and Keil 1979).
blight in a collection of mature trees of more than However, domestic cultivars of P. ussuriensis are
500 cultivars and selections comprising primarily more susceptible, perhaps due to interspecific
of P. communis cultivars, but also including Asian hybridizations with other species, such as with
and Asian  European pear hybrids, approxi- P. pyrifolia. Moreover, the interspecific hybrid,
mately 90% of evaluated genotypes are deemed to P.  bretschneideri, is deemed to be variable.
be susceptible (Oitto et al. 1970). Even among The primary source for fire blight resistance in
those moderately resistant to resistant germplasm, rootstock breeding programs has been an old
some infections have continued to progress for a American P. communis cultivar, ‘Old Home’
few additional years (van der Zwet and Oitto 1972; (Brooks 1984; Jacob 2002). Due to variabilities
van der Zwet et al. 1974a). In a summary of pub- in resistance reactions within each Pyrus species,
lished studies of nearly 400 P. communis and it is difficult to assign a consistent resistance
interspecific hybrids, approximately 41% of these rating to a particular species, although there are
genotypes have been deemed as either susceptible general trends (van der Zwet et al. 1974a).
or variable, 33% as moderately resistant, and only Overall, among cultivated Pyrus species, for
19% as resistant. Moreover, among 48 either fruit or rootstock, P. ussuriensis is deemed
P. ussuriensis and P. pyrifolia cultivars, 31% are the most resistant, followed by P. calleryana,
deemed as resistant, 10% as moderately resistant, P. betulifolia, P.  bretschneideri, P. pyrifolia,
40% as susceptible, and 19% as variable (van der and P. communis.
13 Genetics, Genomics, and Breeding for Fire Blight Resistance … 253

13.2.5 Biotechnological Approaches used as a primary screen for hypersensitive

for Genetic resistance reactions in mutation breeding efforts.
Improvement Genetic transformation efforts undertaken to
enhance fire blight resistance in pears have been
Selections of somaclonal variants and of muta- previously reviewed by Hancock and Lobos
tion breeding have been used to develop methods (2008) and by Dondini and Sansavini (2012).
to isolate clones of fire blight susceptible pear The gene attacin E, a lytic peptide gene derived
cultivars with improved resistance to fire blight. from the silk moth, Hyalophora cercropia L., has
These new clones can be generated using in vitro been introduced into the highly susceptible
micropropagation, callus cultures, and adventi- P. communis cultivar ‘Passe Crassane’ using
tious shoot regeneration protocols. In vitro dis- Agrobacterium-mediated transformation, and
ease resistance, evaluation methods can also be transgenic pear lines with reduced levels of sus-
used for early screening of clones for enhanced ceptibility to fire blight have been obtained
resistance to fire blight. Plantlets of ‘Durondeau’ (Reynoird et al. 1999). In another effort, the
have been regenerated from callus cultures, ini- harpin gene, HrpN, a bacterial inducer of sys-
tiated from root tissues (Viseur 1990). Two temic host resistance, is introduced into ‘Passe
somaclonal variants with reduced susceptibility Crassane’, and transgenic lines with reduced
to fire blight have been isolated and determined susceptibility to E. amylovora have been
to be tetraploids. Gamma and ultraviolet irradi- obtained (Malnoy et al. 2005a). Furthermore, a
ation of in vitro-grown leaf explants, followed by gene encoding a depolymerase derived from the
adventitious regeneration of plantlets, have been phage UEa1h, which degrades the capsular
assessed using four commercially important pear exopolysaccharide (EPS) of E. amylovora, has
cultivars (Pinet-Leblay et al. 1992). The effects also been introduced into ‘Passe Crassane’, and
of irradiation on adventitious shoot regeneration transgenic lines with significantly decreased
from leaf tissues have been evaluated, and LD50 susceptibility to fire blight have been observed
levels established for both irradiation methods. (Malnoy et al. 2005b). Moreover, a plant defen-
The LD50 for gamma irradiation is reported to be sin gene, Rs-AFP2, from radish has been trans-
genotype-dependent. Subsequently, compatible ferred into ‘Burakovka’ for the purpose of
and hypersensitive fire blight-resistant reactions enhancing microbial disease resistance; however,
could be differentiated in an assay of detached results of fire blight disease reactions of trans-
leaves of in vitro-derived pear plantlets of the genic lines have not yet been published (Lebedev
susceptible ‘Doyenné du Comice’ and the resis- et al. 2002).
tant ‘Old Home’ (Pinet-Leblay et al. 1996). This
assay involves infiltration of leaf tissues using a
virulent strain of E. amylovora, a dsp mutant, and 13.3 Genetics of Resistance
a heterologous pathogen, Pseudomonas syringae
pv. tabaci, followed by observations of differ- 13.3.1 Inheritance of Resistance
ential reactions. This assay is based on findings and Susceptibility
that the dsp mutant is known to be avirulent in
compatible (i.e., susceptible) host-pathogen The inheritance of resistance to fire blight is
interactions, but will result in necrosis in quantitative, as it is polygenic or controlled by
incompatible (i.e., resistant) interactions. Find- multiple genes acting with additive effects.
ings from infiltration of ‘Old Home’ leaf tissues However, there is some evidence for presence of
have suggested that fire blight resistance of ‘Old gene(s) with major effects. In a study involving
Home’ is characterized by hypersensitivity due to crosses among P. communis, P. ussuriensis, and
observed necrosis in these tissues. Pinet-Leblay P. pyrifolia parents, segregation for resistance of
et al. (1996) have proposed that this assay can be young seedling progenies artificially inoculated
254 R. L. Bell

with E. amylovora is found to be continuous, and similar to that predicted by Allard (1960) for
in many cases with normal distribution, regard- monogenic inheritance with narrow-sense heri-
less of the parental phenotype or species source tability of 50%. Furthermore, crosses with a
for resistance (Layne et al. 1968). Therefore, it P. calleryana parent and two P. pyrifolia parents
has been concluded that resistance is primarily have similarly provided evidence for presence of
polygenically inherited, with either moderate or monogenic resistance. Interestingly, a dominant
high heritability, and that either the same or gene for susceptibility has also been proposed
similar genes for resistance may be present in (Thompson et al. 1975). However, this finding is
each of these Pyrus species. Moreover, the par- based on classifying disease ratings of seedlings
ental phenotype, and to a lesser extent the species for field resistance into two discrete classes, but
source for resistance, significantly influences the no bimodal distribution of all ratings has been
proportion of seedlings obtained in each resis- demonstrated.
tance class. In other words, there is variability for Subsequent studies revealed that narrow-sense
transmitting resistance to their progenies in this heritability, estimated from parent-offspring
species. In a few seedling progenies, a skewed regression, was 0.52 for epiphytotic fire blight
segregation pattern has been observed, thus of mature seedling trees and of their parents (Bell
suggesting presence of major genes for resis- et al. 1977). Moreover, there were small differ-
tance, with resistance being dominant. Some ences between estimates within crosses of spe-
distributions could be due to monogenic inheri- cies involving parents of P. ussuriensis and
tance with low heritability or expressivity. Fur- P. pyrifolia ancestries. In this study, general
thermore, U-shaped distributions are attributed to combining ability was highly significant, while
monogenic inheritance, with moderate heritabil- specific combining ability was less significant. In
ity and dominance of resistance. In other inter- a later study by Quamme et al. (1990), it was
specific crosses, it has been noted that there is reported that general combining ability was sig-
quite a bit of variability in transmission of fire nificant, but specific combining ability was
blight resistance (van der Zwet et al. 1974a). non-significant. Bagnara et al. (1993) also found
Quamme and Bonn (1981) have concluded that that heritability was 50%, with observed differ-
general combining ability is greater than specific ences between crosses accounting for the highest
combining ability, and therefore inheritance of amount of variance. Therefore, they suggested
fire blight resistance is polygenic with a high increasing the number of crosses used in such
additive genetic variance. Dondini et al. (2002b) studies. Due to the high environmental variance
have also concluded that based on continuous and non-additive effects, they also concluded that
distribution of infection in young seedlings, parents should be selected based on their breed-
resistance from ‘Harrow Sweet’ and US309 is ing values. However, they also noted that sus-
polygenically inherited. Earlier, Decourtye ceptible parents such as ‘Bartlett’, ‘Max Red
(1967) has also proposed presence of major Bartlett’, ‘Coscia’, and ‘Bella di Guigno’ could
genes derived from parents used in his popula- also yield some resistant seedlings. In a later
tions. Similarly, Thompson et al. (1962) have study with some different parents, Bagnara et al.
concluded that resistance is inherited in a poly- (1996) found that the narrow-sense heritability
genic fashion, but with evidence for major gene was approximately 50%, and that both general
inheritance from P. ussuriensis. Likewise, Bok- combining ability and specific combining ability
szczanin et al. (2012) have found evidence for were significant. Similar to their previous study,
monogenic resistance from two P. ussuriensis they also observed that susceptible parents could
parents used in hybridizations with ‘Doyenné du yield resistant offspring, but also that some
Comice’, a susceptible P. communis cultivar. resistant parents could also produce susceptible
A continuous, but skewed distribution of disease offspring. Subsequently, Durel et al. (2004)
reactions in progeny of ‘Doyenné du analyzed data from the French INRA pear
Comice’  P. ussuriensis var. ovoidae 8 is breeding program at Angers, wherein a
13 Genetics, Genomics, and Breeding for Fire Blight Resistance … 255

population consisting of more than 17,000 clones within a Pyrus species (Thompson et al.
seedlings generated from 173 progenies, pro- 1962; Layne et al. 1968; van der Zwet et al. 1974b),
duced over 10 years by crossing 23 resistant it is suggested that conducting progeny tests may
parents with 23 susceptible parents, was evalu- serve as a useful step prior to committing resources
ated for fire blight resistance. Phenotypic data for growing and evaluating large progenies, in
consisted of five semi-quantitative classes of spite of the moderately high narrow-sense heri-
disease progression. This analysis used a tability and significant general combining ability
maximum-likelihood (ML) procedure combined (Bell et al. 1977). It is proposed that either test
with a pedigree matrix to compute heritability crosses or sub-cross generations between each
and best linear unbiased predictors (BLUP) for backcross generation, particularly in an inter-
parents and ancestors. This method was used in specific scheme, are recommended for recovering
part to compensate for inbreeding due to the desirable recessive alleles in homozygous geno-
recurrent use of some parents, such as ‘Williams’ types, and to identify heterozygous parents or to
(syn. ‘Bartlett’) in these crosses. The distribution accumulate polygenes in individuals (Lespinasse
was found to be skewed, with a large proportion and Aldwinckle 2000). However, it can also be
of seedlings scored as highly susceptible. argued that because of the moderately high
Narrow-sense heritability was estimated to be narrow-sense heritability that genetic advances
0.40 ± 0.04, which was slightly lower than those can be made when selection is based on phenotypic
estimates of Bell et al. (1977) and Bagnara et al. values (Quamme et al. 1990; Lespinasse and
(1993, 1996). This was perhaps due to the dif- Aldwinckle 2000).
ferent parental structure of the population, envi- It has been proposed that population size
ronmental effects, differences in resistance should be determined by considering the heri-
scoring methods, non-normal distribution of tability of each trait of interest, genetic and
data, and/or the pedigree-based methodology, environmental variances, and phenotypic and/or
which was not used in these earlier studies. In genetic correlations among traits of interest.
any case, BLUP values ranged from 1.91 for Variances within and between families should be
‘Campas’ to 5.42 for ‘Baurotard’ (Durel et al. computed from an appropriate genetically
2004). Therefore, it was proposed that the use of diverse population. For example, large negative
the pedigree method should provide more accu- correlations between fire blight resistance and
rate estimations of heritability and parental fruit quality traits would have detrimental effects
breeding values. on simultaneous selection for such target traits.
Undesirable fruit traits such as grittiness, poor
flavor, and small fruit size are often associated
13.3.2 Breeding Strategy with P. pyrifolia or P. ussuriensis. In a study of
large numbers of parents and seedling popula-
Parental selection for fire blight resistance is of the tions, derived from P. communis progenies and
utmost importance. The range of resistance within interspecific progenies involving P. communis
a species and the polygenic nature of inheritance crossed with either P. ussuriensis, P. pyrifolia, or
renders accurate determination of the fire blight P. calleryana, it is observed that phenotypic
resistance phenotype of each prospective parent (Bell et al. 1976) and genetic (Bell, unpublished
important. In addition, evaluation methods must data) correlations between seven fruit quality
be capable of detecting small differences, as well as traits and fire blight resistance, while generally
either minimizing or quantifying environmental negative, are small and usually statistically
variance (Lespinasse and Aldwinckle 2000). This non-significant. When using these Asian pear
is particularly important for identifying QTLs species and P.  bretschneideri as sources of fire
linked to resistance. As there are differences in blight resistance, larger population sizes are
transmission of resistance among individual required to increase the likelihood of identifying
256 R. L. Bell

selections carrying all desirable traits. It has been 13.4 Genomics

suggested that individual seedling population
sizes of at least 100 seedlings are required. 13.4.1 Mapping of Quantitative Trait
For European pear markets, the melting tex- Loci
ture of the fruit is a highly desired ideotype for
pear cultivars. For breeding for this type of fruit, One of the most important advances in genetics
it is preferable that hybridization is conducted is the development of genetic linkage maps uti-
among P. communis germplasm, especially as lizing DNA sequence-based markers, such as
cultivars and selections transmitting high levels microsatellites or SSRs, among other marker
of fire blight resistance have also been identified. types. Studies of linkages of these markers to
This is particularly true as the probability of QTLs have been used to investigate the genetic
combining fire blight resistance with high fruit architecture controlling fire blight host resistance.
quality is greater than that observed in an inter- In the first study of pear, a seedling population
specific Pyrus hybridization program that may of 99 individuals, derived from a cross between
require several backcross generations. However, two P. communis cultivars, ‘Passe Crassane’
this is not necessarily the case in the New Zeal- (susceptible) and ‘Harrow Sweet’ (resistant), has
and pear breeding program, as it is desirable to been used (Dondini et al. 2004). Various markers,
combine the aromatic flavor of P. communis including SSRs, microsatellite-anchored fragment
along with the fine and juicy texture of the best length polymorphisms (MFLPs), amplified frag-
Asian pear germplasm (White and Brewer ment length polymorphisms (AFLPs), resistance
2002b). gene analogs (RGAs), and AFLP-RGAs have been
Interspecific hybridization schemes have been used to build linkage maps for the two parents, and
evaluated for transferring fire blight resistance have identified four loci linked to fire blight
from Asian species into a P. communis genetic resistance from ‘Harrow Sweet’ (Dondini et al.
background (Layne et al. 1968; Layne, unpub- 2004). Furthermore, it has been found that ‘Har-
lished, as cited in Bell et al. 1996a). However, no row Sweet’ linkage group (LG) 2, HS2, is divided
single crossing scheme has generated a clearly into two sections, HS2a and HS2b, by
superior proportion of fire blight-resistant seed- 32 centiMorgans (cM) due to incomplete marker
lings. Nevertheless, crosses between two mod- coverage. Thus, disease incidence, severity (mea-
erately resistant parents have transmitted sured as percent lesion length), and ISV, a
resistance to a higher proportion of seedlings weighted mean index based on both incidence and
than crosses between either moderately resistant severity (Le Lézec et al. 1985), have been calcu-
parents with susceptible parents or those between lated for experiments repeated in three years. It is
susceptible parents. Thus, specific parental worth pointing out that disease resistance reactions
combinations are likely to be more important have been classified into five resistance classes. It
than the Pyrus species used as a source of is observed that analysis of the distribution of these
resistance to fire blight. phenotypic disease resistance reactions has indi-
It is important to note that multistage selection cated that fire blight resistance is under polygenic
is recommended to increase frequencies of control. Furthermore, interval mapping has iden-
accumulations of desirable alleles into a single tified four regions of ‘Harrow Sweet’ (HS) that are
genotype, thus requiring a large number of significantly associated with fire blight resistance,
crosses (Lespinasse and Aldwinckle 2000). while no associations have been detected for
Therefore, simultaneous multi-trait selection ‘Passe Crassane’. Interestingly, the most signifi-
should be also conducted (Bagnara et al. 1996). cant association is detected on LG HS2a with SSR
13 Genetics, Genomics, and Breeding for Fire Blight Resistance … 257

marker CH03H03-1 and AFLP marker M59P38-3. presence of any of the favorable alleles on HS2.
Moreover, the percent phenotypic variance Therefore, it was hypothesized that the favorable
explained by the AFLP marker is 24.6 for inci- allele could be traced back to ‘Early Sweet’, the
dence, 16.6 for severity, and 16.4 for ISV. pollen parent of Purdue 80-51, the seed parent of
Whereas, it is observed that on LG HS2b, the ‘Harrow Sweet’. However, the favorable allele of
markers AFLP-RGA B3M55-5 and SSR AT000420-SSR on HS4 was detected in ‘Bartlett’,
CH03D10 are significantly linked to fire blight the pollen parent of ‘Harrow Sweet’. Fortunately,
resistance, with AFLP-RGA B3M55-5 accounting an analysis of fire blight resistance in the progeny
for 11.8, 9.9, and 9.6% of variances for incidence, of ‘Angelys’  ‘Harrow Sweet’ validated pres-
severity, and ISV, respectively. In addition, ence of the HS2 QTL.
AFLP-RGA T2E32-1 and SSR CH01F02 on HS4 In another study, a seedling population of 155
are found to be linked to fire blight resistance, individuals, derived from a cross between P. com-
wherein AFLP-RGA T2E32-1 accounts for 9.5, munis ‘Doyenné du Comice’ (susceptible) and
8.7, and 12.0% of variances for incidence, sever- P. ussuriensis Maxim. No. 18 (resistant), was
ity, and ISV, respectively. Finally, it has been evaluated for fire blight disease resistance (Bok-
found that SSR CH05A03 on HS9 accounts for szczanin et al. 2009). In this study, disease severity
6.9, 8.4, and 8.5% of the variance for incidence, was calculated as percentage lesion length of total
severity, and ISV, respectively. Interestingly, it shoot length, and seedlings were classified into five
has been reported that detection of the two disease resistance classes, with each class of 20% in
AFLP-RGAs may indicate presence of major size. Transgressive segregation for fire blight
genes for resistance (Dondini et al. 2004). resistance was observed in this population. A puta-
Subsequently, Le Roux et al. (2012) repeated tive QTL on LG 11 of the P. ussuriensis parent
the analysis of the ‘Passe Crassane’  ‘Harrow linked to SSR RLG1, located at 0 cM, was found.
Sweet’ cross using additional SSR markers and Moreover, SSR CH03d02a, located at 22 cM, was
were able to combine HS2a and HS2b from the also significantly associated with resistance, and
previous study (Dondini et al. 2004) into a single another QTL linked to SSR CH02c02b on LG 4 of
contiguous linkage group. A single major QTL ‘Doyenné du Comice’ was also identified. These
that was significantly (p = 0.0001) linked to the findings suggested that resistance genes could also
SSR TsuENH001 and located at 30.1 cM was be found in susceptible germplasm.
identified by interval mapping. This SSR marker, In a subsequent analysis of the above popu-
flanked by TsuENH017 at 17.0 cM and NH033b lation, wherein AFLP markers were included, a
at 36.7 cM, accounted for 32.3, 28.9, and 28.1% of QTL on LG 9 of P. ussuriensis No. 18,
phenotypic variances for incidence, severity, and accounting for 61.9% of the phenotypic variance,
ISV, respectively. Furthermore, on HS04 linkage was found (Bokszczanin et al. 2011). Further-
group, markers SSR CH01d07, located at more, additional QTLs on LGs U11, U_a, U_e,
23.4 cM, and AT000420-SSR, located at and U_g, accounting for a total of 31.5% of the
41.7 cM, were found to be associated with fire phenotypic variance, were discovered. The QTLs
blight disease frequency, but only at the 0.005 of LGs U_e and U_g were found to be linked to
level of significance. In addition, as reported pre- AFLP-RGA markers, thus confirming presence
viously (Dondini et al. 2004), the AFLP-RGA of resistance genes in these linkage groups. In
T2E32-1, mapped close to AT000420-SSR and addition, four QTLs identified on LGs K3, K4,
located at 45.2 cM, was found to be associated K11, and K_a of ‘Doyenné du Comice’, collec-
with disease severity and IVS, but only at the 0.005 tively accounting for 25.6% of the phenotypic
level of significance. Thus, it was concluded that variance, were also discovered. This finding
associations at the 0.005 level represented only further confirmed earlier conclusions of Bok-
putative QTLs. Interestingly, an analysis of ‘Bar- szczanin et al. (2009) as the susceptible pear
tlett’ and ‘Old Home’, the only available ancestors cultivar ‘Doyenné du Comice’ contributed QTLs
in the pedigree of ‘Harrow Sweet’, did not reveal of small effects for resistance to fire blight.
258 R. L. Bell

An interspecific seedling population of on LG 9 of PremP003, and resistance was asso-

PremP003 (P.  bretschneideri Rehd.  ciated with the C allele. This QTL accounted for
P. communis L.)  ‘Moonglow’ (P. communis) 14.8 and 13.9% of the observed phenotypic
was artificially inoculated with E. amylovora in variance for disease severity and AUDPC,
France in 2013, and in New Zealand in both respectively (Montanari et al. 2016). Compar-
2013 and 2014 (Montanari et al. 2016). A total of isons with the QTL located on LG 9 of ‘Harrow
85 seedlings were evaluated in France in 2013, Sweet’ (Dondini et al. 2004) were conducted
while 90 seedlings were evaluated in New using a map generated by Celton et al. (2009a,
Zealand in 2013, and 105 seedlings in 2014, with b). It was found that the QTL of ‘Harrow Sweet’
85 seedlings common to both years. Disease was linked to SSR CH05a03, and although it was
progress was measured weekly for four weeks. closely mapped to SSRs CH05c07 and NB130b
Infection length as a percentage of shoot length of PremP003, it was located on a different region
(PLL) and area under disease progress curve of LG 9. However, the QTL on LG 9 in the New
(AUDPC) were computed. Analysis of phenotypic Zealand experiment mapped close to SSR
distributions detected some transgressive segre- CH03a03. Therefore, it was concluded that this
gation, consistent with polygenic control of fire latter QTL could not be verified as to whether or
blight resistance. Furthermore, QTL mapping was not it was the same QTL detected in the French
conducted, utilizing PLL at 28 dpi and AUDPC, experiment (Montanari et al. 2016).
using data for each location, and pooled for all Interestingly, three QTLs for fire blight
years and locations. Previously, genetic marker resistance, mapped to LGs 7, 12, and 15, were
maps using single nucleotide polymorphisms only discovered in the New Zealand experiment.
(SNPs) and SSRs for the two parents have been These QTLs might be strain-specific, as they
developed (Montanari et al. 2013); therefore, these were not detected in inoculation experiments in
genetic maps were used in this study. A major France where a different strain of E. amylovora
QTL associated with both PLL and AUDPC was was used. Furthermore, when fire blight inocu-
located on LG 2 of ‘Moonglow’, accounting for lation data from both French and New Zealand
12.9–34.4% of the phenotypic variance, and experiments were combined, a minor QTL linked
found to be stable between the two environments. to the “C” allele of ss475876971 was located on
In addition, associated SNP markers were identi- LG 10 of PremP003, and it was found to be
fied, including the “C” allele of ss527789653, epistatic with the locus on LG 2 (Montanari et al.
located at 15 cM, for data collected in France, and 2016). However, as phenotypic segregation of
the “G” allele of ss52779655, located at 17 cM, seedlings at the two locations was different, it
for data collected in New Zealand. However, was proposed that this QTL required further
when data from both environments were pooled, it verification. Yet, another minor QTL linked to
was observed that the “C” allele of ss527789653 the “T” allele of ss47589592 was located on LG
accounted for 66.3 and 66.5% of the globalR2 for 15 of PremP003. Overall, these minor QTLs
PLL and AUDPC, respectively (Montanari et al. accounted for 8.1–14.8% of the observed phe-
2016). Previously, a QTL for fire blight resistance notypic variance. In addition, there was a good
was discovered in ‘Harrow Sweet’ (Dondini et al. correlation between QTL results for severity and
2004). Therefore, it was suggested that the high for AUDPC. However, no homologies could be
effect of this QTL indicated presence of major detected between these minor QTLs and QTLs
genes located in this region. In fact, it was noted detected in other pear populations reviewed
that chromosome 2 of P.  bretschneideri was herein.
rich in resistance gene paralogues (Wu et al. It has been reported that the QTL on LG 2 of
2013), and that P. communis might also possess ‘Moonglow’, associated with a 176 bp allele of
such genes. CH02f06 and a 179 bp allele of TsuENH017,
Based on data collected in France, a QTL was inherited from its pollen parent, ‘Roi Charles
peak, co-located with ss475879846, was detected de Wurtemburg’. This QTL, along with a QTL
13 Genetics, Genomics, and Breeding for Fire Blight Resistance … 259

on LG 2 of ‘Harrow Sweet’, mapped by Le Roux in new rosaceous cultivars’, has been initiated in
et al. (2012), co-located with TsuENH017, thus the USA, and involving various international
indicating that it was stable in different genetic collaborators (Iezzoni et al. 2017). The major
backgrounds. However, fire blight resistance was goal for pear is to discover and/or validate QTLs
associated with different alleles of TsuENH017. in three populations segregating for fire blight
Noting that the allelic profile of this SSR in resistance.
‘Moonglow’ was the same as that identified for The genomics of host resistance to fire blight
the fire blight-resistant ‘Old Home’ (179:189), in pear genomics has been reviewed by Yama-
reported by Le Roux et al. (2012), it was moto and Chevreau (2009). Additionally, aspects
hypothesized that part of the ‘Old Home’ fire of genomics of Malus and Pyrus, as well as those
blight resistance was linked to a 179-bp allele. In of the bacterial pathogen E. amylovora have also
addition, there was colinearity between these been reviewed by Malnoy et al. (2012).
regions, and that the two pear cultivars had the
same haplotypes, with one haplotype associated
with resistance in ‘Moonglow’, while the other 13.4.2 Resistance Gene Analogues
haplotype associated with susceptibility in ‘Old
Home’. It was hypothesized that the two pear It has been reported that disease resistance genes
cultivars must carry the same QTL, and that from different plant species conferring resistance
pending further validation in other genetic against various pathogens have conserved
backgrounds, this QTL was a good candidate for regions involved in pathogen recognition and
marker-assisted breeding (MAB) (Montanari defense response (Staskawicz et al. 1995). Pri-
et al. 2016). mers can be designed for these regions, and used
Although the minor QTL on LG 9 of in PCRs to amplify similar fragments, known as
PremP003 was associated with a 141 bp allele of resistance gene analogues (RGAs), in other plant
CH05c07 and a 90 bp allele of NB130b, both species. RGAs have been identified in fire
alleles inherited from ‘Xue Hua Li’, neither of blight-resistant pear genotypes, including ‘Har-
these favorable alleles were found on LG 9 of the row Sweet’, ‘Old Home’, and US309 (Dondini
fire blight-resistant ‘Harrow Sweet’ (Dondini et al. 2002a). In fact, primers have been designed
et al. 2004; Le Roux et al. 2012). The origins of for the P-loop and for GLPL motifs, and then
QTLs mapped onto LGs 7, 12, and 15 could not used to generate PCR products. All these primers
be determined. have amplified a major 500 bp band in all pear
Overall, it is concluded that these data support genotypes. This band must have resulted from
the hypothesis of polygenic control for fire blight co-migration of more than 80 fragments, which
resistance. Furthermore, it is also concluded that have been subsequently cloned, and grouped by
a high broad-sense heritability supported the cluster analysis. After sequencing of 15 colonies,
reliability of these detected QTLs. However, it is followed by FASTA analysis, it has been shown
reported that the globalR2 is less than that of H2, that these sequences have 58–65% homology to
and this is due either to small population sizes or known resistance genes or RGA sequences.
to presence of additional QTLs in regions of Alignments among pear RGAs have revealed a
these maps that are not covered by markers. high degree of sequence variability, but most of
Therefore, it is proposed that pre- and these sequences are found to belong to the
post-zygotic incompatibilities may have pre- TIR-NBS-LRR family. Therefore, it has been
vented saturation of the parental genetic maps proposed that these RGA sequences can serve as
due to linkages to a lethal gene. genetic markers to search for polymorphisms
All results of the above-mentioned QTL between fire blight-resistant and susceptible
studies are summarized in Table 13.1. parents, and to establish linkages with fire blight
A new project, entitled ‘RosBREED2: Com- resistance. An analysis of the phylogeny of
bining disease resistance and horticultural quality RGAs in Rosaceae species, including 34 from
260 R. L. Bell

Table 13.1 Quantitative trait loci for fire blight resistance in pear
Mapping n Trait Linkage Marker type Marker Marker Percent Reference(s)
population group position r2
(cM)
‘Passe 99 ISV, 28 HS2a AFLP M59P38-3 9.0 16.4 Dondini et al.
Crassane’  HS2b AFLP-RGA B3M55-5 10.7 9.6 (2004)
‘Harrow Sweet’
HS4 AFLP-RGA T2E32-1 9.8 12.0
HS9 SSR CH05A03 21.9 8.5
ISV, 28 HS2 SSR TsuENH001 30.1 28.1 Le Roux
HS4 AFLP-RGA T2E32-1 45.2 13.3 et al. (2012)

‘Doyennédu 155 PLL, 28 DC4 SSR CH02c02b 56.0 - Bokszczanin

Comice’  Pu11 SSR CH03d02a 22.0 - et al. (2009)
P. ussuriensis
No. 18
PremP003  85 AUDPC, M2 SNP ss527789563 15.0 34.4 Montanari
‘Moonglow’ 28 et al. (2016)
P9 SNP ss475879846 35.0 13.9
90, M2 SNP ss527789655 17.0 17.7
105
P7 SNP ss475876829 48.0 7.2
P12 SNP ss475880537 48.0 10.3
P13 SNP ss527788568 23.0 10.0
Trait: ISV = Index of varietal susceptibility (Thibault et al. 1987), 28 = days after inoculation, PLL = percent lesion
length, AUDPC = area under the disease progress curve; Linkage group: HS = ‘Harrow Sweet’, DC = ‘Doyenné du
Comice’, Pu = P. ussuriensis No. 18, M = ‘Moonglow’, P = PremP003

Pyrus, has found that three clades contain RGAs Bagnara GL, Rivalta L, Laghi M, Quarta R, Lecomte P
of Pyrus, Malus, and Prunus, thus indicating a (1993) Cross combinations for fire blight resistance in
pear. Acta Hortic 338:369–374
monophyletic origin and conservation of these Bagnara GL, Rivalta L, Laghi M, Quarta R (1996)
RGAs in these three genera of Rosaceae (Per- Evaluation of fire blight resistance in pear: combining
azzolli et al. 2014). ability and breeding strategy. Acta Hortic 411:383–
392
Bell RL (1991) Pears (Pyrus). Acta Hortic 290:657–700.
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 Functional Genomics
14
Songling Bai and Yuanwen Teng

Abstract 14.1 Introduction

Several closely related species of commercial
importance in the genus Pyrus are cultivated With the release of draft genome sequences for
throughout the world. In eastern Asia, specif- the Asian pear, Pyrus  bretschneideri cv.
ically in China, Japan, and Korea, the East Dangshansuli (Wu et al. 2013a), and the Euro-
Asian pear, including the Chinese white pear pean pear, P. communis cv. Bartlett (Chagné
(P. pyrifolia white pear group, also referred to et al. 2014), increased efforts have been under-
as P.  bretschneideri), the Chinese sand taken to pursue functional genomics studies in
pear (P. pyrifolia), the Japanese pear (P. pyri- pear. Many large-scale studies have identified
folia), the Ussurian pear (P. ussuriensis), and numerous candidate genes related to various
the Xingiang pear (P. sinkiangensis), is cul- traits of horticultural importance associated with
tivated, while in the rest of the world, the tree growth and development, as well as with
European pear (P. communis) is more com- various flowering, fruiting, and fruit quality
monly grown. Whole-genome sequences have characters (Nashima et al. 2013b; Xie et al. 2013;
been released for both P.  bretschneideri cv. Wang et al. 2014; Nham et al. 2015; Yang et al.
Dangshansuli (also known to belong to 2015; Reuscher et al. 2016; Zhang et al. 2016;
P. pyrifolia white pear group) and P. commu- Shi et al. 2017; Wang et al. 2017; Zhang et al.
nis cv. Bartlett. As a result of these draft pear 2017). These studies were followed by more
genome sequences, major advances have been in-depth studies to investigate functions of some
made in pursuing functional genomics studies of these important genes using various functional
in pear. genetic analysis approaches (Huang et al. 2015;
Jin et al. 2016; Niu et al. 2016; Tuan et al. 2016;
Li et al. 2017a).
In this chapter, we will cover genomic data-
bases and tools that have been developed for the
pear genome that are critical in pursuing func-
tional genomics studies. This will be followed by
S. Bai
Y. Teng (&)
a review of recent advances in our knowledge of
The State Agricultural Ministry Key Laboratory of gene functions related to important horticultural
Horticultural Plant Growth, Development and traits of the pear, such as vegetative/reproductive
Quality Improvement, Department of Horticulture, phase transition, grafting, fruit coloration, and
Zhejiang University, 310058 Hangzhou, Zhejiang,
China
development of stone cells, among others.
e-mail: [email protected]

© Springer Nature Switzerland AG 2019 265

S. S. Korban (ed.), The Pear Genome, Compendium of Plant Genomes,
https://doi.org/10.1007/978-3-030-11048-2_14
266 S. Bai and Y. Teng

14.2 Databases for Genomic genome/12793?genome_assembly_id=40827).

Resources This draft genome sequence consists of 2192
scaffolds with 47,086 predicted proteins.
14.2.1 The Expressed Sequence Tag
(EST) Database for Pear 14.2.1.2 A Whole-Genome
Sequence Database
The Genome Database for Rosaceae (GDR, for the European Pear
https://www.rosaceae.org) is a resource for Genome sequences of the European pear cv.
expressed sequence tags (ESTs), genome Bartlett (P. communis), published by Chagné
sequences, and data mining tools for various et al. (2014), have been submitted to the GDR,
members of the Rosaceae family (Jung et al. which also has several useful applications, such
2014). Prior to completion of draft whole- as BLAST and GBrowser, among others (https://
genome sequences for the pear genome, the www.rosaceae.org/organism/Pyrus/communis).
GDR has included a small set of pear ESTs. The Recently, Li et al. (2017b) have reassembled
latest version (v5) of this database includes 1760 these sequences, and this dataset has been
EST reads, yielding 259 assembled contigs and deposited in yet another database, referred to as
964 singlets. The use of this EST database has ‘Bartlett V1.1’ (http://peargenome.njau.edu.cn).
been limited following the release of
whole-genome draft sequences for Pyrus. 14.2.1.3 The KEGG Database
The KEGG database (http://www.kegg.jp/ or
14.2.1.1 A Whole-Genome http://www.genome.jp/kegg/) covers an ency-
Sequence Database clopedia of genes and genomes. The primary
for the Chinese White objective of the KEGG database project is to
Pear assign functions to genes and genomes, both at
Wu et al. (2013a) published the first draft gen- the molecular and at the higher structural/
ome sequence of the Chinese white pear cv. organismal levels. Molecular-level functions are
Dangshansuli (P.  bretschneideri, also reported stored in the KO (KEGG orthology) database,
to belong to the white pear group of P. pyrifolia). wherein each KO is defined as a functional
The draft genome size of this Asian pear is orthologue of genes and proteins (Kanehisa et al.
estimated to be 512 Mb and corresponding to 2017). The KEGG orthology of the Chinese
97.1% of the estimated genome size. This gen- white pear is available based on predicted
ome sequence database is hosted at the Nanjing genes/proteins in NCBI (http://www.kegg.jp/
Agriculture University (http://peargenome.njau. dbget-bin/www_bget?gn:T03446).
edu.cn). At present, this database provides gen-
ome sequences of this Asian pear, as well as that
of the European pear, P. communis cv. Bartlett 14.3 Functional Genomics Studies
(Chagné et al. 2014). In this database, genome in Pear
sequences for the Asian pear are assembled into
2103 scaffolds with a total of 42,812 gene loci 14.3.1 Phase Transition of Annual
identified. Growth
Wu et al. (2013a) also published pseudo-
molecule sequences in (GIGA)nDB (http:// Similar to other woody perennial trees, the life
gigadb.org/dataset/100083) and assigned pre- cycle of pear is different from that of annual
dicted gene loci into pseudomolecules. plants. Pear trees have long juvenility periods,
The NCBI database has also presented pear and it takes 8–10 years for European pear seed-
genome data based on publicly available raw ling trees to reach reproductive maturity (Layne
sequence data (https://www.ncbi.nlm.nih.gov/ and Quamme 1975). Once reproductive maturity
14 Functional Genomics 267

is reached, flower buds are formed on lateral 14.3.1.2 The Regulation

buds, on 2-year-old wood of European pears and of Endodormancy
on 1-year-old wood of some Asian pears, on an It has been reported that dormancy-associated
annual basis. Floral bud formation and differen- MADS-box (DAM) genes encode members of
tiation for the following growing season are ini- MADS-box transcription factors that have been
tiated soon after completion of shoot elongation implicated to play important roles in dormancy in
in late spring or early summer (Ito et al. 1999). a mutant peach (Prunus persica) genotype (Bie-
During the fall season, leaves wilt and drop, and lenberg et al. 2008). Subsequently, two research
pear trees enter endodormancy during which groups have independently identified three pear
formed buds are repressed by internal cues and DAM genes (Saito et al. 2013; Niu et al. 2016).
are not capable of sprouting, even under suitable Although these two groups have identified the
growth conditions until the chilling requirement same set of DAM genes from two different pear
is fulfilled (Lang et al. 1987). After fulfillment of cultivars, they have used different nomenclatures
the chilling requirement, these buds can poten- for these genes. As a result, this has created some
tially begin to sprout and grow, but low tem- level of confusion. For example, the DAM1 gene
peratures during the winter will hinder bud identified by Niu et al. (2016) is a homologue of
growth (Faust et al. 1997). With elevated tem- PpMADS13-2, from P. pyrifolia, previously
peratures in early spring, buds will expand, identified by Saito et al. (2013).
proceed to sprout, and bloom; this is followed by DAM genes belong to the flower regulator
development of new leaves, shoot elongation, group of genes that include the SHORT VEGE-
along with early fruit development (Saito et al. TATIVE PHASE and AGMOUS-LIKE 24 with an
2015b). Thus, vegetative and reproductive EAR motif, functioning as transcriptional
growth proceeds simultaneously within the same repressors. Some reports have proposed that pear
year; thereby, annual growth is highly regulated DAM genes repress growth by targeting one of
and coordinated and involves many complex the two FLOWERING LOCUS T (FT) homo-
regulatory pathways. logues, specifically that of PpFT2a (Fig. 14.1).
However, this proposed hypothesis lacks critical
14.3.1.1 Induction of Flower Bud evidence; in particular, there is no CArG motif
Initiation identified in the promoter region of PpFT2.
Floral bud induction is an important event, sig- Therefore, it cannot yet be excluded that
naling the beginning of a new reproductive cycle. PpDAMs bind to other related motifs to repress
Although pear is a long-day plant, the detailed transcription of PpFT2.
floral bud induction pathway, by environmental Several lines of evidence have supported the
cues, has not yet been well characterized. It is proposal that C-repeated binding factor
reported that pear Flowering Locus T homo- (CBF) proteins from P. pyrifolia, specifically
logues, PpFT1a and PpFT2a, are involved in the PpCBF2 proteins, directly induce expression of
induction of flower bud initiation, but are not the PpDAMs by binding to CRT/DRE motifs
determinants of flowering (Bai et al. 2017b). (Fig. 14.1) (Saito et al. 2015a; Niu et al. 2016).
Instead, the transcriptional drop in expression of However, expression patterns of PpCBF2 and
Terminal Flower Like 1 (TFL1) homologues, PpDAM are found to be inconsistent, thereby
PpTFL1-1a and PpTFL1-2a, prior to flower bud suggesting that other members of the CBF group
initiation, is in fact the primary trigger for flower or other transcriptional factors (TFs) are poten-
bud initiation. Furthermore, several hormone- tially involved in the regulation of DAM genes
related transcription factors are potentially (Saito et al. 2015a; Niu et al. 2016).
involved in PpTFL1-mediated floral induction It has long been known that abscisic acid
(Bai et al. 2017b). (ABA) content in plant tissues is significantly
268 S. Bai and Y. Teng

Fig. 14.1 An illustration of

the regulation of pear bud
dormancy

correlated with endodormancy establishment and 14.3.2 Fruit Development

release. The expression pattern of PpCYP707A3,
encoding for cytochrome P450, in P. pyrifolia is As fruit growth and development are of particular
highly associated with chilling accumulation (Li interest, there have been increasing functional
et al. 2018), and that PpDAM1 directly upregu- genomics studies to understand the functional
lates expression of PpNCED3, coding for the roles of genes involved in fruit development, as
enzyme 9-cis-epoxycarotenoid dioxygenase well as of various fruit quality traits. In pear,
(Tuan et al. 2017). Simultaneously, the ABA there are several cultivated pear species, includ-
response element (ABRE)-binding transcription ing P. pyrifolia, P.  bretschneideri, P. sinkian-
factor, PpAREB1 (=PpABF2), which binds to gensis, P. ussuriensis, and P. communis, that
three ABRE motifs in the promoter region of produce fruits of commercial importance with
PpDAM1, negatively regulates its activity. In varying fruit development and fruit quality traits.
turn, this forms a feedback regulation mechanism For example, P. ussuriensis and P. communis
between PpDAMs and each of the ABA meta- bear climacteric fruits requiring post-harvest
bolism and the signaling pathway during ripening, while fruits of other pear species are
endodormancy in pear (Tuan et al. 2017). readily edible at maturity following harvest. In
Based on degradome sequence data, it is addition, fruits of P. communis pears are mostly
reported that miR6390 targets PpDAM genes gourd-shaped, have soft and smooth flesh with
(Niu et al. 2016). Furthermore, miR6390 and few stone cells, high sugar and acid contents,
PpDAM have shown contrasting expression pat- along with a strong aroma. Likewise, fruits of
terns, thus indicating that miR6390 might play a P. ussuriensis usually have good aroma and
critical role in dormancy release via degradation strong flavor. In contrast, fruits of Asian pears are
of PpDAM transcripts (Niu et al. 2016). How- mostly round in shape, have crisp flesh, high
ever, additional studies are required to verify the stone cell contents, low aroma and flavor, and
role of miRNAs in regulating pear tree with some species having high sugar and low
dormancy. acid contents.
14 Functional Genomics 269

To better understand fruit development char- dehydrogenase-like (SDH-like) and ATP-dependent

acteristics in different pear species, Zhang et al. 6-phosphofructokinase (ATP-PFK). Although the
(2016) compared transcriptomes of developing detailed regulatory pathway for stone cell formation
fruits of five different pear species and identified has not yet been characterized, these large-scale
differentially expressed genes related to fruit transcriptome data provide solid basis for further
quality and development. In addition, several studies. For further detailed information on stone cell
ethylene synthesis genes and polyphenol development, please see Chap. 11 in this volume.
oxidase-related genes were identified as Some physiological and molecular mechanism
co-expressed genes, thus suggesting their poten- studies have been conducted to investigate dif-
tial functions during fruit ripening. ferent fruit development characteristics, as well as
Stone cells are peculiar cells in pear fruits. fruit quality traits. For example, it has been
During the development of pear fruits, stone cells observed that fruit texture is influenced by ACO
are mainly formed following rapid cell division. (coding for 1-aminocyclopropane-1-carboxylate
Fruits of some pear cultivars have high stone cell oxidase) and then by XTH (coding for xyloglucan
contents, which significantly influence their endotransglucosylase/hydrolase)-related genes,
quality. Stone cells are particular types of par- thereby contributing to cell wall disassembly and
enchyma cells that differentiate into cells with loosening (Zhang et al. 2016). In another exam-
thickened secondary cell walls that are highly ple, it has been found that fruit ripening of the
lignified, and referred to as sclerenchyma cells. European pear ‘Bartlett,’ while still hanging on
Zhang et al. (2016) have identified several genes, the tree, can be enhanced by spraying trees with
such as 4CL (encoding 4-coumarate CoA ligase), ethylene, as this contributes to fruit softening
C3H (encoding p-coumarate 3-hydroxylase), (Murayama et al. 2006). It has since been dis-
CA5H (encoding coniferyl aldehyde covered that endo-PG genes play various impor-
5-hydroxylase), and CAD (encoding cinnamyl tant roles in many different fruit maturation
alcohol dehydrogenase) with relatively high characteristics (Hiwasa et al. 2004; Murayama
levels of expression at early stages of fruit et al. 2006). A microarray analysis study has
development for all tested pear cultivars. Fur- revealed that a cupin family protein gene and two
thermore, genes regulating hydroxycinnamoyl unannotated genes in P. communis, but absent in
transferases (HCT), which reduce the H-lignin Japanese pear (P. pyrifolia), may be involved in
content, have also been identified and found to be the ripening process specific to P. communis
expressed at early stages of fruit development. (Nashima et al. 2013a).
Specifically, caffeoyl-CoA o-methyltransferase
(CCOMT)-related genes are specifically expres-
sed in P. ussuriensis, and they are likely related to 14.3.3 Red Coloration of Fruit
high contents of stone cells in flesh tissues of
these fruits. Similarly, Zhang et al. (2017) have Red pears are attractive, deemed to have better
reported that by comparing transcriptomes of nutritional value, and have gained more con-
fruits of two pear cultivars with different stone cell sumer preference. To date, red-colored pear
contents, more than 7000 differentially expressed cultivars (or sports) have been identified in both
genes have been identified, including many lignin Asian pears and European pears.
biosynthesis-related genes. These include genes Development of red coloration depends on
coding for coumaroylquinate 3-monooxygenase accumulation of anthocyanins in peels of pear
(C3H), shikimate O-hydroxycinnamoyl transferase fruits. Anthocyanin is synthesized in the cytosol
(HCT), ferulate 5-hydroxylase (F5H), cinnamyl and then transported to the vacuole by a glu-
alcohol dehydrogenase (CAD), and peroxidase tathione S-transferase (GST) (Tanaka et al.
(POD), as well as genes related to carbon metabo- 2008). The biosynthesis of anthocyanin involves
lism, such as those coding for sorbitol several well-characterized enzymes, including
270 S. Bai and Y. Teng

chalcone synthase (CHS), chalcone isomerase been found that during the red color fading phase
(CHI), flavanone 3-hydroxylase (F3H), flavonoid of ‘Red Bartlett,’ the structural gene LDOX and
3′-hydroxylase (F3′H), dihydroflavonol six GST family genes are downregulated, while
4-reductase (DFR), anthocyanidin synthase FLS, LAC, POD, and five light-responding genes
(ANS), and UDP-glucose: flavonoid are significantly upregulated. Additionally, 45
3-O-glucosyltransferase (UFGT) (Fig. 14.2). genes encoding transcription factors MYB,
Spatial and temporal expression of genes coding bHLH, WRKY, NAC, ERF, and zinc finger have
for these enzymes are regulated at the transcrip- been identified among 947 DEGs. Based on this
tional level by various TFs, particularly those of wealth of information, a detailed regulatory
the well-studied MYB-bHLH-WD40 pathway is emerging and under current
(MBW) complex, which is composed of MYB, development.
basic Helix-loop-Helix (bHLH), and WD40 Traditional Asian pear fruits usually have
(Broun 2005; Hichri et al. 2011). In many hor- smooth green (or yellow) and brown-russet skin
ticultural crops, R2R3 MYB proteins have been colors, but in recent years, development of
reported as important TFs for activation of red-colored Asian pear is rapidly increasing.
anthocyanin biosynthesis genes (Kobayashi et al. Several genes involved in the regulation of
2004; Takos et al. 2006; Espley et al. 2009; anthocyanin biosynthesis have already been
Medina-Puche et al. 2014). Similarly, a set of identified (Table 14.1). The red pear cultivar
pear MYB genes, namely PcMYB10, PyMYB10, ‘Bayuehong’ is a progeny of European pear
and PyMYB114, contribute to the anthocyanin ‘Clapp’s favorite’ and ‘Zaosu’ pear, and the latter
biosynthesis in the fruit peel (Fig. 14.2 and cultivar is a hybrid of ‘Pingguoli’ (P. pyrifolia)
Table 14.1). and ‘Mishirazu’ (P. communis). ‘Bayuehong’
Although cultivars with red-colored fruits develops red color on the sunny side of the fruit
have been discovered in both European and peel. Based on genetic analysis, an R2R3 MYB
Asian pears, their genetic regulation of the red transcription factor, PpMYB114, is found to be
coloration is different. For European pear ‘Max responsible for regulating red coloration of
Red Bartlett,’ which is a somatic mutant of ‘Bayuehong’ (Yao et al. 2017). It is reported that
‘Bartlett,’ the red coloration of fruit peel depends PpMYB114 interacts with an ERF transcription
on active transcription of the PcMYB10 gene, factor, PpERF3, and PpbHLH3 to co-regulate
although it is not quite clear as to why PcMYB10 anthocyanin biosynthesis (Yao et al. 2017). In
is the one that is transcribed (Pierantoni et al. another pear cultivar, ‘Red Zaosu’, a red-colored
2010). Another study has mapped the red locus somatic mutant of ‘Zaosu’, PbMYB10b
to linkage group (LG) 4, a locus different from (=PpMYB114) is identified as an activator of the
that of PcMYB10 (Dondini et al. 2008), thus anthocyanin and proanthocyanin pathways, and
suggesting an unknown upstream regulator of PbMYB9 is found to be an activator of proan-
PcMYB10 corresponds to the main regulator of thocyanin, anthocyanin, and flavanol pathways
red coloration of ‘Red Bartlett.’ In addition, (Zhai et al. 2016). As red color developmental
DNA methylation levels of the promoter of patterns of Asian pears differ from those of
PcMYB10 are correlated with red coloration of European pears (Qian et al. 2013), germplasm
‘Max Red Bartlett’ (Wang et al. 2013). resources are deemed highly useful for studying
For some red European pear cultivars, such as the regulatory mechanism(s) of pear fruit
‘Max Red Bartlett,’ red coloration peaks during coloration.
early stages of fruit development, and then fades There are various approaches for studying
to red-green at maturity. This reduces the com- functions of genes in pears. For one, transient
mercial value of these cultivars. Wang et al. expression of pear genes can aid in studying
(2017) have identified 947 differentially expres- functions of these genes. Interestingly, this is
sed genes by comparing transcriptomes of fruit widely used for the study of anthocyanin pro-
peels of ‘Red Bartlett’ and ‘Starkrimson.’ It has duction. In fact, it has been observed that
14 Functional Genomics 271

Fig. 14.2 Genes involved in

the anthocyanin biosynthesis
of pear fruits

overexpression of the PpMYB114/bHLH/ERF3 pear fruit alter anthocyanin accumulation in

complex in tobacco leaves and in strawberry fruit peel (Zhai et al. 2016 and Ni et al. 2019;
can significantly induce synthesis of antho- Bai 2019). Therefore, such transient assays
cyanin. This confirms the important roles of serve as good preliminary tests prior to pur-
PpMYB114 and PpERF3 in the biosynthesis of suing development of stable transgenic plants
anthocyanin (Yao et al. 2017). Moreover, for further testing.
transient overexpression of some other genes, In another approach, virus-induced gene
including PbMYB10b, PbMYB9, and PbMYB3, silencing (VIGS) assays have been used for
several EFR genes, and BBX family genes in studying gene functions during anthocyanin
272 S. Bai and Y. Teng

Table 14.1 Genes involved in the regulation of anthocyanin biosynthesis in pear

Gene name Asian/European Gene family Function(s) Reference(s)
pear
MYB10 Both MYB Directly activates Feng et al. (2010),
structural genes Pierantoni et al. (2010),
Wang et al. (2013)
WD40 Asian pear WD40 Forms the MWB Qian et al. (2017)
complex
bHLH3/33 Asian pear bHLH Forms the MWB Qian et al. (2017)
complex
ERF3 Asian pear AP2/ERF Interacts with Yao et al. (2017)
MYB114
HY5 Asian pear bZIP Directly activates Tao et al. (2018)
structural genes and
MYB10
SPL Asian pear SPL Interacts with MYB10 Qian et al. (2017)
to destabilize the
MBW complex
miR156 Asian pear miRNA Contributes to SPL Qian et al. (2017)
degradation
COP1 Asian pear F-box Destabilizes HY5 and Tao et al. (2018)
MYB10
CRY1 Asian pear Cryptochrome Destabilizes COP1 Tao et al. (2018)
CRY2 Asian pear Cryptochrome Destabilizes COP1 Tao et al. (2018)
MYB114/MYB10b Asian pear MYB Directly activates Zhai et al. (2016), Yao
structural genes et al. (2017)
MYB9 Asian pear MYB Directly activates Zhai et al. (2016)
structural genes
PyMADS18 European pear MADS Not yet clarified Wu et al. (2013b)

accumulation. Although the host efficiency of the pathways in response to light (Qian et al. 2013).
tobacco rattle virus (TRV) has not yet been well RNA-Seq analysis of peels of bagged red pear
characterized in pear and in other rosaceous fruits of P. pyrifolia has identified a total of 8870
plants, the TRV-based VIGS system has been non-redundant differentially expressed genes,
used in many studies. For example, using VIGS, including HY5, CRY-DASH, and a CO-like
it has been reported that silencing of PpMYB114, transcription factor. This has indicated that
PpbHLH, and PpERF3 inhibits biosynthesis of other light-responsive transcriptional factors are
anthocyanin in ‘Red Zaosu’ (Yao et al. 2017). also involved in anthocyanin accumulation in red
Bagging of fruit is an efficient and common Asian pears (Bai et al. 2017a).
method used to improve color development in
many fruit crops. However, it has been reported
that light reactions of Asian and European red 14.3.4 Fruit Russet
pears are quite different. It has been observed that
removing bags prior to maturation efficiently Fruit russeting is a unique feature of some
induces anthocyanin accumulation in Asian red important commercial pear cultivars, and so this
pear, but not in European pear fruits, thus sug- trait is of particular interest. Fruit russeting is
gesting presence of different signal transduction characterized by a corky and netlike texture of
14 Functional Genomics 273

the fruit peel. It is known that the peel is made up It has been found that some pear endogenous
of cuticle lamellae, epidermal cell layers, and mRNAs are capable of transport through the
cork cambium, wherein the cork cambium forms phloem, including NAM/ATAF1/2/CUC2 PRO-
a thick-walled cell layer; i.e., cork layer, in a TEIN, GA IINSENSITIVE, WUSHEL
mature pear fruit. In Asian sand pears, P. pyrifo- RELATED HOMEOBOX T1, and KNOTTED1
lia, there are variations in peel colors, including (Zhang et al. 2012, 2013; Duan et al. 2015,
russet, green, and mixtures of russet and green. 2016). This transport involves the movement
The russet peel of sand pear is attributed to protein binding protein 2C (Duan et al. 2015) and
accumulation of a cork layer. This is an impor- the polypyrimidine tract binding protein (Duan
tant horticultural trait as the cork layer can pro- et al. 2016), which directly bind to mRNAs to
tect fruit from external stresses caused by assist in the movement. Although the detailed
diseases, insects, unfavorable weather condi- mechanism for the mutual regulation in the graft
tions, and shipping hazards. Wang et al. (2014) system, these advances have helped researchers
have compared transcriptomes of peel russet in using transportable mRNAs in pursuing the
formation in two pear genotypes of contrasting development of new pear breeding efforts.
peel colors, and have identified candidate genes
for suberin, cutin, and wax biosynthesis in russet
peels. They have proposed that genes encoding 14.5 Abiotic Stresses
putative cinnamoyl-CoA reductase (CCR), cin-
namyl alcohol dehydrogenase (CAD), and per- Abiotic stresses affect growth, development,
oxidase (POD) are involved in lignin productivity, as well as various economic traits
biosynthesis and in pigmentation of russet peels of pears. To cope with abiotic stress, pears have
of sand pears. evolved sophisticated mechanisms to respond to
such stresses, ranging from perception of stress
signals to modification of physiological and
14.4 Scion–Rootstock Interactions biochemical responses. Unlike model species,
there are only a few studies focusing on the
As with other fruit trees, pear trees are propa- function of a particular gene on stress response;
gated by grafting. Grafting is a practice involving this is partially due to lack of reliable approaches
fusion of tissues, vascular tissues, from two for investigating abiotic stress in pear. However,
genetic systems, the scion and the rootstock. The several exciting and conclusive studies have used
newly established communication between the heterologous ectopic expression approaches, and
rootstock and the scion can induce alterations in have obtained some interesting findings,
traits of the two fused genetic partner systems, although such approaches may potentially lead to
including stress tolerance, dwarfing, fruit devel- false positive results. For example, ectopic
opment, and other phenotypic changes. To date, expression of PubHLH1, from P. ussuriensis, in
the detailed mechanism for regulation of one transgenic tobacco has conferred enhanced tol-
grafting partner by the other partner is not well erance to cold stress (Jin et al. 2016). While,
characterized. A well-accepted hypothesis is that overexpression of PbrMYB21, from P. betulae-
matter exchanges through the vascular bundle folia, in tobacco has conferred enhanced dehy-
play important roles. In the past two decades, dration and drought tolerance (Li et al. 2017a).
macromolecules, including proteins, mRNAs, Furthermore, using a VIGS assay, it has been
and siRNAs, have been identified in the phloem further confirmed that PbrMYB21 positively
sap, shedding light on the study of the mecha- regulates drought stress (Li et al. 2017a). In
nism of the mutual effects on the graft system. addition, ectopic expression of a novel NAC
Among these macromolecules, mRNAs have transcription factor, PbeNAC1, in tobacco leads
been the main focus of study thus far. to enhanced cold and drought tolerance.
274 S. Bai and Y. Teng

ICE1 is an important gene in the functions. In recent years, some important genes
cold-responsive pathway. In a recent study, have been identified by using genetic analysis
Huang et al. (2015) have reported that PuICE1 of along with a heterologous transgenic system.
P. ussuriensis can be upregulated by various Such a strategy will be more likely used in future
abiotic stresses, such as cold and dehydration. pear functional genomics studies. On the other
Using transgenic tomato plants overexpressing hand, there has been success in using a homol-
PuICE1, it has been demonstrated that this gene ogous transgenic system in European pear
confers enhanced tolerance to cold. In fact, the (Freiman et al. 2012). The expanded use of these
PuHHP1 protein physically interacts with systems will significantly accelerate our func-
PuICE1 and regulates the transcriptional activity tional genomics studies in pear in the future.
of PbDREBa, which further confers tolerance to
multiple other stresses (Huang et al. 2015).
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Zhai R, Wang ZM, Zhang SW, Meng G, Song LY, Zhang WN, Duan XW, Ma C, Harada T, Li TZ (2013)
Wang ZG, Li PM, Ma FW, Xu LF (2016) Two MYB Transport of mRNA molecules coding NAC domain
transcription factors regulate flavonoid biosynthesis in protein in grafted pear and transgenic tobacco. Biol
pear fruit (Pyrus bretschneideri Rehd.). J Exp Bot Plantarum 57(2):224–230
67(5):1275–1284 Zhang WN, Gong L, Ma C, Xu HY, Hu JF, Harada T,
Zhang JY, Cheng X, Jin Q, Su XQ, Li ML, Yan CC, Li TZ (2012) Gibberellic acid-insensitive mRNA
Jiao XY, Li DH, Lin Y, Cai YP (2017) Comparison of transport in Pyrus. Plant Mol Biol Rep 30(3):614–623
the transcriptomic analysis between two Chinese white
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https://doi.org/10.1371/journal.pone.0187114
 Whole-Genome Duplications in Pear
and Apple 15
Hao Li, Chien-Hsun Huang and Hong Ma

Abstract apple, and of other fleshy-fruit-producing

Whole-genome duplications (WGDs) are genera of the subtribe Malinae, following
widespread in angiosperms, and are proposed divergence of dry-fruit-bearing lineages of
to have contributed to angiosperm diversifica- Maleae. Moreover, over 1000 gene duplicates
tion. Pear (Pyrus) and apple (Malus) belong to from the Malinae WGD have been mapped to
the large and diverse Maleae tribe, and their syntenic blocks in the apple genome, thus
genome sequences have extensive syntenic supporting the hypothesis that syntenic blocks
blocks covering much of the chromosomes, found in apple (and pear) have been generated
thus providing strong support for WGDs. by the Malinae WGD, dated in late Eocene
Comparative analyses further indicate that at (*38–42 million years ago). Further, nearly
least a single WGD is shared by both pear and two-thirds of gene duplicates, initially retained
apple, and it has likely occurred following following the Malinae WGD, have been lost
pear/apple lineage split from that of straw- in the apple genome, with relatively rapid
berry (Fragaria). Furthermore, phylogenomic losses in early Oligocene. Finally, the
analysis of thousands of nuclear genes, from Malinae-WGD-generated duplicates are
public genome datasets and from over 120 enriched in GO categories for transcriptional
transcriptomic datasets, has uncovered strong regulation, including members of the
evidence of presence of thousands of gene MADS-box gene family, possibly contribut-
duplicates for a WGD in the ancestor of pear, ing to the evolution of fleshy fruits in Malinae.
There is also supporting evidence for this
finding provided by functional analysis of
several apple MADS-box genes.
H. Li
H. Ma (&)
Department of Biology, Huck Institutes of the Life
Sciences, Pennsylvania State University, University
Park, PA 16802, USA
e-mail: [email protected] 15.1 Introduction
H. Li
C.-H. Huang
State Key Laboratory of Genetic Engineering and Pear is one of the oldest and most widespread
Collaborative Innovation Center for Genetics and fruits of the world, and it has been cultivated for
Development, Ministry of Education Key Laboratory more than 3000 years, with thousands of culti-
for Biodiversity Science and Ecological Engineering,
Institute of Plant Biology, Institute of Biodiversity
vars that are available nowadays (Lombard and
Sciences, Center for Evolutionary Biology, School Westwood 1987). Fruits are the defining char-
of Life Sciences, Fudan University, Shanghai acteristics of angiosperms, and contribute to
200438, China

© Springer Nature Switzerland AG 2019 279

S. S. Korban (ed.), The Pear Genome, Compendium of Plant Genomes,
https://doi.org/10.1007/978-3-030-11048-2_15
280 H. Li et al.

angiosperm evolutionary success by protecting As Pyrus and Malus, among other genera of
and dispersing seeds. Moreover, fruits are also Maleae, have a basic chromosome number of
economically and ecologically important by x = 17, it has been proposed, based on morpho-
providing foods and nutrition to humans and to logical characters, that the ancestor of Maleae is
animals. Fruits have a wide variety of morpho- derived from allopolyploidization between
logical types and often exhibit important features ancestors of two other subfamilies, the Spi-
that distinguish one species from another (Sey- raeoideae (x = 9) and the Amygdaloideae (x = 8)
mour et al. 2013). (Evans and Campbell 2002). However, as of yet,
The pear belongs to the angiosperm family there is no molecular supporting evidence for this
Rosaceae, which is a moderately large family proposed hypothesis. Furthermore, all Maleae
with three subfamilies, 16 tribes, *100 genera, genera bearing pome-like fleshy fruits form a
and *3000 species (Hummer and Janick 2009; monophyletic group and are members of the
Phipps 2014). In many angiosperm families, subtribe Malinae. Whereas, the genera of Vau-
there are generally only one or few types of quelinia, Kageneckia, and Lindleya produce dry
morphologically similar fruits. For example, dehiscent fruits and form early divergent lineages
Brassicaceae species (cabbage, radish, and their (Potter et al. 2007; Xiang et al. 2017) (Fig. 15.1).
relatives) produce silique or silicle types of As of to date, whole-genome sequences of at
dehiscent dry fruits. Also, members of Fabaceae least 11 Rosaceae species have been published,
(such as soybean and peanut), Poaceae (such as including those of Pyrus  bretschneideri,
rice and corn), and Vitaceae (grape) produce Pyrus communis, Malus  domestica, Prunus
legumes (bean pods), caryopsis (grain), and avium, Prunus mume, Prunus persica, Fragaria
berries, respectively. On the other hand, Rosa- vesca, Rosa roxburghii, Rosa multiflora, Rosa
ceae species have highly distinctive types of chinensis, and Rubus occidentalis (Velasco et al.
fruits, including fleshy pomes (with a relatively 2010; Shulaev et al. 2011; Zhang et al. 2012; Wu
soft core and multiple seeds such as pear and et al. 2013; Chagné et al. 2014; Lu et al. 2016;
apple), drupes (with a hard central shell and a VanBuren et al. 2016; Daccord et al. 2017;
single seed such as peach, cherry, and plum), dry Shirasawa et al. 2017; Verde et al. 2017; Naka-
achene (with a thin wall and a single seed, such mura et al. 2018; Raymond et al. 2018). Among
as strawberry), and aggregate fruits (such as these, the Asian pear (P.  bretschneideri) and
raspberry and blackberry). Many of the fleshy apple (M.  domestica), hereafter referred to as
fruit-bearing species have been domesticated and pear and apple, respectively, unless otherwise
produce economically important fruits (Potter noted, have been extensively investigated, along
et al. 2007). molecular and genomic evolution levels, as well
Within Rosaceae, pear and apple belong to a as in breeding and cultivation efforts.
large tribe, known as Maleae, which corresponds Genome-wide analyses have provided strong
to the subfamily Maloideae, as described in early evidence supporting the hypothesis that several
classifications using morphological characters. whole-genome duplication (WGD) events have
Maleae consists of more than 30 genera, occurred during the evolution of Rosaceae, thus
including Pyrus (pear), Malus (apple and facilitating their adaptive radiation
crabapple), Docynia, Eriolobus, Sorbus (rowan (Rousseau-Gueutin et al. 2009; Lo et al. 2010;
and mountain-ash), Cydonia (quince), Considine et al. 2012; Burgess et al. 2014;
Chaenomeles, Photinia, Rhaphiolepis, Eri- Fougere-Danezan et al. 2015).
obotrya (loquat), Crataegus (hawthorn), Mespi- Events of WGDs contribute to the recovery of
lus, Amelanchier (serviceberry), Vauquelinia, duplicates of all genes at the same time, thereby
and Kageneckia, among others, and at least 500 resulting in initial doubling of chromosome
species (Schulze-Menz 1964; Xiang et al. 2017) numbers; however, over time, they are often
(Fig. 15.1). followed by chromosomal rearrangements and
15 Whole-Genome Duplications in Pear and Apple 281

1 Malus domestica
2 Malus baccata
4 Docynia delavayi
Clade A
Eriolobus trilobatus
3 Pyrus bretschneideri
5 Pyrus betulifolia
Sorbus aria
Sorbus alnifolia
Sorbus torminalis Clade B
6 Sorbus commixta
Sorbus aucuparia
Cydonia oblonga Malinae
Pseudocydonia sinensis Maleae
7 Chaenomeles japonica
Sorbus keissleri Clade C
Photinia villosa
8 Stranvaesia amphidoxa
Rhaphiolepis indica
Clade D
Eriobotrya japonica
Crataegus cuneata
Mespilus germanica
Clade E
Amelanchier alnifolia
Malacomeles denticulata
Vauquelinia californica
Kageneckia oblonga

Fig. 15.1 Phylogenetic relationships of 25 Maleae clades, Clades A to E, and represented in five different
species. The phylogeny is based on a recently published colors. Red colored numbers at nodes indicate ancestor
phylogenetic tree of the Rosaceae family using hundreds nodes of apple and pear, and progressively with additional
of nuclear genes from over 120 species (Xiang et al. genera within Malinae
2017). The 23 Malinae species used are divided into five

loss of many duplicate copies (Pontes et al. 2004; Maere and Van de Peer 2010; Schranz et al.
Madlung et al. 2005; Albalat and Canestro 2012; Fawcett et al. 2013).
2016). Importantly, those retained duplicates In this chapter, the syntenic evidence for
provide abundant genetic materials for functional WGDs in published pear and apple genomes will
gene evolution, such as subfunctionalization, be presented (Wu et al. 2013; Daccord et al.
involving division of original functions into two 2017), and this WGD will be linked to one of
duplicates (Cusack and Wolfe 2007), neofunc- two WGDs that have likely occurred in the
tionalization, involving acquisition of a new common ancestor of pear, apple, and other
function in a duplicate copy (Blanc and Wolfe members of Malinae (Xiang et al. 2017), here-
2004), and gene conservation induced by dosage after referred to as the Malinae WGD. Interest-
effects, contributing to increased production of a ingly, comparisons of these genomic and
beneficial gene product (Freeling 2009; Bekaert phylogenomic/phylotranscriptomic studies have
et al. 2011; Hudson et al. 2011). These various allowed for analysis of the origin of the WGD, as
processes contribute to genomic novelty, organ- revealed by pear and apple genome sequences.
ismal complexity, speciation, and adaptive radi- Furthermore, this has also provided valuable
ation (Stebbins 1940; Levin 1983; Soltis et al. information on chromosomal distribution of
2003; Rieseberg and Willis 2007; Maere and Van duplicates in the apple. Subsequently, detailed
de Peer 2010; Mayrose et al. 2011; Arrigo and analyses of gene duplicates from the Mali-
Barker 2012). Consequently, such changes may nae WGD have provided new knowledge of
allow organisms to benefit from either new eco- patterns of gene retention and losses, as well as
logical opportunities or to respond new envi- of rates of such losses during the evolutionary
ronmental challenges (Ohno 1970; Hahn 2009; history of Malinae. Furthermore, comparative
282 H. Li et al.

analyses allowed for GO annotation of duplicates along with strikingly similar patterns to those
in apple and helped in unraveling the evolu- detected in the pear genome (Table 15.1).
tionary history of Malinae MADS-box genes that Wu et al. (2013) performed comparative
potentially contribute to the development of analysis of pear and apple genome sequences
pome fruits. (Velasco et al. 2010) and found that WGD events
in pear and apple, supported by extensive syn-
tenic blocks described above, must have occurred
15.2 Genome Sequences of Pear in their common ancestor (Wu et al. 2013).
and Apple Reveal Extensive Besides this WGD, they also proposed an earlier
Syntenic Evidence for WGD WGD in pear and apple, which might correspond
to a well-known paleohexaploidization event that
Previous analyses of chromosome numbers and took place about *140 million years ago (Mya),
genome sequences of Rosaceae species have although it has much less support in pear and
supported suggestions that members of this apple genomes (Wu et al. 2013). In addition, the
family must have undergone either one or more strawberry genome seems to lack large-scale
WGDs (Dickinson et al. 2007; Velasco et al. within-genome duplication (Shulaev et al. 2011).
2010; Wu et al. 2013; Chin et al. 2014; Zhao Furthermore, an analysis of syntenic blocks
et al. 2016). In particular, it has been proposed, between pear (x = 17) and strawberry (x = 7)
based on chromosome numbers, that there is a revealed that there is generally a two-to-one
WGD event that is shared by Maleae members correspondence between chromosomes of pear to
(Vamosi and Dickinson 2006; Dickinson et al. those of strawberry (Wu et al. 2013; Chagné
2007). Furthermore, analysis of the pear genome et al. 2014; Li et al. 2017). In other words, the
has revealed numerous duplicate genes (par- proposed older WGD in pear and apple, which
alogs), which are aligned in 870 collinear regions could also have been shared by strawberry, is not
of different lengths (Wu et al. 2013), and forming obvious in the comparative analysis between
large syntenic blocks covering major portions of pear and strawberry genomes, consistent with the
chromosomes (Table 15.1). Specifically,  90% relatively weak evidence for this event (Wu et al.
in lengths of each of the following four chro- 2013). Moreover, an observed ancestral chro-
mosome pairs are covered by syntenic blocks: mosome reconstruction for Rosaceae suggests
Chr03 and Chr11, Chr05 and Chr10, Chr09 and that the ancestor for this family has nine chro-
Chr17, as well as Chr13 and Chr16. Moreover, mosomes (Wu et al. 2013).
large fragments of chromosomes or chromoso- In summary, the above findings in genomes of
mal arms in seven additional pairs are syntenic, the pear and apple indicate that these two fruit
including Chr01 and Chr07 (upper region), crop species have similar and extensive dupli-
Chr02 (upper) and Chr15 (middle upper), Chr02 cated genomic regions that have likely resulted
(lower) and Chr07 (upper), Chr04 (lower) and from the same WGD event that has occurred
Chr12 (lower), Chr06 (lower) and Chr14 (lower), prior to the divergence of pear and apple.
Chr08 and Chr15 (upper and lower), and Chr12 Therefore, these resources offer opportunities for
(upper) and Chr14 (upper) (Table 15.1). Fur- unraveling the roles that duplication events of
thermore, a recent high-quality apple genome genomes and genes might have played in the
sequence has also provided strong and convinc- evolution of these two tree fruit species. How-
ing support for WGD, with syntenic blocks ever, these studies have not yet provided a more
covering most regions of all 17 chromosomes precise timing of the WGD that is shared by the
(Velasco et al. 2010; Daccord et al. 2017), and pear and the apple.
15 Whole-Genome Duplications in Pear and Apple 283

Table 15.1 Summary of synteny blocks between chromosome pairs in apple and peara

Pear Chr. A Pear Chr. B Apple Chr. A Apple Chr. B

Chr01 (upper) Chr15 (middle lower)
Chr01 Chr07 (lower)
Chr01 (lower) Chr07 (lower)
Chr02 (upper) Chr15 (middle upper) Chr02 (upper) Chr15 (middle upper)
Chr02 (lower) Chr07 (upper) Chr02 (lower) Chr07 (upper)
Chr03 Chr11 Chr03 Chr11
Chr04 (upper) NA Chr04 (upper) Chr13 (lower)
Chr04 (middle) Chr06 (middle)
Chr04 (lower) Chr12 (lower) Chr04 (lower) Chr12 (lower)
Chr05 Chr10 Chr05 Chr10
Chr06 (upper) NA Chr06 (upper) Chr16 (lower)
Chr06 (lower) Chr14 (lower) Chr06 (lower) Chr14 (lower)
Chr08 Chr15 (upper & lower) Chr08 Chr15 (upper & lower)
Chr09 Chr17 Chr09 Chr17
Chr12 (upper) Chr14 (upper) Chr12 (upper) Chr14 (upper)
Chr13 Chr16 (upper) Chr13 (upper) Chr16 (upper)
a
Synteny blocks were summarized from previous studies (Wu et al. 2013; Daccord et al. 2017). Blocks cover  90% of
chromosome length in both of chromosomes are in red. White and blue backgrounds are used only for indicating
different Chromosomes. Chr., chromosome; upper, the upper region of a chromosome; lower, the lower region of a
chromosome; middle, the middle region of a chromosome not including either end of the chromosome; middle upper,
the middle region of a chromosome adjacent to the upper region of the chromosome; middle lower, the middle region of
a chromosome adjacent to the lower region of the chromosome; upper and lower, both of the upper and lower regions of
a chromosome, but not including the middle region of the chromosome

tribes, as well as well-resolved relationships

15.3 Phylogenomic Analyses among subfamilies and tribes using hundreds of
of Multiple Species Place nuclear genes from 125 transcriptomics and
Two WGD Events Close genomic datasets (Xiang et al. 2017). A portion
to the Origin of Maleae of this newly established phylogeny is presented
herein for tribe Maleae and its large subtribe
As mentioned in the previous section, presence Malinae (Fig. 15.1). In this phylogenetic tree,
of extensive syntenic chromosomal blocks in Malinae is divided into five clades, designated
pear and apple genomes supports incidence of a herein as Clades A to E, with pear and apple
WGD, which has likely occurred prior to the belonging to Clade A (Fig. 15.1).
divergence of these two fruit tree species, but Using the new Rosaceae phylogeny as a refer-
following their split from strawberry (Wu et al. ence (Xiang et al. 2017), WGD can be detected
2013). In order to accurately place the WGD using phylogenomic analysis of thousands of gene
event in the evolutionary history of these two families obtained from many species with avail-
fruit tree species, it is necessary to establish a able transcriptome datasets (Xiang et al. 2017) (see
well-resolved phylogeny of Rosaceae and to Fig. 15.2a for the two WGDs detected in Maleae,
analyze sequences of many more members of denoted with circles 1 and 2, and see below for
Rosaceae. Recently, a well-resolved Rosaceae additional description). This phylogenomic
phylogeny has been reconstructed, with highly approach has been effectively used to detect strong
supported clades for each of the subfamilies and support for incidence of WGDs in common
284 H. Li et al.

(a) (b) (c)

Fig. 15.2 WGDs supported by multiple gene duplication possible topologies for each of duplicated gene trees are
events shared by Maleae members determined by phy- illustrated. c By mapping of duplication in gene trees with
logenomic methods. a Two WGD events on the backbone respect to species trees, numbers of gene duplication
of the Maleae phylogeny are marked by red circles, events at each node, with strong bootstrap (>50 bp), are
numbered 1 and 2, while two other possible WGD events determined. The number of counts is then divided into
shared by either pear (Pyrus) or apple (Malus) genera are three types for additional detailed information. Both
noted with blue circles. Notations written below the percentages and actual gene pair numbers, of each type at
phylogenetic tree refer to geological ages (million years) nodes marked by numbers 1 or 2, are shown. These
and correspond to geological periods estimated by results are obtained from a recent study (Xiang et al.
molecular clock analysis (Xiang et al. 2017). b Three 2017)

ancestors of angiosperms (Jiao et al. 2011), in duplications present in each gene tree in between
Asteraceae (Huang et al. 2016), and in other nodes on the species tree. When large numbers of
groups (Jiao et al. 2012; Cannon et al. 2015; Li gene duplication events are detected before a
et al. 2015; Yang et al. 2015). It is important to specific node on a species tree, it is proposed that
point out that this approach has been deemed a WGD event is responsible for incidence of such
reliable (Kellogg 2016). One of the advantages of gene duplications at nearly the same time. To
this phylogenomic approach is the ability to place assess the strength of support for such a WGD,
occurrence of WGD events relative to the species topologies of the gene tree adjacent to the node of
phylogeny and in between nodes of species duplication can be further assessed (Fig. 15.2b).
divergence. In addition, it is possible to estimate For example, presence of a node with three or
the timing of WGD events along a geological time more species in the reference species tree allows
scale, particularly when the species phylogeny for classification of observed topologies into three
corresponds to molecular clock estimates of types of gene retention in each of duplicated
divergence times. Such information about WGD subclades following the node of interest. These
events can help demonstrate and explain likely would include the following types, wherein type I
effects of WGDs on species and gene function retains both gene copies in both large and small
evolution within the context of geological ages. subclades; whereas, types II and III lack gene
The basic approach is to construct thousands duplicates for whole small or large subclades,
of gene trees using sequences from whole gen- respectively (Fig. 15.2b). Among these, type I
omes or transcriptomes, and then to compare topology provides the strongest evidence among
topologies of these gene trees with that of the the three types due to the presence of more genes
reference species tree, thereby mapping gene to infer an accurate phylogeny.
15 Whole-Genome Duplications in Pear and Apple 285

In the phylogenomic analysis of Rosaceae 15.4 Possible Effects of the Two

species, a total of 9482 gene family trees with WGD Events Near the Origin
greater than 85% taxon coverage were used to of Maleae on Evolution
detect gene duplications (Xiang et al. 2017). When of Fruit Tree Species
a node was found with >50 bootstrap support
values along with the same two species found in The two WGD events shared by Maleae/Malinae
each of its duplicated subclades, a gene duplica- might have facilitated the evolutionary process of
tion was mapped and counted to the corresponding these species and contributed to multiple mor-
position of the reference species tree. These find- phological variations of members of Maleae.
ings provided evidence for a duplication event Recent molecular phylogenetic analyses of
(Fig. 15.2a, circle numbered 1) shared by all Rosaceae have expanded the subfamily Amyg-
Maleae members with 8.12% (375 pairs) of gene daloideae to include Maleae and others, in
families showing duplication, and among these, addition to peach and plum. The ancestral fruit
7.64% (353 pairs) had strong support (type I) type of the expanded Amygdaloideae was pro-
(Fig. 15.2c). Strikingly, a stronger signal was posed to be a follicetum with several to many
detected for a WGD event (circle numbered 2) carpels (Xiang et al. 2017). This ancestral fruit
shared by members of Malinae (all Maleae mem- type further evolved into one with five carpels for
bers, except for the early divergent Vauquelinia the common ancestor of Maleae and its sister
and Kageneckia), as supported by 50.12% (3201 tribe Gillenieae. Subsequently within Maleae,
pairs) of gene families that were duplicated at this following divergence of dry-fruit producing lin-
node, with 38.86% having a type I topology. eages (i.e., Kageneckia), additional changes have
As described in the previous section, the likely led to the evolution of fleshy pome fruits.
common ancestor of pear and apple must have These likely changes in fruit structure include
experienced a WGD event following divergence partial ‘sinking’ of the ovary into the hypanthium
from strawberry (Wu et al. 2013). Based on and their fusion (Xiang et al. 2017), as well as
analysis of the apple genome, this WGD has transformation of the fruit type from one with
been dated 30–45 Mya (Velasco et al. 2010). thin and non-fleshy hypanthium/pericarp to that
With more than 120 genomic and transcriptomic with fleshy tissues. In Maleae, five carpels were
datasets, phylogenomic findings support inci- fused together as a coccetum (such as that found
dence of two WGD events that must have in Vauquelinia), while the hypanthium became
occurred successively near the origin of Maleae, urceolate (cup-like) and further closed-up with
around 38–42 Mya and 48–55 Mya, respectively carpels, evolving into either partially inferior,
(Fig. 15.2a). In addition, evidence for incidence such as that of Crataegus, or fully inferior
of polyploids has also been previously reported ovaries, such as those of pear (Pyrus) and apple
to occur within Maleae for members of the (Malus).
genera Sorbus, Crataegus, and Amelanchier Molecular clock analysis, using nuclear gene
(Vamosi and Dickinson 2006; Dickinson et al. sequences with the newly established phylogeny
2007). All these members are included in the as a reference (Xiang et al. 2017), supports the
subtribe Malinae and are represented by the clade proposal that the timing of fruit character transi-
marked with number 2 (Fig. 15.2a). Moreover, it tions is correlated with those of WGDs and cli-
is proposed that a recent WGD may have mate events. Molecular clock estimates indicate
occurred within Pyrus, with 15.39% (585 pairs) that the tribe Maleae has split from Gille-
gene families duplicated before speciation of nieae *54 Mya, just after the Paleocene–
P.  bretschneideri and P. betulifolia (Xiang Eocene boundary, with further incidents of
et al. 2017); however, further analysis using divergence within Maleae beginning soon after-
genomic datasets is needed to confirm occur- ward. The earlier WGD (Fig. 15.2a, circle num-
rence of this event. bered 1) shared by all Maleae members is
286 H. Li et al.

estimated to have occurred in early Eocene, at the node for Malinae (Xiang et al. 2017). For
which has been the hottest period since the this analysis, a gene family is defined as a group
Cenozoic Era, including both the Paleocene– of homologous genes derived from the same
Eocene Thermal Maximum (PETM) and the ancestral gene after divergence of Malinae from
Early Eocene Climate Optimum (EECO) (Zachos its sister lineage, Vauquelinia. These gene fami-
et al. 2008). lies have a duplication detected just before the
Within Maleae, after the separation of Kage- node of Malinae, thus supporting incidence of a
neckia (with a follicetum fruit type), the ancestor Malinae WGD (Fig. 15.2a, circle numbered 2).
of Vauquelinia and other genera have likely To detect chromosome distribution of the
produced the coccetum fruit type, with a short lag Malinae-WGD-derived gene duplicates in apple,
period from the early WGD event. Whereas, the we have analyzed 1043 gene families, each with
second WGD is shared by the fleshy-fruited an apple gene in each of two duplicated clades. It
genera of Maleae (all in Malinae) (Fig. 15.2a, is revealed that longest synteny chromosomal
circle numbered 2), and it is estimated to have blocks, described above in Sect. 15.2, also con-
occurred in late Eocene when the Earth experi- tain the most duplicated gene pairs derived from
enced a continuous drop in temperature and the Malinae WGD (Fig. 15.3). For example, 120
humidity. This has been closely followed by a gene families contain syntenic gene pairs on
short glaciation period with many extinctions in Chr05 and Chr10, 102 on Chr09 and Chr17, 90
Europe (Zachos et al. 2001; Hooker et al. 2004). on Chr03 and Chr11, and 90 on Chr13 and
The extremely high percentage (50.12%) of gene Chr16. Generally, the more the synteny blocks
pairs retained after the WGD and the rapid taxon cover a chromosome, the more duplicated gene
separation/diversification after the WGD strongly pairs are detected in these blocks, as illustrated
suggest that duplicate genes have contributed to by the above-mentioned four chromosome pairs.
diversification of Maleae genera. Therefore, it is In contrast, chromosome pairs with lower cov-
likely that the new gene copies from the two erage by synteny blocks also contain fewer pairs
Maleae WGDs have allowed Maleae members to of duplicates from the Malinae WGD. Most
evolve into species producing new fruit types duplicated genes, a total of 812 pairs, are located
under selective forces of both dramatic climate within synteny blocks between two different
changes and interactions with animals/insects chromosomes. However, some duplicated gene
feeding on Maleae fleshy fruits. See below for pairs are located within the same chromosome,
additional discussion of genes affected by e.g., Chr05 and Chr05. This latter finding could
WGDs. be attributed to either genome rearrangement or
some other events that may have occurred fol-
lowing the Malinae WGD event. This deserves
15.5 Chromosome Distribution further analysis to achieve a better understanding
of Malinae-WGD-Derived of this observed phenomenon.
Duplicated Genes in Apple Furthermore, analysis of a recent apple gen-
ome sequence (Daccord et al. 2017) has detected
Phylogenomic analysis of thousands of gene gene numbers and duplicated gene pairs in syn-
trees with sequences from pear, apple, and mul- teny blocks between different chromosome pairs
tiple other members of Maleae has provided of apple, as presented in Table 15.2. These
strong support for presence of a WGD event results reveal that, despite chromosome rear-
shared by members of Malinae (Xiang et al. rangements and additional gene duplications,
2017) (Fig. 15.2a). However, is this WGD the WGD-derived duplicated gene pairs identified in
same as the one revealed by extensive syntenic each synteny block account for about 30–50% of
blocks in pear and apple genomes? To address all genes within the same synteny block
this question, chromosomal distribution of 2985 (Table 15.2). For example, Chr03 has 2529
gene families has been evaluated for duplication genes and Chr11 has 2728 genes, while 1180
15 Whole-Genome Duplications in Pear and Apple 287

Table 15.2 Gene numbers and duplicated gene pair numbers in synteny blocks in applea

Apple Chr. A Gene # in Chr. A Apple Chr. B Gene # in Chr. B Gene pair #
Chr01 (upper) 325 Chr15 (middle lower) 301 128
Chr01 (lower) 1478 Chr07 (lower) 1509 781
Chr02 (upper) 1710 Chr15 (middle upper) 1421 849
Chr02 (lower) 1060 Chr07 (upper) 1084 383
Chr03 2529 Chr11 2728 1180
Chr04 (upper) 401 Chr13 (lower) 88 35
Chr04 (middle) 292 Chr06 (middle) 254 94
Chr04 (lower) 1320 Chr12 (lower) 1363 701
Chr05 3166 Chr10 2961 1461
Chr06 (upper) 177 Chr16 (lower) 156 63
Chr06 (lower) 1433 Chr14 (lower) 1314 773
Chr08 2162 Chr15 (upper & lower) 2074 1078
Chr09 2515 Chr17 2444 1116
Chr12 (upper) 933 Chr14 (upper) 867 394
Chr13 (upper) 2308 Chr16 (upper) 2285 1322
a
Synteny blocks were summarized from a previous study (Daccord et al. 2017). The second column (Gene # in Chr. A)
indicates the total gene number in the synteny block shown in the first column. The fourth column (Gene # in Chr. B)
indicates the total gene number in the synteny block shown in the third column. The fifth column (Gene pair #) indicates
the duplicated gene pair number in the synteny block shown in the row. Synteny blocks and the corresponding
duplicated gene pair numbers were identified by MCScanX (Wang et al. 2012). Other notes are same as Table 15.1

gene pairs support synteny between these two syntenic regions (Fig. 15.3a) suggests that syn-
chromosomes, they account for 46.7% of genes tenic regions are the result of the Malinae WGD.
present on Chr03 and for 43.3% of those on To test this hypothesis, the average Ks value
Chr11. Chromosome pairs with higher synteny (ratio of observed synonymous changes to pos-
block coverage have most of the duplicated gene sible synonymous changes used as a measure of
pairs, such as Chr05 and Chr10 (1461), Chr13 evolutionary age) between paralogs is estimated.
and Chr16 (1322), Chr03 and Chr11 (1180), and For 90 pairs of apple genes on Chr03 and Chr11,
Chr09 and Chr17 (1116). The number of gene detected by phylogenomic analysis and due to
pairs detected between syntenic blocks in the the Malinae WGD, the Ks value is estimated to
apple genome is much larger than the 2985 gene be 0.28, which is very close to the Ks value of
families with duplication from the Mali- 0.24 for all apple paralogs (1180 pairs) found
nae WGD, as revealed by phylogenomic analysis between Chr03 and Chr11. This supports the
of multiple species in the Maleae tribe. The hypothesis that these two types of paralogs are
reason for this observed difference is likely due probably generated by the same WGD event.
to an incomplete transcriptome sequencing used As mentioned above, among 2985 gene fam-
to obtain gene sequences for most of the species ilies with two duplicated Malinae clades, 1043
included in the analysis, and criteria used for at gene families have two duplicates in the apple
least 85% species coverage of gene families, as genome, but the remaining 1942 gene families,
well as other requirements limiting the number of with an ancestral Malinae duplication, must have
genes used in this analysis. Nevertheless, the undergone loss of at least one duplicate in apple.
wide distribution of most duplicates in apple, Furthermore, 947 gene families have retained
from the Malinae WGD, detected in apple one duplicate in the apple genome. Thus, their
288 H. Li et al.

(a)
140
120
100
GF counts

80
60
40
20
0

(b)
16
14
12
10
GF counts

8
6
4
2
0
05—05
00—06
00—15
08—08
12—15
02—02
00—03
00—05
00—10
01—01
06—06
00—09
03—03
07—07
10—10
14—14
02—14
05—12
09—14
17—17
00—07
00—11
00—12
00—17
04—07
10—14
00—00
00—02
00—08
00—14
02—08
02—11
03—16
04—09
04—14
08—13
09—09
10—11
10—12
10—15
13—13
Chromosome pairs

Fig. 15.3 Counts of gene families of synteny analyses in the previous study. Red color,
Malinae-ancestor-derived duplicated apple gene pairs in synteny blocks cover  90% of chromosome length in
corresponding chromosome pairs. Data of chromosome both chromosomes (see Table 15.1); yellow color, syn-
synteny blocks are derived from a recently published teny blocks cover <90%, but  30% of chromosome
apple genome (Daccord et al. 2017). A total of 58 length in both chromosomes; blue color, within one
chromosome pairs are shown. a 17 chromosome pairs that chromosome or synteny blocks cover <30% of chromo-
are supported by synteny analyses in the previous study, some length in both chromosomes. GF, gene family
and b 41 chromosome pairs that are not supported by

chromosome distribution is investigated. If the Therefore, loss of duplicates derived from the
gene loss rate is proportional to chromosome Malinae WGD must have been uneven among
size, longer chromosomes with more genes different chromosomes of the apple genome.
should have more of these 947 single-copy
genes. Based on the physical map of the apple,
Chr15 is the longest among all 17 chromosomes 15.6 Retention of Duplicates
(Daccord et al. 2017), and it is found to carry and Their Loss Rates During
most of the single-copy genes (Fig. 15.4). the Evolution of Pear
However, other relatively long chromosomes, and Apple
such as Chr05 and Chr13, are found to have
similar numbers of single-copy genes, when The pear and apple are closely related, belonging
compared to those found on shorter chromo- to the same small clade, when compared with
somes, such as Chr10 and Chr07 (Fig. 15.4). other genera in Malinae (Fig. 15.1) (Xiang et al.
15 Whole-Genome Duplications in Pear and Apple 289

Fig. 15.4 Chromosome 180

distribution of retained genes
160
in 947 gene families with one
duplicate retained in apple. 140
Every gene in each
120
orthogroup is accounted for.

Gene counts
Gene location data are derived 100
from a recently published
80
apple genome (Daccord et al.
2017) 60

40

20

Chromosome

2017). Therefore, distribution of duplicates on Following analysis of gene families having 2, 1,

chromosomes of pear, which has undergone the or 0 duplicate(s) in pear and/or apple, it is
same Malinae WGD as that of apple, is probably determined that 2106 gene families (70.6% of the
similar to that found in apple. As almost total) have at least one detected duplicate in pear
two-thirds (1942) of the total (2985) detected and/or apple, while only 215 gene families have
gene duplicates in Malinae have lost at least one no detected duplicate in both pear and apple
copy in apple, it would be of interest to deter- (Fig. 15.5). This suggests that the vast majority
mine the evolutionary timeline of these losses in of such genes have important functions in pear
pear and apple. Using sequence datasets gener- and/or apple. Furthermore, as about two-thirds of
ated for many genera in Malinae (Xiang et al. all 2985 families have likely experienced gene
2017), we have investigated the retention/loss loss during the evolution of pear and apple,
number and loss rate of duplicates during dif- 70.3% in pear and 65.1% in apple, a single gene
ferent periods of evolution. Those duplicates copy might be sufficient for undertaking their
found in apple and pear, as well as those detected functions in pear and apple. It is likely that, as
at eight ancestral nodes, Nodes 1 to 8 (Fig. 15.1), domesticated species, pear and apple might have
wherein Node 8 represents the most recent experienced relaxed selection pressure under
common ancestor of Malinae, are presented in human cultivation and might have lost some of
Tables 15.3 and 15.4. For any specific node, and those genes retained in wild relatives in other
if any descendant lineage contains a duplicate, Malinae genera.
then it is assumed that the node would also have We have further analyzed the rate of duplicate
this gene. On the other hand, if none of descen- gene loss over time during the period of evolu-
dant lineages of a node has a specific duplicate, tion from the Malinae ancestor (Fig. 15.1; Node
then it is assumed that the node lacks a copy. 8) to extant pear and apple. Those gene families
These findings have revealed that losses are with losses have been divided into two types. In
distributed into eight successive periods along one type, ‘one-duplicate loss’ refers to events
the backbone, from the Malinae ancestor to the wherein a duplicate number has changed
extant pear and apple (Tables 15.3 and 15.4). between two adjacent nodes from either 2 to 1 or
Among 2985 gene families with two dupli- from 1 to 0; and a second type, ‘two-duplicate
cates in the Malinae ancestor (Node 8 in loss’ refers to events wherein a duplicate number
Fig. 15.1), 886 gene families have two detected has changed from 2 to 0 between two adjacent
duplicates in pear and 1043 in apple, accounting nodes (Fig. 15.6a). The average loss rates of
for 29.7 and 34.9% of the total, respectively. ‘one-duplicate loss’ and ‘two-duplicate loss’
290 H. Li et al.

Table 15.3 Summary of gene families with duplicates lost in any of the 6 nodes during evolution of peara

Rest # of duplicates Node 8 Node 7 Node 6 Node 5 Node 4 Node 3 Pear GFs #
No duplicate lost 2 2 2 2 2 2 2 886
2 2 2 2 2 2 1 490
Lost after Node 3
2 2 2 2 2 2 0 53
2 2 2 2 2 1 1 603
Lost after Node 4 2 2 2 2 2 1 0 161
2 2 2 2 2 0 0 95
2 2 2 2 1 1 1 309
2 2 2 2 1 1 0 67
Lost after Node 5
2 2 2 2 1 0 0 70
2 2 2 2 0 0 0 27
2 2 2 1 1 1 1 89
2 2 2 1 1 1 0 26
Lost after Node 6 2 2 2 1 1 0 0 15
2 2 2 1 0 0 0 10
2 2 2 0 0 0 0 10
2 2 1 1 1 1 1 26
2 2 1 1 1 1 0 3
2 2 1 1 1 0 0 5
Lost after Node 7
2 2 1 1 0 0 0 1
2 2 1 0 0 0 0 0
2 2 0 0 0 0 0 0
2 1 1 1 1 1 1 21
2 1 1 1 1 1 0 10
2 1 1 1 1 0 0 1
Lost after Node 8 2 1 1 1 0 0 0 5
2 1 1 0 0 0 0 1
2 1 0 0 0 0 0 1
2 0 0 0 0 0 0 0
Total: 2985
a
Data here are derived from the previous WGD results (Xiang et al. 2017). Different background colors are used only
for indicating different loss time. Blue, gene families retained 2 duplicates in the ancestor at this node; green, gene
families retained 1 duplicate in the ancestor at this node; red, gene families retained 0 duplicate in the ancestor at this
node. GFs, gene families

events are 114.5 and 9.3 per million years, Previous analyses have also revealed that
respectively. Furthermore, both ‘one-duplicate highly redundant gene pairs must have undergone
loss’ and ‘two-duplicate loss’ events have higher either relatively less negative selection or neutral
average rates in the period between Nodes 5 and selection, and are usually lost more rapidly than
4 than those in other periods (Fig. 15.6b; orange more divergent gene pairs during the early period
bars). The timing of these duplicate loss events following duplication (Ohno 1970; Lynch and
between Nodes 5 and 4 is estimated to be about Force 2000; Conant and Wolfe 2008; Li et al.
30–31 Mya, corresponding to early Oligocene 2016). Taken together, these findings suggest that
(Xiang et al. 2017). Geological studies have many duplicated gene pairs might have experi-
indicated that during this time period, global enced limited diversification following duplica-
temperature and humidity have become steady tion, thereby retaining two partially redundant
following the dramatic drop in late Eocene, as copies in the Malinae ancestor of pear and apple
mentioned in a previous section. In addition, this (Fig. 15.1; Node 8). Subsequently, many such
period coincides with the expansion of angios- duplicates must have been lost quickly between
perms (Zachos et al. 2001; Hooker et al. 2004), Nodes 5 and 4, but less rapidly during other
which is consistent with evolution of highly periods, thereby facilitating likely adaptation of
diverse Malinae genera. different genera to various new environments.
15 Whole-Genome Duplications in Pear and Apple 291

Table 15.4 Summary of gene families with duplicates lost in any of the 7 nodes during evolution of applea

Rest # of duplicates Node 8 Node 7 Node 6 Node 5 Node 4 Node 2 Node 1 Apple GFs* #
No duplicate lost 2 2 2 2 2 2 2 2 1043
2 2 2 2 2 2 2 1 357
Lost after Node 1
2 2 2 2 2 2 2 0 53
2 2 2 2 2 2 1 1 377
Lost after Node 2 2 2 2 2 2 2 1 0 93
2 2 2 2 2 2 0 0 45
2 2 2 2 2 1 1 1 213
2 2 2 2 2 1 1 0 63
Lost after Node 4
2 2 2 2 2 1 0 0 35
2 2 2 2 2 0 0 0 9
2 2 2 2 1 1 1 1 324
2 2 2 2 1 1 1 0 61
Lost after Node 5 2 2 2 2 1 1 0 0 39
2 2 2 2 1 0 0 0 22
2 2 2 2 0 0 0 0 27
2 2 2 1 1 1 1 1 96
2 2 2 1 1 1 1 0 18
2 2 2 1 1 1 0 0 10
Lost after Node 6
2 2 2 1 1 0 0 0 6
2 2 2 1 0 0 0 0 10
2 2 2 0 0 0 0 0 10
2 2 1 1 1 1 1 1 22
2 2 1 1 1 1 1 0 5
2 2 1 1 1 1 0 0 6
Lost after Node 7 2 2 1 1 1 0 0 0 1
2 2 1 1 0 0 0 0 1
2 2 1 0 0 0 0 0 0
2 2 0 0 0 0 0 0 0
2 1 1 1 1 1 1 1 20
2 1 1 1 1 1 1 0 5
2 1 1 1 1 1 0 0 6
2 1 1 1 1 0 0 0 1
Lost after Node 8
2 1 1 1 0 0 0 0 5
2 1 1 0 0 0 0 0 1
2 1 0 0 0 0 0 0 1
2 0 0 0 0 0 0 0 0
Total: 2985
a
Data here are derived from the previous WGD results (Xiang et al. 2017). Different background colors are used only
for indicating different loss time. Blue, gene families retained 2 duplicates in the ancestor at this node; green, gene
families retained 1 duplicate in the ancestor at this node; red, gene families retained 0 duplicate in the ancestor at this
node. GFs, gene families

families, 1294 have annotation information in

15.7 Gene Ontology Annotations agriGO v2 (Tian et al. 2017). Interestingly,
of Duplicated Genes among ‘molecular function’ categories, five sig-
and Evolutionary History nificant GO terms have been detected. These are
of MADS-Box Genes Related related to catalytic activity (GO:0003824) and
to Fruit Development in Pear binding (GO:0005488), particularly phosphatase
and Apple activity (GO:0016791), transcription factor
activity, and RNA binding (GO:0008135)
To gain a better understanding of functions of (Fig. 15.7). Furthermore, among ‘biological
duplicated genes from the Malinae WGD, we process’ categories, 23 GO significant terms have
have further assessed Gene Ontology (GO) an- been detected, and most are related to cellular
notations of 2452 gene families retaining at least process (GO:0009987) and metabolic process
one duplicate in apple, with one representative (GO:0008152) (Fig. 15.8). However, no signifi-
apple gene from each family. Among these gene cant term has been detected within the ‘cellular
292 H. Li et al.

component’ category. These findings suggest that

Duplicates in pear 2 2 Duplicates in apple gene families supporting the Malinae WGD
1 574 1 include many genes homologous to those known
383 241 to be involved in various transcription regulatory
0 0
86 908 71 networks, cellular processes, as well as metabolic
processes, and potentially play important roles in
260 247
the adaptation of Malinae species. Duplicates
215 from the Malinae WGD could have diverged
Orthogroup #
sufficiently to either have different expression
patterns or even gain new functions. In turn, this
Fig. 15.5 Counts of gene families with either 2, 1, or 0 has allowed different lineages represented by
duplicate(s) present in genomes of pear and apple. pear, apple, and other genera to adapt to new
Numbers written outside of the purple box represent
retained duplicate(s) in either pear or apple. Whereas, environments during their evolution.
numbers written within the purple box represent number Anatomical structures of pome fruits of pear,
of gene families with corresponding duplicates present in apple, and of other Malinae genera are derived
pear and apple, as indicated by numbers written outside of from fusion of the ovary with a floral tube (hy-
the box. The colors used to indicate either 2, 1, or 0
number of duplicates are the same as those used in panthium) consisting of lower portions of sepals,
Tables 15.3 and 15.4 petals, and stamens (Pratt 1988). In several plant

(a) (b)
GF counts per million years
GF counts

Fig. 15.6 Counts of gene families with either one or two Node 5 (33–31 Mya). Orange color, gene loss events
apple/pear duplicate(s) lost between two adjacent ances- occuring between Nodes 5 and 4 (31–30 Mya). Green
tral nodes (a) and rates of their loss per million years (b). color, gene loss events occurring between Node 4 and
A ‘one-duplicate loss’ refers to an event wherein duplicate apple, including orthogroups with duplicate loss during
numbers have changed either from 2 to 1 or from 1 to 0 transition from either Node 4 to Node 2 (30–22 Mya),
between two adjacent nodes. A ‘two-duplicate loss’ refers Node 2 to Node 1 (22–12 Mya), or Node 1 to apple
to an event wherein a duplicate number changed from 2 to (12 Mya). Yellow color, gene loss events occurring
0 between two adjacent nodes. Blue color, gene loss between Node 4 and pear, including orthogroups with
events that must have occurred between Nodes 8 and 5, as duplicate loss during transition from Node 4 to Node 3
shown in Fig. 15.1, including orthogroups with duplicate (30–14 Mya) and from Node 3 to pear (14 Mya). Ages of
loss during transition from either Node 8 to Node 7 (36– each node are derived from an estimation presented in our
34 Mya), Node 7 to Node 6 (34–33 Mya), or Node 6 to previous study (Xiang et al. 2017). GF, gene family
15 Whole-Genome Duplications in Pear and Apple 293

(a) 2 25 (b)
Percentage of genes
18
17

1 12 12
208

131
111
67
66

GO annotation

Fig. 15.7 GO analysis of 1294 apple genes from 2452 genes in all apple genes. Numbers written above each bar
gene families among ‘molecular function’ categories. GO represent gene number in either 1294 input apple genes or
annotation results and the hieratical graph are derived in all 26,714 apple genes in agriGO v2. b A hieratical
from agriGO v2 (Tian et al. 2017). a A GO abundance graph of GO annotations. Yellow-green colored boxes
chart of all GO significant terms. Blue color, percentage indicate five GO significant terms shown in (a)
of genes in 1294 apple genes; orange color, percentage of

species, including Arabidopsis thaliana and To determine whether or not the Malinae WGD
Antirrhinum majus, among others, flower and has influenced the copy number of these
fruit development are controlled by members of MADS-box genes during evolution of apple and
the MADS-box gene family. Specifically, AGA- pear, we have reconstructed phylogenetic rela-
MOUS (AG), SHATTERPROOF1/2 (SHP1/2), tionships of FUL, AP1, AG, SHP, and SVP
and FRUITFULL (FUL) [related to APETALA1 genes. Sequences of these genes have been
(AP1)] genes are important for ovary and fruit obtained from 28 Rosaceae species, including 25
development in several plant species (Seymour members of Maleae (Fig. 15.1), Prunus mume,
et al. 2013). Molecular studies have revealed that Prunus persica, and Fragaria vesca, as well as
these genes are also important for fruit develop- four other eudicots, used as outgroups, including
ment in apple. First, apple genes closely related Glycine max, Medicago truncatula, Arabidopsis
to AG and FUL are differentially expressed dur- thaliana, and Brassica rapa. Phylogenetic anal-
ing development of the pome fruit (Yao et al. yses of these genes have indicated that duplicates
1999). Moreover, genes related to SHORT of FUL, AP1, AG, SHP, and SVP genes, due to
VAGETATIVE PHASE (SVP), contributing to the Malinae WGD, are often retained in apple
enlarged sepals when overexpressed, are and/or pear, as well as in other Malinae species
expressed in the apple fruit (Masiero et al. 2004). (Fig. 15.9). This has suggested that the
 491
294

460

418
8452
398
8010

7408

6496

218

158 3322
142 142
133 2382

Percentage of genes
2126 109 2151 106
1646
1316 1543
57 67
57
712 40 37 43 812 714
15 13 13 13 450 395 15 13 437
106 81 81 81 94 81

GO annotation

Fig. 15.8 GO abundance chart of all significant terms of 1294 apple genes among ‘biological process’ categories. GO annotation results and a hieratical graph are derived
from agriGO v2 (Tian et al. 2017). Blue color, percentage of genes in 1294 apple genes; orange color, percentage of genes in all apple genes. Numbers above each of the bars
H. Li et al.

refer to gene numbers in either 1294 input apple genes or in all 26,714 apple genes in agriGO v2
15 Whole-Genome Duplications in Pear and Apple 295

Fig. 15.9 Evolutionary

(a) Species tree (b) FUL
history of FUL, AP1, AG, MdoFUL1
SHP, and SVP MADS-box Malus domestica (Mdo) SarFUL1a
Pyrus bretschneideri (Pbr)
genes in Maleae. The PviFUL1
Sorbus aria (Sar)
phylogeny is based on a RinFUL1
Photinia villosa (Pvi)
recent study (Xiang et al. MdeFUL1a
Stranvaesia amphidoxa (Sam)
2017). a Species tree of 13 MdoFUL2
WGD Rhaphiolepis indica (Rin) WGD
Rosaceae species, including PbrFUL
Eriobotrya japonica (Eja)
11 Maleae species. b–f Gene SarFUL2a
Crataegus cuneate (Ccu)
trees of FUL, AP1, AG, SHP, PviFUL2
Malacomeles denticulate (Mde)
and SVP, respectively. Red Vauquelinia californica (Vca) RinFUL2a
MdeFUL2
circle, Malinae WGD Kageneckia oblonga (Kob)
Prunus mume (Pmu) VcaFUL1
Prunus persica (Ppe) VcaFUL2
KobFUL

(c) AP1 (d) AG

MdoAP1-1
MdoAG1
SarAP1-1 PbrAG1
PviAP1-1 SarAG1
CcuAP1 PviAG1
WGD MdoAG2
WGD RinAP1 PbrAG2
MdoAP1-2 SarAG2a
PbrAP1 PviAG2
CcuAG2a
SarAP1-2
PmuAG
PmuAP1 PpeAG
PpeAP1

(e) SHP (f) SVP

MdoSVP1
MdoSHP1 PbrSVP1
SarSHP1a SarSVP1
SamSVP1
PviSHP1 EjaSVP1
CcuSHP1 CcuSVP1
WGD
WGD MdoSVP2
MdoSHP2 PbrSVP2
PbrSHP SarSVP2
SamSVP2a
SarSHP2 EjaSVP2
VcaSHP MdeSVP2a
VcaSVP
PpeSHP KobSVP

Malinae WGD must have contributed to expan- multiple Maleae species and others, while using a
sion of these genes, and that these MADS-box well-resolved phylogeny of Rosaceae as a refer-
gene duplicates may have potentially contributed ence, have provided valuable information on
to the evolution of pome fruits in Malinae. WGD and gene duplication in these species. Both
Overall, recently published genome sequences extensive syntenic chromosome blocks in pear
of pear and apple in Maleae, along with phy- and apple along with thousands of gene duplicates
logenomics analyses of thousands of genes from support existence of a WGD event that must have
296 H. Li et al.

occurred in the ancestor of Malinae. Subse- Chagné D, Crowhurst RN, Pindo M, Thrimawithana A,
quently, about 30% of duplicated gene pairs from Deng C, Ireland H, Fiers M, Dzierzon H, Cestaro A,
Fontana P, Bianco L, Lu A, Storey R, Knabel M,
this WGD are retained, and these can be mostly Saeed M, Montanari S, Kim YK, Nicolini D, Larger S,
detected in synteny blocks in both pear and apple Stefani E, Allan AC, Bowen J, Harvey I, Johnston J,
genomes. These duplicated genes are involved in Malnoy M, Troggio M, Perchepied L, Sawyer G,
transcription regulatory networks and in other Wiedow C, Won K, Viola R, Hellens RP, Brewer L,
Bus VGM, Schaffer RJ, Gardiner SE, Velasco R
cellular or metabolic processes. In addition to (2014) The draft genome sequence of European pear
these retained duplicates, about two-thirds of (Pyrus communis L. ‘Bartlett’). PLoS ONE 9: e92644
duplicates resulting from the WGD event that Chin S-W, Shaw J, Haberle R, Wen J, Potter D (2014)
must have occurred in the Malinae ancestor have Diversification of almonds, peaches, plums and cher-
ries—molecular systematics and biogeographic his-
been lost during subsequent evolution, with the tory of Prunus (Rosaceae). Mol Phylogenet Evol
highest rate of loss occurring about 30–31 Mya. 76:34–48
These losses may correspond to differential Conant GC, Wolfe KH (2008) Turning a hobby into a job:
adaptation of various genera to new environ- how duplicated genes find new functions. Nat Rev
Genet 9:938–950
ments, including the divergence of apple and pear. Considine MJ, Wan YZ, D’Antuono MF, Zhou Q,
Han MY, Gao H, Wang M (2012) Molecular genetic
Acknowledgements We thank Ji Qi for his constructive features of polyploidization and aneuploidization
comments and technical supports on WGD analysis, and reveal unique patterns for genome duplication in
Qichao Lian and Duoyuan Chen for their technical diploid Malus. PLoS ONE 7:57–65
assistance. This work was supported by a grant received Cusack BP, Wolfe KH (2007) When gene marriages don’t
from the National Natural Science Foundation of China work out: divorce by subfunctionalization. Trends
(31670209), funds provided by the State Key Laboratory Genet 23:270–272
of Genetic Engineering at Fudan University, as well as Daccord N, Celton JM, Linsmith G, Becker C, Choisne N,
support provided by the Biology Department and the Schijlen E, van de Geest H, Bianco L, Micheletti D,
Huck Institutes of the Life Sciences at the Pennsylvania Velasco R, Di Pierro EA, Gouzy J, Rees DJG,
State University. Guerif P, Muranty H, Durel CE, Laurens F,
Lespinasse Y, Gaillard S, Aubourg S, Quesneville H,
Weigel D, van de Weg E, Troggio M, Bucher E
(2017) High-quality de novo assembly of the apple
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 Future Breeding Strategies
16
Kamila Łucja Bokszczanin

Abstract will provide holistic approaches for discovery

Pear breeding is considered as one of the most of gene function, elucidate mechanisms of
important sectors of temperate fruit breeding. gene function, support genotyping, and accel-
While this follows breeding efforts for apple, erate the breeding cycle. Furthermore, nan-
new technologies and approaches are awaiting otechnologies utilized in gene transfer,
pear breeders on the horizon. New plant phenotyping, detection of pathogens, and
breeding techniques, tested for their efficacy sequencing will also contribute to faster, more
in other fruit trees, as well as conventional precise and specific high-quality monitoring,
methods will be presented in this chapter. and consequently breeding of cultivars resis-
Moreover, the potential combination of these tant to biotic and abiotic stresses.
approaches toward development of ‘smart’
pear cultivars will be also described. Further-
more, as there is an observed trend of elevated
consciousness of the health benefits of organ- 16.1 Directions and Strategies
ically grown crops among consumers world- for Pursuing Pear Breeding
wide, the issue of organic pear breeding
strategies pear will also be discussed. Based In order to fulfill consumer needs and render
on the principles of organic plant breeding, any cultivars successful in markets, it is crucial to set
breeding technique is evaluated against four specific directions and conceptualize strategies
mandatory criteria, and must meet genome- for breeding. This involves deep knowledge of
and cell-level integrity, capability for propa- the global pear fruit industry, including those of
gation, as well as preservation against crossing extended networks of fruit growers, breeders, and
barriers. Thus, the use of molecular markers as pear marketers, as well as an understanding of
diagnostic tools is not excluded in organic fruit industry preferences, in particular for char-
breeding. For future pear breeding strategies, acteristics of new cultivars. Most pear breeding
the merger of different ‘omics’ technologies programs worldwide have focused on efforts to
combine superior pear fruit quality, high pro-
ductivity, precocious fruit bearing, long
postharvest storage life, along with multiple
K. Ł. Bokszczanin (&) disease resistance (e.g., resistance to pear scab
Department of Pomology, Faculty of Horticulture,
Biotechnology and Landscape Architecture, Warsaw
and black spot diseases) and pest resistance, as
University of Life Sciences, Nowoursynowska 159 well as of self-compatibility. Developing pear
str., Warsaw, Poland cultivars with early ripening or attractive fruit
e-mail: [email protected]

© Springer Nature Switzerland AG 2019 301

S. S. Korban (ed.), The Pear Genome, Compendium of Plant Genomes,
https://doi.org/10.1007/978-3-030-11048-2_16
302 K. Ł. Bokszczanin

Fig. 16.1 Directions for pear breeding

appearance is also important. Moreover, avoiding superior lines during early stages of development.
inbreeding depression is essential for future These innovation-driven newest developments,
breeding, and therefore expanding the pool of among others in plant biotechnology, will allow
genetic resources used in pear breeding programs for pursuing advanced ‘smart’ breeding efforts for
is critical. pear. However, there are some limitations for
Overall, the following traits of interest for pear genetic engineering (GE) in certain regions of the
breeding have emerged in recent years. These world, especially in the European Union whereby
traits include blushed-fruit cultivars, solid colored cultivation of genetically modified organisms
fruit cultivars, in particular green-colored fruit (GMOs) or GMO crops is restricted solely to
cultivars, as well as a distinctive flavor profile of MON810 maize. Nevertheless, some of these new
fruits, crunchiness and sweetness of fruits, along genetic technologies, including GE, are receiving
with resistance to various diseases and pests, acceptance in other parts of the world, such as in
particularly for fire blight, pear psylla, fruit skin North America. More recently, the powerful
browning, along with early fruit ripening, as well CRISPR technology, referring to ‘clusters of
as cold-hardy dwarfing rootstocks (Fig. 16.1). regularly interspaced short palindromic repeats
These breeding goals could be achieved using (CRISPR)/cas9 associated protein,’ for gene
various spectra of methods, individually or in editing will also have a significant impact on
combination, including advanced biotechnology genetic enhancement efforts in various crops,
techniques, hybridization, and mutagenesis. including pears (Malnoy et al. 2016).
These breeding strategies will greatly benefit from However, it is also important to take into
the use of ‘omics’ technologies and nanotech- consideration the growing importance and inter-
nology for phenotyping, as well as for selection of est in organic cultivation of fruit trees, including

Pear Breeding Technologies

Genome-wide association studies (GWAS) Marker assisted selection (MAS) Nanotechnology

Linkage mapping (LM) X-omics : genomics, transcriptomics, epigenomics, metabolomics, bioinformatics

New Plant Breeding Techniques (NPBT) Gene transfer Mutagenesis Protoplast fusion

Fig. 16.2 Available technologies for future pear breeding strategies. Technologies allowed in organic breeding are in
green boxes
16 Future Breeding Strategies 303

Organic Pear Breeding Technologies

Genome-wide association studies (GWAS) Marker assisted selection (MAS) Nanotechnology

Linkage mapping (LM) X-omics : genomics, transcriptomics, epigenomics, metabolomics, bioinformatics

Fig. 16.3 Technologies for future pear organic breeding strategies

pear, and related organic pear breeding strategies. serious vulnerability to diseases, pests, and cli-
Although GE has opened up new pathways for matic changes. It is quite common that wild rel-
genetic improvement, worldwide standards for atives have not been largely exploited as sources
organic agriculture (OA) do not allow for GE or of desirable traits, including disease resistance,
any products derived from GE. Instead, alterna- fruit quality, and rootstock characteristics. It is
tive breeding approaches are pursued based on important to expand the genetic base, and to have
norms and standards for OA, not only at the access to large and wide collections of diverse
technical level, but also at social and organiza- germplasm to avoid such vulnerabilities.
tional levels, by including other value-chain There have been successful examples of using
players and consumers (Nuijten et al. 2016). MAS in tree fruit breeding programs (Migicov-
All available pear breeding technologies are sky et al. 2016). In order to establish
presented in Fig. 16.2, while those specific for genotype-phenotype relationships and advance
organic breeding are presented in Fig. 16.3. MAS in apple, over 24,000 phenotype scores
were extracted from the USDA-Germplasm
Resources Information Network (GRIN) data-
16.2 Conventional Pear Breeding base, and these were linked to over 8000 single
nucleotide polymorphisms (SNPs) from 689
16.2.1 Marker-Assisted Selection apple accessions obtained from the USDA apple
(MAS) germplasm clonal collections maintained at
Geneva, NY (Migicovsky et al. 2016).
Although marker-assisted selection (MAS) has
been conceptualized three decades ago ( Smith
and Simpson 1986), it remains an important tool 16.2.2 Genome-Wide Association
for fruit breeding. As the fields of molecular Studies (GWAS)
genetics and genomics have advanced, they have and Genomic Selection
become valuable tools for improving breeding (GS)
efficiency by allowing for early screening and
selection of progenies and/or seedlings possess- High-throughput genotyping technologies, such
ing traits of interest at the seed or seedling stage. as DNA chips (Gupta et al. 2008), and genotyping
The benefits of MAS for a plant breeder are using next generation sequencing (NGS) (Davey
greatest when the targeted species has a long et al. 2011) have enabled new genomic-based
generation cycle, and it is expensive to grow and strategies, such as genome-wide association
maintain. Thus, MAS holds particular promise studies (GWAS). This is an approach for detecting
for fruit trees, such as pears, as they have gen- target genes or quantitative trait loci (QTL) based
eration cycles of 5–7 years to reach maturity, and on associations between genome-wide markers
they are costly to establish and grow in the field. and phenotypes caused by linkage disequilibrium
Often, well-characterized perennial tree (LD) between molecular markers and either causal
germplasm beneficial to breeders is limited in its genes or QTLs. The GWAS approach is an alter-
genetic diversity. A narrow genetic base in fruit native to bi-parental QTL mapping in long-lived
breeding programs can certainly contribute to perennials. It does not require establishment of
304 K. Ł. Bokszczanin

segregating populations, which is time- Japanese pear cultivars have been genotyped for
consuming and costly. Moreover, the high map- 162 DNA markers resulting in significant asso-
ping resolution offered by GWAS is amplified in ciations for harvest time, black spot resistance,
many perennials due to a relatively rapid LD and numbers of spurs (Iwata et al. 2013).
decay in highly diverse perennial crops. The cor- It is noteworthy to point out that inclusion of a
relation between a molecular marker and a causal large number of unrelated individuals in GWAS
variant is related to the level of LD between these anticipates that a large number of recombination
two, wherein the higher the LD, the more likely events must have occurred in the history of the
the marker will serve as an indicator for presence target genetic material under study. Whereas, in
of the causal variant. While rapid LD decay results linkage mapping (LM), it is only those recom-
in high mapping resolution, it also means that a bination events captured through the develop-
very high density of markers is required for ment of a bi-parental cross that are exploited,
effective GWAS, as correlations among markers thus resulting in recovery of a relatively large
surrounding the causal variant decay very quickly. proportion of DNA of shared co-ancestry among
In some cases, generating sufficient coverage for individuals. One of the main advantages of
GWAS by saturating the genome with molecular GWAS over traditional LM is its superior map-
markers may be prohibitively expensive due to ping resolution. Markers detected in GWAS are
rapid LD decay. The costs of marker discovery deemed to be closely linked to causal genes and
and genotyping are likely to continue to decrease, major QTL controlling important agronomic
thus rendering GWAS more affordable in the traits. In some cases, the likely causal genetic
future. variant itself can be identified through GWAS
The rapid decay of LD observed in apple (Migicovsky et al. 2016). However, in LM, large
suggests that millions of SNPs may be required genomic intervals, often spanning millions of
for pursuing a well-powered GWAS (Migicov- nucleotides, are identified, thus rendering it dif-
sky et al. 2016). However, rapid LD decay also ficult or unlikely to identify the causal genetic
promises to enable extremely high-resolution variant.
mapping of causal variants, which holds great In instances wherein a trait of interest
promise for advancing MAS. A GWAS of 36 co-segregates well with, for example, a wild
apple phenotypes has confirmed presence of an relative species, yet it is completely absent in the
association between fruit color and an MYB1 cultivated germplasm, then a different breeding
locus, as well as between the transcription factor approach than that of GWAS is needed. Specif-
NAC18.1 and harvest date and fruit firmness ically, when phenotypes are well-segregated for a
(Migicovsky et al. 2016). As a result, harvest trait of interest, GWAS is of no use. Instead, a
time and fruit size have been predicted with bi-parental cross between wild and cultivated
relatively high accuracies (r > 0.46) using individuals must be made to genetically map the
genomic prediction (Migicovsky et al. 2016). In trait of interest. LM in the resulting bi-parental
turn, a high LD has been attributed to genetic population allows for such co-segregating traits
bottlenecks during domestication and breeding of to be genetically mapped. However, it has been
Japanese pear (Iwata et al. 2013). A genetic observed that in fruit crops, such as apple, pear,
bottleneck increases the extent of LD by elimi- and grape, wild and domesticated germplasm
nating recombinant lineages (Iwata et al. 2013). share segregating polymorphisms, and these are
Even when loci remain polymorphic during not readily or easily differentiated. In such
bottlenecks, the numbers of allelic combinations instances, confounding effects of co-ancestry
across loci can be greatly reduced, thereby may not be strong enough, and GWAS may be
leading to extensive haplotype structure (Ham- the genetic mapping approach of choice. Addi-
blin et al. 2011). In a pear GWAS program, 76 tionally, when a trait of interest does not
16 Future Breeding Strategies 305

co-segregate well with its ancestry, but rather it is 2013). In fact, GS can avoid issues of uncertainty
differentially expressed in two populations, it in QTL identification and effect estimation,
may be possible to perform GWAS using wild which can be problematic in MAS, by simulta-
and domesticated plant materials. neously estimating effects of all marker loci. This
Although a simple distinction between simultaneous estimation of genomic effects pro-
GWAS and LM is useful, unfortunately, experi- vides further benefits as effects that are too small
mental designs blur this distinction, and they to be declared ‘statistically significant’ can be
tend to exploit the benefits of both approaches, captured by markers. Due to these features, GS is
thus uncovering numerous genotype-phenotype proposed as efficient, even for low-heritability
associations. For example, a Multi-parent polygenic traits (Lorenz et al. 2011); whereas,
Advanced Generation InterCross (MAGIC) MAS is deemed unsuitable for improvement of
population is generated by intercrossing multiple such traits (Iwata et al. 2013). In Japanese pear,
parental lines rather than a single bi-parental Pyrus pyrifolia, genome-wide predictions for GS
cross. In another strategy to increase recombi- have been determined to be accurate at high
nation frequency in a progeny for enhanced probability levels (p = 0.75) for harvest time, at
mapping is to utilize inbred offsprings (Cavanagh medium probability levels (p = 0.38–0.61) for
et al. 2008). However, development of inbred resistance to black spot (incited by Alternaria
lines in perennial fruit trees is rather not feasible, gaisen Nagano), firmness of flesh, fruit shape in
thereby necessitating implementation of other longitudinal section, fruit size, fruit acid content,
mating designs. For example, a factorial mating and numbers of spurs, and at low levels (p < 0.2)
design consisting of four female parents and two for all soluble solids content and for tree vigor
pollen parents has been used in an apple study (Iwata et al. 2013).
(Kumar et al. 2012). This family-based design It has been proposed that both GWAS and
has allowed for identification of molecular GS will be useful in accelerating genetic
markers linked to several fruit quality traits, improvement of Japanese pear (Iwata et al.
including fruit firmness, internal browning, and 2013). In fact, significant associations have been
titratable acidity, that are useful in MAS (Kumar detected for harvest time, black spot resistance,
et al. 2013). Therefore, alternative mating and numbers of spurs (Iwata et al. 2013).
designs serve as promising tools for enhancing However, accumulating large data sets sufficient
mapping resolution when performing LM for conducting such analyses is rather difficult
between wild and domesticated crops. for fruit trees due to their long juvenility periods,
In another alternative strategy for MAS, large plant sizes, and at times difficulties in
selection of either elite or desirable lines is based phenotyping. Therefore, collecting and main-
on genomic predictions of breeding values, and taining a genetically diverse pear collection,
this is referred to as genomic selection (GS). GS including wild relatives, are a valuable resource
allows for selection of superior genotypes based for developing new and enhanced pear cultivars.
on genomic estimated breeding values (GEBV), For instance, several Asian pear species are
as it derives information based on genome-wide known to serve as candidates for fire blight
markers. Thus, GS is more effective than MAS, resistance (incited by Erwinia amylovora [Bur-
particularly for traits controlled by large numbers rill] Winslow et al.), carrying both polygenic and
of genes. Furthermore, GS is similar to GWAS as presumably monogenic resistance, depending on
it utilizes LD between markers on one hand and the genotype (Bokszczanin et al. 2012). More-
causal genes and QTL on the other. However, over, transgressive segregation for fire blight
unlike GWAS, GS is designed to detect genes resistance has been observed within progenies of
and QTL and aims to predict the genetic poten- crosses among fire blight susceptible, moder-
tial; e.g., breeding values, of breeding lines ately susceptible, and resistant pear parents
without locating genes and QTL (Iwata et al. (Bokszczanin et al. 2012).
306 K. Ł. Bokszczanin

16.2.3 Mutagenesis One of the main problems of mutagenesis is

the induction of chimeral mutants. The risk of
Based on EU definition of genetically modified incidence of such chimeral mutants can be
organisms (GMOs), mutagenesis is not regarded reduced by irradiating in vitro-grown buds
as a process that results in the development of (Decourtye 1982; Broertjes 1982; Lacey and
GMOs. Thus, mutagenesis is deemed as an Campbell 1982). Predieri and Zimmerman
alternative strategy for introducing genetic vari- (2001) have irradiated in vitro-grown shoots of
ability in cultivars or in parental germplasm used six European pear cultivars using gamma rays
in cross-hybridizations. In fact, mutagenesis has (3.5 Gy). Subsequently, mutant trees have been
been successfully implemented in pear breeding selected for improved characters related to
programs, either for directly enhancing cultivars reproductive growth, such as early bearing and
for specific traits or for yielding valuable mutants consistent annual productivity. Furthermore,
that can be used either in cross-hybridizations or variations in overall fruit characters, such as
for pursuing biotechnology studies for genetic amounts of russeting, fruit shape, and fruit size,
enhancement (Fujimaki 1996; van Harten 1998). have also been observed in these mutants (Pre-
Most often, irradiation treatments have been dieri and Zimmerman 2001).
used to induce mutations in fruit trees. Among
traits affected by mutagenesis, plant size, ripen-
ing time, fruit color, and self-fertility have been 16.3 ‘Smart’ Breeding
reported (Spiegel-Roy 1990). Moreover, irradia-
tion was used to obtain dwarfing rootstocks of Several new plant breeding techniques (NPBTs),
apple (Przybyla 1988). Several forms of muta- representing significant advances toward crop
tions have been induced in European pear improvement, are currently being implemented
(P. communis), including variations in bloom in breeding programs. Although NPBTs make
time, blossom color, ripening time, fruit color, use of genetic modification technology, the
and compact growth habit (Predieri and Zim- resulting end-products do not contain any foreign
merman 2001). As an alternate strategy, mutation genes. Consequently, NPBT products are genet-
breeding for Japanese pear was initiated by the ically similar to or may be even indistinguishable
Institute of Radiation Breeding using gamma from conventionally bred plants. These strategies
irradiation. Since the 1980s, several induced include cisgenesis and intragenesis, as well as
mutants with some levels of resistance to black gene editing techniques. Products from NPBTs
spot disease have been selected from ‘Nijisseiki’, may be grouped into three classes as follows:
‘Osanijisseiki’, ‘Shinsui’, and ‘Kisui’ using (1) plants that carry a new DNA fragment, often
chronic or acute gamma irradiation (Masuda a new gene and/or regulatory element; (2) plants
et al. 1997). Among these selected mutants, four that do not carry a new DNA fragment, but carry
cultivars were named and released, including a mutation or a native DNA modification; and
‘Gold Nijisseiki’ (Kotobuki et al. 1992), ‘Osa (3) plants that do not carry a new DNA fragment
Gold’ (Masuda et al. 1998), ‘Kotobuki Shinsui’ or any native DNA modification.
(Kitagawa et al. 1999), and ‘Shizukisui’ (Sawano
et al. 2011). ‘Gold Nijisseiki’ demonstrated
levels of resistance to black spot that were 16.3.1 Techniques to Shorten
intermediate between those known for ‘Chojuro’ the Juvenility Period
and ‘Nijisseiki.’ Moreover, this resistance was
found to be inherited by offsprings, as well as Induced early flowering has been applied to fruit
detection of incomplete recessive mutations that trees to accelerate breeding efforts. Fruit species,
were induced in L-II histogenic cell layers such as pear and apple, have a long generation
(Sanada et al. 1994). cycle (5–7 years). Thus, fruit breeding is a
16 Future Breeding Strategies 307

long-term endeavor, particularly when novel be used to shorten the juvenility period in fruit
traits from related wild species are introgressed trees, among other plants. VIGS involves the use
into a domesticated cultivar, as multiple breeding of a viral vector to infect a plant with a particular
cycles are required to remove genetically linked gene, resulting in an RNA-mediated defense
undesirable traits, derived from wild species. response to silence expression of a target gene
A member of the APETALA1/FRUITFULL within a plant (Lu et al. 2003). It has been
group of MADS-box genes, isolated and cloned reported that the apple latent spherical virus
from silver birch (Betula pendula), designated as (ALSV) does not induce disease symptoms in an
BpMADS4, has been found to drastically reduce infected plant, and can be used as a vector for
the juvenility period when introduced into apple, VIGS (Igarashi et al. 2009). When ALSV has
thereby promoting flower induction in seedlings been used to express an Arabidopsis thaliana
within the first year of growth (Flachowsky et al. florigen while also silencing expression of a
2011). An early flowering transgenic apple line Malus  domestica TERMINAL FLOWER
expressing the BpMADS4 gene has been devel- (TFL) gene, MdTFL1-1, in apple or a P. com-
oped, thereby affording future efforts opportuni- munis TFL gene, PcTFL1-1, in pear, flowering of
ties to exploit this technology in combination these regenerated fruit trees can be reduced down
with MAS to pyramid disease resistance genes to 3 months or less. In a test orchard, it has been
for apple scab, powdery mildew, and fire blight reported that neither transmission via an insect
(Flachowsky et al. 2011). Schlathölter et al. vector nor horizontal transmission via pollen has
(2018) have already been successful in obtaining been detected (Nakamura et al. 2011). In another
null apple segregants carrying both heterozygous study, Kishigami et al. (2014) have reported that
resistance to fire blight (caused by E. amylovora) approximately 99% of seedlings from
and homozygous resistance to the Rvi6 gene for ALSV-infected trees can be deemed virus-free.
scab (incited by Venturia inaequalis). They have Finally, ALSV can be eliminated from an
also used a rapid crop cycle breeding approach, infected tree by using high temperature, allowing
based on overexpression of the birch MADS4 for vegetative propagation of such a tree, thus
transcription factor, in apple (Schlathölter et al. resulting in fruit deemed exempt from restric-
2018). While transgenic lines expressing this tions on GMOs (Yamagishi et al. 2016). There-
BpMADS4 gene are helpful in drastically reduc- fore, VIGS is a promising method for reducing
ing the generation time in fruit breeding efforts, it the juvenile phase period of fruit trees, such as
is often desirable to develop a cultivar that does pear, allowing for a shorter generation time, and
not contain a transgene, so as it is not deemed a facilitating backcrossing, when deemed neces-
GMO crop. Such a desired outcome can be sary, for breeding elite selections with wild rel-
facilitated by using a transgene that is dominant atives (Migicovsky and Myles 2017).
and heterozygous, thus yielding only 50% off-
spring carrying the desired gene in each genera-
tion. Therefore, once the rapid cycling of 16.3.2 Grafting of Scion Cultivars
generations is completed, a non-GMO tree pos- onto a Genetically
sessing desirable traits from wild relatives, but Modified
not the transgene, can then be easily selected (GM) Rootstock
(Flachowsky et al. 2011). Nevertheless, ‘Arctic’
apple, a genetically engineered apple for There are several available approaches wherein
non-browning of fruit flesh, has been approved GM rootstocks can be useful for improving
for commercial production in the USA, and it is performance of non-genetically modified
currently being grown in Midwest orchards. (GM) scion cultivars. Using genetic modification
An alternative to developing transgenic fruit technologies, characteristics of a rootstock, such
trees expressing such a MADS-box gene from as rooting ability, adaptation to heavy soils, or
birch, virus-induced gene silencing (VIGS) can resistance to soil-borne diseases and pests, can be
308 K. Ł. Bokszczanin

improved. This would, in turn, enhance perfor- overcome hybridization barriers. For example, in
mance of a non-GM scion cultivar. instances of either wide crosses or interspecific
In another application of GM technology, hybridizations, wherein distantly related parents
rootstocks can be used as target materials for belonging to different species or even genera,
gene silencing through RNA interference (RNAi) post-zygotic barriers, such as endosperm abor-
(Kalantidis 2004). Small interfering RNAs (siR- tion, can be overcome by using embryo rescue.
NAs) are natural silencing signals in plants; thus, In fact, rescue of hybrid embryos from intra- and
siRNAs can be generated in transgenic plants inter-specific crosses, commonly used in apple
using RNAi-expression vectors. The efficacy of breeding programs, is aimed at increasing seed
RNAi to confer virus resistance in wild-type germination efficiency, as well as recovery of
sweet cherry (Prunus avium) has been demon- higher numbers of individuals obtained via sex-
strated in scions grafted onto a GM rootstock ual hybridization.
(Zhao and Song 2014). For this, a Prunus Genes used in cisgenesis strategies are intro-
necrotic ringspot virus (PNRSV)-resistant trans- duced either as extra copies of the desired gene
genic cherry rootstock has been developed by or as natural dominant variants of the desired
introducing an RNAi vector expressing siRNAs gene with improved characteristics to confer
against the PNRSV coat protein (Song et al. resistance or enhanced resistance to a particular
2013). Subsequently, a non-GM sweet cherry disease or some other desired trait. For example,
scion cultivar has been grafted onto this trans- cisgenic apple lines have been developed with
genic rootstock. The transfer of PNRSV- enhanced resistance to fire blight disease using
targeting siRNA signal molecules from the the cisgene FB_MR5 from the wild apple M. 
rootstock to the non-transgenic scion has been robusta 5, and introducing it into the fire blight
confirmed, and enhanced PNRSV resistance of susceptible cultivar Gala Galaxy (Kost et al.
grafted sweet cherry scions has been observed. 2015). By the way, fire blight disease is one of
These findings have demonstrated, for the first the most serious diseases of pear, and therefore,
time, transfer of transgene-derived siRNAs from such a strategy should be explored to introduce
a GM rootstock to a non-GM scion in grafted fire blight resistance into susceptible pear
trees, and that these transferred siRNAs could cultivars.
enhance virus resistance of these grafted scions
(Schaart et al. 2016).
Therefore, this approach could be explored in 16.3.4 Intragenesis
pear to develop GM pear rootstocks with resis-
tance to fire blight, among other diseases, as well Intragenesis is similar to cisgenesis, as all ele-
as with adaptation to cold temperatures for ments introduced via genetic modification are
enhanced cold hardiness, or for dwarfing. Then, derived either from within the species of interest
these rootstocks could be used for grafting of or from a cross-compatible species. However,
non-GM scion cultivars. intragenesis differs from cisgenesis by allowing
use of new gene combinations generated by
in vitro rearrangements of functional genetic
16.3.3 Cisgenesis elements. These new combinations of functional
elements, such as regulatory elements or trans-
Cisgenesis refers to the development of plants posable elements, will offer new opportunities
via genetic modification strategies using only for genetic enhancement. For example, such
those genes derived from either the species itself opportunities may deal with temporal and spatial
or from a species that can intercross with this activation of a desirable gene of interest in a
species using conventional methods. It is target tissue, or organ, of a plant.
important to note that conventional methods may Efforts to either enhance or regulate gene
include such technologies as embryo rescue to expression by introducing a stronger promoter
16 Future Breeding Strategies 309

will drive gene expression to enhance expression technology is faster, cheaper, more accurate, and
of a trait of interest, such as plant disease resis- more efficient than other existing genome-editing
tance or fruit color pigmentation, among others. methods. Mutations resulting from ODM can be
It is important to point out that intragenesis also obtained using traditional mutagenesis;
cannot be achieved through conventional breed- however, the advantage of ODM over traditional
ing, as new combinations are unlikely to arise in mutagenesis is that it does not produce thousands
such a breeding scheme (Holme et al. 2013). of other mutations (Limera et al. 2017).
Therefore, pear breeding efforts can be certainly The use of such gene editing technologies in
advanced further via the use of intragenesis for pear is currently ongoing, and will have a sig-
genetic enhancement of disease resistance or of nificant impact in pursuing genetic improvement
fruit quality traits. efforts to address various important traits that
will enhance pear genotypes for such traits.

16.3.5 Gene Editing

16.3.6 RNA-Dependent DNA
Gene editing, also known as genome editing, Methylation (RdDM)
involves a group of technologies that allow for
targeted DNA insertion, deletion, or alteration of An RNA-dependent DNA methylation (RdDM)
a particular gene or segment of a genome. Sev- approach involves design of recombinant genes
eral approaches for genome editing have been that produce RNA molecules matching either the
developed, using a sequence-specific nuclease target gene or its promoter region, and their sub-
technology (SSN). These nucleases are synthetic sequent introduction into plant cells. Such RNA
proteins that bind to a specific DNA target molecules are recognized by the RNA-induced
sequence and induce a break in the DNA (a silencing complex (RISC), thereby resulting in
‘lesion’). Such a DNA break is subsequently methylation of the corresponding DNA, which in
repaired by the plant’s native DNA repair turn blocks expression of the target gene.
machinery. There are three types of SSN appli- This approach has been recently used in
cations, including SSN-1 which results in gene devising strategies to accelerate apple breeding.
knockout, SSN-2 which results in a targeted It is reported that significant changes in 24
mutation, and SSN-3 which results in gene nucleotide (nt) sRNAs, that are the hallmarks of
replacement. Interestingly, accurate native DNA the RdDM pathway, are suggestive of a corre-
machinery leads to either a single base substitu- lation between epigenetic modifications and
tion (SSN-2) or introduction of a new DNA floral transition (Guo et al. 2017). Therefore,
fragment (SSN-3); whereas, non-accurate repair differentially expressed miRNAs and siRNAs
machinery results in a deletion (SSN-1). between vegetative and floral buds have been
Gene editing techniques include zinc finger identified following small RNA (sRNA)
nucleases (ZFN), transcription activator-like sequencing data analysis. Bioinformatics analy-
effector nucleases (TALEN), clustered regularly sis of these sRNAs has shed new light of our
interspaced short palindromic repeats/CRISPR- understanding of floral transition in woody plants
associated protein 9 (CRISPR/Cas9), and (Guo et al. (2017). This is quite helpful in pur-
oligonucleotide-directed mutagenesis (ODM), suing similar studies in pear.
also known as the rapid trait development system Elucidation of the mechanism regulating floral
(RTDS). Recently, it has been demonstrated that transition is critical for both pear and apple
CRISPR/Cas9 can be used in apple to modify the breeding, as well as for their cultivation (Ban-
genome (Nishitani et al. 2016). The gerth 2009). Furthermore, as for other crops, pear
CRISPR-Cas9 system has generated a lot of improvement and breeding strategies can benefit
excitement in the scientific community, as this from the use of epi-marks of promoter regions of
310 K. Ł. Bokszczanin

a gene(s) for ‘fine-tuning’ gene expression in exclude cultivars and derived organic products
pear cultivars (Gallusci et al. 2016). developed via new generations of GE and gene
editing techniques (Nuijten et al. 2016). In Eur-
ope, a position paper of the International Feder-
16.4 Organic Breeding ation of Organic Agriculture Movements
(IFOAM) EU GROUP (Nuijten et al. 2016) has
Nowadays, the importance of organic farming urged that cultivars derived from NPBT that
has gained more attention. Worldwide standards engineer living organisms in cells and/or nuclei
for OA do not allow GE or any products derived through technical, chemical, or biotechnological
from GE. As organic certification is based on the intervention should be designated as GE. Thus,
farming process rather than on end-products as these cultivars are subject to risk assessment, and
such, this may also impact breeding as an activity if authorized for release, these are subject to
within the agricultural industry, as the breeding mandatory traceability and labeling requirements
activity will be evaluated for compliance with that apply to other GE techniques.
organic rules and values (Van Bueren et al. 2003, Based on the principles for organic plant
2010; Nuijten et al. 2016). A notable difference breeding, as described by the European Consor-
between EU and US regulations is that in EU tium for Organic Plant Breeding (ECO-PB)
legislation of GE, both the process and the pro- (2012), and in the IFOAM Norms for organic
duct of GE are taken into consideration, while in production and processing in 2014 (IFOAM
the USA, it is only the final product that is 2014), any breeding technique is evaluated
evaluated (Araki and Ishii 2015). However, in against four mandatory criteria that must be met.
the USA, the National Organic Standards Board These criteria include the following:
has decided to update organic standards to (i) genome-level integrity, (ii) cell-level integrity,

Table 16.1 Criteria for evaluation of breeding technologies along with principles for organic plant breeding according
to the European Consortium for Organic Plant Breeding (ECO-PB) and the International Federation for Organic
Agriculture Movements (IFOAM) Norms of 2014
Breeding Genome-level Cell-level Ability for Preservation Breeder’s Farmers rights on
technology integrity integrity propagation of crossing privilege is farmer-sown
barriers affected seeds are affected
Chemical No No Yes Yes No No
mutagenesis,
irradiation
Cisgenetics No No Yes Yes Yes (patent) Yes (patent)
Cytoplast fusion Yes No Case-specific No Possibly Possibly
Marker-assisted Yes Yes Yes Yes No No (patent?)
selection
Minichromosomes No No Yes No Yes (patent) Yes (patent)
Oligo directed No No Yes Yes No No
mutagenesis
Reverse breeding No No Yes Yes No No
RNA Interference No No Yes Yes Yes (patent) Yes (patent)
(RNAi)
Transgenetics No No Possibly No Yes (patent) Yes (patent)
Zinkfinger No No Yes Possibly Yes (patent) Yes (patent)
Nuclease III
Zinkfinger No No Yes Yes Yes (patent) Yes (patent)
Nucleases I and II
16 Future Breeding Strategies 311

(iii) ability for propagation, and (iv) preservation Nanopore-based DNA sequencing protocols
of crossing barriers. Farm-saved seed is pre- allow for single molecule electrical detection of a
ferred, but it is not an exclusive criterion. DNA sequence, and have potentials for low
Table 16.1 summarizes such techniques and sample preparation work, high-speed, and
assesses their validity for use in organic pear low-cost (Branton et al. 2008). These advances
breeding. offer dramatic forward steps in improving this
inexpensive and potentially more rapid alterna-
tive to NGS technologies (Khiyami et al. 2014).
16.5 Advanced NGS Methods Recently, the development of the newest
and Nanodiagnostics OxfordTM nanopore technology has provided
to Accelerate Pear Breeding novel improvements in molecular sensing, such
as real-time data streaming, improved simplicity,
16.5.1 NGS-Based Methods efficiency and scalability of workflows, as well as
direct analysis of the molecule of interest. These
Current advances in genomics, including DNA platforms, along with new bioinformatic tools,
sequencing, are the most important tools in plant have provided complete annotated sequences.
breeding and biotechnology. For the first time,
important genes for a trait can be accurately
identified and at low cost in almost any organ- 16.5.2 Nanotechnology
ism. Rapid developments in NGS technologies
over the last decade have opened up many new It is common knowledge that conventional or
opportunities for discovery of relationships traditional plant breeding methods are
between genotypes and phenotypes. time-consuming. Nanodiagnostic tools, including
Third generation systems (TGS) will quickly microfluidics, nanofluidics, nanomaterials, and
become more common in plant research, as bioanalytical nanosensors, among others, offer
additional breeding materials are sequenced. The opportunities for advancing and enhancing plant
transition of high-throughput-sequencing data breeding programs. These tools can potentially
into useful information for breeders is one of the overcome problems in dealing with issues, such
main goals, and it has been documented in many as biotic and abiotic resistance, production, and
successful collaborations. Currently, TGS are prevention protocols, and are likely to be used in
being introduced to streamline sequencing pro- field-based assays for transgene expression
tocols. Several platforms such as Helicos Helis- assays, among others (Stewart 2005). Nanodi-
cope® (Thompson and Steinmann 2010), agnostic methods, among other nanotechnology
Complete Genomics® (Drmanac et al. 2010), tools, enable higher precision breeding as they
Nanopore® (Greninger et al. 2015), and Pacific offer new opportunities for selecting and trans-
Biosciences SMRT® (Eid et al. 2009) have ferring genes, while reducing the time required to
incorporated new modifications. First, poly- remove redundant genes, and also allowing a
merase chain reaction (PCR) is no longer breeder to access useful genes from distant plants
required before sequencing, and secondly, the (Abd-Elsalam and Alghuthaymi 2015). It has
signal is captured in real time. This indicates that been demonstrated that a honeycomb meso-
the signal is either a fluorescent signal (Pacbio) porous silica nanoparticle (MSN) system with
or an electric current (Nanopore), and it is 3-nm pores can transport DNA and chemicals
monitored during the enzymatic reaction of into isolated plant cells and intact leaves (Torney
adding nucleotides to the complementary strand. et al. 2007). Nanofluidics, such as the Open
Additionally, all of these platforms process mil- Array or the Fluidigm Dynamic Array tech-
lions of sequence reads in parallel with very long nologies supply automated PCR mixes for
reads, and in some cases, up to 10 kb in length mega-molecular breeding assays. Moreover,
(English et al. 2012). nanotechnology can specifically target specific
312 K. Ł. Bokszczanin

plant pathology problems in agriculture, such as technologies include RNA-Seq, massive analysis
in plant-pathogen interactions, and provide new of cDNA ends (MACE), miRNA-Seq
strategies for crop disease control (Khiyami et al. (smallRNA-Seq). Whereas, examples of geno-
2014). mics technologies include exome sequencing,
Nanoparticles and quantum dots (QDs) have whole genome sequencing, de novo-sequencing,
emerged as essential tools for fast detection of and target enrichment. Examples of epigenomics
particular biological markers with high accura- technologies include methyl-Seq and bisulfite-
cies. Biosensors, QDs, nanostructured platforms, seq.
nanoimaging, and nanopore DNA sequencing Undoubtedly, there will be new technologies
tools offer opportunities for improving sensitiv- that will become available in the near future as
ity, specificity, and speed of pathogen detection, well. Deep sequencing of transcriptomes is also a
facilitating high-throughput analysis, and powerful tool for analysis of precise levels of
high-quality monitoring, and crop protection. expression for each gene in a sample. It consists
This is of particular benefit for all crops, but in in quantifying short cDNA reads, obtained by
particular for long-lived tree fruit crops, such as NGS technologies, in order to compare whole
pears. Furthermore, nanodiagnostic kits can transcriptomes among genotypes grown under
easily and readily detect potential serious plant different environmental conditions. Whereas,
pathogens, thus allowing experts to help farmers miRNAs are non-coding short RNAs involved in
in averting disease epidemics. In addition, using the regulation of different physiological pro-
nanotools or nanoparticles for gene transfer in cesses, which can be identified by
plant cells may lead to advances in developing high-throughput sequencing of RNA libraries
new disease-resistant pear cultivars, as this will obtained by reverse transcription of purified short
minimize expenses for use of agrochemicals RNAs, and by in silico comparisons with known
required for plant disease control, and in allevi- miRNAs of other plant species.
ating environmental concerns (Taylor et al. 2005; Altogether, NGS techniques and their appli-
Sekhon 2014). cations have increased the resources available for
plant breeding efforts of pear trees, among other
tree species, thereby closing earlier gaps of
16.5.3 Omics Technologies genetic tools available for perennial trees in
comparison with annual plant species. The use-
Nowadays, X-omics approaches accelerate the fulness of X-omics platforms in Solonaceae has
breeding process, as they complement research been demonstrated by one such example of elu-
efforts of targeted studies, yielding knowledge of, cidating the pollen thermotolerance mechanism
thus far, unrecognized genes, proteins, and (Bokszczanin et al. 2013). Thus, X-omics will
metabolites. The collection of such new knowl- have similar impacts in efforts to expand
edge will provide significant support for knowledge of critical traits of pear and corre-
improvement of breeding programs and facilitate sponding genetic improvement efforts of this
the development of new better cultivars. Within important economic tree fruit crop.
this context, there is a special role for ‘-omics’
technologies in dissecting genetic mechanisms
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