JCRP_2015_Advancements in Molecular Marker Developments in Peanuts

8/18/2019 JCRP_2015_Advancements in Molecular Marker Developments in Peanuts

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Review

Advancements in molecular marker development and theirapplications in the management of biotic stresses in peanuts

Gyan P. Mishra*, T. Radhakrishnan, Abhay Kumar, P.P. Thirumalaisamy, Narendra Kumar,Tejas C. Bosamia, Bhagwat Nawade, Jentilal R. Dobaria

Directorate of Groundnut Research, Post Box No. 05, Junagadh 362 001, Gujarat, India

a r t i c l e i n f o

Article history:

Received 31 March 2015

Received in revised form

17 July 2015

Accepted 20 July 2015

Available online xxx

Keywords:

Genomics

Diseases and pests

Marker assisted selection

Linkage-mapping

Transgenics

a b s t r a c t

Peanut is grown extensively in different parts of world, where various biotic and abiotic factors limit its

productivity and quality. The major fungal biotic constraints to peanut production include rust ( Puccinia

arachidis Speg.), stem-rot (Sclerotium rolfsii), collar-rot ( Aspergillus niger Van Teighem), aa-root ( Asper-

gillus avus), and late leaf spot (Phaeoisariopsis personata Ber. and M A Curtis), while viral disease con-

straints are peanut bud necrosis disease (PBND) caused by peanut bud necrosis virus (PBNV) and peanut

stem necrosis disease (PSND) caused by tobacco streak virus (TSV). Since, only a few sources of resistance

are available in cultivated peanut for some diseases, which has resulted in the limited success of con-

ventional breeding programmes on disease resistance. Moreover, even marker assisted breeding in

peanut is in the nascent stage and identication of some major quantitative trait loci (QTLs) for a few

fungal disease resitance genes has only recently been reported. Substantial efforts are underway to

develop PCR-based markers for the construction of high-density genetic linkage maps. This will enable

the breeders to effectively pyramid various biotic stress resistance genes into different agronomically

superior breeding populations, in a much shorter time. It is expected that the availability of various cost-

effective genomic resources (SNPs, whole genome sequencing, KASPar, GBS etc.) and more effective

mapping populations (NAM, MAGIC etc.) in the coming years will accelerate the mapping of complex

traits in peanut. This review provides an overview of the current developments and future prospects of

molecular marker development and their applications for improving biotic-stress resistance in peanut

crop.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Peanuts ( Arachis hypogaea L.), also known as groundnuts, are

grown in more than 120 countries with different agro-climatic

zones between latitudes 40 S and 40 N on approximately

21e24 M ha of land annually (Sarkar et al., 2014). It is cultivated

predominantly by small farms under low input conditions andranks third and fourth as a source of protein and edible oil,

respectively (Bhauso et al., 2014). Several biotic stresses are known

to limit peanut productivity, and their severity and extent of dis-

tribution vary with the cropping system, growing season, and re-

gion. Among biotic stresses, several diseases including rust

(Puccinia arachidis Speg.), early leaf spot (ELS, Cercospora arach-

idicola), late leaf spot (LLS, Phaeoisariopsis personata Ber. and M A

Curtis), and aatoxin contamination by Aspergillus avus and Aspergillus parasiticus are global constraints against peanut pro-

duction (Subrahmanyam et al., 1984; Waliyar, 1991). Rust, stem-rot

(Sclerotium rolfsii), collar-rot ( Aspergillus niger Van Teighem), and

leaf spots are also quite serious and together may cause the loss of

50e60% of pod yield in India (Dwivedi et al., 2003; Subrahmanyam

et al., 1985). In the peanut growing regions, high yielding, well-adapted cultivars contain multiple resistances to biotic stresses

that can provide enhanced and sustainable peanut production

(Dwivedi et al., 2003).

The world's largest peanut germplasm collection with more

than 15,000 accessions is housed at the International Crops

Research Institute for the Semi-Arid Tropics (ICRISAT) in India

(Gowda et al., 2013). These accessions have many differences in

their vegetative, reproductive, physiological, and biochemical traits.

The global Arachis gene pool possesses the source of resistance to

many biotic stresses, including rust, ELS, LLS, Groundnut Rosette

Disease [GRD, caused by a complex of three agents: groundnut* Corresponding author.

E-mail address: [email protected] (G.P. Mishra).

Contents lists available at ScienceDirect

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2015 Elsevier Ltd. All rights reserved.

Crop Protection 77 (2015) 74e86

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rosette virus (GRV), its satellite RNA (sat RNA), and a groundnut

rosette assistor virus (GRAV)], Peanut Bud Necrosis Virus (PBND), A.

avus induced aatoxin contamination, bacterial wilt (Ralstonia

solanacearum), leafminer ( Aproaerema modicella), Spodoptera, jas-

sids (Empoasca kerri Pruthi), thrips (Frankliniella schultzei Trybom)

and termites (Odontotermes sp.) (Rao et al., 2002; Basu and Singh,

2004; Amin et al., 1985; Rao et al., 2014).

Since the 1960s, interspecic hybridization has received much

attention in peanuts because several wild Arachis species show a

very high level of resistance to many biotic stresses, such as rust,

ELS, LLS, and stem rot (Holbrook and Stalker, 2003; Singh et al.,

1984). However, success in transferring the resistance to culti-

vated peanuts has been limited mainly because of cross compati-

bility barriers, linkage drag, and long periods required for

developing stable tetraploid interspecic derivatives (Wynne et al.,

1991; Singh et al.,1997). Moreover, the partial and polygenic nature

of biotic stresses makes the identication of resistant and suscep-

tible lines very tedious using conventional screening techniques

(Leal-Bertioli et al., 2009). Because of the frequent occurrence of

multiple diseases, peanut yields are often signicantly lower than

their potential (Holbrook and Stalker, 2003). In the future, cultivars

with multiple disease and pest resistances will be needed, which

appears to be a very dif cult endeavour for this crop species (Basuand Singh, 2004).

Marker-assisted selection (MAS) offers great promise for

improving the ef ciency of conventional plant breeding ( Janila

et al., 2013), including the potential to pyramid resistance genes

in peanuts (Mishra et al., 2009; Varshney et al., 2014; Pandey et al.,

2012). For any molecular breeding program, assessment of genetic

diversity and development of genetic linkage maps are two very

important steps (Dwivedi et al., 2003). Abundant polymorphisms in

wild Arachis species have been observed, but progress in the mo-

lecular breeding of cultivated peanuts is greatly constrained due to

the low level of detectable molecular genetic variation (Mondal

et al., 2005; Herselman, 2003; Raina et al., 2001; He and Prakash,

2001). Therefore, the use of more robust assays such as single

nucleotide polymorphisms (SNPs), competitive allele-specic PCR (KASPar) and genotyping by sequencing (GBS) approaches are

needed. However, cost-effective SNP genotyping platforms are not

readily available for tetraploid peanuts, but a large number of

robust markers such as SSRs and SNPs (including KASPar) would be

valuable. SSRs are still considered the marker of choice in peanuts

(Pandey et al., 2012), and a wide range of genotypes have been used

for mapping (Table 1) of many important biotic and abiotic traits

using SSR markers (Table 2).

Despite being an important oilseed crop, very limited work in

the area of molecular genetics and breeding of peanuts has been

performed (Dwivedi et al., 2002; Raina et al., 2001). However, over

the last decade, signicant developments have been made in the

use of various molecular approaches for biotic stress management

in peanuts, and new efforts such as functional genomics are likelyto play key roles in the future (Wang et al., 2011; Varshney et al.,

2014; Gajjar et al., 2014). Recently, Kanyika et al. (2015) has

identied 376 polymorphic SSR markers in 16 African groundnut

cultivars with a wide range of disease resistance. These identied

markers can be used to improve the ef ciency of introgression of

resistance to multiple important biotic constraints into farmer-

preferred varieties of Sub-Saharan Africa. In this review, we made

an attempt to capture the recent updates in molecular marker

development and their applications in the management of various

biotic stresses in peanut.

2. Markers associated with rust and LLS resistance gene(s)

Rust and leaf spots areeconomically very important foliarfungal

diseases of peanuts that often occur together and not only reduce

the yield but also adversely affect the fodder and seed quality

(Subrahmanyam et al., 1985; Waliyar, 1991). Despite the economic

importance of rust and LLS, very limited work has been carried out

on hostefungus interaction, fungal genetic diversity, and physio-

logical specialization (Mondal and Badigannavar, 2015). Several

studies have emphasized the application of different types of mo-

lecular markers, construction of peanut linkage maps, or tagging of

important agronomic traits, such as disease resistance (Wang et al.,

2011; Gajjar et al., 2014). Recently, many DNA markers have been

found to be putatively linked to rust and LLS resistance genes(Mondalet al., 2012a; Khedikar et al., 2010; Shoba et al., 2012; Sujay

et al., 2012) (Table 2), a few of which have been validated and used

in the breeding programme (Sujay et al., 2012; Gajjar et al., 2014;

Varshney et al., 2014). Location of markers on the various linkage

groups in Table 2, is derived after doing intensive meta-analysis of

all the published literature, including the most comprehensive and

consensus linkage maps available in peanut.

Validation of other linked markers will accelerate the process of

introgression of disease resistance into preferred peanut genotypes

(Sujay et al., 2012; Gajjar et al., 2014). Near isogenic lines (NILs)

developed for rust resistance were thoroughly screened with both

foreground and background molecular markers (Yeri et al., 2014).

For the identication of LLS resistance, Luo et al. (2005b) identied

genes in the resistant genotype that were more highly expressedthan in the susceptible genotype (in response to Cercosporidium

personatum infection) by microarray analysis and validated them by

real-time PCR. In a recombinant inbred line (RIL) population (VG

9514 TAG 24), two transposable element (TE) markers, TE 360

and TE 498, were found to be associated with the rust resistance

gene. These two markers need further validation before they could

be effectively applied for MAS of rust resistance in different back-

grounds (Mondal et al., 2013).

3. Soil-borne fungal diseases and associated markers

(Collar rot, Stem rot, Aspergillus spp., Bacterial wilt and Scle-

rotinia blight)

Among soil-borne diseases, collar rot ( A. niger ) and stem-rot (S.rolfsii) are very important (Farr et al., 1989; Kolte, 1984). The search

for peanut cultivars resistant to S. rolfsii originates all the way back

Table 1

List of a few genotypes, used for mapping of various resistance gene(s) (Dwivedi et al., 2003; Shoba et al., 2012;

Sujay et al., 2012).

Traits Genotypes

Early leaf spot ICG 405, ICG 1705, ICG 6284, TMV 2

Late leaf spot GPBD 4, ICGV 99001, ICGV 99004, COG 0437, TAG 24, TMV 2

Rust GPBD 4, ICGV 99003, ICGV 99005, TG 26, TMV 2

Rosette disease ICG 6323, ICG 6466, ICG 11044, JL 24

Bacterial wilt ICG 7893, ICG 15222, and Chico

Aatoxin production U 4-7-5, 55-437, J 11

G.P. Mishra et al. / Crop Protection 77 (2015) 74e86 75


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to 1918 (McClintock, 1918), but a high degree of resistance is yet to

be found. Recently, Thirumalaisamy et al. (2014) has reported some

sources for stem rot resistance in peanut, but the genetics of

resistance and markers linked with the resistance gene(s) are still

lacking. Thus, there is an urgent need to nd molecular markers

linked with the stem rot resistance QTLs, for its use in marker-

assisted breeding programme.Very few published reports pertaining to the molecular char-

acterization of S. rolfsii are available. Variations in the internal

transcribed spacer (ITS) regions have revealed 12 sub-specic

groupings, some of which correlated with their mycelial compati-

bility groups (MCGs) (Harlton et al., 1995). Punja and Sun (1997)

detected 68 MCGs while comparing 128 isolates of S. rolfsii from

36 host species collected from 23 geographic regions. Cilliers et al.

(2000) found Amplied Fragment Length Polymorphism (AFLP) as

a suitable technique for the assessment of genetic variability be-

tween isolates and MCGs of S. rolfsii. Genetic variability among the

virulent isolates of S. rolfsii was studied using molecular techniques,

such as RAPD, ITS-PCR and RFLP (Prasad et al., 2010). Several mo-

lecular markers associated with pod rot resistance and suscepti-

bility in mutants and Giza5 peanut genotype were reported by

Azzam et al. (2007) using RAPD markers, although these markers

still need to be validated.

A. avus and A. parasiticus infect peanut seeds and produce af-

latoxins, which are harmful to both domestic animals and humans

(Waliyar et al., 2015; Singh et al., 2015a,b; Liu et al., 2014). The

progress in peanut breeding for resistance to aatoxin is slow due

to the lack of a cost-effective method for resistance identication in

breeding materials or segregating progeny (Lei et al., 2006). Barros

et al. (2007) and Singh et al. (2015a,b) analysed Aspergillus isolates

collected from peanut elds in Argentina and India using AFLP,

where different levels of polymorphism in different groups of iso-

lates were recorded. Guo et al. (2008) has identified up-regulation

of 8 resistance-related genes for Aspergillus infection. Lei et al.

(2006) reported an AFLP (E45/M53-440) converted SCAR marker

(AFs-412) to be closely linked to resistance of A. avus infection.

Screening of isolates using gene-specic PCR was found to be quite

effective for the identication of gene defects in A. avus, which

could be used as bio-control agents in peanut growing areas (Dodia

et al., 2014). Moreover, to date, the genetic basis of S. rolfsi and

A. avus stress resistance traits in peanuts is not fully understood.

Therefore, extensive efforts are required to

rst identify the resis-tance sources and then to nd the markers associated with the

resistance genes/QTLs for their future use in marker assisted

breeding (Mishra et al., 2014).

Bacterial wilt (R. solanacearum) is an important constraint to

peanut production in several Asian and African countries, and

planting of bacterial-wilt resistant cultivars is the most feasible

method fordisease control. Genetic diversityanalysis using SSR and

AFLP markers among selected peanut genotypes identied certain

genotypes and primer combinations for developing mapping

populations and breeding for high yield, resistant cultivars ( Jiang

et al., 2007a). In an attempt to understand the molecular mecha-

nism of bacterial wilt resistance, 25 differentially expressed

candidate genes were identied by studying the differences in gene

expression between inoculated and control seeds. Conrmation of

the functions of these genes needs to be validated through trans-

genic studies (Ding et al., 2012). For Sclerotinia blight (Sclerotinia

minor ) resistance, Kelly et al. (2010) characterized 96 US peanut

mini-core collections using a resistance-associated SSR marker, and

39 accessions as new potential sources of resistance were

identified.

4. Molecular studies on resistance to viral diseases

(Groundnut rosette disease, PBND and PSND)

Groundnut rosette disease is the most destructive viral disease

of peanuts causing serious yield losses. Single recessive gene con-

trol for resistance to the aphid vector ( Aphis craccivora) was

observed in the breeding line ICG 12991, which was mapped3.9 cM

Table 2

SSR markers and its linkage group known to be associated to the rust and/or LLS resistance gene(s).

S. No. Primers Linkage group Cross/genotypes Resistance Reference

1 seq3A01238a a07 ICGV 99003 TMV 2 Rust Varma et al., 2005

2 seq5D05274 b07and a07 TMV 2 COG 0437 (F2)

and ICGV 99005 TMV 2

Rust and LLS Shoba et al., 2012; Varma et al., 2005;

Shirasawa et al., 2013

3 seq16F01271 b03 ICGV 99005 TMV 2 Rust Varma et al., 2005

4 seq17F06152 b04 ICGV 99005 TMV 2 and 22 genotypes Rust and LLS Varma et al., 2005; Mace et al., 2006;

Shirasawa et al., 20135 seq13A07265 b01 ICGV 99005 TMV 2 and 22 genotypes Rust and LLS Varma et al., 2005; Mace et al., 2006;


6 seq2F05280 b02 22 genotypes Rust and LLS Mace et al., 2006; Shirasawa et al., 2013

7 seq8E12200 a01 22 genotypes Rust resistance Mace et al., 2006

8 seq16C06263 b03 22 genotypes Rust Mace et al., 2006

9 seq13A10250 b04 22 genotypes Rust Mace et al., 2006

10 seq2B10290 b03 22 genotypes LLS Mace et al., 2006

11 IPAHM103160 a03 and b03 TAG 24 xGPBD 4 Rust Khedikar et al., 2010

12 PM384100 e TMV 2 COG 0437 (F2) LLS Shoba et al., 2012

13 PM137150 b06 TMV 2 COG 0437 (F2) LLS Shoba et al., 2012; Shirasawa et al., 2013

14 PM03168 a03 and b03 TMV 2 COG 0437 (F2) LLS Shoba et al., 2012; Shirasawa et al., 2013

15 PMc588183 e TMV 2 COG 0437 (F2) LLS Shoba et al., 2012

16 PM375102 a04 TMV 2 COG 0437 (F2) LLS Shoba et al., 2012

17 seq8D09190 b10 and a09 TAG 24 GPBD 4 LLS Sujay et al., 2012; Shirasawa et al., 2013

18 GM1536410 b03 TG 26 xGPBD 4 Rust Sujay et al., 2012

19 GM2301137 b03 TG 26 xGPBD 4 Rust Sujay et al., 2012

20 GM2079418

b03 TG 26 xGPBD 4 Rust Sujay et al., 2012

21 PM50110 b05 20 genotypes Rust Mondal and Badigannavar, 2010

22 PM35124 a06 and b04 20 genotypes Rust and LLS Mondal and Badigannavar, 2010;


23 seq4A05 and

gi56931710

a03 RILs from VG 9514 TAG 24 Rust Mondal et al., 2012a

a Subscript numerical values are for linked band size.



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from an AFLP marker on LG 01 (Herselman et al., 2004). Peanut bud

necrosis disease (PBND) caused by peanut bud necrosis virus

(PBNV) and vectored by Thrips (Vijayalakshmi et al., 1995) i s a

major viral disease of peanuts. In 2000, a new threat to peanuts

emerged in the form of a viral disease named peanut stem necrosis

disease (PSND) caused by the peanut stem necrosis virus (PSNV),

which results in lethal necrosis (Rao et al., 2000). Tobacco streak

virus (TSV) was found to be associated with the disease (Reddy

et al., 2002). Despite several years of effort, a conrmed source of

genetic resistance/tolerance to TSV has not been identied in the

gene pool of cultivated peanuts. However, genetically engineered

resistance has been actively investigated in recent years as an

alternative to cope with this type of situation (Mehta et al., 2013).

Kamdar et al. (2014) studied 115 resistant and one susceptible line

to PBND using SSR, but no clear differentiation was observed.

Similarly, SSR-based diversity analysis among 15 peanut genotypes

(resistant and susceptible to PBND) grouped together based on

their origin rather than PBND resistance (Srinivasaraghavan et al.,

2012). In the future, the availability of more cost effective

genomic resources, such as SNPs and whole genome sequencing,

will hasten complex trait mapping efforts (especially for viral dis-

eases) in this crop (Pandey et al., 2012).

5. Molecular studies on nematode resistance

In many peanut production areas across the world, the peanut

root-knot nematode causes signicant economic losses (Holbrook

and Stalker, 2003). Many sources of resistance were identied in

the germplasm, and lot of molecular work has been performed to

nd the linked gene(s)/QTLs (Mishra et al., 2009). For the root-knot

nematode (Meloidogyne arenaria), two dominant genes viz . Mae

(restricted egg number) and Mag (restricted galling) conditioning

resistance were identied, and a RAPD marker (Z3/265) was found

to be linked to these genes (Garcia et al., 1996).

Different types of DNA markers associated with root-knot

nematode resistance were also identied (Burow et al., 1996;

Garcia et al., 1996; Choi et al., 1999; Church et al., 2000; Wanget al., 2008a) and are currently being used for the development of

resistant peanut cultivars. A DNA marker with 6% recombination

frequency to the resistance gene has been developed to screen the

segregating populations for nematode resistance (Chu et al., 2007).

The RFLP marker R2430E was found to link to a locus for resistance

of the peanut root-knot nematode M. arenaria (Neal) Chitwood race

1 (Pípolo et al., 2014). Genes controlling the resistance to nema-

todes were introgressed through interspecic hybridization and

resulted in the release of the rst nematode-resistant peanut

cultivar COAN (Simpson and Starr, 2001). Other works on the

development of nematode-resistant peanut cultivars are presented

in other sections of this review.

6. Single nucleotide polymorphism (SNP) technologies for biotic stresses

Recent developments in SNP technologies for peanuts have

indicated that, in the near future, additional options may be

available for the rapid identication of large numbers of poly-

morphic markers in peanuts (Kanazin et al., 2002; Khera et al.,

2013). A single base pair extension (SBE) was found to be an ef -

cient method for high-throughput SNP mapping in peanuts, and

ve candidate genes for resistance were identied on the genetic

map (Alveset al., 2008). Recently, Khera et al.(2013) useda set of96

informative SNPs to develop competitive allele-specic PCR (KAS-

Par) assays in peanuts, designated as GKAMs (Groundnut KASPar

Assay Markers), through which 90 GKAMs were validated for

different biotic stresses. A comprehensive list of the various

markers associated with different diseases and pests is given in

Table 3.

7. Advancements in linkage mapping

Peanut is a self-pollinated allotetraploid ( 2n ¼ 4x ¼ 40, AABB)

crop having ten basic chromosomes, harboring about 2813 Mb of

DNA content (Arumuganatham, 1991). Peanut genome is nearly 20

times larger than that of Arabidopsis thaliana and is somewhat

similar to Gossypium hirsutum and Zea mays. Variation in genome

size among accessions of A. hypogaea (2n ¼ 4x ¼ 40) and Arachis

duranensis (2n ¼ 2x ¼ 20) (Singh et al., 1996) and between

A. hypogaea and A. monticola (Temsch and Greilhuber, 2000) has

also been reported. The rst RFLP-based genetic linkage map of

peanuts was constructed using an F2 population (Halward et al.,

1993). A list of a few classical and the most recent genetic linkage

maps, including consensus maps, are given in Table 4.

Compared to that of many other legume crops, the development

of molecular markers in cultivated peanuts has progressed at a

relatively slow pace. Although many markers have been developed

in both wild and cultivated peanuts, the genetic linkage map is not

yet saturated. There is a need to saturate the linkage mapto provide

suf cient markers for marker-assisted breeding (Dwivedi et al.,2003). Constant increases in peanut marker identities and linkage

groups together with the availability of genome sequence data in

the recent past have opened up the possibility of integrating new

markers to some LGs and identifying closely linked markers

(Mondal et al., 2012a). Efforts are currently underway for applying

genomics to peanut biotic stress resistance breeding through

mapping and MAS for different biotic stresses (Pandey et al., 2012).

8. QTL analysis for biotic stress resistance

In peanuts, many markers were recently found to be associated

with QTLs for various biotic stresses, namely, rust and LLS (Sujay

et al., 2012; Mondal and Badigannavar, 2010), Cylindrocladium

black-rot and ELS (Stalker and Mozingo, 2001), nematodes (Chuet al., 2007; Nagy et al., 2010), tomato spotted wilt virus (TSWV)

(Qin et al., 2012), aphid vector of GRD (Herselman et al., 2004). The

majority of the identied QTLs are not major and account for less

than 10% of the phenotypic variation explained (PVE). Major QTLs

identied for rust and LLS in the germplasm source (Khedikar et al.,

2010; Sujay et al., 2012) and for nematode resistance (Nagy et al.,

2010; Simpson, 2001) were of a wild species origin.

In peanuts, candidate genome regions controlling disease

resistance were identied by placing the 34 sequence-conrmed

candidate disease resistance genes and 05 QTLs against LLS on

the genetic map of the A-genome of Arachis. Upon grouping, these

genes and QTLs were found to be present on the upper and lower

regions of LG 04 and 02, respectively, indicating the prominence of

these regions for imparting disease resistance (Leal-Bertioli et al.,2009). QTL analysis in a ‘Tifrunner GT-C20’-derived mapping

population has identied 54 QTLs in the F2 map, including 02 for

thrips, 15 for TSWV, and 37 for LS. However, in the F5 map, 23 QTLs

could be identied, including 01 for thrips, 09 for TSWV, and 13 for

LS. This is the rst QTL study reporting novel QTLs for thrips, TSWV,

and LS, which needs renement in the future (Wang et al., 2013,

2014). Using RIL population (VG 9514 TAG 24), 13 main and 31

epistatic QTLs for total developmental period (TDP) were detected

for bruchid resistance. Two years screening identied two common

main QTLs, qTDP-b08 for TDP (57e82% PVE) and qAE2010/11-a02

for adult emergence (13e21% PVE) (Mondal et al., 2014).

Shoba et al. (2013) reported Ah 4-26 and PM 384 as the closest

markers for QTLs of 100 kernel weight and LLS severity, respec-

tively. Because PMc 588 and Ah 4-26 are the

anking markers for



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PM 384, they can therefore be used for marker-assisted breeding of

LLS resistance. A genetic map derived from a SunOleic97R NC94022 cross (Qin et al., 2012) was developed, and multiple

phenotyping data have identied a total of 155 QTLs, of which, 01

and 03 major QTLs for TSWV and LLS resistance, respectively, were

identied (Guo et al., 2013).

A QTL region with 82.62% phenotypic variation for rust resis-

tance (Sujay et al., 2012) was validated and introgressed in some

susceptible varieties using a marker-assisted backcrossing (MABC)

approach by employing four markers, namely IPAHM103, GM2079,

GM1536 and GM2301 (Varshney et al., 2014). It is expected that

multi-location testing of the promising introgressed lines will

result in the identication of entries for its possible release as va-

rieties with enhanced disease resistance. This result emphasized

the utility of using markers for QTL selection through MAS for

improving the rust resistance of any elite varieties (Varshney et al.,2014).

To date, neither background nor genome-wide selection has

been performed in peanuts, and this analysis must await the

development of high-throughput, economical assays for a large

number of markers (Holbrook et al., 2011). Details of some QTLs

that have been identied to be associated with biotic stress-related

traits in peanuts are given in Table 5. Identication of major QTLs

for various biotic stress resistances in peanuts will enable ef cient

MABC for the development of resistant cultivars. Recently, 39

marker trait associations (MTAs) for A. avus, ELS, LLS, and GRD

were identied using genome wide association studies (GWAS). Of

these MTAs, 01 was for Aspergillus (24.69% PV), 31 for GRD

(10.25e39.29% PV), 06 for ELS (9.18e10.99% PV), and 01 for LLS

(18.10% PV). It is expected that upon validation these MTAs may be

deployed for marker-assisted improvement of peanut genotypes

(Pandey et al., 2014).

9. Biotic stresses and gene expression studies

(Transcriptome analysis, microRNAs, epigenetic regulation,

microarrays)

In cultivated peanuts, which do not have whole genome

sequence information available yet, transcriptome data are an

alternative source of information, and ESTs can be used to identify

candidate genes (Pandey et al., 2012). During recent years, a wealth

of genomic data has been generated in peanut by high throughput

transcriptome sequencing (Chopra et al., 2014, 2015). However, the

available transcriptome sequences are not complete; many have

low N50 values, ranging from500 to 750 bp (Guimar~aes et al., 2012;

Chopra et al., 2014). On the basis of gene annotations, Bosamia et al.(2015) has identied 2784 SSR containing sequences, of which,

2027 (72.81%) were annotated and assigned in 4124 gene ontology

terms and 31.91% sequences were found to be associated with

response to stimulus encompassing biotic as well as abiotic

stresses.

Transcriptome analysis of cDNA collections from Arachis sten-

osperma infected with C. personatum revealed a number of tran-

scription factor families and defence-related genes. Additionally,

the expression of ve A. stenosperma resistance gene analogues

(RGAs) and four retrotransposon (FIDEL-related) sequences were

also analysed by qRT-PCR, which was used to design EST-SSRs of

which 214 were shown to be polymorphic (Guimar~aes et al., 2012).

Bertioli et al. (2003) generated 78 RGAs based on the nucleotide-

binding site (NBS) regions from A. hypogaea and other wild

Table 3

List of different markers associated with various biotic stresses.

Markers Disease/Causal organism Reference

RAPD Nematode Garcia et al., 1996; Burow et al., 1996

RFLP Nematode Choi et al., 1999; Church et al., 2000

AFLP Bacterial wilt Jiang et al., 2003; Ren et al., 2008

AFLP Rosette disease Herselman et al., 2004

AFLP A. avus Lei et al., 2005

SCAR A. avus Lei et al., 2006RAPD Pod-rot Azzam et al., 2007

AS-PCR a Nematode Chu et al., 2007

RAPD Rust Mondal et al., 2007

AFLP and SSR Bacterial wilt Jiang et al., 2007a; Jiang et al., 2007b

AFLP Rust Hou et al., 2007

AFLP LLS Xia et al., 2007

SSR Sclerotinia blight Kelly et al., 2010

RAPD and ISSR Rust and LLS Mondal et al., 2008

SSR Nematode Wang et al., 2008a

ISSR Rust and LLS Mondal et al., 2009

SSR A. avus Hong et al., 2009

Microarray A. avus Guo et al., 2011

EST-SSR Rust Mondal et al., 2012b

SNP Rust and Leaf spots Khera et al., 2013

TEa Rust Mondal et al., 2013

a TE: Transposable Element; AS-PCR: Allele Specic-PCR.

Table 4

List some classical and latest genetic linkage maps.

Populations Marker(s)/Loci Map distance (cM) References

F2 RFLP 1063.0 Halward et al., 1993

Back Cross RFLP 2210.0 Burow et al., 2001

Arachis duranensis 1724 marker loci 1081.3 Nagy et al., 2012

02 RILs 324 marker loci 1352.1 Qin et al., 2012

02 RILs 225 SSR 1152.9 Sujay et al., 2012

10 RILs, 01 BC 897 marker loci 3863.6 Gautami et al., 2012

03 RILs 3693 marker loci 2651.0 Shirasawa et al., 2013



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relatives. A collection of peanut nucleotide binding site leucine-rich

repeat (NBSeLRR) resistance gene candidates (RGCs) were identi-

ed by mining the GenBank databases (Radwan et al., 2010).Further, Yuksel et al. (2005) also isolated 234 RGAs based on the

primer sequence information from NBS-LRR and LRR-‘Toll’-like

motif (LRR-TM) classes. On the similar note, Proite et al. (2007)

identied 35 putative non-redundant RGAs and 26 pathogenesis

related ESTs from an accession of A. stenosperma, resistant to

different foliar diseases.

At present, a total of 281,754 ESTs are available for Arachis spp.

on the NCBI website (www.ncbi.nlm.nih.gov, accessed January 24,

2015), of which the contribution of each species is as follows: A.

hypogaea (205,442), A. duranensis (35,291), Aipaensis ipaensis

(32,787), A. stenosperma (6264), Alberta magna (750), A. hypogaea

subsp. fastigiata (745), A. appressipila (400) and A. diogoi (75). Many

of these have been generated for the identication of biotic stress

resistance genes (e.g., 21,777 ESTs were identi

ed from developingseeds against Aspergillus infection (Guo et al., 2008), and 8000 ESTs

from the root tissues of A. stenosperma resistant to M. arenaria

(Proite et al., 2007). Guo et al. (2009) identied 17,376 ESTs from the

leaves of TSWV and leaf spot resistant and susceptible cultivars, of

which 5717 had unknown functions. In response to A. avus

infection under drought stress, 29 protein spots showing differen-

tial expression between resistant and susceptible cultivars were

identied and further validated using RT-PCR analysis (Wang et al.,

2010). In addition, some defense-related transcripts, such as puta-

tive oxalate oxidase (EU024476) and NBS-LRR domains, were also

identied.

Large numbers of available ESTs in the public databases were

used for EST-SSR marker development. From 16424 unigenes, using

MISA (MIcroSAtellite identication tool) search, a total of 2456

novel EST-SSR primer pairs were designed; of which 366 unigenes,

having relevance to various stresses and other functions were PCR

validated using a set of 11 diverse peanut genotypes (both resistant

and susceptible to various diseases). Of these, 340 primer pairs

yielded clear and scorable PCR products and 39 primer pairs

exhibited polymorphisms which are of potential use for the resis-

tance breeding programme (Bosamia et al., 2015). Out of 411 sor-

ghum EST-SSR (SbEST-SSR) markers tested in peanuts, 39% could be

successfully amplied, of which 14% showed polymorphism among

resistant and susceptible cultivars for rust and LLS diseases ( Savadi

et al., 2012). Out of 259 EST-SSR markers, which were developed

using A. hypogaea ESTs (NCBI database), SSR_GO340445 and

SSR_HO115759 were found to be closely linked to the rust resis-

tance gene (Mondal et al., 2012b). Chen et al. (2008a, 2008b, 2011)

identified and cloned the resistance gene to TSWV and character-

ized two peanut oxalate oxidase genes.

In response to A. avus infection, many genes were found to beup-regulated in peanuts, of which PR-10 and pathogenesis-induced

protein (PIP) genes were cloned (Xie et al., 2009a,b). Resveratrol

imparts resistance in plants against both UV radiation and fungal

infection; the gene that synthesizes resveratrol synthase has been

cloned from peanuts, and expression analysis indicates that it is

expressed in the root (Zhou et al., 2008; Han et al., 2010). Lipid

transfer proteins (LTP), which were reported to be involved in

disease resistance in plants, have also been cloned from peanuts

(Zhao et al., 2009).

Transcriptome analysis of the migratory plant parasitic nema-

tode Ditylenchus africanus (peanut pod nematode) from mixed

stages revealed the involvement of putative proteins in develop-

mental and reproductive processes, as well as the role of unigenes

in oxidative stress and anhydrobiosis (Haegeman et al., 2009). Eightdifferentially expressed genes were identified in A. stenosperma

roots in response to M. arenaria infection using in silico ESTs and

microarray analysis (Guimar~aes et al., 2010, 2011).

From non-protein coding genes, microRNAs were transcribed by

RNA polymerase II (Kim, 2005), and understanding of its functional

and regulatory roles could open new windows for crop improve-

ment including disease tolerance. Zhao et al. (2010) and Pan and Liu

(2010) have identied 89 peanut miRNAs belonging to 14 new

miRNA and 22 conserved miRNA families, which can provide the

basis for peanut miRNA research for resistance to different stresses.

Relationships between functional molecules and plant phenotypes

can be studied more specically using proteomics (Wang et al.,

2011), and proteins that may play roles in aatoxin resistance in

imbedded peanut seeds have been discovered through proteomic

studies (Wang et al., 2008b).

In eukaryotes, epigenetic regulation is known to contribute to

gene silencing and plays critical roles in development and genome

defence against viruses, transposons, and transgenes (Lister et al.,

2008; Gehring et al., 2006; La et al., 2011). It has been observed

that bacterially infected plant tissue shows a net reduction in DNA

methylation, which may affect the disease resistance genes

responsible for surveillance against pathogens (Alvarez et al., 2010).

An investigation regarding epigenetic regulation mechanisms of

the peanut allergen gene Arah3 in developing peanut embryos

demonstrated an association between the loss of histone H3 from

the proximal promoter and high expression of Arah3 during em-

bryo maturation (Fu et al., 2010). For better understanding and

management of various bioticstressesin peanuts, there is an urgent

Table 5

List of QTLs identified for various biotic-stress related traits in peanut.

Traits No of QTLs identied Phenotypic variance explained (PVE) % Reference

LLS 28 10.07e67.8 Khedikar et al., 2010;

Sujay et al., 2012

Leaf rust 13 2.54e82.62 Khedikar et al., 2010; Sujay et al., 2012

Aspergillus avus 6 6.2e22.7 Liang et al., 2009

Bruchid resistance 13 13e82 Mondal et al., 2014

Aphid vector of rosette disease 8 1.18e

76.16 Herselman et al., 2004In F 2 map 54 (Total) e Wang et al., 2013, 2014

TSWV 15 4.40e34.92

Leaf spot 37 6.61e27.35

Thrips 02 12.14e19.43

In F 5 map 23 (Total) e

Thrips 01 5.86

TSWV 09 5.20e14.14

Leaf spot 13 5.95e21.45

LLS 05 4.2e43.8 Leal-Bertioli et al., 2009

TSWV 01 16.7 Guo et al., 2013

LLS 03 12.42e20.59


http://www.ncbi.nlm.nih.gov/http://www.ncbi.nlm.nih.gov/http://www.ncbi.nlm.nih.gov/


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need to determine the epigenetic regulatory mechanisms of

different biotic stress resistant genes and promoters.

For global gene expression proling, microarray analysis is a

powerful tool (Casson et al., 2005). Because a peanut gene chip is

commercial unavailability, high-throughput gene expression anal-

ysis is currently very limited. However, peanut microarrays have

been specically designed to solve particular issues, such as the

characterization of A. parasiticus infection-induced changes in gene

expression and gene expression proling in different peanut tissues

(Luo et al., 2005a; Payton et al., 2009). Approximately 25,000 ESTs

from cDNA libraries were constructed using the bacterial wilt

resistant peanut leaf before and after bacterial infection (Huang

et al., 2008). Shan et al. (2007) used soybean gene chips to anal-

yse the differential gene expression of peanut varieties that are

resistant and susceptible to A. avus infection. Using microarray

analysis, Guoet al. (2011) identied62 genes in Aspergillus resistant

peanut cultivars that were up-expressed in response to Aspergillus

infection, and 22 putative resistance genes that were constitutively

overexpressed. In addition to microarray-based gene expression

analysis, the expression and regulation of individual genes has also

been studied in peanuts (Wang et al., 2011). There is a need for the

development of a comprehensive genome-scale platform for

developing Aspergillus-resistant cultivars through targeted marker-assisted breeding and genetic engineering.

10. Transgenic approach for biotic stress management

(Fungal, viral, bacterial and insect-pest resistance)

The lack of available resistance genes within crossable germ-

plasms of peanuts necessitates the use of genetic engineering

strategies to impart genetic resistance against various biotic

stresses (Vasavirama and Kirti, 2012). Transgenic peanut lines

possessing fungal resistance genes offer an alternative to traditional

resistance and fungicide applications in managing fungal diseases

(Chenault et al., 2005). Vasavirama and Kirti (2012) generated

transgenic peanuts using a double gene construct with SniOLP

(Solanum nigrum osmotin-like protein) and Rs- AFP2 (Raphanussativus antifungal protein-2) genes under separate constitutive 35S

promoters, which then showed enhanced disease resistance to LLS.

Transgenic peanuts with rice chitinase-3 overexpression (with the

CaMV 35S promoter) exhibited a resistance for leaf spot that was

higher than that of the control, and a good correlation was observed

between chitinase activity and fungal pathogen resistance (Iqbal

et al., 2012).

Similarly, peanuts transgenic for the chitinase gene (Rchit ) from

rice showed 2- to 14-fold higher chitinase activity than the wild

type, and a signicant negative correlation was observed between

the chitinase activity and the frequency of infection to the three

tested pathogens ( A. avus, LLS and rust) (Prasad et al., 2013).

Transgenic peanut lines containing antifungal genes (rice chitinase

and/or alfalfa glucanase), when evaluated for their reaction toSclerotinia blight, showed varying degrees of resistance (Chenault

et al., 2005). Signicantly reduced lesion size was recorded in

transgenic plants expressing a barley oxalate oxidase gene

compared to the controls, which means that oxalate oxidase can

confer enhanced resistance to Sclerotinia blight in peanuts

(Livingstone et al., 2005). A non-heme chloroperoxidase gene (cpo-

p) from Pseudomonas pyrrocinia, a growth inhibitor of mycotoxin-

producing fungi, introduced into peanuts resulted in transgenic

plants that showed inhibition of A. avus hyphal growth and

reduced aatoxin contamination of peanut seeds (Niu et al., 2009).

The epidemiology of PBNV is yet to be fully understood for

peanut, including the search for linked markers for resistance

gene(s) and the development resistant transgenic lines for its

management. To this end, attempts have been made to develop

transgenic peanuts expressing PBNV genes using viral coat protein

(CP) genes (Satyanarayana et al., 1995). Evaluation of transgenic

peanuts with CP genes showed that resistance could be achieved

against PSND (Mehta et al., 2013). Transgenic peanut lines con-

taining the CP gene of the peanut stripe virus (PStV) were less

susceptible to PStV. TSWV has a broad host range, and it spreads

through ubiquitous thrips. The nucleocapsid (N) protein gene of the

lettuce isolate of TSWV was inserted into the peanut genome,

which showed divergent levels of gene expression (Yang et al.,

1998). Transgenic peanuts expressing the N protein of TSWV and

subjected to natural infection of the virus under eld conditions

resulted in a signicantly lower incidence of spotted wilt compared

to that of non-transgenic lines (Yang et al., 2004). Increased insect

tolerance of the transgenic plants was recorded when the cowpea

trypsin inhibitor gene was transfected into peanuts (Xu et al., 2003;

Zhuang et al., 2003). The transgenic lines developed against various

diseases in peanuts are compiled and presented in Table 6, but to

date no transgenic peanut cultivars have been released

commercially.

During the last decade, strong public opposition to genetically

modied (GM) food crops, especially in European countries, are

juxtaposed with issues such as freedom to operate related to

patented technologies (Holbrook et al., 2011). However, during lasttwo years, many patents have been approved in various countries

for eld trials of GM food crops. Two genetically modied types of

corn, namely ‘TC1507’ and ‘SmartStax’ developed by the US com-

panies Pioneer and Monsanto, respectively, won EU approval for

their cultivation in EU countries in February of 2014 and November

of 2013, respectively (Mcmanus, 2014). In India, the Union Ministry

of Environment and Forests (MoEF) has approved eld trials for GM

food crops (rice, wheat, maize, castor and cotton) to determine

their suitability for commercial production (Business Standard,

2014). Altogether, the possibility is good that various transgenic

peanut crops will be used in the next few years.

11. Achievements and future prospects

The dynamic challenges of peanut farming demand a quick

response from breeders to develop newcultivars, a process that can

be aided by the application of molecular markers (Chu et al., 2011).

‘NemaTAM ’ is the rst root-knot nematode-resistant peanut variety

developed using the MABC approach in the USA (Simpson et al.,

2003). The ‘Tifguard High O/L’ cultivar, with nematode resistance

and a high oleic:linoleic acid (high O:L) ratio in seeds, was devel-

oped using Tifguard (nematode-resistant cultivar) as the recurrent

female parent and Georgia-02C and Florida-07 (high O:L cultivars)

as donor parents after three rounds of accelerated backcrossing

using MAS (Chu et al., 2011).

A QTL region explaining up to 82.62% of the phenotypic varia-

tion for rust resistance was introgressed from cultivar ‘GPBD 4’ into

three rust susceptible varieties (ICGV 91114, JL 24 and TAG 24)through MABC using four markers (IPAHM103, GM2079, GM1536,

GM2301). This has generated several promising introgression lines

with enhanced rust resistance and higher yields. These linked

markers may be used to improve rust resistance in peanut breeding

programmes across the world (Varshney et al., 2014). Through

validating LLS and rust resistance-linked markers in different

peanut genotypes, six superior genotypes were identied. More-

over, three lines that are superior for productivity traits and equal

for disease resistance to GPBD 4 (the resistant parent) are being

included in variety release trials for multi-location evaluation

(Sukruth et al., 2015). Gajjar et al. (2014) studied 22 SSR markers

reportedly linked to rust and LLS disease resistance on 95 diverse

genotypes for marker validation, from which 16 SSRs could be

validated.



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Because of evolutionary selection pressure and their high

genetic-diversity, wild peanut species are considered to be excel-

lent sources of various disease resistance genes that remain under-

utilized (Stalker and Simpson, 1995). A dense genetic linkage map

should enable breeders to effectively pyramid genes for various

biotic stress resistances, such as rust, LLS, and nematode resistance,

into agronomically superior lines in a much shorter time period

than would be possible by conventional techniques (Gajjar et al.,

2014). RILs are being developed to map various genes/QTLs un-

derlying these traits. Currently, more than 14,000 SSR markers are

available from different sources; however, substantial efforts are

still required to develop suf cient PCR-based markers for the

construction of a high-density genetic linkage map and for routine

applications in the molecular breeding of peanuts (Mishra et al.,2014; Bosamia et al., 2015).

The International Peanut Genome Initiative with a time line of

2012e2016 is working on different aspects of biotic stress man-

agement at the genome level, including screening peanut acces-

sions for new sources of disease resistance, discovering new

sources of disease resistance, identifying a core set of informative

markers for their deployment in resistance breeding programmes,

searching and validating markers that can be used in pre-breeding

for disease and pest resistance, placing candidate genes for disease

resistance on the Arachis genetic map, and identifying core sets of

markers for QTLs associated with biotic stress resistance (http://

www.peanutbioscience.com/peanutgenomeinitiative.html ).

Nevertheless, the genomic progress made in peanuts over the last

few years has been quite encouraging, and it is expected that aclearer genomic picture of peanuts will be available by the year

2016 for researchers to utilize.

Recently, the International Peanut Genome Initiative (IPGI) has

sequenced the genome of two wild species, A. duranensis (AA

genome) and A. ipaensis (BB genome), supposedly the ancestral

parents of cultivated peanut, which is an allotetraploid species. The

hope is that the available sequence information will provide re-

searchers with approximately 96 percent of all peanut genomic

information and provide the molecular map needed to more

quickly breed drought-resistant, disease-resistant, lower-input and

higher-yielding varieties (http://www.icrisat.org/newsroom/news-

releases/icrisat-pr-2014-media13.htm ; Mondal and Badigannavar,

2015).

However, compared to other crops such as rice, soybeans and

chickpeas, the ongoing molecular breeding programme for peanuts

is relatively slow primarily due to low genetic diversity among the

cultivated gene pool, introgression barriers between wild and

cultivated lines, non-availability of genome sequence information

until last year and inadequate funding. Because a draft sequence of

the Arachis genome (AA and BB) is available, the expectation is that

in the next few years the quality of peanuts will be improved

through the use of modern biotechnological approaches ( Janila

et al., 2013). Ongoing research efforts across the world have not

only resulted in better understanding of the peanut genome but

also facilitated ongoing marker-assisted peanut breeding programs

(Holbrook et al., 2011). The use of biotechnological interventions in

peanut breeding for the development of resistance cultivars against

various biotic stresses is quite promising. Despite substantial ad-vancements in the biotic management strategies, the global peanut

production is still threatened by a multitude of insect pests and

diseases. The situation demands judicious blending of conventional

and modern crop improvement technologies for more ef cient and

rapid tackling of these problems (Krishna et al., 2015). An outline of

the tentative biotechnological interventions for peanuts is pre-

sented in Fig. 1.

12. Conclusions

We have reviewed the recent biotechnological developments

for the biotic stress management of peanut crops with special

preference given to genomic approaches. The goal of these studies

is to nd more effective and economical high-throughput tools tomanage various biotic stresses in peanuts. Conventional breeding

along with phenotyping tools have largely been used in peanut

improvement programs, and low genetic variability has been

considered one of the major bottlenecks. Therefore, mutations

were used to introduce variability, and wide-hybridizations were

attempted to determine the variability from wild Arachis species.

For the creation of newsourcesof genetic diversity, approaches that

are being explored include the development of transgenics, TILLING

(targeting-induced local lesions in genomes) populations and

synthetic allotetraploids (Holbrook et al., 2011). Additionally, a few

other robust mapping populations such as NAM (nested association

mapping) and MAGIC (multi-parent advanced generation inter-

cross) are being developed by various institutions working on

peanut crops. To date, the genetic basis of many important traits in

Table 6

Transgenics developed for various biotic stress resistances in peanut.

Genes Resistance Reference

Coat protein PBND Satyanarayana et al., 1995

cry1AC Elasmopalpus lignoscellus Singsit et al., 1997

Nucleocapsid (N) Tomato spotted wilt virus Yang et al., 1998; Magbanua et al., 2000; Yang et al., 2004

C owpea trypsin i nhi bitor gene Insec t toleran ce Xu et al., 2003; Zhuang et al., 2003

Coat protein Peanut stripe potyvirus Higgins et al., 2004

Chitinase and glucanase Sclerotinia blight Chenault et al., 2005Barley oxalate oxidase Sclerotinia blight Livingstone et al., 2005; Partridge-Telenko et al., 2011

cry1EC Spodoptera litura Tiwari et al., 2008; Tiwari et al., 2011

cry1X Helicoverpa armigera and Spodoptera litura Entoori et al., 2008

cry1EC and Chi11 Spodoptera litura and Phaeoisariopsis personata Beena et al., 2008

Coat Protein gene PStV PStV Hapsoro et al., 2008

Mustard defensin LLS Anuradha et al., 2008

Chloroperoxidase (cpo-p) A. avus Niu et al., 2009

Rice chitinase-3 Leaf spot Iqbal et al., 2012

Rchit and CHI Fusarium wilt and Cercospora arachidicola Rohini and Sankara, 2001; Iqbal et al., 2011

SniOLP and Rs- AFP2 LLS Vasavirama and Kirti, 2012

Rice chitinase gene (Rchit ) A. avus, LLS and rust Prasad et al., 2013

Coat protein PSND Mehta et al., 2013

cry1AcF Spodoptera litura Keshavareddy et al., 2013

cry8Ea1 Holotrichia parallela Geng et al., 2013

Tfgd2-RsAFP2 fusion gene ELS and LLS Bala et al., 2015


http://www.peanutbioscience.com/peanutgenomeinitiative.htmlhttp://www.peanutbioscience.com/peanutgenomeinitiative.htmlhttp://www.icrisat.org/newsroom/news-releases/icrisat-pr-2014-media13.htmhttp://www.icrisat.org/newsroom/news-releases/icrisat-pr-2014-media13.htmhttp://www.icrisat.org/newsroom/news-releases/icrisat-pr-2014-media13.htmhttp://www.icrisat.org/newsroom/news-releases/icrisat-pr-2014-media13.htmhttp://www.peanutbioscience.com/peanutgenomeinitiative.htmlhttp://www.peanutbioscience.com/peanutgenomeinitiative.html


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