Molecular

Markers For
Biotic and Abiotic
Stress Resistance
LANDMARKS IN MOLECULAR BIOLOGY

1900 Rediscovery of Mendelian principles

1909 The term ‘gene’ coined by Johansen
1940 One gene-one enzyme theory- Beadle & Tatum
1943 DNA as genetic material- AVERY
1953 Blueprint of DNA – Watson &Crick
1955 DNA polymerase – Kornberg
1958 Semi conservative replication of DNA – Meselson &Stahl
1961 Triplet codon, Operon concept
1962 Central dogma: gene-m RNA-rotein
(Watson,Crick&Wilson)
1973 DNA Cloning :Cohen &Boyer
1975 DNA sequencing :Sanger &Maxam &Gilbert
1988 Polymerase Chain Reaction method published :Mullis
2001 First draft of human genome
2003 Rice genome project to be completed
50 years of double helix
DNA to protein
 Molecular markers
RFLP
RAPD
SSRs
AFLP
ISSR

to generate reliable molecular profiles of the genetic

resources, including elite breeding materials.
The information is used for gaining a better
understanding of the genetic variation in many gene
pools.

It is essential to understand

• the various types of analysis suited to different

situations
• the nature and number of molecular markers needed for
 Molecular markers
are heritable differences in nucleotide sequences of
DNA from two different individuals,

Advantages

 Follow a simple Mendelian inheritance.

• Numerous markers can be obtained
• Relatively large no. of alleles can be found
• Molecular markers are genetically silent on their effect
on the genotype
• Genotypes of most molecular markers can be detected
in early developmental stages
• These differences are detected using two basic
techniques viz.,
(a) Southern hybridization ex. RFLP
(b) Polymerase chain reaction ex. RAPD
Restriction Fragment Length Polymorphism (RFLP)

• The first marker developed (Grodzicker et al., 1974).

• Screening of RFLP markers directly at DNA level, co-
dominance and phenotypically neutral nature,
absence of epistatic effects and virtually
limitless number are the advantages over
morphological or isozyme markers.
• Relatively slow process, less cost effectiveness,
fewer numbers of discriminating loci, requirement
of large quantities of DNA samples and use of
radioactive isotopes are the inherent drawbacks
of RFLP.
• Their inability to detect single base changes also
restricts their use in detecting point mutations
occurring within the regions at which they are
detecting polymorphism.
•RFLP profile for 13 different Seiridium isolates belonging to five different
species.
•Two subgroups of S. cupressi can be distinguished.

•Lane M; 100 bp ladder marker.

 Principle of PCR

It is in vitro amplification of a specific target DNA

sequence in a cyclic process using two oligonucleotide
primers.

It consists of repetitive cycles of

1. DNA denaturation,
2. Primer annealing
3. Extension by DNA polymerase.

Two primers flank the DNA fragment to be amplified and

hybridize to opposite strands of the target sequence
such that the synthesis proceeds across the region
between the primers.
 Taq DNA polymerase

 isolated from a thermophilic

microorganism Thermus aquaticus found in
hot springs, growing at 70-75 C.
 T opt (temperature optimum) - 75 0– 80 0C
 Taq remains stable for hours at higher
temperature

Taq polymerase was named as the molecule of the

year 1989 (by the journal Science).
Equipment
 A thermal cycler that heats and cools the
reaction mixtures to the desired temperature for
each stage of amplification process
 Maintains the required temperature for a fixed
period.
 Temperature cycling parameters
DNA denaturation - 94 0C for 1 m
Primer annealing by cooling to 40-
600C for I m
(depending upon the primer)
Primer extension at 720 C for I m
 Upto 10 Kb can be amplified through PCR.
 Advantages of PCR
1 Specificity
2. High sensitivity : a single copy of target DNA can be
amplified to 1000s
3. Exact copy number
4. Speed : within a few hours
5. Simplicity:

Once the reaction conditions and primer sequences

have been optimised, preparation of reaction mixtures and
purification of amplified sample is very simple.

The product of one round of amplification serves as the

template for the successive cycle
the amount of the desired product will be doubled.
End result is an exponential increase in the total number of
copies of the target DNA with a theoritical abundance
of 2n where n is the number of cycles performed.
12
 PCR Amplification

PCR carried out on the DNA from which the gene has to
be isolated
Annealing Temperature
Ta = 2(A+T) + 4( C+G) or using computer programmes

Amplified product is resolved on agarose gel and product

eluted from the gel and purified.

PCR product cloning

Cloning can be done using vectors viz. pUC , bluescript
PGEM

Purified PCR product is ligated with PGEM vector using

ligase enzyme at 40C overnight.
 Sequence-tagged sites (STS)
 RFLP probes specifically linked to a desired
trait can be converted into PCR-based STS
markers based on nucleotide sequence of the
probe giving polymorphic band pattern, to
obtain specific amplicon.
 Tedious hybridization procedures involved in
RFLP analysis can be overcome.
 When these markers are linked to some
specific traits, (ex. powdery mildew resistance
gene), they can be easily integrated into plant
breeding programmes for marker-assisted
selection of the trait of interest.
Segregation of dominant length polymorphisms
of sequence-tagged-site (STS) markers
 RAPD

 Involves the use of single short random

nucleotides that expose polymorphism
randomly distributed throughout the genome
leading randomly amplified polymorphic DNA.
 Dramatic reduction in the amounts of DNA
required, generation of fingerprints without the
need of DNA sequence information,
 Ease of the technique
 RAPD assay has been used by several groups as
efficient tools for identification of markers
linked to agronomically important traits,
which are introgressed during the development
of near isogenic lines.
 Repeatability and stability are potential
snags.
 Are dominant markers and hence have

limitations in their use as markers

for mapping, which can be overcome
to some extent by selecting those
markers that are linked in coupling
(Bernardo, 1992).
With OPA 15 lane p: mother plant (tomato)
1-11: regenerants
Arrows show polymorphism
 Sequence characterized amplified regions
for amplification of specific band (SCAR)
Michelmore et al (1991) and Martin et al .(1991)
 RAPD marker termini are sequenced and longer primers
are designed (22–24) nucleotide bases long) for specific
amplification of a particular locus.

 Similar to STS markers in construction and application.

 The presence or absence of the band indicates variation

in sequence and are better reproducible than RAPDs.

 SCARs are usually dominant markers

 Some of them can be converted into codominant markers

by digesting them with restriction enzymes and
polymorphism can be deduced by gel electrophoresis.
 Advantages
 Compared to arbitrary primers, SCARs
are used in mapping studies
 Codominant SCARs are informative for

genetic mapping than dominant

RAPDs),
 For map-based cloning - as they can be

used to screen pooled genomic

libraries by PCR, physical mapping,
locus specificity, etc.
 Cleaved amplified polymorphic sequences
(CAPs):
 These polymorphic patterns are generated by
restriction enzyme digestion of PCR products.
 Such digests are compared for their differential
migration during electrophoresis.
 PCR primer for this process can be synthesized
based on the sequence information available in
databank of genomic or cDNA sequences or
cloned RAPD bands.
 Are codominant in nature.
 This approach is widely used in tagging disease
resistance genes.
Two codominant CAPS markers,

SCAR181067 (a) and SCBC792779 (b)

 Microsatellites and minisatellites
 Microsatellites was coined by Litt and Lutty (1989)
 Minisatellites was introduced by Jeffrey (1985).
 Both are multilocus probes and belong to the repetitive DNA
family.
 Minisatellites are tandem repeats with a monomer repeat length of
about 11–60 bp
 Microsatellites or short tandem repeats/simple sequence repeats
consisting of 1 to 6 bp long monomer sequence that is repeated
several times.
 These loci contain tandem repeats that vary in the number of
repeat units between genotypes and are referred to as variable
number of tandem repeats (VNTRs).
 Microsatellites and minisatellites form ideal marker system
creating complex banding patterns by simultaneously detecting
multiple DNA loci.
Sequence-tagged microsatellite site markers (STMS)

 The sequences flanking specific microsatellite loci

are used for designing primers to amplify individual
microsatellite loci
 The technique is described as sequence tagged
microsatellite loci (STMS) (Beckmann and Soller,
1990).
 Considering their species specificity, it has become
necessary to develop such markers for each crop.
 Microsatellites are ideal for multiplexing. When two
or more such primers produce products that differ in
size, more such pairs can be used in the same PCR
reaction, thereby two or more than two
polymorphisms could be resolved simultaneously in
the same line. This would further reduce the cost.
FOR GENE TAGGING
Polymorphism detected by the new STMS primer NKSCSSR 63
and NKSCSSR 64
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Lane1:Co 7201, 2.7201-134,3. 7201-157, 4.7201-136,5.7201-138, 6.7201-149,7.7201-153,8.Co1148,

9.1148-240,10.1148-250,11.1148-251,12.1148-237,13.1148-239,14.1148-252,15. Co775, 16. 775-
165,17.775-170,18.775-176,19.775-166,M- 50 bp ladder
 Inter-simple sequence repeat markers (ISSR)
 Primers based on microsatellites are utilized to
amplify inter-SSR DNA sequences.
 Microsatellites anchored at the 3’ end are used
for amplifying genomic DNA which increases
their specificity.
 Mostly dominant markers, though occasionally a
exhibit codominance.
 An unlimited number of primers can be
synthesized for various combinations of di-,tri-,
tetra- and pentanucleotides etc. with an anchor
made up of a few bases
 Exploited for a broad range of applications in
plant species.
Amplified Fragment Length Polymorphism (AFLP):
 A technique based on the detection of genomic
restriction fragments by PCR amplification and
can be used for DNAs of any origin or complexity.
 Is based on PCR amplification of restriction
fragments generated by specific restriction
enzymes and oligonucleotide adapters of few
nucleotide bases (Vos et al. 1995).
 Generates a large number of restriction fragment
bands facilitating the detection of
polymorphisms.
 The number of DNA fragments, which are
amplified, can be controlled by choosing the
different base number and composition of
nucleotides in adapters.
 Advantages
 Widely used for developing polymorphic
markers.

 High reproducibility, rapid generation and high

frequency of identifiable AFLP polymorphisms
make AFLP analysis an attractive technique for
gene tagging.

 Similar to RAPDs, the bands of interest obtained

by AFLP can be converted into SCARs.
 Steps:
(a) Restriction of DNA using a rare cutting and a
commonly cutting restriction enzyme
simultaneously (ex. Mse I and Eco RI) followed by
ligation of oligonucleotide adapters, of defined
sequences including the respective restriction
enzyme sites.
(b) Selective amplifications of sets of restriction
fragments, using specifically designed
primers. To achieve this, the 5' region of the primer
is made such that it would contain both the
restriction enzyme sites on either sides of the
fragment complementary to the respective adapters,
while the 3' ends extend for a few arbitrarily
chosen nucleotides into the restriction fragments.
(c) Gel analysis of the amplified fragments.
AFLP
PROFILE
 Single nucleotide polymorphisms (SNPs):
 SNPs are an alteration of one nucleotide in a DNA
sequence.
 Their frequent occurrence provides a large source of
genetic markers that are more likely to be located
close to target gene of interest. SNP markers are
increasingly being used in humans and in crop plants.
 SNP markers have been tested in hexaploid wheat and
allele proportions can be identified indicating their
suitability for use with the highly polyploidy genome
of sugarcane.
 Sequencing of ESTs have provided several SNPs (ex.
EST sequencing identified at least 400 SNPs.
 Analysis and identification of SNPs call upon the
capabilities of Pyrosequencer, which delivers real time
detection of DNA sequencing events This provides an
accurate means of identifying the expected proportion
of alleles.
SNP
 Detection of a SNP site by an appropriate
restriction endonuclease

• Recognition sequence has been altered or introduced by

the SNP

• In combination with a PCR assay, the corresponding SNP

can be analysed as a cleaved amplified polymorphic
sequence (CAPS) marker

•The costs of a CAPS assay is generally low, especially

when it relies on commonly used restriction
enzymes.
Illustration of possible scenarios at an EcoRI recognition
site (G↑AATTC) between two aligned sequences.
Conversion of the barley SNP marker GBS0734 into an EcoRV CAPS marker.
(a) Relevant part of the multiple sequence alignment of SNP marker GBS0734.
The recognition site of EcoRV (GAT↑ATC) is affected by one SNP at position
151 (C→T transition)
 Candidate genes as markers
A candidate gene is a gene located in a
chromosome region suspected of being
involved in the expression of a trait such as a
disease, whose protein product suggests that
it could be the gene in question.

A candidate gene can also be identified by

association with the phenotype and by linkage
analysis to a region of the genome.
 Candidate genes are potentially an extremely useful
source of markers for MAS because they may
be the genes responsible for expression of the
trait.
 If so, there will be no recombination between the
candidate gene and the trait and thus is the
perfect indirect selection tool.
 Further research to determine the association of the
trait and the presence of the gene would show
whether the association is coincidental, in which
case candidate gene is still a useful marker.
 This would show whether the gene is implicated in
the expression of the trait, in which case the gene
could provide valuable information on the
underlying genetics, biochemistry and physiology of
the trait itself (CSIRO project report, 2004).
 Steps

•Choose candidate genes based on biological system

(physiology, biochemistry) and/or QTL
information
• Amplify part or all of the gene using consensus or
degenerate primers
• Identify polymorphisms in the candidate genes or
flanking sequence (length, DNA sequence, restriction
sites)
• Scale up genotyping using allele specific primers or
sequencing
• Identify and genotype a population
• Analyze association between allele (haplotype) and
phenotype
• Validate
• Software
 Advantages:

 Complementary to QTL analysis

 Can be used with many different kinds of
populations
 Modest cost - sequencing, primers,
polymerase, DNA extraction
 SNPs and Indels provide an inexhaustable
supply of polymorphic markers
 Adaptable to breeding program
 Disadvantages:

 Requires apriori knowledge of genes and a putative

function
 Population structure may result in false positives
 Non-trait genes may influence target trait making it
difficult to select candidates
 Difficult to narrow down candidates from 100s to a
few with high linkage disequilibrium
 Proof of gene identity difficult due to variation in
closely linked genes
 Can be complicated by pleiotropy and gene
interactions
Large data sets of DNA and protein sequences generated.
Biologists sought for automatic information storage
and retrieval systems.

Major public databanks taking care of DNA and protein

sequences

1. Genbanks of USA (http://www.ncbi.nlm.nih.gov)

2. EMBL (European Molecular Biology Laboratory) in
Europe (http://www.ebi.ac.uk)
3. DDBJ (DNA Data Bank in Japan
(http://www.ddbj.nig.ac.jp).
4. The Institute of Genome Research (TIGR) and are
available in the website www.tigr.org.

Size of databases
23,000 Mb (EMBL), 29000 Mb (Genbank) and 27,000 Mb
(DDBJ) DNA base pairs.
Sequences available in public database
Human genome (3 billion bp),
rice (450Mb),
Arabidopsis (130 Mb)

Gene prediction
GENSCAN developed by MIT, USA.
(http://www.genes.mit.edu/GENSCAN.html).
On-line analysis of DNA sequences can be performed here
free of charge.
Gene annotation
The output obtained from the GENSCAN is used for gene
annotation by using Basic Local Alignment Search Tools
(BLAST) - to find the match to the query sequence with
the sequences available in the Genbank.
Genome sequences of any organism can be used for the in-
silico mapping of different genes as well
as designing gene specific primers for gene
cloning.
 Genomics QTL approach

A QTL (Quantitative Trait Locus) is defined as a region of the

genome that is associated with an effect on a quantitative trait.
The term coined by Geldman (1971).

Not necessarily genes themselves, QTLs are stretches of

DNA that are closely linked to the genes that underlie
the trait in question.
QTLs can be molecularly identified to help map regions of the
genome that contain genes involved in specifying a
quantitative trait.
This can be an early step in identifying and sequencing
these genes

Stress tolerance is generally a quantitative character

governed by QTLs.
Principle of QTL mapping:

Identification of markers linked to QTLs that

influence the trait.
Where such linkage occurs, the marker locus and the
QTL will co-segregate

QTL mapping :

is the statistical study of the alleles that occur in a

locus and the phenotypes (physical forms or
traits) that they produce.
1. A set of genetic markers must be developed for the
species in question
2. Identify the marker that is significantly more likely to
co-occur with the trait through statistical
association.
 Requirements for QTL mapping

o A suitable mapping population generated from

phenotypically contrasting parents

o A saturated linkage map based on molecular markers
o A reliable phenotypic screening of mapping
population
o Appropriate statistical packages to analyze genotypic
information in combination with phenotypic
information for QTL detection.
Analysis generally reveals regions of DNA that are very
close to the genes in question rather than finding
the specific gene in question.

Where such linkage occurs, the marker locus and the

QTL will co-segregate.

In order to find a QTL linked to a marker,

the mapping population has to be partitioned
into different genotypic classes based on the
genotype at the marker locus
 Indicator traits :
Ex. Drought:
Provide convenient selection criteria for breeding programs.
In maize the silk-tassel interval was identified as a
highly indicative secondary trait for drought-resistant
breeding.

Need of special experimental designs:

Genetic dissection of complex traits like drought resistance

could be achieved with special experimental
designs.

Plant-wise drought treatment protocol provides a

useful method for independent evaluation of the individual
components contributing to drought resistance.
In several crop species, genetic maps have allowed
to identify chromosomal regions controlling some
traits related to stress response.

Consistency of the above QTLs in different

genetic backgrounds and their association with
plant performance under field conditions and
used in MAS
Applications of molecular markers in biotic
and abiotic stresses
 In tagging of genes
The very first reports on gene tagging from tomato:
- identification of markers linked to genes
 resistance to Fusarium oxysporum (the 12 gene)

(Sarfatti et al.,1989),
 leaf rust resistance genes lr 9 and 24

(Schachermayr et al.1994
 root knot nematodes (Meliodogyne sp.) (mi gene)

(Klein-Lankhorst et al.1991).
 Advantages

• Molecular markers that flank a gene determining a trait

can be used to track down the gene in segregating
generations derived from appropriate crosses.

• This would permit the identification of putative resistant

plants in the absence to disease tests.

• Molecular markers would enable an effective selection

for horizontal resistance even in the presence of vertical
resistance genes, which is not possibly by conventional
approaches.
 Marker-assisted selection plant breeding

 A breeding strategy in which selection for a gene is

based on molecular markers closely linked to the
gene of interest rather than the gene itself
 Markers are used to monitor the incorporation of
the desired allele from the donor source.
 This is the basis for molecular breeding, wherein
plant breeding programmes are supported by the
use of DNA-based markers.
 Molecular breeding requires genetic maps,
molecular markers linked to agronomic traits, high
throughput, automated diagnostic technique and a
modification in breeding practice that takes full
advantage of the information provided by such
diagnostic assays.
Promise of MAS
 Indirect selection of desirable plants, free of
environmental, pleiotrophic or epistatic
effects,
 The ability to discriminate between homo and
heterozygote,
 Pyramiding of genes
 Monitoring the introgression
 Identification of recombinants possessing
least amount of linkage drag and donor DNA
flanking the gene of interest.
 Reports on actual application of MAS are still
limited.
 The predictive value of molecular markers

used in MAS depends on their

- inherent repeatability,
- map position
- linkage with economically important
traits
Success stories
 Identification of homozygous or heterozygous resistant
plants against barley mild mosaic virus (BaMMV) and
barley yellow mosaic virus BaYMV
A single recessive gene (ym4) confers complete immunity
is located on chromosome 3 and is mapped with RFLP
markers.
Two RFLP markers (MWG10 and MWG 838) flanking this
gene are 1.6 cM apart and are thus, highly attractive for
the breeding programmes.
One of the markers (MWG 838) was converted into a PCR
based STS marker.
Based on this marker, plants possessing homozygous or
heterozygous resistance against BaMMV/ BaYMB were
selected and are being used actively in commercial
breeding programmes ((Tuvesson et al., 2006).
 Barley stripe rust (caused by Puccinia striiformis)
 Two RFLP and One AFLP markers were used to map
on chromosome 3 and 4, the QTL conferring
resistance to barley stripe rust.
 These QTL were introgressed through marker-
assisted back crossing into elite breeding material
lacking resistance.
 The selected backcross lines were converted into
doubled haploids.
 These doubled haploid lines after phenotyping for
stripe rust resistance were characterized with
different markers to confirm the target gene and to
identify lines with maximum percentage of recurrent
parent genome.
 The procedure allowed rapid development of
resistant germplasm of barley on commercial scale
(Toojinda et al., 1998).
Resistance to soybean cyst nematode (SCN )

 Resistance is primarily due to the gene rgh 1.

 The microsatellite marker sat 309 is located at 2

cM from this gene.

 Selection based on sat 309 is 99% accurate in

predicting SCN susceptible lines.

Soybean insect resistance
 QTL for soybean insect resistant (SIR) QTL
were identified in soybean germplasm with
RFLP markers (James et al., 2001).
 Introgression of these QTL into registered

cultivars was detected with SSR markers.

There was high proportion of introgression
with minimum linkage drag.
 SCAR/STS marker linked to the translocated
segment on 4 AL of bread wheat carrying the Lr28
gene has been tagged (Naik et al .1998).
 ISSR marker (AC)8 YT has been found to be linked
to the gene for resistance to fusarium wilt race 4 in
repulsion at a distance of 5.2 cM in chickpea
(Ratnaparkhe et al.1998).
 In common bean (Phaseolus vulgaris L), RAPD
markers identified were significantly correlated with
yield and MAS improved the performance by 11%
under stress condition and 8% under non stress
conditions (Schneider et al. 1997).
 Pearl millet

 Parents : thermo-tolerant drought sensitive inbred

pollinator lines
thermosusceptible drought tolerant breeding line
 These parental lines, skeleton maps and mapped progenies from
the mapping population were used in a series of marker assisted
backcross breeding,
 Resulted into transfer of drought tolerance QTL identified on
linkage group 2 of thermosusceptible, drought tolerant parent to
thermo-tolerant drought sensitive background.
 The progeny at BC4F1 generation segregated for the target QTL and
its flanking markers based on marker genotypes of the non-
recurrent parents.
 Based on these markers, plants possessing both temperature and
drought tolerance were selected.
 These are being used as elite drought tolerant male parent of
several hybrid cultivars of pearl millet in India (Yadav et al, 1999).
  PROBLEMS

 The association between the marker and a QTL

would appear valid only in rare cases.
 QTL needs to be verified before applying in MAS.
 It is hence necessary to identify markers that can
explain significant portions of the combining ability
so as to derive universally applicable markers and
adopting direct use of markers specific for QTL
 For adopting MAS for QTL, it is imperative to
understand that MAS combined with phenotypic
selection will be superior initially, but will become
inferior when QTL approaches fixation (Hospital et
al. 1992).
MAS produced rapid responses early in the selection
process; however, the rate of these responses diminished
greatly within three to five cycles.
The gains from MAS ranged from 44.7 to 99.5% of the
maximum potential, depending on the genetic model
considered.
Linkage distance between markers and quantitative trait loci
(QTLs) was the factor which most limited the
responses from MAS.
Flanking QTLs within two marker loci produced 38% more
gain than selection based on single markers if
markers were loosely-linked to a QTL (20% recombination).
Flanking markers were much less advantageous when
markers were closely-linked to a QTL (5% recombination),
producing an advantage over single markers of only 11%
 Markers were most effective in fully exploiting
the genetic potential when fewer QTLs
controlled the trait.
 Large QTL numbers exacerbated the problem of
marker-QTL recombination by requiring more
generations for fixation.
 In annual crop species, MAS may offer a primary
advantage of enabling two selection cycles per
year versus the 2 years per cycle required
 MAS thus appears to allow very rapid gains for
the first 2–3 years of recurrent selection, after
which time conventional methods might replace
MAS to achieve further responses.
 Gene pyramiding:
In resistance breeding, molecular markers can be
useful in gene pyramiding
- involves bringing two or more different
genes conferring resistance to
the same pathogen in the same
line.

A few success stories:

• STS markers have been used for MAS to pyramid
bacterial leaf blight genes in rice viz. Xa4, xa5, xa13
and xa 21 in all possible combinations. The derived
lines showed a wide spectrum or higher level of
resistance to the disease resistance genes.

• Two rice varieties Angke and Conde developed by

MAS for bacterial leaf blight resistance have been
 Using three near isogenic lines (NILs), resistance
gene for rice blast Pi-1, Pi-z5 and Pi-ta were
characterized. The three genes were mapped with
RFLPs to obtain closely linked DNA markers for
MAS.

 F1 obtained by crossing between carrier isogenic

line with the recurrent parent were subjected to
Southern analysis to select plants with two genes
in homozygous condition.

 Crossing such lines with the plants carrying the

third resistant gene followed by selection was done
to achieve durable resistance against rice blast
(Hittalmani et al., 2000).
 Soybean

Simple sequence repeat markers were used to

create isogenic lines of the susceptible
cultivar Essex containing one, two, or three
Rsv loci
Following MAS and three near-isogenic lines,
each containing a different SMV-resistance
gene, the pyramided lines with two or three
genes could be generated with high levels of
resistance to SMV (Saghai Maroof et al.
2008).