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Molecular tagging of a rust resistance gene in cultivated
groundnut (Arachis hypogaea L.) introgressed from
Arachis cardenasii
Suvendu Mondal •A. M. Badigannavar •
S. F. D’Souza
Received: 5 May 2010 / Accepted: 9 March 2011 / Published online: 27 March 2011
Springer Science+Business Media B.V. 2011
Abstract Rust is a serious and the most prevalent
groundnut disease in tropical and subtropical growing
regions of the world. A total of 164 recombinant
inbred lines derived from resistant (VG 9514) and
susceptible (TAG 24) cultivated groundnut parents
were screened for rust resistance in five environments.
Subsequent genotyping of these lines with 109 simple
sequence repeat (SSR) markers generated a genetic
linkage map with 24 linkage groups. The total length
of the linkage map was 882.9 cM with an average of
9.0 cM between neighbouring markers. The markers
pPGPseq4A05 and gi56931710 flanked the rust resis-
tance gene at map distances of 4.7 cM and 4.3 cM,
respectively, in linkage group 2. The significant
association of these two markers with the rust reaction
was also confirmed by discriminant analysis. The
informative SSR markers classified rust-resistant and
susceptible groups with 99.97% correctness. The SSR
markers pPGPseq4A05 and gi56931710 were able to
identify all the susceptible genotypes from a set of 20
cultivated genotypes differing in rust reaction. Tag-
ging of the rust resistance locus with linked SSR
markers will be useful in selecting the rust resistant
genotypes from segregating populations and in intro-
gressing the rust resistance genes from diploid wild
species.
Keywords Recombinant inbred lines
Puccinia arachidis SSR markers Linkage map
Discriminant analysis
Introduction
Groundnut (Arachis hypogaea L.) is an important
oilseed crop in India. It ranks fifth in the world among
oilseeds with an area of 24.59 million hectares,
production of 38.20 million tonnes (with shell) and
productivity of 1.55 tonnes/hectare (FAOSTAT
2008); however, the productivity is only 1.07 tonnes/
hectare from a cultivated area of 6.85 million hectares
in India (FAOSTAT 2008). This lower productivity is
accounted for by the rain-fed cultivation in semi-arid
areas of the country, where the crop faces severe
damage from foliar diseases like rust and late leaf spot.
Groundnut rust is caused by a basidiomycetes fungus,
Puccinia arachidis Speg.; the main symptom is the
appearance of orange coloured pustules or uredinia on
the lower surface of the leaves, which upon rupture
release masses of reddish-brown urediniospores. The
rust commonly develops in a radiating pattern from a
single spot in the field. The yield losses due to rust
range from 10 to 52%, in addition to a decline in seed
quality (Subrahmanyam et al. 1995).
Electronic supplementary material The online version of
this article (doi:10.1007/s11032-011-9564-z) contains
supplementary material, which is available to authorized users.
S. Mondal (&)A. M. Badigannavar S. F. D’Souza
Nuclear Agriculture and Biotechnology Division,
Bhabha Atomic Research Centre, Mumbai 400085, India
e-mail: suvenduhere@yahoo.co.in
123
Mol Breeding (2012) 29:467–476
DOI 10.1007/s11032-011-9564-z
Resistance to rust is reported to be controlled by
two or more recessive genes (Knauft 1987)orin
some cases by one partial dominant gene (Singh et al.
1984; Wynne et al. 1991; Mondal et al. 2007). Due to
its economic importance, much attention has been
given to developing cultural and chemical control
measures. However, such measures not only increase
the production cost but also increase the risk for food
safety and environmental pollution. Hence a major
thrust in groundnut breeding programs worldwide has
been to develop resistant and/or tolerant cultivars.
The development of disease-resistant varieties not
only requires characterization of the resistance, but
also thorough and repeated screening under disease
epiphytotics, which is laborious and error-prone, as
the environment influences the disease development.
Identification of linked markers would help to
identify the rust-resistant genotypes even in the
absence of disease epiphytotics.
Besides the tremendous morphological and agro-
nomic variability in cultivated groundnut germplasm,
it exhibits a low level of genetic polymorphism as
detected by different molecular markers such
as random amplified polymorphic DNA (RAPD)
(Mondal et al. 2005), inter simple sequence repeat
regions (ISSR) (Raina et al. 2000), amplified frag-
ment length polymorphisms (AFLPs) (Herselman
2003) and simple sequence repeats (SSRs) (He
et al. 2005; Moretzsohn et al. 2005). Recent devel-
opment of new SSR markers from enriched genomic
(Gimenes et al. 2007; Cuc et al. 2008) and cDNA
libraries (Proite et al. 2007; Guo et al. 2008) has
facilitated the detection of moderate to high levels of
variation in the cultivated groundnut (Varshney et al.
2009a; Mondal and Badigannavar 2010). Very few
genes for resistance to agronomically important pests
and pathogens like aphids (Herselman et al. 2004),
Aspergillus flavus (Yong et al. 2005) and nematodes
(Burow et al. 1996; Garcia et al. 1996) have been
tagged using AFLP and RAPD markers. Resistance to
rust, late leaf spot and Sclerotinia minor were
associated with SSR loci (Mace et al. 2006; Cham-
berlin et al. 2010; Mondal and Badigannavar 2010).
Use of molecular markers tightly linked to genes
controlling the trait of interest would be of great help
in marker-assisted selection (MAS). SSR markers are
preferred over RAPD and AFLP markers in MAS
because of their co-dominant nature, universality and
high reproducibility.
Non-parametric statistical analysis such as dis-
criminant analysis can be used as a complementary
approach to traditional mapping in a crop for which
saturated linkage maps are unavailable or difficult to
obtain (Alwala et al. 2009). This is a multivariate
approach that associates an individual with a descrip-
tive class (Fisher 1936). Discriminant analysis was
used to associate several molecular markers with
agronomic traits in rice and wheat (Capdevielle et al.
2000; Fahima et al. 2002; Zhang et al. 2005) and with
sugar-related traits in sugarcane (Alwala et al. 2009).
Aluko (2003) and Alwala et al. (2008) found some
common markers that were associated with important
agronomic traits in rice and resistance to Aspergillus
flavus in maize, respectively, using both discriminant
analysis and traditional quantitative trait locus (QTL)
mapping approaches.
Arachis cardenasii Kaprov. & W.C. Greg. is a
diploid (2n =2x =20) wild groundnut species with
‘A’ genome. Accessions of A. cardenasii are reported
to have resistance to nematodes (Meloidogyne are-
naria), early leaf spot (Cercospora arachidicola),
rust, late leaf spot (Phaeoisariopsis personata) and
peanut bud necrosis diseases (Varman 1999; Reddy
et al. 2000; Holbrook and Stalker 2003). Introgres-
sion of useful genes from diploid wild species into
A. hypogaea (2n =4x =40) is difficult due to its
ploidy barrier. However, this can be overcome
through chromosome doubling of interspecific F1
plants and subsequent backcrossing with tetraploid
cultivated species followed by selfing. A high level of
resistance to rust was successfully transferred to
A. hypogaea from A. cardenasii by developing a
tetraploid breeding line VG 9514 (Varman 1999).
Earlier, we had identified two RAPD markers (J7
1350
and J7
1300
) linked to a rust resistance gene by
analyzing 117 F
2
plants from a cross between VG
9514 and TAG 24 (Mondal et al. 2007). The marker
J7
1350
was located 18.5 cM away from the rust
resistance gene in the coupling phase and J7
1300
was
tightly linked to the resistance gene in the repulsion
phase. Universality and reproducibility are prime
concerns for any molecular markers to be applied for
MAS. In this context, we employed SSR markers to
find an association with the rust resistance gene from
VG 9514 using both genetic linkage maps and
discriminant analyses. These linked markers were
also validated in an additional 20 cultivated ground-
nut genotypes differing for rust reaction.
468 Mol Breeding (2012) 29:467–476
123
Materials and methods
Plant material
A recombinant inbred line (RIL) population consist-
ing of 164 lines (F
2:8
) from a cross between VG 9514
and TAG 24 was used in this study. VG 9514 is a
rust-resistant genotype developed by crossing cv. CO
1 and A. cardenasii (Varman 1999). TAG 24 is a well
adapted, high-yielding, early-maturing variety grown
extensively in India but susceptible to rust (Patil et al.
1995). A set of 20 cultivated groundnut genotypes
differing in rust resistance (Mondal and Badiganna-
var 2010) was taken to validate the association of rust
resistance with SSR markers (Table 1).
Field evaluation for rust resistance
The F
2:8
seeds from RILs and the parents were grown
in five environments with two replications using a
spreader row technique (Subrahmanyam et al. 1995).
These five environments were the 2008 and 2009 rainy
seasons at Trombay (E1and E2, respectively), the 2009
summer season at Gauribidanur (E3), and the 2008
rainy season and 2009 summer season at Dharwad (E4
and E5, respectively). Each RIL was planted in 3-m
rows with 50 910 cm spacing. A spreader row of the
susceptible parent, TAG 24, was planted after every
four lines of the test population and around all sides of
the experimental plot. Inoculum was prepared by
suspending the diseased parts of TAG 24 plants from
the previous rainy season (June to September) in water
and agitating the plant tissue vigorously to dislodge
urediniospores. The urediniospore suspension was
then sprayed over the test plants and spreader rows
in the evening of alternate days between 70 and
90 days after sowing (DAS). Rust was scored at
harvest on 10 plants of each RIL and recorded using
the modified 1–9 scale (Subrahmanyam et al. 1995),
comprising a disease score of 1 for 0%, 2 for 1–5%, 3
for 6–10%, 4 for 11–20%, 5 for 21–30%, 6 for
31–40%, 7 for 41–60%, 8 for 61–80% and 9 for
81–100% disease severity. Plants with a disease score
of 1– 3 were designated as being resistant, and those
with a score of 4–9 were deemed to be susceptible,
according to Pande and Rao (2001). Pearson correla-
tion analysis of the disease scores on RILs among the
environments was performed using PAST software
ver. 1.81 (Hammer et al. 2008).
Development of new EST-SSR markers
Sequence information of 745 newly submitted ground-
nut expressed sequence tags (ESTs) from NCBI
(http://www.ncbi.nlm.nih.gov) were used to identify
di-, tri-, tetra-, penta- and hexanucleotide repeats
using Websat software (http://wsmartins.net/websat)
(Martins et al. 2009). All these EST sequences were
developed through a forward and reverse subtractive
library from groundnut leaves inoculated with Cer-
cosporidium personatum (Nobile et al. 2008). After
finding the SSRs, the selected EST sequences were
retrieved from NCBI to design primer pairs using
Primer 3.0 software available at http://www.ncbi.nlm.
nih.gov/tools/primer-blast/index.cgi. The following
set of parameters was used in designing the SSR
primer pairs: product length 100–300 bp; melting
temperature (T
m
) 50–60C; primer length 20–25
nucleotides; and at least 50% GC content. The SSR
markers developed in this study were named with
prefix ‘Cer’ (from Cercosporidium) followed by an
ascending number (e.g. Cer 1 …Cer 21).
DNA isolation and SSR analysis
Total genomic DNA was isolated from fresh leaf
tissue of each RIL and parents at 30 DAS using a
modified CTAB method (Mondal et al. 2005).
Integrity and quality of isolated DNA were checked
via 0.8% agarose gel electrophoresis with kDNA as a
standard. The DNA samples were quantified using a
UV–Vis spectrophotometer (Jasco, Cambridge, UK)
and adjusted to a final concentration of 10 ng/ll. A
total of 1,093 SSRs earlier reported (Hopkins et al.
1999; Ferguson et al. 2004; He et al. 2003; Palmieri
et al. 2002; He et al. 2005; Moretzsohn et al. 2004;
2005;2009; Gimenes et al. 2007; Martins et al. 2006;
Wang et al. 2007; Cuc et al. 2008; and Liang et al.
2009) along with 21 newly synthesized EST-SSR
primer pairs were screened between the parents. The
polymorphic primer pairs were used for genotyping
of all the 164 RILs. Each 15 ll PCR reaction volume
contained 10 mM Tris–HCl (pH 9.0), 50 mM KCl,
1.5 mM MgCl
2
, 0.2 lM of each primer, 0.20 mM
dNTP (Promega, Madison, USA) and 0.5 U Taq
DNA polymerase (Promega). SSR amplification was
performed under the following conditions: initial
denaturation at 94C for 5 min with 35 cycles of
94C for 30 s, 50/52C for 30 s, 72C for 1 min, with
Mol Breeding (2012) 29:467–476 469
123
a final extension at 72C for 10 min. Gel electropho-
resis and its documentation was carried out as per
Mondal and Badigannavar (2010).
Linkage analysis
The SSR band scoring, rust reaction, plant habit and
testa colour (indicator traits) were recorded in binary
fashion. A v
2
test was performed to test the null
hypothesis of 1:1 segregation for these traits and
markers. The linkage analysis was performed using
MapMaker/EXP v3.0b (Lincoln et al. 1993). Mini-
mum LOD score of 3.0 and maximum recombination
fraction of 0.44 were set as thresholds for linkage
group determination with the ‘group’ command.
Multipoint analysis was performed to confirm the
exact marker order in the linkage group by comparing
the log-likelihood of the possible orders through the
‘compare’ command. The map distance was
expressed in centimorgans (cM) using the Kosambi
(1944) map function. Graphical representation of
linkage groups was obtained by using Mapchart
version 2.1 (Voorrips 2002).
Discriminant analysis
Association of markers with rust reactions in RILs
was analysed by discriminant analysis, which is used
to classify cases into the values of a categorical
dependent variable, usually a dichotomous one
(Fisher 1936). Discriminant analysis has two steps:
(1) an Ftest (Wilks’ lambda) is used to test if the
discriminant model is significant, and (2) if the Ftest
shows significance, then individual independent
variables are assessed to see which differ significantly
in mean (by group) and these are used to classify the
Table 1 List of cultivated groundnut genotypes used for validating the association of two SSR markers (gi56931710 and
pPGPseq4A05) with rust resistance
No. Genotype Pedigree Rust
a
gi56931710 pPGPseq4A05
2004 2005
1 VG 9514 Arachis cardenasii XCO1 11– –
2 GPBD 4 KRG 1 9ICGV 86855 3 2 – –
3 GBFDS 272 Not known 1 1 – –
4 NCAc 343 NC Bunch 9PI 1216067 2 3 ??
5 Mutant 28-2 EMS mutant of VL 1 2 2 – –
6 DTG 57 TAG 24 9GPBD 4 3 2 – –
7 DTG 60 TG 26 9Mutant 28-2 1 2 – –
8 DTG 58 TG 26 9Mutant 28-2 1 2 – –
9 DTG 27 TG 49 9B 37c 1 1 – –
10 TDG 56 GPBD 4 9TG 49 2 3 ??
11 TFDRG 5 TAG 24 9VG 9514 1 1 – ?
12 TMV 2 Mass selection from Gudhiatham bunch 8 9 ??
13 SB XI Ah 4213 9Ah 4354 8 8 ??
14 JL 24 Selection from EC 94943 9 8 ??
15 TAG 24 TGS 2 9TGE 1 8 9 ??
16 TG 37A TG 25 9TG 26 9 7 ??
17 TG 39 TAG 24 9TG 19 8 9 ??
18 TG 40 TAG 24 9TG 19 8 9 ??
19 TPG 41 TG 28A 9TG 22 8 9 ??
20 TG 42 TG 19 9TG 26 8 9 ??
Rust scores between 1 and 9 indicate rust severity between 0 and 81–100%, respectively
‘?’ and ‘–’ indicate the presence and absence of band of the respective SSR marker in corresponding genotype, respectively
a
Rust reaction is as per Mondal and Badigannavar (2010)
470 Mol Breeding (2012) 29:467–476
123
dependent variable. A parametric discriminant anal-
ysis (PROC STEPDISC, SAS Institute ver. 9.1.3,
forward method, select up to 15 markers, minimum
criteria set with default SLENTRY =0.15) was
performed to identify markers that best differentiated
resistant and susceptible genotypes in RILs. The
nonparametric method within the PROC DISCRIM
procedure (SAS) was used to construct and validate a
class prediction function and to predict group mem-
bership. Further, percent correct classification was
calculated with the crossvalidate option providing a
better assessment of classification accuracy.
Results and discussion
Rust reaction in RIL population
The rust-resistant parent VG 9514 is a Virginia bunch
type (A. hypogaea ssp. hypogaea var. hypogaea) with
red testa (Varman 1999), whereas TAG 24 is a
Spanish bunch type (A. hypogaea ssp. fastigiata var.
vulgaris) with rose testa (Patil et al. 1995). The
mapping population derived from these parents
consisting of 164 RILs was fitted for 1:1 segregation
for both plant habit and testa colour (Table 2). The
expected segregation of these traits in the present
study confirms the suitability of this RIL population
for performing genetic linkage map analysis.
VG 9514 was immune to rust (average score 1.2
with 0–1% disease severity) while TAG 24 was
susceptible (average score 8.8 with 80–100% disease
severity) over the different locations and seasons.
Screening of RILs for rust disease in five environ-
ments revealed that the frequency distribution of
resistant and susceptible plants fitted a 1:1 ratio well
(Table 2). The rust resistance gene in VG 9514 was
introgressed from a diploid wild species, A. cardena-
sii, and this resistance was reported to be controlled
by a single dominant gene (Varman 1999; Mondal
et al. 2007). Earlier reports also described the
dominant or partially dominant nature of rust resis-
tance in crosses involving wild species and wild
derivatives (Singh et al. 1984; Wynne et al. 1991).
Pande and Rao (2001) identified wild Arachis species,
including A. cardenasii, as good sources of rust
resistance genes. Rust reactions in our RIL popula-
tion showed strong (82–96%) positive associations
(P\0.0001) among the five environments (Table 3).
Development and screening of new EST-SSR
markers
Of the 745 newly submitted groundnut EST
sequences (Nobile et al. 2008), only 21 (2.8%) had
SSR motifs. Among them, ten were trinucleotides,
nine were dinucleotides, and one each hexanucleotide
and compound repeat motifs. These 21 SSRs were
tested for amplification and polymorphism (Elec-
tronic Supplementary Material). Although all the 21
primer pairs produced positive PCR amplifications,
only two (Cer 2 and Cer 14) were found to be
polymorphic (9.5%) between VG 9514 and TAG 24.
SSR genotyping in RILs
The availability of greater numbers of genomic and
EST-SSR primer pairs in groundnut allowed us to
screen 1,114 SSR primer pairs between VG 9514 and
TAG 24. Of them, only 105 (9.43%) were found to be
polymorphic based on 3.5–4.0% MetaPhor agarose
gel electrophoresis. The low level of polymorphism
Table 2 Segregation of
rust resistance, plant habit
and testa colour in a RIL
population of cultivated
groundnut
a
E1, E2, E3, E4 and E5 are
the five environments as
described in ‘Materials and
methods’ section
Traits Phenotypic classes Observed
ratio
v
2
(1:1) Pvalue
Plant habit Virginia bunch versus Spanish bunch 76:88 0.88 0.35
Testa colour Red versus Rose 86:78 0.40 0.53
Rust resistance at different environments
a
E1 Resistant versus Susceptible 89:75 1.19 0.27
E2 88:76 0.88 0.35
E3 89:75 1.19 0.27
E4 90:74 1.56 0.21
E5 90:74 1.56 0.21
Mol Breeding (2012) 29:467–476 471
123
in cultivated groundnut was also reported in earlier
studies involving RAPD (Subramanian et al. 2000),
AFLP (Herselman 2003) and SSR markers (He et al.
2003;2005; Krishna et al. 2004). The lack of
polymorphism in cultivated groundnut was attributed
to its recent (based on evolutionary time scale) origin
from a single event of hybridization followed by
chromosome duplication, which created a ploidy
barrier for the transfer of genes from most of its wild
diploid species (Young et al. 1996).
Of the 105 primer pairs, 11 showed dominant
inheritance. The markers gi56931710 and pPGPseq
4A05 amplified the band in TAG 24 whereas the other
nine dominant markers did so only in VG 9514. Of the
105 polymorphic primer pairs, four (TC11A04,
RN13D04, TC6E01 and Ah282) pairs generated dupli-
cate polymorphic markers between the parents. A
segregation pattern of two parental genotypes for most
of the SSR markers among RILs was 1:1 except for 22
(20.2%) markers, which showed segregation distortion
(P\0.05). A large amount of segregation distortion
(about 44%) was detected in the SSR-based linkage map
of an interspecific cross for AA genome (Moretzsohn
et al. 2005), while in cultivated groundnut it was 21.7%
(Hong et al. 2008). Fifteen of the distorted SSR loci
showed an excess of TAG 24 alleles and the remaining
loci had an excess of VG 9514 alleles. Of the 22
distorted loci, seven were mapped to linkage group 7;
two each mapped to linkage groups 3, 12, 14 and 16; one
each to linkage groups 5, 10, 11 and 24; and three loci
remained unlinked. Grouping of distorted markers in
different linkage groups represents real associations
among the segregating loci, since a chromosomal
segment showing distorted segregation will also cause
skewed segregation ratios in neighbouring segments
(Garcia et al. 2005).
Genetic linkage map construction and tagging
of rust resistance gene
The 164 RILs were genotyped with 105 polymorphic
SSR primer pairs resulting in 109 segregating loci (four
primer pairs produced duplicate markers) that were used
to construct a genetic linkage map in the cultivated
groundnut.A linkage map was constructedwith 95 SSR
markers, genes for rust resistance, plant habit and testa
colour that were mapped in 24 linkage groups (Fig. 1),
whereas 14 markers remained unlinked. The linkage
analysis revealed a total Kosambi map distance of
882.9 cM with an average of 9.0 cM between neigh-
bouring markers. Previously, two independent research
groups had developed a SSR linkage map in cultivated
groundnut. In them, 135 SSR loci were mapped in 22
linkage groups which covered a map distance of
1,270.5 cM (Varshney et al. 2009b). Similarly, Hong
et al. (2008), 2010 generated a linkage map of 679 cM
with 131 SSR loci in 20 linkage groups and a composite
linkage map of 885.4 cM, respectively, in cultivated
groundnut. Generation of a saturated map in cultivated
groundnut is restricted by its narrow genetic base and a
limited number of polymorphic markers.
The loci associated with plant habit and testa colour
were located 15.6 cM apart in linkage group 1 with five
SSR markers. Among the five SSRs, S 93 was found
linked to the testa colour gene at 10 cM (Fig 1).
Linkage group 2 had the rust resistance gene flanked by
two SSR markers, pPGPseq4A05 (4.7 cM distant from
the rust resistance gene) and gi56931710 (4.3 cM
distant from the rust resistance gene) (Fig. 1). These
SSR markers were not present in the previously
reported SSR-based linkage maps involving wild and
cultivated groundnut (Moretzsohn et al. 2005; Hong
et al. 2008;Varshneyetal.2009b;Hongetal.2010). In
addition, two other SSR markers, IPAHM 103 and S
17, were also mapped in linkage group 2. Recently,
Khedikar et al. (2010) identified 12 QTL for rust with a
major QTL linked to SSR marker IPAHM 103. This
confirms the linkage of the rust resistance gene with
marker IPAHM 103 in linkage group 2 of cultivated
groundnut in the present study.
SSR marker association based on discriminant
analysis
Multivariate statistics such as discriminant analysis
was used recently to associate categorical molecular
Table 3 Pearson correlations of rust scores of RILs of the
cultivated groundnut among the different environments
E1
a
E2 E3 E4 E5
E1 1.00
E2 0.965** 1.00
E3 0.948** 0.967** 1.00
E4 0.822** 0.847** 0.855** 1.00
E5 0.870** 0.877** 0.902 0.853** 1.00
** Indicates significance at P=0.01
a
E1, E2, E3, E4 and E5 are the five environments as described
in ‘Material and methods’
472 Mol Breeding (2012) 29:467–476
123
marker data with agronomic traits in different crops
(Capdevielle et al. 2000; Fahima et al. 2002; Zhang
et al. 2005; Alwala et al. 2008). For rust resistance,
discriminant analysis identified a minimum of ten of
the most informative SSR markers that could cor-
rectly classify about 99.97% (or\0.03% error rate) of
the genotypes into resistant and susceptible groups
(Table 4). Of the ten most informative markers, the
markers gi56931710, pPGPseq4A05 and IPAHM 103
were mapped in the same linkage group (group 2)
with rust resistance gene in this study (Fig 1). Among
the other informative marker associations, PM 50 and
pPGPseq8E12 were also found significantly associ-
ated with the rust resistance gene (Table 4). Earlier,
Mace et al. (2006) and Mondal and Badigannavar
(2010) independently identified the association of
pPGPseq8E12 and PM 50, respectively, with rust
resistance in cultivated groundnut based on Kruskal–
Wallis one-way ANOVA. Moreover, the present
PM179
TC11D09
S89
TC1E01
S93
testacolor
pltype
1
pPGPseq4A05
rust
gi56931710
IPAHM103
S17
2
AHBGS21003A02*
Ah282_b
Cer2
gi52221650*
3
gi56932168
TC11A04_a
IPAHM165
PM137
4
TC3H07
IPAHM659
PM35
pPGPseq5D03
TC11A04_b
RN34H10
PM377*
5
TC5D06
PM343
EM87
TC1D02
pPGPseq3B12
IPAHM468
6
pPGPseq4E08*
S31*
PM375*
IPAHM219*
IPAHM229*
IPAHM171*
IPAHM407*
7
EM106
EM31
IPAHM287
PM36
gi30420446
PM50
PM183
8
0
10
20
30
40
50
60
70
80
90
IPAHM475
EE16
IPAHM23
gi42560331
IPAHM531
IPAHM176
TC5D06
9
RM14E11
PM3
gi30420088*
pPGPseq2C11
pPGPseq2H08
10
TC6E01_a*
TC6E01_b
Ah4-2 6
TC2B09
Ah242
11
RN32H04*
TC4G10_a*
TC4G10_b
EM30
12
RN13D04_a
RN13D04_b
pPGPseq2E06
13
pPGPseq4E08*
RM3H09*
14
IPAHM123
TC9C08
15
TC11B11*
Ah4-4*
16
0
10
20
30
40
50
60
70
80
90
gi30420405
pPGPseq7G02
lec1
pPGPseq14H06
AHBGS11002C11
17
TC7H11
IPAHM352
pPGPseq4H11
18
TC7E04
TC11F02
19
Ah3
pPGPseq8E12
TC3H02
20
AHBGSc1003E10
pPGPseq2B10
21
TC2D06
pPGPseq16F10
22
PM238
pPGPseq12A07
23
RN9C02*
pPGPseq15C12
24
0
10
20
30
40
50
PM179
TC11D09
S89
TC1E01
S93
testacolor
pltype
1
pPGPseq4A05
rust
gi56931710
IPAHM103
S17
2
AHBGS21003A02*
Ah282_b
Cer2
gi52221650*
3
gi56932168
TC11A04_a
IPAHM165
PM137
4
TC3H07
IPAHM659
PM35
pPGPseq5D03
TC11A04_b
RN34H10
PM377*
5
TC5D06
PM343
EM87
TC1D02
pPGPseq3B12
IPAHM468
6
pPGPseq4E08*
S31*
PM375*
IPAHM219*
IPAHM229*
IPAHM171*
IPAHM407*
7
EM106
EM31
IPAHM287
PM36
gi30420446
PM50
PM183
8
0
10
20
30
40
50
60
70
80
90
IPAHM475
EE16
IPAHM23
gi42560331
IPAHM531
IPAHM176
TC5D06
9
RM14E11
PM3
gi30420088*
pPGPseq2C11
pPGPseq2H08
10
TC6E01_a*
TC6E01_b
Ah4-2 6
TC2B09
Ah242
11
RN32H04*
TC4G10_a*
TC4G10_b
EM30
12
RN13D04_a
RN13D04_b
pPGPseq2E06
13
pPGPseq4E08*
RM3H09*
14
IPAHM123
TC9C08
15
TC11B11*
Ah4-4*
16
0
10
20
30
40
50
60
70
80
90
gi30420405
pPGPseq7G02
lec1
pPGPseq14H06
AHBGS11002C11
17
TC7H11
IPAHM352
pPGPseq4H11
18
TC7E04
TC11F02
19
Ah3
pPGPseq8E12
TC3H02
20
AHBGSc1003E10
pPGPseq2B10
21
TC2D06
pPGPseq16F10
22
PM238
pPGPseq12A07
23
RN9C02*
pPGPseq15C12
24
0
10
20
30
40
50
Fig. 1 A SSR-based linkage map of rust resistance gene in cultivated groundnut. *Indicates the distorted markers that are assigned to
the linkage map
Table 4 Association of SSR markers with rust resistance in
cultivated groundnut as revealed by discriminant analysis
Marker Linkage group Pr [F Wilk’s kPr \k*
gi56931710
a
LG 2 \0.0001 0.290 \0.0001
pPGPseq4A05
a
LG 2 \0.0001 0.247 \0.0001
IPAHM 103
a
LG 2 0.0008 0.230 \0.0001
IPAHM 171a LG 7 0.0027 0.218 \0.0001
RM14E11_1 Unlinked 0.0172 0.210 \0.0001
pPGPseq4C01 LG 14 0.0151 0.202 \0.0001
IPAHM 352 LG 20 0.0312 0.196 \0.0001
PM 50 LG 8 0.0423 0.191 \0.0001
PM 377 LG 5 0.0259 0.185 \0.0001
S 89 LG 1 0.0248 0.179 \0.0001
pPGPseq8E12 LG 22 0.0168 0.166 \0.0001
*k=Wilk’s lambda used to test the significance of the
discriminant model
a
Indicates linkage of these markers with rust resistance gene
as detected through linkage analysis in the present study
Mol Breeding (2012) 29:467–476 473
123
analysis also revealed the association of one unlinked
marker (RM14E11_1), which was previously mapped
in LG_AhXIV in cultivated groundnut (Varshney
et al. 2009b). The other SSR markers associated with
rust resistance through the discriminant analysis
approach could be due to non-random association
of loci (Zhang et al. 2005). Thus both linkage and
discriminant analyses confirmed the tagging of the
rust resistance gene with SSR markers gi56931710,
pPGPseq4A05 and IPAHM 103.
Validation of linked markers
The two linked SSR markers were validated by
testing their amplification in a set of 20 cultivated
groundnut genotypes (Table 1). Among the geno-
types, the linked SSR marker gi56931710 was absent
in all the 11 rust-resistant genotypes except for NCAc
343 and TDG 56, while it was present in all the nine
susceptible genotypes (Table 1). Further, marker
pPGPseq4A05 was also absent in eight of the 11
resistant genotypes (except for NCAc 343, TDG 56
and TFDRG 5) and was present in all the susceptible
genotypes. The non-occurrence of VG 9514 (resis-
tant) allele of gi56931710 and pPGPseq4A05 in the
genotype NCAc 343 can be explained by the exotic
nature of the genotype in the Indian groundnut
breeding programme or by an unrelated genotypic
background. The absence of resistant alleles in TDG
56 and TFDRG 5 may be due to the occurrence of
crossing over between the marker and the resistance
gene during their development (Table 1). The similar
marker reactions in other unrelated resistant geno-
types signify the suitability of gi56931710 and
pPGPseq4A05 in MAS for rust resistance in culti-
vated groundnut.
This result will help groundnut breeders to select
the rust-resistant genotype from the segregating
population even in the absence of a rust disease
nursery or rust epiphytotics, and will further help to
introgress the rust resistance gene from wild acces-
sions to elite groundnut varieties.
Acknowledgments The authors thank Dr. Ashok M.
Badigannavar for help in discriminant analysis and critical
comments. Technical help from Ramani, Pratibha and Shakthi
during their project work at our laboratory is duly acknowledged.
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