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crop science, vol. 57, m ay–june 2017 www.crops.org 1121
ReseaRch
P or groundnut (Arachis hypogaea L.), an important self-
pollinating tetraploid (AA BB, 2n = 4x = 40) oilseed crop, is
grown in more than 108 countries representing tropical, subtropical
and warm temperate regions of the world, extending from 40° N
to 40° S. It ranks sixth among the oilseed crops and is cultivated on
25.7 million ha area, with a total production of 42.4 million t and
average productivity of 1.65 t ha−1 globally (FAO, 2014). Peanut is
mainly cultivated for its seeds, which are rich in oil, protein, min-
erals, and vitamins and are consumed in a variety of forms. About
two-thirds of global produced peanut is crushed for extracting veg-
etable oil, whereas the remaining one-third is used in the form of
edible products. Peanut cake obtained after oil extraction is used as
protein-rich meal for livestock or for making other food products.
The peanut production is adversely aected by biotic stresses
such as diseases [rust (Puccinia arachidis Speg.), early leaf spot (Cerco-
spora arachidicola S. Hori), late leaf spot [LLS; Phaeoisariopsis personata
(Berk. & M.A. Curtis) Arx], peanut bud necrosis virus, rosette dis-
ease, and bacterial wilt (Pseudomonas solanacearum (Smith) Smith)],
insect pests [leaf miner (Aproaerema modicella Deventer), tobacco
caterpillar/tobacco armyworm (Spodoptera litura Fab.), cotton leaf-
worm (Spodoptera littoralis Boisduval), termites (Microtermes spp.,
Odontotermes spp., Macrotermes spp., Ancistrotermes latinotus Hol-
gren), corn earworm (Helicoverpa zea Bodd ie), lesser cornstalk
borer (Elasmopalpus lignosellus Zeller) and southern corn rootworm
(Diabrotica undecimpunctata howardi Barber)], abiotic stresses (drought,
Harnessing Genetic Diversity of Wild Arachis
Species for Genetic Enhancement
of Cultivated Peanut
Shivali Sharma,* Manish K. Pandey, Hari K. Sudini, Hari D. Upadhyaya, and Rajeev K. Varshney
ABSTRACT
Peanut (Arachis hypogaea L.) is an important
self-pollinating tetraploid (AABB, 2n = 4x = 40)
legume grown for the high-quality edible oil and
easily digestible protein in its seeds. Enormous
genetic variability is present in the genus Arachis
containing 79 wild species and cultivated
peanut. Wild species offer signicant variability,
particularly for biotic and abiotic stresses, and
can be used to develop cultivars with enhanced
levels of resistance to key stresses. However,
utilization of these species requires use of ploidy
manipulations, bridge crosses, and embryo or
ovule rescue. For efcient use of diploid wild
species from section Arachis, several synthetics
(amphidiploids and autotetraploids) have been
developed using A- and B-genome accessions
with high levels of resistance to multiple
stresses. These synthetics are used in crossing
programs with cultigens to develop prebreeding
populations and introgression lines (ILs) with
high frequency of useful genes and alleles
into good agronomic backgrounds. Evaluation
of two such populations derived from ICGV
91114 ´ ISATGR 121250 (a synthetic derived
from A. duranensis Krapov. & W.C. Greg. ´
A. ipaensis Krapov. & W.C. Greg.) and ICGV
87846 ´ ISATGR 265-5 (A. kempf-mercadoi
W.C. Greg. & C.E. Simpson ´ A. hoehnei Krapov.
& W.C. Greg.) resulted in the identication of ILs
with high levels of late leaf spot (LLS) and rust
resistance and signicant genetic variability
for morphoagronomic traits. Genotyping of
these ILs with markers linked to rust and LLS
resistance provided evidence that introgression
of possible novel alleles and resistance sources
from different wild species other than the
commonly used A. cardenasii Krapov. & W.C.
Greg. will be benecial for peanut improvement.
S. Sharma, M.K. Pandey, H.K. Sudini, H.D. Upadhyaya, and R.K.
Varshney, ICRISAT, Patancheru-502324, Hyderabad, India. Received
10 Oct. 2016. Accepted 2 Feb. 2017. *Corresponding author (Shivali.
sharma@cgiar.org). Assigned to Associate Editor Zhanguo X in.
Abbreviations: DAS, day s after sow ing; g ´ e, genot ype ´ env ironment;
IL, introgression line; LLS, late leaf spot; RBD, randomized block
design; REML, residual maximum likelihood.
Published in Crop Sci. 57:1121–1131 (2017).
doi: 10.2135/cropsci2016.10.0871
© Crop Science Societ y of America | 5585 Guilford Rd., Madison, WI 53711 USA
This is an open access article distr ibuted under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Published online June 16, 2017
1122 www.c rops .org crop scie nce, v ol. 57, may–ju ne 2017
salinity, and high temperature), and aatoxin contamina-
tion. Among diseases, foliar fungal diseases such as LLS and
rust are widespread and major constraints for production,
resulting in yield losses and poor seed quality (Subrah-
manyam et al., 1980; McDonald et al., 1985; Miller et al.,
1990; Grichar et al., 1998). The severity of other diseases
varies across regions. For instance, peanut bud necrosis virus
is important in South Asia, rosette disease in Africa, and
bacterial wilt in Southeast Asia. Both LLS and rust, due
to chlorotic lesions, result in the reduction of the green
leaf area available for photosynthesis and stimulate leaet
abscission leading to extensive defoliation (McDonald et
al., 1985), thereby aecting the seed quantity, quality, and
fodder value of the plants (Gupta et al., 1987).
Although chemical control measures are available to
control these diseases, they increase the cost of production
and thus are not economical to the smallholder farmers.
Host-plant resistance to develop resistant cultivars is the
most eective and economic way to minimize the yield
losses due to these diseases. Breeding eorts have led to
the development of high-yielding peanut cultivars with
moderate levels of resistance to LLS. However, develop-
ing new high-yielding cultivars with high levels of LLS
resistance and acceptable market traits remains a focus for
peanut improvement globally. Although sources of resis-
tance have been identied for foliar diseases, complete or
high levels of resistance to these diseases are not available
in cultivated genepool (Waliyar et al., 1993; Singh et al.,
1997; Fávero et al., 2009; Sudini et al., 2015). The genus
Arachis, containing 80 species classied into nine sections,
provides sucient genetic variability and new and diverse
sources of resistance genes, such as immune to highly resis-
tance sources for LLS and rust (Subrahmanyam et al., 1985;
Stalker and Moss, 1987; Pande and Rao, 2001; Fávero et al.,
2009; Michelotto et al., 2015) for peanut improvement. A
few interspecic derivatives with high levels of LLS resis-
tance were developed, most of which carry LLS resistance
derived from A. cardenasii Krapov. & W.C. Greg. Keeping
in mind the frequent breakdown of foliar disease resistance,
such as the breakdown of the Yr17 gene conferring resis-
tance against yellow rust (caused by Puccinia striiformis f. sp.
tritici) in wheat (Triticum aestivum L.) cultivars (Bayles et al.,
2000; El-Jarroudi et al., 2011), it would be necessary to
include other species as donors to broaden the genetic base
of foliar disease resistance in peanut.
In the genus Arachis containing cultivated peanut,
its tetraploid progenitor A. monticola Krapov. & Rigoni,
and 29 diploid wild Arachis species (including two diploid
progenitor species, A. ipaensis Krapov. & W.C. Greg. and
A. duranensis Krapov. & W.C. Greg.) (Krapovickas and
Gregory, 1994; Valls and Simpson, 2005) is of particu-
lar interest to peanut breeders, as the wild species in this
section are crossable with cultivated peanut. However,
frequent use of these wild species for peanut improvement
is hindered due to the dierences in ploidy levels. Such
crossing programs involving tetraploid cultivated peanut
and diploid wild Arachis species would require several
generations of selng in the segregating material to select
desirable tetraploid recombinants. To overcome these
diculties, synthetic peanuts have been developed by
doubling the chromosome number of the hybrid derived
from two diploid (2n = 2x = 20) wild Arachis species
(Mallikarjuna et al., 2011). These tetraploid synthetic pea-
nuts (2n = 4x = 40) can be used freely in the crossing
programs to transfer useful genes and alleles from wild
species into cultivated genetic backgrounds. In previous
studies, the development and use of synthetic amphidip-
loids such as TxAG-6 (Simpson et al., 1993) in breeding
programs has resulted in the release of two cultivars, Coan
(Simpson and Starr 2001) and NemaTAM (Simpson et al.,
2003), carrying genes for root-knot nematode (Meloido-
gyne arenaria) resistance from A. cardenasii (Simpson and
Starr, 2001, Simpson et al., 2003), as well as led to the
development of backcross progenies having high yield
and higher seed weight (up to 95 g) (Upadhyaya 2008).
Use of the synthetics derived from dierent wild Arachis
species having resistance or tolerance to important biotic
and abiotic stresses would not only help in diversifying
the sources of resistance but would also enable the pyra-
miding of resistance genes from dierent species into a
common genetic background to develop new cultivars
with enhanced levels of resistance or tolerance to stresses.
The present investigation was undertaken to use syn-
thetics derived from LLS- and rust-resistant diploid wild
Arachis accessions for introgressing high-level resistance
into peanut cultivars. The objectives of the study were to
develop introgression lines (ILs) having high levels of foliar
disease resistance and to diversify the sources of resistance
in cultivated backgrounds for peanut improvement. The
present study holds a great signicance in enhancing and
diversifying the sources of LLS and rust resistance using
synthetics derived from dierent diploid wild Arachis spe-
cies. Such an attempt provides sucient useful genetic
variability in peanut breeding pipelines to develop new
cultivars with a broad genetic base and enhanced levels
of foliar disease resistance, especially for diversifying LLS
resistance derived from dierent wild species other than
the commonly used A. cardenasii.
MATERIALS AND METHODS
Plant Material and Population Development
Two peanut cultivars, ICGV 91114 and ICGV 87846, and two
synthetic tetraploids were used in this study. The amphidiploid
ISATGR 121250 (AABB, 2n = 4x = 40) (Mallikarjuna et al.,
2011) was derived from a cross between LLS- and rust-resistant
A-genome species A. duranensis (ICG 8123) and B-genome spe-
cies A. ipaensis (ICG 8206) (Subrahmanyam et al., 1985; Pande
and Rao, 2001). The autotetraploid ISATGR 265-5 (AAAA;
crop science, vol. 57, may–jun e 2017 www.crops.org 1123
the crop growth. Subsequent irrigations were provided as and
when needed in the rainy season. At 30 DAS, LLS- and rust-
infected potted plants of TMV 2 from greenhouse were placed
randomly throughout the infector rows of the experimental plot.
Further, articial inoculation was done at 50 DAS by spraying
the test plants and infector rows with a conidial suspension of
LLS and urediniospores of rust pathogens at a concentration of 5
´ 104 spores mL−1 to ensure uniform and heavy disease pressure
in the experimental plot. After inoculation, sprinkler irrigation
was provided daily, for 30 min in the evening for 30 d, to create
a congenial environment for disease development. Obser vations
on diseases score for LLS and rust were recorded on 75 and 90
DAS on each row following a 1-to-9 scale (where 1 = no disease
and 9 = 81–100% disease severity), as described by Subrahman-
yam et al. (1995). Besides test material, t wo synthetics and four
wild parents were also screened under eld as well as greenhouse
conditions for conrming LLS and rust resistance. The synthet-
ics and wild Arachis parents have small pods, and it is dicult
to plant them directly in the eld along with cultivated mate-
rial. Therefore, plants of each synthetic and its wild parent were
raised in pots with one plant per pot. For each synthetic and wild
parent, ve pots were used for screening. These pots were placed
at random in rows in the eld, along with the test material, in
the 2014 rainy season for screening against LLS and rust. Besides
eld screening, these synthetics and their diploid wild parents
were also screened for LLS in the greenhouse in the 2014 rainy
season, following similar methodology and two to three arti-
cial inoculations at a weekly inter val to ensure very high disease
pressure. Observations on disease score in the greenhouse were
recorded 75 d after rst inoculation following a 1-to-9 scale. The
genotypes were classied as resistant (score of 1–3), moderately
resistant (score of 4–5), susceptible (score of 6–7), and highly sus-
ceptible (score of 8–9) (Sudini et al., 2015).
The ILs in both populations had one or more undesir-
able traits, such as procumbent growth habit, late owering
and maturity, and small, highly beaked and reticulated pods.
Therefore, within LLS- and rust-resistant ILs (score 2.0–3.0),
further selection was made to select the plants with the lowest
LLS and rust scores coupled with acceptable plant type, such as
erect growth habit, early maturity, and acceptable pod traits.
Using these criteria, 17 LLS- and rust-resistant ILs with good
agronomic backgrounds were selected. Single-plant progenies
of these selected 17 ILs were reevaluated to conrm resistance
during the 2015 and 2016 rainy seasons in a randomized block
design (R BD) with three replications, along with four peanut
cultivars, GPBD 4, ICG 1311, J 11, and JL 24, as checks using the
infector-row technique. The methodolog y used in the 2014 rainy
season screen was followed to conrm resistance under similar
eld conditions in both years. The plot size was a two-row plot
per genotype in the 2015 rainy and a four-row plot per genotype
in the 2016 rainy season. Each row was 4 m long in a ridge-fur-
row system. The infector row, TMV 2, was repeated after every
ve rows (Fig. 1). In both seasons, observations on diseases score
were recorded on 75 and 90 DAS for each genot ype in each rep-
lication, following a 1-to-9 scale as described by Subrahmanyam
et al. (1995). In the 2015 rainy season, besides disease score, data
were also recorded for morphoagronomic traits on ve randomly
selected competitive plants per genotype per replication. Data
on days to rst owering, days to 50% owering, and growth
2n = 4x = 40) was derived from a cross between two LLS- and
rust-resistant A-genome species, A. kempf-mercadoi (ICG 8164)
and A. hoehnei (ICG 8190) (Subrahmanyam et al., 1985). ICGV
91114 is a Spanish bunch-type (A. hypogaea ssp. fastigiata var.
vulgaris), widely adapted, high-yielding, drought-tolerant, LLS-
and rust-susceptible, early-maturing (90–95 d) peanut cultivar.
ICGV 87846 is a Virginia bunch-type (A. hypogaea ssp. hypo-
gaea var. hypogaea), drought-tolerant, moderately resistant to
LLS (score 5.0 at 90 d after sowing [DAS]) and rust (score 4.0
at 90 DAS), dual-purpose cultivar having high pod yield and
released as Co 6 in Tamil Nadu, India. Using these two peanut
cultivars as recipients and synthetics as donors for LLS and rust
resistance, two F1 crosses, ICGV 91114 ´ ISATGR 121250
and ICGV 87846 ´ ISATGR 265-5, were generated. Both
F1 crosses were backcrossed twice with respective cultivated
recipient parents to generate two advanced backcross (BC2F1)
populations, followed by selng for three seasons to generate
BC2F4 populations. The BC2F4 population derived from ICGV
91114 ´ ISATGR 121250 containing 437 ILs was designated as
Pop I, and the population from ICGV 87846 ´ ISATGR 265-5
containing 598 ILs was designated as Pop II.
Evaluation for LLS, Rust,
and Yield-Related Traits
Pop I containing 437 ILs, cultivated parent ICGV 91114, Pop
II containing 598 ILs, and cultivated parent ICGV 87846 were
screened in a disease screening nursery in an Alsols (Alsol-
Patancheru soil series: Udic Rhodustolf) precision eld under an
infector row system during the 2014 rainy season at ICRISAT,
Patancheru, India. The disease screening experiments were con-
ducted in augmented designs. Six peanut cultivars—GPBD 4,
ICGS 76, J 11, JL 24, Tifrunner, and ICG 1311—were used as
checks, and each check was repeated three times after 25 entries
in Pop I and 34 entries in Pop II. Of these, the GPBD 4 is LLS
and rust resistant and is used as a national resistance check for both
the diseases in eld tr ials of the All India Coordinated Research
Projects (AICRP) on peanut. This cultivar was derived from the
cross K RG 1 ´ CS 16 (ICGV 86855) and is a second-cycle deriv-
ative of interspecic hybridization between A. hypogaea and A.
cardenasii. ICGS 76 is a high-yielding, medium-duration cultivar
with moderate resistance to LLS (score 5.0) and rust (score 4.0); J
11 and JL 24 are high-yielding, short-duration cultivars and both
are susceptible to LLS and rust; ICG 1311 is a germplasm line sus-
ceptible to LLS and moderately resistant to rust; and Tifrunner is
a runner cultivar and is moderately resistant to both LLS and rust
(score 5.0). In each population, ILs, cultivated parents, and checks
formed the test material. After every ve rows of test material,
an infector row of susceptible cultivar TMV 2 (national suscep-
tibility check for LLS and rust in eld trials of the AICRP on
peanut) was planted to ensure uniform spread of disease inocu-
lum. In both populations, the plot size was a single 4-m-long row
per genotype in a ridge-furrow system. Row-to-row distance
was 60 cm, and plant-to-plant distance within a row was 10 cm.
Standard package of practices were adopted to raise a healthy
crop that included 60 kg P2O5 as basal application, seed treatment
with Mancozeb at 2 g kg−1 seed before sowing, pre-emergence
application of Pendimethalin at 1 kg a.i. ha−1, irrigation soon
after planting, g ypsum application at 400 kg ha−1 at the peak
owering stage, and protection against insect pests throughout
1124 www.c rops .org crop scie nce, v ol. 57, may–ju ne 2017
habit were recorded on plot basis, while branching pattern,
number of pods per plant, pod yield per plant, number of seeds
per plant, seed yield per plant, 100-seed weight, shelling per-
centage, pod reticulation, pod constriction, and pod beak were
recorded on ve selected plants. Morphoagronomic performance
of these selected 17 ILs, along with four peanut cultivars (GPBD
4, ICG 1311, J 11, and JL 24) as checks, was reevaluated during
the 2015–2016 post-rainy season in RBD with three replications,
following the same methodology under similar eld conditions.
DNA Isolation and Genotyping with Linked
Markers to LLS and Rust Resistance
Fresh leaves from 25-d-old seedlings of the selected 17 ILs were
collected, and DNA was extracted using a modied cetyltri-
methylammonium bromide (CTAB) extraction method (Cuc
et al., 2008). After isolation of DNA, its quality and quantity
was checked on 0.8% agarose gels, and DNA concentration,
followed by normalization to ~5 ng ml−1, was used in geno-
typing with linked markers. IPAHM103, GM1536, GM2301
and GM2079 for both rust and LLS resistance and Seq8D09,
GM1009, GM1573, and GM2032 for LLS resistance were used
for genotyping the selected ILs. These markers were used for
amplication with polymerase chain reaction (PCR) follow-
ing conditions mentioned in Khedikar et al. (2010) and Sujay
et al. (2012). Genotyping of eight linked markers for LLS and
rust resistance was performed on a total 17 selected ILs in the
genetic backgrounds of ICGV 91114 (PBGNIL-1, PBGNIL-
2, PBGNIL-3, PBGNIL-4, PBGNIL-5, and PBGNIL-6) and
ICGV 87846 (PBGNIL-7, PBGNIL-8, PBGNIL-9, PBGNIL-
10, PBGNIL-11, PBGNIL-12, PBGNIL-13, PBGNIL-14,
PBGNIL-15, PBGNIL-16, and PBGNIL-17). In addition to
the above ILs, two cultivated parents (ICGV 91114 and ICGV
87846), two synthetics (ISATGR 121250 and ISATGR 265-5),
four diploid wild Arachis accessions (ICG 8164, ICG 8190, ICG
8123, and ICG 8206), and four peanut cultivars ( J 11, JL 24, ICG
1311, and GPBD 4) were also included in the genotyping panel.
Statistical Analysis
The disease score data for LLS and rust for Pop I and Pop II
screened in the 2014 rainy season and the replicate-wise data on
disease score of LLS and rust and agronomic traits, such as days
to rst owering, days to 50% owering, number of pods per
plant, pod yield per plant, number of seeds per plant, seed yield
per plant, 100-seed weight, and shelling percentage, were used
for statistical analysis of each environment and season using the
residual maximum likelihood (REML) method and consider-
ing genotypes as random eects using GenStat software (VSN
International, 2015). Variance components due to genotypes
(s2
g) and their standard errors were determined. Environment-
wise best linear unbiased predictors for the genotypes were
calculated. The signicance of variance components was tested
using respective standard errors. For the pooled analysis, seasons
were considered as xed eects. The variances due to genotype
(s2
g) and genotype ´ environment (g ´ e) interaction (s2
g ´ e)
and their standard er rors were determined. The signicance of
environment was assessed using the Wald statistic that asymp-
totically follows a c2 distribution.
RESULTS
Late Leaf Spot Resistance
The REML analysis indicated signicant variations
(P £ 0.05) for LLS resistance among genotypes in
Pop I at 90 DAS and in Pop II at both stages (75 and
90 DAS) (Table 1). In both populations, most of the ILs
and all control cultivars had a disease score of £3.0 at 75
DAS, whereas the disease score was greater as the plants
grew older, and considerable variability was observed at
90 DAS. At 90 DAS in Pop I, only 18 ILs were found
resistant (score 2–3) and 260 ILs were found moderately
resistant (score 4–5) (Table 2). This was probably due
to the increase of inoculum rather than the age of the
plants (Sudini et al., 2015). Similarly, in Pop II at 90 DAS,
335 ILs were found resistant (score 2–3) and 238 ILs were
Fig. 1. Confirmation of late leaf spot and
rust resistance in selected introgression
lines and the resistant and susceptible
control cultivars using an infector-row
technique during the 2016 rainy season
at ICRISAT, Patancheru, India.
crop science, vol. 57, may–jun e 2017 www.crops.org 1125
cultivars, GPBD 4 was resistant (score 2–3); ICGS 76, Tif-
runner, and ICG 1311 were found moderately resistant
(score 4–5); and the remaining all were susceptible (score
6–7) in both populations. The cultivated parent ICGV
91114 was susceptible (score 6) and ICGV 87846 was found
moderately resistant (score 4). Synthetics ISATGR 1212 and
ISATGR 265-5 and their diploid parents, ICG 8123 and
ICG 8206 and ICG 8164 and ICG 8190, respectively, were
highly resistant for rust (score 1–2).
Confirmation of LLS and Rust Resistance
Given the disease score and morphoagronomic traits, such
as growth habit and pod traits, six ILs from Pop I and
11 ILs from Pop II possessing low disease score for LLS
(score 2–3) and rust (score 1–3) at 90 DAS were selected
for rescreening to conrm the resistance. The REML
analysis indicated signicant variation among genotypes
for LLS and rust resistance at both stages in both years
separately, as well as in pooled data (P £ 0.05) (Table 3).
Signicant g ´ e interactions were observed for LLS and
rust. Given the pooled disease score, LLS and rust resis-
tance were conrmed in all of these selected accessions at
75 and 90 DAS, wherein 15 ILs were found resistant for
LLS (score 2–3 at both 75 and 90 DAS) and rust (score
1–3 at both 75 and 90 DAS) (Fig. 2), and two ILs (IL
PBGNIL-2 and IL PBGNIL-6) were moderately resistant
to LLS and rust (Table 4). The best LLS- and rust-resistant
found moderately resistant (score 4–5) (Table 2). Among
the control cultivars, GPBD 4 was resistant (score 2.0–3.0),
ICGS 76 and Tifrunner were moderately resistant (score
5.0), and the remaining were susceptible (score 6–7). The
cultivated parent ICGV 91114 was susceptible (score 6) and
ICGV 87846 was moderately resistant (score 5). Synthet-
ics ISATGR 1212 and ISATGR 265-5 and their diploid
parents (ICG 8123 and ICG 8206 and ICG 8164 and ICG
8190, respectively) were found resistant for LLS (score 2–3)
under eld conditions. Under very high disease pressure
in the greenhouse, however, ISATGR 265-5 was moder-
ately resistant (score 5.0), whereas its diploid wild parents,
ICG 8164 (score 3.0) and ICG 8190 (score 2.0), continued
to be LLS resistant. Another synthetic, ISATGR 121250
(score 5.0), and its diploid wild parents, ICG 8123 (score
5.0) and ICG 8206 (score 4.0), were found moderately
resistant under greenhouse conditions.
Rust Resistance
For rust resistance, REML analysis indicated signicant
variation (P £ 0.05) among the genotypes in Pop I and
Pop II at both stages (Table 1). In both populations, disease
score varied from 1.0 to 4.0 at 75 DAS. At 90 DAS, 240 ILs
in Pop I and 573 ILs in Pop II were found resistant (score
1–3), 164 ILs in Pop I and 24 ILs in Pop II were moderately
resistant (score 4–5), and the remaining ILs in Pop I and
Pop II were susceptible (Table 2). Among the control
Table 1. Variance components due to peanut genotypes (s2
g), and their SEs for late leaf spot (LLS) and rust in Pop I and Pop II,
screened during the 2014 rainy season at ICRISAT, Patancheru, India.
Disease Stage
Pop I Pop II
Range Mean s2
gSE Range Mean s2
gSE
DAS†
LLS 75 1–4 2.6 0.0 9 0.055 2–3 2.0 0.02* 0.0 04
90 2–8 5 .1 0.50* 0 .13 4 2–7 3.4 1.01* 0 .105
Rust 75 1–4 2.2 0.16 * 0.039 1– 3 1.1 0.07* 0.0 08
90 2–7 3.6 0.68* 0.235 1–7 1. 9 0. 51* 0.049
* Significant at the 0.05 probability level.
† DAS, days after sowing.
Table 2. Number of introgression lines (ILs) having late leaf spot (LLS) and rust resistance in Pop I and Pop II, screened during
the 2014 rainy season at ICRISAT, Patancheru, India.
Population
No. of ILs
screened Disease Stage Resistant
Moderately
resistant Susceptible
Highly
susceptible
DAS†
Pop I 437 LLS 75 431 (1–3, 2.6)‡6 (all 4.0) – –
90 18 (2–3, 2.9) 260 (4–5, 4.7) 156 (6–7, 6.1) 3 (all 8.0)
Rust 75 431 (1– 3, 2.2) 6 (all 4.0) – –
90 240 (2–3, 2.6) 164 (4–5, 4.4) 33 (6–7, 6.2) –
Pop II 598 LLS 75 598 (2–3, 2.0) –––
90 335 (2–3, 2.6) 238 (4–5, 4.3) 25 (6–7, 6.1) –
Rust 75 598 (1–3, 1.1) – – –
90 573 (1–3, 1.8) 24 (4–5, 4.0) 1 ( 7. 0 ) –
† DAS, days after sowing.
‡ Range an d average disease score given in the pare nthesis.
1126 www.c rops .org crop scie nce, v ol. 57, may–ju ne 2017
control cultivar, GPBD, was also found resistant at 75 and
90 DAS (Table 4) (Fig. 2).
Agronomic Evaluation
The REML analysis indicated signicant (P £ 0.05) vari-
ations among genotypes for days to rst owering in both
seasons separately, as well as in the pooled data (Table 5).
In the 2015 rainy season, genotypic variance (s2
g) was
signicant for most of the traits except 100-seed weight
and shelling percentage, whereas in the 2015–2016 post-
rainy season, s2
g was signicant for four traits (days to
rst owering, days to 50% owering, number of pods per
plant, and 100-seed weight) (Table 5). The Wald statistics
indicated a nonsignicant eect of seasons for most of the
traits except seed yield per plant, 100-seed weight, and
shelling percentage. Signicant g ´ e interactions were
observed for most of the traits except 100-seed weight and
shelling percentage.
Agronomic performance of these selected LLS- and
rust-resistant ILs was compared with the best LLS-resis-
tant control cultivar, GPBD 4. Promising ILs with superior
performance over GPBD 4 for various yield-related traits
in the 2015 rainy and 2015–2016 post-rainy seasons and
across seasons is given in Table 6. Most of the ILs performed
better than or at par with the control cultivar. In the 2015
rainy season, seven ILs owered at par with GPBD 4, of
which two ILs (PBGNILs 2 and 4) owered earlier than
GPBD 4 on per-se basis. Five ILs (PBGNILs 7, 11, 15,
1, and 8) were signicantly superior (36–43 pods plant−1)
to GPBD 4 (26 pods) for pod number, one (PBGNIL-1)
for seed number (53 vs. 36 seeds plant−1 in GPBD 4), and
one (PGNIL-10) for 100-seed weight (51.5 vs. 35 g in
Table 3. Variance components due to genotype (s2
g), genotype ´ environment (s2
g ´ e) interactions, and their standard errors
(SE) for late leaf spot (LLS) and rust in selected introgression lines rescreened during the 2015 and 2016 rainy seasons at
ICRISAT, Patancheru, India.
Tra i t s Stage
2015 r ain y 2016 rai ny Pooled
s2
gSE s2
gSE s2
gSE s2
g ´ e SE
DAS†
LLS 75 1.01* 0.330 1. 02 * 0.346 0.84* 0.304 0 .16* 0.072
90 1.61* 0.535 2.54* 0.825 1.73 * 0.616 0.35* 0 .1 3 4
Rust 75 0.54* 0.1 8 2 0. 91* 0.297 0.56* 0.210 0 .16* 0.063
90 1.92* 0.612 2.8 6* 0.916 1. 88* 0.684 0. 51* 0 .16 9
* Significant at the 0.05 probability level.
† DAS, days after sowing.
Fig. 2. Symptoms of late leaf spot and rust resistance in the introgression line (top and bottom left) and the resistant (bottom middle) and
susceptible control cultivars (bottom right) at ICRISAT, Patancheru, India.
crop science, vol. 57, may–jun e 2017 www.crops.org 1127
Table 4. Disease reaction and allelic pattern for selected introgression lines and cultivated and wild parents for late leaf spot
(LLS) and rust at ICRISAT, Patancheru, India.
Genotypes/
introgression lines Pedigree information
LLS disease reaction
Rust disease
reaction
Linked markers for rust
and LLS resistance†
LLS 75
DAS‡
LLS 90
DAS
Rust 75
DAS
Rust 90
DAS GM1536 GM2079 SEQ8D09
————————— 1 – 9 s c a l e § —————————
IC GV 91114 72-R Virginia ´ Chico) F2-P1-B1-NIB1-
B1-B1-NIB1-B1-B1-B1-B1-B1) ´
[( R o b u t 33-1-18 -17 ) ´ NC Ac 1705]
3 6 4 6 − − −
IS ATG R 12125 0 ICG 8123 ´ ICG 8206 3 5 1 2 − − +
IC G 8123 A. duranensis 2511
ICG 8206 A. ipaensis 2 5 1 1 − −
PBGNIL-1 IC GV 91114 ´ [IC GV 91114 ´
(IC GV 91114 ´ 265-5)]
2 3 2 2 + + +
PBGNIL-2 4 5 3 4 − − −
PBGNIL-3 2 3 2 2 + + +
PBGNIL-4 2 3 2 2 + + +
PBGNIL-5 2 3 2 2 + + +
PBGNIL-6 3 5 2 4 − + −
ICGV 878 46 CS 9 ´ [(Robut 33-1 ´ NC Ac 316)
F2-B2-B1-B1-NIB1-B1-B1-B1-B1-B1-
B1-B1)]
4 5 3 4 − −
ISATGR 265-5 ICG 8164 ´ ICG 819 0 3 5 1 1 − − +
IC G 816 4 A. kempf-mercadoi 2311 +
IC G 819 0 A. hoehnei 2211 +
PBGNIL-7 ICGV 87846 ´ [ICGV 87846 ´ (ICGV
87846 ´ 26 5-5)]
2 2 1 2 + + +
PBGNIL-8 2 2 1 2 + + +
PBGNIL-9 2 3 2 2 + + +
PBGNIL-10 2 3 1 2 + + +
PBGNIL-11 2 2 1 2 + + +
PBGNIL-12 2 3 2 2 + + +
PBGNIL-13 2 3 2 2 + + +
PBGNIL-14 2 3 2 2 + + +
PBGNIL-15 2 3 2 2 + + +
PBGNIL-16 2 3 1 2 + + +
PBGNIL-17 2 3 1 2 + + +
GPBD4 KRG 1 ´ CS 16 (ICGV 86855) 2 3 2 2 + + +
J11 Ah 4213 ´ Ah 4354 5 6 3 6 − − −
JL 24 Selection from EC 94943 (introduction
fr o m Taiw a n)
5 7 3 6 − − −
IC G 13 11 A germplasm line 5 6 3 5 − − +
† (−) indicates s uscepti ble alle le simil ar to JL 24, (+) indicates resistance allele s imilar to GPBD4, and no sy mbol indicates a different allele than JL 24 and GPBD 4
‡ DAS, days after sowing.
§ Diseas e reactio n score, where 1 = no dise ase and 9 = 81–100% diseas e severit y.
Table 5. Variance components due to genotypes (s2
g), genotype ´ environment (s2
g ´ e) interactions and their standard errors
(SE) for yield-related traits in the selected introgression lines evaluated during the 2015 rainy and 2015–2016 post-rainy seasons
at ICRISAT, Patancheru, India.
Tra i t
2015 rainy season
2015–2016 post-rainy
season Pooled
s2
gSE s2
gSE s2
gSE s2
g ´ e SE
Days to first flowering 4.29* 1.434 1.10 * 0.528 1.12 * 0.537 1.6 2 * 0.401
Days to 50% flowering 7. 4 6 * 2.427 1.3 4* 0.647 1.3 0 0.69 0 2.79* 0.599
No. of pods per plant 83.08* 30.280 65.30* 26.570 26.57 15 .70 0 58.97* 16.200
Pod weight per plant (g) 43.03* 17.6 70 11.74 8.990 1. 96* 5.030 25.66* 9.340
No. of seeds per plant 116.8 0 * 48.500 79.40 42.000 36.2 25.600 88.00* 3 3 .10 0
Seed weight per plant (g) 18.47* 8.000 4.92 3.210 2.47 2.630 8.59* 3.940
100-seed weight (g) 55.50 29.3 00 22.43* 9.770 23.66 12 .5 4 0 6.75 10.810
Shelling (%) 7.74 9.970 22.98 16 .4 00 24 .01 12.3 9 0 6.32 13. 09 0
* Significant at the 0.05 probability level.
1128 www.c rops .org crop scie nce, v ol. 57, may–ju ne 2017
GPBD 4). Similarly, in the 2015–2016 post-rainy season,
almost all of the ILs performed better than or similar to
GPBD 4, and a few ILs were signicantly better than the
control cultivar. For example, three ILs (PBGNILs 4, 13,
and 5) were signicantly superior (38–53 pods plant−1 ) to
GPBD 4 (27 pods) for pod number, and one (PBGNIL-
4) for seed number (62 vs. 39 seeds plant−1 in GPBD 4)
(Table 6). In combined analysis, seven ILs (PBGNILs 11,
4, 13, 1, 7, 15, and 8) were signicantly better than GPBD
4 for pod number (32–40 vs. 26 pods plant−1 in GPBD 4),
one (PBGNIL-13) for pod yield per plant (25 vs. 19 g in
GPBD 4), one (PBGNIL-1) for seed number per plant (49
vs. 37 seeds plant−1 in GPBD 4), one (PBGNIL-13) for seed
yield per plant (17 vs. 12 g in GPBD 4), and one (PBGNIL-
10) for 100-seed weight (42 vs. 31 g in GPBD 4).
Confirmation of Resistance Alleles and
Identification of Novel Alleles for Resistance
Eight linked markers to LLS and rust resistance were
genotyped among a set of 29 genotypes to check the
loci variation for these marker alleles among the newly
developed ILs. Three markers—GM1536, GM2079, and
Seq8D09—gave clear peak pattern and amplied in all
genotypes. The markers GM1536 and GM2079 were
found to control both the foliar fungal diseases, whereas
the marker Seq8D09 represents another genomic region
controlling only LLS resistance (Sujay et al., 2012). The
markers GM1536, GM2079, and Seq8D09 produced resis-
tance alleles of 473, 403, and 132 bp, respectively, in GPBD
4, a resistant parent of the mapping population (TAG 24
´ GPBD 4) used earlier to conduct genetic mapping. A
majority of the ILs had similar markers alleles to GPBD
4 except two ILs, PBGNIL-2 and PBGNIL-6. The IL
PBGNIL-2 showed moderate resistance to both the dis-
eases but carried susceptible alleles (485 bp from GM1536,
409 bp from GM2079, and 135 bp from Seq8D09). Simi-
larly, another IL, PBGNIL-6, carried susceptible alleles
for markers GM1536 and Seq8D09 and a novel allele of
418 bp from marker GM2079, despite having a moderate
level of disease resistance. Therefore, these two ILs could
be important sources of novel alleles for resistance to both
the foliar fungal diseases and should be used in further
genetic and breeding applications.
DISCUSSION
Lack of availability of high levels of resistance for foliar
fungal diseases in cultivated peanut necessitates the exploi-
tation of new sources to enhance the levels of resistance in
new peanut cultivars. Several studies have reported high
levels of resistance against two of the most devastating
foliar fungal diseases, LLS and rust, in wild Arachis spe-
cies (Subrahmanyam et al., 1985; Pande and Rao, 2001).
The currently well-exploited resistance alleles present
in popular peanut cultivar GPBD 4 were traced back to
Table 6. Late leaf spot (LLS)- and rust-resistant introgression lines better or significantly better for yield-related traits compared
with LLS-resistant control cultivar, GPBD 4, in the 2015 rainy and 2015–2016 post-rainy seasons and pooled analysis.
Tra i t s 2015 rainy season 2015–2016 post-rainy season Pooled
Days to first flowering PBGNIL-2, PBGNIL-4 PBGNIL-2, PBGNIL-1, PBGNIL-15 PBGNIL-2
Days to 50% flowering PBGNIL-2 PBGNIL-2, PBGNIL-1, PBGNIL-15,
PBGNIL-11
PBGNIL-2, PBGNIL-1
Pod no. PBGNIL-7, PBGNIL-11, PBGNIL-15,
PBGNIL-1, PBGNIL-8, PBGNIL-13,
PBGNIL-12
PBGNIL-4, PBGNIL-13, PBGNIL-5,
PBGNIL-11, PBGNIL-1, PBGNIL-3,
PBGNIL-17, PBGNIL-8, PBGNIL-6,
PBGNIL-2, PBGNIL-15, PBGNIL-7,
PBGNIL-12
PBGNIL-11, PBGNIL-4, PBGNIL-13,
PBGNIL-1, PBGNIL-7, PBGNIL-15,
PBGNIL-8, PBGNIL-12, PBGNIL-5
Pod yield per plant (g) PBGNIL-8, PBGNIL-7, PBGNIL-1,
PBGNIL-15, PBGNIL-13, PBGNIL-11,
PBGNIL-14, PBGNIL-9, PBGNIL-12
PBGNIL-4, PBGNIL-13, PBGNIL-5,
PBGNIL-15, PBGNIL-6, PBGNIL-3,
PBGNIL-1, PBGNIL-11
PBGNIL-13, PBGNIL-15, PBGNIL-1,
PBGNIL-8, PBGNIL-7, PBGNIL-11,
PBGNIL-4, PBGNIL-14, PBGNIL-5,
PBGNIL-12
Seed no. PBGNIL-1, PBGNIL-7, PBGNIL-8,
PBGNIL-11, PBGNIL-13, PBGNIL-15,
PBGNIL-12
PBGNIL-4, PBGNIL-13, PBGNIL-5,
PBGNIL-1, PBGNIL-11, PBGNIL-6,
PBGNIL-3, PBGNIL-7, PBGNIL-2,
PBGNIL-8, PBGNIL-15, PBGNIL-12,
PBGNIL-17, PBGNIL-9
PBGNIL-1, PBGNIL-13, PBGNIL-4,
PBGNIL-7, PBGNIL-11, PBGNIL-8,
PBGNIL-5, PBGNIL-15, PBGNIL-12
Seed yield per plant (g) PBGNIL-13, PBGNIL-7, PBGNIL-1,
PBGNIL-15, PBGNIL-8, PBGNIL-11,
PBGNIL-12
PBGNIL-13, PBGNIL-5, PBGNIL-4,
PBGNIL-15, PBGNIL-2, PBGNIL-6,
PBGNIL-14, PBGNIL-12, PBGNIL-1,
PBGNIL-3
PBGNIL-13, PBGNIL-15, PBGNIL-1,
PBGNIL-7, PBGNIL-12, PBGNIL-8,
PBGNIL-11, PBGNIL-5
100-seed weight (g) PBGNIL-10, PBGNIL-13, PBGNIL-15,
PBGNIL-12, PBGNIL-14, PBGNIL-8,
PBGNIL-7, PBGNIL-11, PBGNIL-6,
PBGNIL-9
PBGNIL-14, PBGNIL-15, PBGNIL-2,
PBGNIL-13, PBGNIL-10
PBGNIL-10, PBGNIL-13, PBGNIL-15,
PBGNIL-14, PBGNIL-12, PBGNIL-6
Shelling (%) PBGNIL-13, PBGNIL-10 PBGNIL-10, PBGNIL-2, PBGNIL-9,
PBGNIL-15, PBGNIL-14, PBGNIL-13,
PBGNIL-5, PBGNIL-12, PBGNIL-1
PBGNIL-10, PBGNIL-13, PBGNIL-2,
PBGNIL-9
crop science, vol. 57, may–jun e 2017 www.crops.org 1129
A. cardenasii (Varshney et al., 2014). Keeping in mind the
presence of enormous genetic variability and high levels
of resistance or tolerance to important biotic and abiotic
stresses in dierent wild Arachis species, it is important to
exploit these diverse sources to develop new peanut cul-
tivars. Such an attempt would play an important role in
improving peanut production and productivity glob-
ally, especially under disease pressure by avoiding disease
development and epidemics. In peanut, a few successful
examples are available wherein genes from wild Arachis spe-
cies were successfully used for genetic improvement, such
as introgression of root-knot nematode resistance from the
amphidiploid TxAG-6 (Burow et al., 2014), rust and LLS
resistance in GPBD 4 from CS 16 (ICGV 86855) (Gowda
et al., 2002), and identication of quantitative trait loci for
rust resistance (Leal-Bertioli et al., 2015). In the present
study, we aimed to introgress high levels of LLS and rust
resistance from diploid wild Arachis species into cultivated
peanut to develop ILs with enhanced levels of resistance in
good agronomic backgrounds and to diversify the sources
of LLS and rust resistance by using wild species, rather than
the commonly used A. cardenasii, for peanut improvement.
To overcome the ploidy level dierence between the dip-
loid wild Arachis species and tetraploid cultivated peanut,
two tetraploid synthetics derived from the chromosome
doubling of F1 crosses between diploid wild Arachis species
were used as donors. Further, to minimize the linage drag,
an advanced backcross approach was used for population
development, with the aim to recover the recombinants
with a small segment introgressed from wild species in the
genetic background of cultivated types.
The synthetic ISATGR 121250 was derived from
F1 crosses between A. duranensis (ICG 8123) and
A. ipaensis (ICG 8206), the two progenitor species of cul-
tivated peanut. Another synthetic, ISATGR 265-5, was
derived from F1 crosses between nonprogenitor species,
A. kempf-mercadoi (ICG 8164) and A. hohnei (ICG 8190)
(Mallikarjuna et al., 2011). These four accessions (ICG
8123, ICG 8206, ICG 8164, and ICG 8190) were reported
to possess high levels of LLS and rust resistance (Subrah-
manyam et al., 1985; Pande and Rao, 2001). In this study,
two advanced backcross populations were developed using
two synthetics, ISATGR 121250 and ISATGR 265-5, as
donors for introgressing LLS and rust resistance into two
popular peanut cultivars, ICGV 91114 and ICGV 87846.
Screening of these two advanced backcross populations,
followed by rescreening, resulted in the identication
of 15 ILs with high levels and two ILs with moderate
levels of LLS and rust resistance in acceptable agronomic
backgrounds. These results indicated that LLS and rust
resistance in these ILs were introgressed from wild Arachis
species A. duranensis, A. ipaensis, A. kempf-mercadoi, and A.
hohnei. Because the levels of LLS and rust resistance in
these ILs was found better than GPBD 4 and were derived
from wild Arachis species other than the commonly used
A. cardenasii, these ILs provide new and diverse sources of
LLS and rust resistance for peanut improvement.
Besides LLS and rust resistance, these accessions also
exhibited good agronomic performance, such as early
owering, high number of pods per plant, pod yield per
plant, number of seeds per plant, seed yield per plant,
100-seed weight, and shelling percentage, compared with
the best LLS- and rust-resistant peanut cultivar, GPBD
4, in rainy and post-rainy seasons. However, the vari-
able performance of these ILs in rainy and post-rainy
seasons was mainly due to the signicant g ´ e interac-
tion observed for most traits. Most of the ILs were erect
(15 ILs) and decumbent (2 ILs) in growth habit, with
sequential (14 ILs) and alternate (3 ILs) branching patterns,
slight (9 ILs) to moderate (8 ILs) pod beaks, slight (13 ILs)
to moderate (4 ILs) pod constriction, and moderate (all 17
ILs) pod reticulations (data not given). It is interesting to
note that the majority of favorable alleles conferring dis-
ease resistance were contributed by the wild Arachis species
through synthetics, whereas favorable alleles for agronomic
traits were mostly contributed by the cultivated parent after
backcrossing and recombination. All of these ILs exhibited
high LLS and rust resistance and good agronomic per-
formance and could be evaluated across multilocations to
identify promising and stable high-yielding LLS- and rust-
resistant ILs, either for direct release as cultivars in specic
regions or for use as new and diverse sources of variation
in the breeding programs developing new disease-resistant
peanut cultivars with a broad genetic base.
An eort was made to genotype these promising ILs
by using linked markers for LLS and rust resistance. These
markers were identied in the mapping population devel-
oped from the cross TAG 24 ´ GPBD 4 (Khedikar et al.,
2010; Sujay et al., 2012) and have also been deployed suc-
cessfully in breeding programs to improve rust resistance
in three elite and popular cultivars using a marker-assisted
backcrossing (MABC) approach (Varshney et al., 2014).
In these studies, the resistance source for LLS and rust was
GPBD 4, and the resistance alleles were traced back to
A. cardenasii. In the present study, genotyping results
showed that two ILs, namely PBGNIL-2 and PBGNIL-6,
carry dierent alleles than the known resistance source,
GPBD 4, and showed moderate levels of resistance for
both foliar fungal diseases. Because these two ILs were
derived from a cross involving LLS- and rust-susceptible
peanut cultivar ICGV 91114, and ISATGR 121250 derived
from LLS- and rust-resistant A. duranensis (ICG 8123) ´
A. ipaensis (ICG 8206), the results indicated that the resis-
tance in these two ILs may be dierent from the commonly
used A. cardenasii, and they therefore provide novel sources
introgressed from A. duranensis and A. ipaensis. However,
it is important to mention that the markers used for geno-
typing were associated markers identied in a genetic
1130 www.c rops .org crop scie nce, v ol. 57, may–ju ne 2017
study and not the gene-based functional markers, which
provide allele mining very precisely. Nevertheless, the
genome sequence availability of both the diploid progeni-
tors (Bertioli et al., 2016; Chen et al., 2016) may facilitate
detailed genetic and sequence analysis of the highly resis-
tant ILs, along with other sources of resistance, for faster
discovery of genes using next-generation sequencing
approaches (Pandey et al., 2016). Pyramiding of diverse
resistance alleles will enhance the stable performance of
newly developed cultivars in farm elds, leading to greater
adoption by the peanut farming community.
In summar y, this research reports development of
highly resistant ILs using diverse genetic sources that so far
remained unused in the peanut breeding programs. These
ILs will not only serve as an alternate source of resistance
in breeding but also help towards diversifying the cur-
rently narrow genetic base of cultivated peanut varieties.
In addition, these ILs may also facilitate further genetic,
breeding, and genomics studies hoping to identify the
right allelic combinations to provide sustained resistance
to LLS and rust in peanut.
Conflict of Interest
The authors declare there to be no conict of interest.
Acknowledgments
This work has been undertaken as part of the CGIAR Research
Program on Grain Legumes. ICRISAT is a member of the
CGIAR. The help extended by Mr. M Srinivas for evaluating
the material for morphoagronomic traits under eld conditions,
Mr. Sube Singh for data analysis, and Ms. Manda Sriswathi for
genotyping-related work is duly acknowledged.
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