INTERNATIONAL CONFERENCE

ON

CURR TTRENDS IN BIOTECHNOLOGY

&
LICATIONS IN AGRICULTURE
.~f ' February 19-21, 2009" '~i

Organized by

Sardar Vallabhbhai Patel University of Agriculture & Technology

Meer~t-25011 0 (India)
i':'1,:',~':",;,
a

}'Society for Recent Development in Agriculture

Meerut-250110 (India)
 Enhancing the value of legume genetic resources using core/mini core and
applied genomic tools
HD Upadhyaya\ SL Dwivedi\ RK Varshneyl, CLL Gowda\ R Bhattacharjee2 ,
DA Hoisington\ and V Vadez 1

1International Crops Research Institute Jor the Semi-Arid Tropics (ICRISAT),

Patancheru PO 502324, India
2 International Institute oj Tropical Agriculture (!ITA), PMB532 0, Iharlan, 0)'0 Statf, Nigaia

ABSTRACT

Grain legumes are rich resource of dietary proteins, minerals, and vitamins; however,
productivity remains low, narrow genetic base of the cultivars being one of the several reasons to low
productivity.

Worldwide approximately half a million legume germplasm acceSSIOns are preserved in

genebanks. However, there has been limited use of these resources in crop breeding. Core collection
(10% of the entire collection), a subset of accessions representing at least 70% of the genetic variation
in the entire collection of the species, has been suggested as a gateway to enhance utilization of
germpJasm. Core and mini core (10% of core) collections have been reported in several legumes that
when evaluated identified new sources of variation for agronomic traits including resistance to biotic
and abiotic stresses.

Legumes genomics resources in the past lagged behind cereals. However, situation dramatically
changed with emergence of Medicago truncatula, Lotus japonicus, and Glycine max as models for
comparative genomics within legume family. Several genomic resources including markers, maps,
transcriptomics, proteomics, metabolomics, and bioinformatics resources have been developed. These
resources in model plants will not only allow investigation of basic processes important to legumes, but
also open the possibility to transfer those processes to- or locate them in other crop species including
several legumes. Several genomic projects are developing tools for less-studied legumes which are
economically important in Africa and Asia.

These genetic and genomic resources represent major milestones in the history of legumes
research, which will help understand the evolutionary events that shaped legume genomes, and provide
avenues for genetic enhancement for sustainable agriculture.

I. Introduction:

Legumes represent the second largest family of higher plants after cereals. Leguminasae
consists of about 20,000 species across 700 genera, traditionally divided into three subfamilies:
Caesalpinoideae, Mimosoideae and Papilionoideae (Doyle and Luckow 2003). The two major groups of
cultivated species in the Papilionoideae are the tropical or 'phaseoloid' legumes (Phaseolus, Vigna,
Glycine, and Cajanus) and the temperate or 'galegoid' legumes (Melilo/us, Trifolium, Me dicago ,
Pisum, Vicia, Lotus, Cicer, Lens, and Lathyrus). Groundnut is somewhat distinct from the phaseoloid

18 International conference on Current trends in Biotechnology, February 19-21,2009

and galegoid groups of grain legumes (Figure 1). Grain legumes are a rich source of protein, lysine,
and essential vitamins and minerals. It also contains beneficial secondary compounds with significant
health-promoting properties.
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Figure 1. Phylogenetic rewtionship between the model legumes and major temperate and tropical legumes
(Crouch and Dwivedi 2005)

The use of plant genetic resources in crop improvement is one of the most sustainable ways to
conserve valuable genetic resources for the future, and simultaneously to increase agricultural
production and food security. Key to successful crop improvement is a continued supply of genetic
diversity in the breeding programs, including new or improved variability for target traits. Collectively,
the CGIAR centers possesses about 600,000 samples, of which, grain legumes represents about 30%,
second only after cereals (65%). The Patancheru-based ICRISAT genebank in India contains 49,344
germplasm accessions of its three legume crops (chickpea, groundnut and pigeonpea), of which, 48,004
belong to cultivated and 1,340 wild species accessions.

The genetic base of legumes is narrow. In addition to bottlenecks associated with the origin and
do~estication events of these crops there has been limited use of germ plasm in breeding programs in
spite of the relatively large collections maintained in the genebanks, and because of the repeated use of
the few germplasmladvanced lines for the development of new cultivars (reviewed in Upadhyaya et al.
2009). The reasons for the underutilization of germplasm include i) lack of accurate and precise large

International conference on Current trends in Biotechnology, February 19-21,2009 19

scale multi-location evaluation of germplasm, (ii) the lack of rational systematic entry points into the
vast international collections, and the lack of robust cost-effective tools to facilitate the efficient
utilization of exotic germplasm in plant breeding programs (Dwivedi et al. 2005). Clearly, there is a
need to identify germplasm with beneficial traits for the diversification of the genetic base in these
crops.

II. Core and mini core collection: Crop germplasm collections held in genebanks are the best
genetic resources for detailed characterization of important traits such as tolerance to biotic and abiotic
stresses, yield, nutrition, and grain quality. However, the size of most crop-related global germ plasm
collections is simply too vast for systematic evaluation in replicated multilocational trials. The
development of core collections (Brown 1989) has been shown to be a particularly powerful strategy
for providing crop breeding programs with a systematic yet manageable entry point into global
germplasm resources. Using passport, characterization and agronomic evaluation data, the core is
constituted from the 10% of the entire germplasm collection, representing over 70% of the collections
variability in that collection. Core collections are a cost-effective means of identifying accessions with
desirable agronomic traits as well new sources of disease and pest resistance or abiotic stress tolerance.
However, in crop species with several thousands of germ plasm accessions, even a core collection
would be unwieldy for evaluation by the breeders in the multi-location replicated trials. To overcome
this, Upadhyaya and Ortiz (2001) suggested mini-core collection approach which is a core of core
(10% of core or 1% of entire collection) representing the species diversity. Mini-core is established
after evaluating the core subset for various morphological, agronomic, and seed quality traits, and
selecting about 10% accessions from the core subset. At both stages in selection of core and mini-core

-
collections, standard clustering procedures are used to separate groups of similar accessions combined
with various statistical tests to identify the best representatives. Conventional core and mini core
collections have been developed in chickpea (Upadhyaya et al. 2001; Upadhyaya and Ortiz 2001),
groundnut (Holbrook et al. 1993; Upadhyaya et al. 2002, 2003; Holbrook and Dong 2005; Dwivedi et
al. 2008a), and pigeonpea (Reddy et al. 2005; Upadhyaya et al. 2006). Core collections have also been
reported in pea, lentil, common bean, and soybean (Dwivedi et al. 2005). More recently,
genotype-based reference sets have also been developed in some legumes including chickpea,
groundnut and pigeonpea (http://www.generationcp.org). Availability of these genetic resources sets
offers immense opportunities to identify new sources of variation for use in crop breeding.

III. New sources of variation: Evaluation of core and mini-core collections has been suggested
as the most efficient and reliable means of carrying out an initial search of germplasm collections for
desirable traits. Such efforts have led to the identification of diverse germplasm with desirable
agronomic traits inchding resistance to biotic and abioitc stresses in chickpea, ground nut, and
pigeonpea (Table 1; ICRISA T Project 2 Archival Report 2008). New sources of variation have also
been reported for agronomic traits including resistance to diseases and pests in pea, lentil, and common
bean (Dwivedi et al. 2005).

20 International conference on Current trends in Biotechnology, Fehruary 19-21, 2009

 IV. Enhancing trait value using wild relatives: When resistance to a particular disease or pest
is not available in the cultivated germplasm, wild relatives of cultigens become often very handy
Resistance to many pests and diseases have been successfully transferred from wild relatives to
agriculturally important crops including legumes (Dwivedi et al. 2005, 2008b). Wild relatives have also
contributed alleles for agronomic traits in tomato and rice (Tanksley and McCouch 1997). Studies at
ICRISAT and elsewhere are in progress to demonstrate this proof of concept in legumes. More
recently, synthetic polyploids have been successful at generating diversity in wheat and Brassica
species. The work is in progress to re-synthesize the cultivated ground nut using ancestral and related
species for the introduction of new genes while minimizing the problem of sterility and suppression of
recombination, both major constraints in the utilization of wild relatives in breeding (ICRISA T Project
2 Archival Report 2008).

V. Genomic resources in model and crop legumes

A. Model legumes: Medicago truncatula and Lotusjaponiclis have emerged as models for plant
genomic research in legumes, relatively with compact genomes of approximately 470 Mbp. Scientists
now study these species to investigate a range of questions from disease resistance to environmental
tolerance and from bacterial and fungal symbiosis to complex secondary metabolism. However, both
belong to temperate legume. For tropical legumes, soybean has emerged as a model genome. M
truncatula (http://www.medicago.org/genoIl1:e/), L. japoniclis (http://www.kazusa.or.jp/lotus/) and
soybean (http://www.phytozome.net/soybean) genomes are currently the subject of independent
large-scale sequencing projects. In addition, large-scale transcriptomics, proteomics, metabolomics,
phenomics, and bioinformatics resources and reverse genetic tools have been developed. The
characterization of these three legume genomes will undoubtedly enhance ongoing comparative
genomic analyses (Szczyglowski and Stougaard 2008; Cannon et al. 2006; Ane et al. 2008; Rose 2008).
The value of model systems will be enhanced by the ability to connect model systems to crops at
structural and functional genome levels, for example, synteny between model and crop species should

Ccnnecting model system; to crop; at

the structural and fl11ctlonal genome
Iel/el

Level r:I conservation decreasES

as the evolutionary dstarce
increases

F19ure 2. 1 ranstational genomlcs ~om model to crop legumes

International conference on CUrrent trends in Biotechnology, ebruary 19-21,2009 21
allow the use of model species as a surrogate genome for map-based cloning of agronomically
important genes in crops with complex genomes (Rose 2008) or identifying legume anchor markers to
link syntenic regions across legumes (Hougaard et al. 2008). Thus, the genetic, genomic, and molecular
tools available to these model plants allow not only investigation of basic processes important to
legumes, but also transfer that processes to other crop species (Ane et al. 2008; Figure 2).
B. Crop legumes: Legumes which form a coherent taxonomic group with frequent and
widespread macro- and micro-synteny, however, have huge variation in nuclear genome size, ranging
from 370 million base pair (Mbp) in Lablab niger to 13,000 Mbp in Vic;a faha (Arumugunathan and
Earle 1991; http://www.rbgkew.org.uklcval/homepage.html). Black gram, mung bean, common bean,
lima bean, tepary bean, and cowpea have the smallest genomes (574 Mbp to 647 Mbp); pigeonpea
(784, 882 Mbp) and chickpea (73 8 Mbp) have slightly larger genomes; soybean has a relatively large
genome (1115 Mbp); while pea and lentil (4063 Mbp to 4397 Mbp) and broad bean (12,603 Mbp) have
massive genome sizes. The whole genome duplication and segmental duplications appear to have
played a significant role in creating new diversity in higher plants including the legumes (AGI 2000;
Vision et al. 2000). Over the years, there have been several publications reporting the development of a
range of genomic resources including markers; ESTs and BAC libraries; genetic, cytogenetic and
physical maps; identification of QTL associated with beneficial traits, and use of these resources in
applied breeding. In recent years, tremendous progress has been made towards developing genetic
markers (especially SSRs and SNPs) and/or construction of high-density genetic linkage maps in
chickpea, ground nut, and pigeonpea (Table 2; Varshney et al. 2009), which will enable researchers to
more rapidly and precisely characterize genetic diversity, identify trait-based genetically diverse
germplasm, target genes underlying key agronomic traits, and develop molecular assays that are both
relevant and of appropriate scale for breeding applications. More importantly, high throughput and cost
effective genotyping platforms, combined with automation in phenotyping methodologies, will increase
the uptake of genomic tools into breeding programs, and thus usher in an era of genomics-enabled
molecular breeding in legumes (Varshney et al. 2009).

Table 1. New sources of variation identified after evaluating core/mini core subsets in chickpea,
groundnut, and pigeonpea
Trait Chickpea Groundnut Pigeonpea . -
Diseases FW-67 GRD- 3 SM -·28
AB-3 ELS-1 F"W - 4
BGM -55 BW -14 PB -78
DRR- 5 Aflatoxin - 5 Multiple resistance -2
Drought 18 18
parS alinity 12 12 16
High temperature 5
Low temperature 158
Early maturity 28 21 20
Seed size 16 7
Grain yield 39 60 54
Protein content 5 5

22 International conference on Current trends in Biotechnology, February 19-21, 2009

 FW= Fusarium wilt, AB=Ascochyta blight, BGM=Botrytis gray mold, DRR=Dry root rot,
GRD=Groundnut rosette disease, ELS=Early leaf-spot, BW=Bacterial wilt, SM=Sterility mosaIC,
PB=Phytophthora blight

Table 2. Genomic resources in chickpea, common bean, cowpea, groundnut and pigeonpea
(Source: Modified from Varshney et al. 2009)
Genomic resource Cowpea Chickpea Groundnut Pigeonpea Common
bean
Ploidy 2n= 22 2n = 16 4n= 40 2n= 22 2n = 22
Genome size (Mbp) 620 740 2890 858 637
SSR 768 -2000 -2700 -3200 -500
BAC 6-l7X 3.8 - lOX 4X -7.4X llX 10-20X
BAC-end sequences 50,120 46,270 41,856 85,785 89,017
(36.7Mhp) (33.2Mbp) (28.6 Mbp) (56.5 Mbp) (62Mbp)
Genetic maps (bc) ++ ++ AA (2X) No ++
genome: ++
Genetic maps (nc) + + BB (2X) No +
genome: ++
AABB (4X): +
Physical map Yes No In progress No Yes
(AAgenome)

bc - broad crosses; nc - narrow crosses

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24 International conference on Current trends in Biotechnology, Fehruary 19-21,2009