American Journal of Plant Sciences, 2012, 3, 1169-1180 Published Online September 2012 (
Transferability of Sorghum Genic Microsatellite Markers
to Peanut
Siddanna B. Savadi1, Bashasab Fakrudin1*, H. L. Nadaf2, M. V. C. Gowda2
1Department of Biotechnology, University of Agricultural Sciences, Dharwad, India; 2Department of Genetics and Plant Breeding,
University of Agricultural Sciences, Dharwad, India.
Received April 14th, 2012; revised May 12th, 2012; accepted May 20th, 2012
Currently development of new marker types has shifted from anonymous DNA fragments to gene-based markers. Sim-
ple Sequence Repeats (SSRs) are useful DNA markers in plant genetic research including in peanut. However, de novo
development of SSRs is expensive and time consuming. Gene-based DNA markers are transferable among related spe-
cies owing to the conserved nature of genes. In this study transferability of sorghum EST-SSR (SbEST-SSR) markers to
peanut was prospected. A set of 411 SbEST-SSR primer pairs were used to amplify peanut genomic DNA extracted
from cultivated peanut where 39% of them successfully amplified. A comparison of amplification patterns between
sorghum and peanut showed similar banding pattern with majority of transferable SbEST-SSRs. Among these transfer-
able SSR markers, 14% have detected polymorphism among 4 resistant and 4 susceptible peanut lines for rust and late
leaf spot diseases. These transferable markers will benefit peanut genome research by not only providing additional
DNA markers for population genetic analyses, but also allowing comparative mapping to be possible between peanut
and sorghum—a possible monocot-dicot comparison.
Keywords: Arachis Hypogea; Transferability; EST-SSR; Polymorphism
1. Introduction
Peanut (Arachis hypogaea L.) is an important oilseed
crop and it has acquired prominence because of its eco-
nomic importance as well as its nutritional value. It is the
third major oilseed crop in the world next only to soy-
bean and cotton. A. hypogaea is believed to have origin-
nated in the Southern Bolivia to Northern Argentina re-
gion of South America. The present day cultivated pea-
nut is an allotetraploid (2n = 4x = 40) while most of wild
relatives are diploid (2n = 2x = 20) in nature. The yield
of the peanut crop has been very low due to biotic and
abiotic stresses and the varietal improvement in peanut
has been difficult due to the limited knowledge on the in-
heritance of important traits and lack of proper under-
standing of genetic diversity and population structure.
Molecular markers have become an important tool in
crop breeding programs for dissecting loci controlling
complex traits: genetic diversity among accession and
evolutionary conservation studies can be done. However,
application of molecular markers in peanut crop im-
provement has been relatively lagging behind chiefly
owing to limited knowledge of genome and seldom mo-
lecular variations revealed by RFLP [1], RAPD [2] and
isozyme markers systems [3]. Indeed, there is an urgent
need to focus efforts on a systematic and comprehensive
examination of the germplasm accessions available in the
peanut employing robust marker types such as SSRs to
reveal polymorphism at the molecular level [4]. Simple
Sequence Repeats (SSRs) or microsatellites have become
one of the most widely preferred molecular marker sys-
tems for genetic analysis for their advantages compared
to other molecular markers: high reproducibility, high
polymorphism, being multi-allelic, co-dominant, higher
relative abundance and extensive genome coverage are
some of the advantages envisaged with SSRs [5]. Previ-
ous studies in peanut have shown that SSR markers could
detect more polymorphism than other molecular markers
like RFLPs [6], AFLPs [7] and RAPDs [8,9].
The de novo development of SSRs markers is a costly
and time-consuming endeavor [5,10], as it involves ap-
proaches, such as genomic library construction, enrich-
ment and screening which are laborious and time con-
suming: this reduces the general utility of this marker
system [11] and also dramatically discounts the advan-
tages [12]. The progress of development or discovery of
new marker types has shifted from anonymous DNA
fragments to gene-based markers, also called as func-
tional markers. Gene based markers are more powerful
*Corresponding author.
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Transferability of Sorghum Genic Microsatellite Markers to Peanut
than others for breeding applications and allele discovery
[13]. ESTs are presently used on a large scale for the
systematic development of gene-based SSR and SNP
markers. EST-SSR markers have been developed for a
number of plant species, such as pigeon pea [14], grape
[15], rice [16], durum wheat [17], rye [18], barley [19],
ryegrass [20], wheat [21], peanut [22] and cotton [23].
EST-SSRs are advantageous over genomic SSRs, as they
can be obtained from public EST databases.and transfer-
able across taxonomic barriers [24]. A putative function
can be deduced for the EST-SSRs as they represent ESTs,
they serve as gene-tagged markers and can be directly
associated with an expressed gene: this offers linking
with putative qualitative or quantitative trait locus alleles.
Thus, EST-SSR markers are superior and more informa-
tive compared to anonymous markers [25]. Comparative
genetic analysis has shown that different plant species
often share orthologous genes for very similar functions
[26] and gene contents and gene orders among different
plant species could be highly conserved [27,28].
As EST-SSR markers are derived from expressed
genes, they are more conserved and have a higher level
of transferability to related species. Study of transferabi-
lity of markers has been attempted in several plant spe-
cies across different taxa [14,15,19,29-32] as well as in
peanut [12,33,34]. However, the conserved nature of
EST-SSRs may also limit their degree of polymorphism.
The feasibility of utilizing EST-SSRs from monocots in
dicots has been investigated. Plant genes display signifi-
cant conservation between the monocots and dicots, thus,
theoretical possibility of transferability from monocots to
dicots is a possibility [35]. Requiring more concerted
efforts in using modern genomic tools, peanut genome
research has made less progress [36,37]. Thus, one of the
pressing needs in peanut genomic research is to take ad-
vantage of progress made in the well characterized other
crops. About 25% SSRs [38] and 34% EST-SSRs [39]
transferability from soybean to peanut has been reported
[40]. A 20% transferability of EST-SSRs from Medicago
to peanut has been reported. In this study, we focused on
analyzing the utility of EST-SSR markers from sorghum
(monocot) to peanut (dicot) experimentally. Sorghum is
considered to be model grass genome where genetic
study has been done at good pace [41] and its genome
sequencing is completed [42], and hence it could be good
source of transferable markers especially the gene-based
2. Materials and Methods
Total DNA from sorghum cultivar E36-1 and a set of
four resistant and four rust and leaf spot diseases suscep-
tible peanut cultivars (Table 1) was isolated following
CTAB protocol of Murry and Thompson (1980) [43]
with suitable modifications. The genomic DNA was used
as the template for all PCR amplifications. Sorghum
EST-SSRs (SbEST-SSRs) developed at IABT, UAS,
Dharwad and synthesized from Sigma-Aldrich pvt. Ltd,
USA, were screened for amplification of peanut DNA
using optimized PCR reaction mixture and touchdown
PCR Profiles. PCR optimization was done using three
different programs of “Touchdown” PCR [44] with base
annealing temperature ranges of 55˚C - 50˚C, 60˚C -
55˚C, and 65˚C - 60˚C. The primers were classified into
three groups based on annealing temperature range re-
quired by them to produce sharp bands without much of
spurious products. In the initial annealing steps, the an-
nealing temperature was decreased by one centigrade
after two subsequent cycles for first 10 cycles. Products
were thereafter amplified for 30 cycles at the appropriate
optimum annealing temperature with a final extension of
20 min. Reaction mixtures of 10 μl containing 10 mM
Tris-HCl (pH 8.8), 50 mM KCl, 10 mM MgCl2, 2.5mM
of each of dNTPs, 20 pM each of forward and reverse
primers, 50 ηg of genomic DNA and 2.5U Taq DNA
polymerase (Fermentas) was used for PCR amplification.
Transference is defined as the positive amplification of
a PCR band of the expected size [45]. SbEST-SSR pri-
mers amplified during primer screening of peanut were
also used for comparing amplification patterns (size and
Table 1. Transferable markers were checked for their ability to detect polymorphism in following eight groundnut cultivars.
Sl No Genotypes Rust Late leaf spot Phenotypes
1 LSVT-I-2003-1 2 2 R
2 ISK-I-2004-4 2 1 R
3 IVT-I-2005-5 2 1 R
4 GPBD-4 3 3 R
5 JL-24 7 8 S
6 TMV-2 7 8 S
7 TAG-24 7 8 S
8 TG-26 7 8 S
Cultivars with a 1 - 3 disease score were designated as resistant and a 4 - 9 score as susceptible according to Pande and Rao (2001).
Copyright © 2012 SciRes. AJPS
Transferability of Sorghum Genic Microsatellite Markers to Peanut 1171
number of bands) to further confirm orthology or trans-
ferability by carrying out amplification in both sorghum
and peanut DNA using optimized PCR reaction mixture
and touchdown PCR profiles. The transferable SbEST-
SSRs were then tested for their ability to detect poly-
morphism in a set of four resistant and four susceptible
breeding lines/cultivars (Table 1) for rust and late leaf
spot diseases of peanut and the ones showing polymer-
phic banding pattern on 4% PAGE gel were considered
as polymorphic markers.
3. Results
3.1. Screening of SbEST-SSRs and Comparison
of Amplification Pattern between Sorghum
and Peanut and Their Polymorphism in
Peanut Cultivars
Out of 411 sorghum EST-SSR primer pairs tested, 161
(39%) were amplifiable in peanut (Table 1) showing
clear sharp bands but other primers gave smear with light
bands or did not amplify under three Touch Down (TD)-
PCR profile conditions (Figure 1). The remaining primer
pairs either recorded no amplification products or pro-
duced a number of faint bands indicating non-specific
amplifications. Out of 161 amplified primers 16 ampli-
fied at 65˚C - 60˚C, 95 at 60˚C - 55˚C and 61 at 55˚C -
50˚C TD-PCR temperature ranges (Table 2). These am-
plifiable markers implied that 39% of primer-binding
sites were conserved between sorghum and peanut ge-
nomes. These primer pairs produced clear PCR bands
and the majority of primers produced multiple bands.
The number of bands amplified by each SbEST-SSR
primer pairs varied from 1 to 16 on 4% polyacrylamide
gel stained following silver staining procedure (Figure 2).
Further, comparison of the kind of amplification pattern
between sorghum and peanut crop species showed simi-
lar banding pattern for many of the SbEST-SSRs; how-
ever, it varied in some of the cases (Figure 3) and the
difference was in number of bands amplified, which were
more or less in either of the crops. Of the161 EST-SSRs
18% were found polymorphic on 4% polyacrylamide gel.
3.2. Structural Analysis and Annotations of
Among these conserved SbEST-SSRs, 91 (56%) dinu-
cleotide repeat motifs appeared to be the most abundant
type, followed by 61 (37.2%) trinucleotides, 8 (6.3%)
consisting of complex nucleotide repeats and then one
tetranucleotide (0.5%) repeats (Table 3). Among these
repeats TC/CT were 35, AG/GA were 28, AGC/CAG/
GCA were 17 and AGC/CAG/GCA were 16. The com-
position of repeat motifs of transferred SbEST-SSRs is
presented in Table 4. Of 161 SbEST-SSR markers, 24
(14%) detected polymorphism on 4% PAGE among 8
accessions of peanut consisting of equal number of resis-
tant and susceptible accessions (Figure 2). Twenty four
polymorphic SbEST-SSRs consisted of 17 (18.7%) dinu-
cleotide repeats, 6 (9.8%) trinucleotide repeats and one
(1.2%) consisted of complex repeat motifs. These results
indicate that there is some kind of correlation between
polymorphism and repeat number. The distribution of
transferred SSRs’ positions was found highest in 5’ESTs
with 67, then 32 in 3’ESTs and 62 in other ESTs. Among
these the SSRs from 3’ESTs were more polymorphic
(21.9%) than 5’ESTs (13.4%) and others (12.9%) (Table
Annotation for the common SbEST-SSRs was per-
formed using the GenBank databases and BLASTX tool
with an expectation value of 1e-5 or better. Eighty three
(52%) of the common ESTs were annotated using BLA-
STX and are listed in Table 5. Most of annotated Sb-
ESTs were related to metabolism, photosynthesis, signal
transduction, growth, and transportation across mem-
branes, stress and defense. The remaining SbESTs when
searched for putative functions resulted in no hits (2.5%),
Table 2. Screening results for 411 SbEST-SSR primers on groundnut genotypes: total number and the percentages of primer
pairs amplified at three different Touch Down P CR pr ofile s.
Sl. No PCR No of Primers Rate 65˚C - 60˚C 60˚C - 55˚C 55˚C - 50˚C
1 Amplified 161 39% 19 (12%) 81 (50%) 61 (38%)
2 Unamplified 250 61% - - -
Total 411 100% - - -
Table 3. Structural details of repeat motifs in transferable SbEST-SSRs and polymorphism revealed by them in groundnut.
Type Dinucleotides Trinucleotides Tetranucleotide Complex
Amplified 91 61 1 8
Per cent Amplification 56.5 37.9 0.6 5
Polymorphic 17 6 0 1
Per cent Polymorphic 18.7 9.8 0 1.2
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Transferability of Sorghum Genic Microsatellite Markers to Peanut
Table 4. Distribution of repeats location or SSRs positions in ESTs of transferable SbEST-SSRs.
Type 3’ESTs 5’ESTs Others
Amplified 32 67 62
Per cent Amplification 19.9 41.6 38.5
Polymorphic 7 9 8
Per cent Polymorphic 21.9 13.4 12.9
Table 5. Description of SbEST-SSR markers transferred to groundnut: NCBI accession ID, repeat motifs, their ability to
detect polymorphism among groundnut accessions and their functional annotation by BLASTX.
1 30936043 iabtgs83 Eukaryotic translation initiation factor 3 2.00E–15(CCG)n (CGC)n (GCC)n M T-III
2 31332261 iabtgs144 Carbamoyl-phosphate synthase
l atp-binding 3 (TCC)n (CTC)n (CCT)n M T-II
3 31331586 iabtgs145 Armadillo/beta-catenin repeat family protein3.00E–12(CCG)n (CGC)n (GCC)n M T-II
4 31330911 iabtgs146 Putative auxin response factor 10 (AGG)n (GAG)n (GGA)n M T-III
5 30976409 iabtgs148 Unknown protein 2.00E–06(CTAG)n M T-III
6 30975909 iabtgs149 Membrane protein-like 3.00E–23(AGC)n (CAG)n (GCA)n M T-II
7 30969042 iabtgs151 Hypothetical protein osj_010305 4.00E–10(AGG)n (GAG)n (GGA)n M T-III
8 30966593 iabtgs152 Heat shock complementing factor1 1.00E–17(AGC)n (CAG)n (GCA)n M T-II
9 30963637 iabtgs155 Dna methylase n-4/n-6 domain protein 3.8 (AGC)n (CAG)n (GCA)n M T-III
10 30946150 iabtgs157 Dnaj heat shock n-terminal
domain-containing 7.00E–12(AGG)n (GAG)n (GGA)n M T-III
11 18061855 iabtgs158 Hypothetical protein osi_023589 8.00E–29(TCG)n (CGT)n (GTC)n M T-III
12 30935909 iabtgs162 Gdsl-motif lipase/hydrolase family protein 1.00E–56(TCG)n (CGT)n (GTC)n M T-II
13 18061163 iabtgs164 Putative vascular plant one zinc finger
protein 2.00E–27(AGC)n (CAG)n (GCA)n M T-III
14 14593539 iabtgs165 Putative peroxidase 9.00E–48(AGC)n (CAG)n (GCA)n M T-II
15 12616750 iabtgs170 Hypothetical protein 8.00E–70(TCG)n (CGT)n (GTC)n M T-II
16 12776313 iabtgs173 Hypothetical protein 0.16 (TCC)n (CTC)n (CCT)n M T-III
17 12501226 iabtgs174 Hypothetical protein (TCG)n (CGT)n (GTC)n M T-II
18 12498888 iabtgs175 Hypothetical protein 1.00E–32(TGG)n (GTG)n (GGT)n M T-III
19 11996438 iabtgs177 Hypothetical protein 9.00E–08(CGG)n (GCG)n (GGC)n M T-III
20 11920940 iabtgs178 No significant similarity found (AGC)n (CAG)n (GCA)n M T-III
21 11679357 iabtgs179 Leucine-rich repeat transmembrane protein5.00E–23(TTC)n (TCT)n (CTT)n M T-III
22 11677886 iabtgs181 Hypothetical protein (AGT)n (TAG)n (GTA)n M T-II
23 9850409 iabtgs188 CBS domain-containing protein 1.00E–65(TCC)n (CTC)n (CCT)n M T-III
24 9299930 iabtgs190 Di-haem cytochrome c peroxidase 3.2 (AGC)n (CAG)n (GCA)n M T-II
25 9299634 iabtgs191 Putative fatty acid elongase 9.00E–25(GCG)n (GGC)n M T-II
26 7553794 iabtgs196 hypothetical protein 2.00E–06(ACG)n (CGA)n (GAC)n M T-III
27 7551234 iabtgs198 AT hook-containing DNA-binding protein 2.00E–10(AGT)n (TAG)n (GTA)n M T-II
28 7535116 iabtgs199 No significant similarity found (TCG)n (CGT)n (GTC)n M T-II
29 7553718 iabtgs200 ripening regulated protein 5.00E–08(TTC)n (TCT)n (CTT)n M T-III
30 30161896 iabtgs201 NADPH-thioredoxin reductase 3.00E–87(CCG)n (CGC)n (GCC)n P T-II
31 31347554 iabtgs202 unnamed protein product 6.00E–42(CCG)n (CGC)n (GCC)n M T-II
32 34446671 iabtgs203 CYP77B1 (cytochrome P450, family 77 4.00E–54(CCG)n (CGC)n (GAC)n (GCC)n M T-III
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Transferability of Sorghum Genic Microsatellite Markers to Peanut 1173
33 30977482 iabtgs205 transporter-related 1.00E–35(AGG)n (GAG)n (GGA)n M T-II
34 34512855 iabtgs207 hypothetical protein OsI_000407 6.00E–75(TTG)n (TGT)n (GTT)n M T-III
35 7536147 iabtgs208 putative xyloglucan endotransglycosylase 9.00E–46(CGG)n (GCG)n (GGC)n M T-II
36 30950922 iabtgs209 3-ketoacyl-CoA synthase 1.00E–51(AGC)n (CAG)n (GCA)n M T-III
37 45949294 iabtgs210 ETHYLENE-INSENSITIVE3-like 1 protein3.00E–45(AGC)n (CAG)n (GCA)n M T-II
38 30968428 iabtgs220 putative copper chaperone 5.00E–27(TC)n (CT)n /(TGC)n (CTG)n (GCT)n M T-II
39 57812929 iabtgs221 ACR4 (ACT REPEAT 4) 2.00E–26(ACT)n (TAC)n (CTA)n/(TC)n (CT)n P T-II
40 37753412 iabtgs222 harpin inducing protein 1.00E–09(TGC)n (CTG)n (GCT)n P T-II
41 9296509 iabtgs223 dehydration responsive element binding 2.00E–22(ACC)n (CAC)n (CCA)n M T-III
42 61099098 iabtgs225 hypothetical protein OsJ_024137 2.00E–17(CGG)n (GCG)n (GGC)n M T-II
43 45993487 iabtgs226 glycoside hydrolase family 28 protein 1.00E–56(TGC)n (CTG)n (GCT)n P T-I
44 9305233 iabtgs227 unknown protein (AGC)n (CAG)n (GCA)n M T-I
45 34510491 iabtgs228 Hypothetical protein (CGG)n (GCG)n (GGC)n P T-II
46 9303289 iabtgs229 Nitrate induced NOI protein 1.00E–40(CCG)n (CGC)n (GCC)n /(AG)n (GA)n M T-II
47 18052712 iabtgs230 ZIM motif-containing protein 0.12 (AGC)n (CAG)n (GCA)n M T-I
48 33110457 iabtgs231 hypothetical protein 1.00E–19(TCG)n (CGT)n (GTC)n P T-I
49 45957262 iabtgs234 leaf senescence related protein-like 9.00E–78(AGC)n (CAG)n (GCA)n M T-II
50 14089228 iabtgs235 Ethylene receptor 3.00E–61(CCG)n (CGC)n (GCC)n M T-I
51 57808251 iabtgs237 CHCH 2.00E–12(CCG)n (CGC)n (GCC)n M T-III
52 45965553 iabtgs242 Cytochrome P450 71E1 3.00E–85(ACG)n (CGA)n (GAC)n M T-II
53 30163708 iabtgs244 phosphoglycerate mutase-like protein 8.00E–52(AGC)n (CAG)n (GCA)n M T-II
54 34443079 iabtgs250 No significant similarity found. (AGG)n (GAG)n (GGA)n M T-III
55 30968371 iabtgs251 putative copper chaperone 5.00E–27(AGC)n (CAG)n (GCA)n M T-II
56 34443334 iabtgs259 No significant similarity found (AAG)n (AGA)n (GAA)n M T-II
57 45990071 iabtgs260 Diadenosine5',5'''-P1 tetraP hydrolase 1.00E–88(CCG)n (CGC)n (GCC)n M T-III
58 34517721 iabtgs263 putative inositol-3-phosphate synthase 3.00E–30(AGC)n (CAG)n (GCA)n M T-III
59 30945913 iabtgs264 chloroplast O2-evolving enhancer protein 18.00E–68(ACG)n (CGA)n (GAC)n M T-III
60 45968979 iabtgs269 EREBP transcription factor 9.00E–46(AGG)n (GAG)n (GGA)n M T-II
61 9303947 iabtgs274 hypothetical protein 3.00E–25(ACC)n (CAC)n (CCA)n M T-II
62 33108470 iabtgs280 PyroP-dependent phosphofructokinase 2.00E–22(AAG)n (AGA)n (GAA)n/(AG)n (GA)n M T-II
63 34440910 iabtgs281 putative 4,5-DOPA dioxygenase extradiol 1.00E–37(AGG)n (GAG)n (GGA)n M T-II
64 45975671 iabtgs282 Plastocyanin precursor 7.00E–19(TCC)n (CTC)n (CCT)n M T-II
65 31332334 iabtgs287 No significant similarity found. (TC)n (CT)n M T-II
66 37711087 iabtgs289 Cyclic beta 1-2 glucan synthetase 3 (AG)n (GA)n M T-II
67 18062597 iabtgs301 No significant similarity found (AT)n (TA)n M T-I
68 30946136 iabtgs304 unknown protein 8.00E–09(TC)n (CT)n M T-III
69 30164571 iabtgs305 No significant similarity found (TC)n (CT)n M T-III
70 7535895 iabtgs307 Mitogen activated protein kinase 6 2.00E–06(TC)n (CT)n P T-II
71 11921286 iabtgs309 AMP-binding protein 1.00E–35(AG)n (GA)n M T-III
72 9851429 iabtgs310 unknown (AC)n (CA)n P T-III
73 30939660 iabtgs314 putative polyprotein 9.00E–14(TG)n (GT)n P T-III
74 30964510 iabtgs322 hypothetical protein 3.00E–15(AG)n (TC)n M T-II
75 11922775 iabtgs323 hypothetical protein 2.00E–26(AT)n (TA)n M T-II
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Transferability of Sorghum Genic Microsatellite Markers to Peanut
76 31329611 iabtgs324 hypothetical protein OsJ_018717 8.00E–58(AG)n (GA)n M T-II
77 18052335 iabtgs327 proline-rich spliceosome-associated factor 8.00E–37(AT)n (TA)n M T-II
78 31383744 iabtgs340 cytochrome P450 monooxygenase
CYP77B5 7.00E–52(AG)n (GA)n P T-II
79 57821918 iabtgs341 Chloroplast phytoene synthase 1 9.00E–38(AG)n (TG)n (GA)n (GT)n M T-III
80 57807306 iabtgs342 Serine/threonine-protein kinase MHK 3.00E–07(AC)n (CA)n M T-III
81 45993419 iabtgs343 APX4_SOLLC L-ascorbate peroxidase 8.00E–33(AG)n (GA)n P T-II
82 37759348 iabtgs345 hypothetical protein HEAR0860 8.7 (AG)n (GA)n M T-III
83 45969660 iabtgs346 E3 ubiquitin ligase 9.00E–06(TC)n (CT)n M T-III
84 34509854 iabtgs349 LHT2 (LYSINE HISTIDINE
85 34517131 iabtgs350 auxin efflux carrier family protein 2.00E–40(TC)n (CT)n M T-III
86 37753820 iabtgs352 Urease accessory protein UreD 2.3 (AT)n (TA)n P T-II
87 37711166 iabtgs353 COG0583: Transcriptional regulator 3.2 (TC)n (CT)n M T-II
88 37706443 iabtgs355 isoprenylcysteine carboxyl
methyltransferase 9.3 (TG)n (GT)n M T-II
89 37705823 iabtgs356 No significant similarity found (TC)n (CT)n M T-III
90 37705032 iabtgs357 SJCHGC01974 protein 2.1 (AC)n (CA)n M T-III
91 34509495 iabtgs359 hypothetical protein 3.00E–05(AG)n (GA)n M T-II
92 34442668 iabtgs360 hydroxycinnamoyl transferase 3.00E–31(AG)n (GA)n M T-II
93 33110250 iabtgs362 NADH-ubiquinone oxidoreductase-protein 3.00E–27(AG)n (GA)n M T-III
94 34443764 iabtgs363 No significant similarity found (AC)n (CA)n P T-I
95 34440481 iabtgs364 hypothetical protein 2.00E–09(AC)n (CA)n M T-III
96 33110768 iabtgs366 putative shrunken seed protein 0.78 (TC)n (CT)n M T-II
97 33107765 iabtgs367 No significant similarity found (AT)n (TA)n M T-III
98 31330527 iabtgs369 No significant similarity found (TC)n (CT)n P T-II
99 30979643 iabtgs371 cation efflux system protein ,RNA helicase 4.4 (TG)n (GT)n M T-II
100 30976566 iabtgs375 hypothetical protein (AT)n (TA)n M T-III
101 30974675 iabtgs377 Plant viral-response family protein 5.00E–06(CG)n M T-III
102 30974381 iabtgs378 putative Myb-like DNA-binding protein 1.00E–07(AG)n (GA)n M T-II
103 30947756 iabtgs381 hypothetical protein 1.6 (AG)n (GA)n M T-II
104 30964406 iabtgs382 Glycyl-tRNA synthetase 0.59 (AG)n (GA)n M T-III
105 30966299 iabtgs383 GHMP kinase family protein 7.00E–39(AG)n (GA)n M T-II
106 30937801 iabtgs384 putative membrane protein 0.9 (TC)n (CT)n M T-II
107 18052318 iabtgs385 hypothetical protein Os01g0260800 0.002 (AT)n (TA)n P T-I
108 30936750 iabtgs386 heat shock protein 2.00E–33(TG)n (GT)n M T-II
109 18065756 iabtgs387 No significant similarity found (TG)n (GT)n M T-II
110 18060131 iabtgs397 hypothetical protein (AG)n (GA)n P T-II
111 18051780 iabtgs399 No significant similarity found (TC)n (CT)n M T-II
112 14513091 iabtgs407 hypothetical protein 1.00E–27(TG)n (GT)n M T-II
113 13587956 iabtgs408 hypothetical protein 4.00E–05(TC)n (CT)n M T-III
114 13469439 iabtgs410 No significant similarity found (AG)n (GA)n M T-II
115 11678719 iabtgs411 putative laccase 3.00E–67(TC)n (CT)n M T-II
116 12776058 iabtgs416 hypothetical protein 2.00E–44(AT)n (TA)n M T-III
117 11409368 iabtgs421 hypothetical protein 2.2 (AC)n (CA)n M T-II
118 9850860 iabtgs425 WRKY transcription factor 16 2.2 (AT)n (TA)n M T-II
Copyright © 2012 SciRes. AJPS
Transferability of Sorghum Genic Microsatellite Markers to Peanut 1175
119 9305649 iabtgs429 No significant similarity found (AT)n (TA)n M T-III
120 7536347 iabtgs430 calcium channel 8.5 (TC)n (CT)n M T-III
121 30973885 iabtgs439 Adenosine 5'-phpsphosulfate reductase 6 2.00E–76(TC)n (CT)n M T-III
122 57815251 iabtgs440 Endoxyloglucan transferase (TG)n (GT)n P T-II
123 30160959 iabtgs441 shaggy-like kinase etha (OSKetha) 9.00E–50(CCG)n (CGC)n M T-I
124 31332570 iabtgs445 para-hydroxybenzoate-polyprenyltransferase3.6 (TC)n (CT)n M T-III
125 33109119 iabtgs447 NADP dependent maleic enzyme 1.00E–14(AG)n (GA)n P T-III
126 7659319 iabtgs449 No significant similarity found (TG)n (GT)n M T-III
127 11922518 iabtgs450 transcription factor MybS3 3.00E–53(TC)n (CT)n P T-III
128 30974889 iabtgs455 Nucellin like aspartic protease 9.00E–43(AG)n (GA)n M T-II
129 33109611 iabtgs456 unknown protein 8.00E–06(TC)n (CT)n M T-I
130 17886525 iabtgs457 hypothetical protein (AG)n (GA)n M T-II
131 61099192 iabtgs458 No significant similarity found (AC)n (CA)n M T-I
132 33108498 iabtgs460 hypothetical protein 0.65 (AG)n (GA)n M T-III
133 33108027 iabtgs464 hypothetical protein (TC)n (CT)n M T-III
134 34442937 iabtgs470 ATTIC21/CIA5/PIC1/(chloropast Import) 3.00E–20(TG)n (GT)n P T-II
135 30937618 iabtgs472 No significant similarity found (TGC)n (CTG)n (GCT)n /(TG)n (GT)n M T-I
136 30942400 iabtgs473 ribosomal-protein-alanine acetyltransferase3.2 (TC)n (CT)n M T-II
137 18070466 iabtgs478 VHS2 protein 1.00E–04(AT)n (TA)n M T-I
138 30939303 iabtgs484 Alpha tubulin 9.00E–08(TC)n (CT)n M T-I
139 12497850 iabtgs487 hypothetical protein 5.1 (AT)n (AC)n /(TA)n (TC)n (CA)n (CT)n M T-II
140 31385392 iabtgs488 Protease inhibitor/seed storage/LTP family 1.00E–15(AG)n (GA)n M T-II
141 30975546 iabtgs491 quinone-oxidoreductase QR1 6.00E–07(TC)n (CT)n M T-II
142 45960926 iabtgs492 No significant similarity found (TC)n (CT)n M T-II
143 30952757 iabtgs493 hypothetical protein 3.00E–07(TG)n (GT)n P T-III
144 12618782 iabtgs496 putative receptor-like kinase 7.00E–10(TC)n (CT)n M T-III
145 9304578 iabtgs499 retrotransposon protein 0.2 (AC)n (CA)n M T-II
146 18066228 iabtgs500 Serine/threonine-protein kinase SSN3 8.5 (TG)n (GT)n M T-I
147 12775763 iabtgs502 putative membrane protein 4.00E–07(TC)n (CT)n M T-III
148 61115436 iabtgs504 No significant similarity found (AT)n (TA)n M T-I
149 8088843 iabtgs505 peptidase C14, caspase catalytic subunit
p20 8.7 (AG)n (GA)n M T-III
150 45961441 iabtgs507 No significant similarity found (AG)n (GA)n M T-III
151 33109939 iabtgs510 prolylcarboxypeptidase-like protein 0.089 (TC)n (CT)n P T-I
152 11678708 iabtgs512 hypothetical protein 0.002 (AGC)n (CAG)n (GCA)n/(AG)n (GA)n M T-II
153 5043542 iabtgs514 hypothetical protein (AT)n M T-II
154 34445779 iabtgs516 Chitin-inducible gibberllin-responsive
protein 4.00E–40(AG)n (GA)n M T-II
155 33108255 iabtgs517 No significant similarity found (TC)n (CT)n M T-I
156 30973999 iabtgs518 hypothetical protein 2.00E–15(TC)n (CT)n M T-III
157 57821918 iabtgs520 No significant similarity found M T-II
158 34515133 iabtgs528 Ubiquitin-conjugating enzyme like 3.00E–12(TC)n (CT)n M T-II
159 33108946 iabtgs529 hypothetical protein (TC)n (CT)n P T-II
160 9852958 iabtgs530 No significant similarity found (AT)n (TA)n M T-I
161 45988690 iabtgs531 RISBZ4 (CGG)n (GCG)n (GGC)n/(AG)n (GA)n M T-I
-T-I, T-II and T-III represent the three Touchdown PCR thermal profiles viz.60˚C - 55˚C, 55˚C - 50˚C and 50˚C - 45˚C respectively.
Copyright © 2012 SciRes. AJPS
Transferability of Sorghum Genic Microsatellite Markers to Peanut
Figure 1. Amplification pattern generate d by sorghum EST-SSR (SbEST-SSRs) during primer screening in groundnut using
genomic DNA. 1-40: Amplification products of SbEST-SSR pr imers in groundnut during primer scr ee ning.
Figure 2. The figure is a composite of multiple polyacrylamide gels which illustrates the multiple and polymorphic bands
generated using the SbEST-SSR primers in eight groundnut accessions. S1 - S4: Four rust and leafspot susceptible genotypes
JL-24, TMV-2, TAG-24 and TG-26; R1-R4: Four rust and leafspot susceptible genotypes LSVT-I-2003-1, ISK-I-2004-4,
IVT-I-2005-5and GPBD-4.
Copyright © 2012 SciRes. AJPS
Transferability of Sorghum Genic Microsatellite Markers to Peanut
Copyright © 2012 SciRes. AJPS
Figure 3. Comparison of amplification patterns in both groundnut and sorghum using SbEST-SSR marker s. G1 S1 - G15 S15:
Comparison of PCR amplification in groundnut (G) and sorghum (S) with SbEST-SSRs.
no significant homology (9.3%), or hypothetical proteins
(37%) (Table 1). In major cases of ESTs putative func-
tions matched both monocots (rice, maize) and dicots
(Arabidopsis, soybean) indicating their common sharing.
4. Discussion
Despite the tremendous diversity, plant geneticists have
found that plants exhibit extensive conservation of both
gene content and gene order [27]. Sequence similarity of
the barley ESTs with 379,944 ESTs of the two model
dicot species, Arabidopsis and Medicago suggested theo-
retical transferability of barley markers into dicot species
although at low frequency [24] and EST-SSR by virtue
of the sequence conservation of the transcribed regions
of the genome are more likely to function in distantly re-
lated species than SSR primer pair derived from genomic
In the present study 161 out of 411 SbEST-SSRs am-
plified in peanut. So, about 39% transferability or con-
servation of SSR motifs and flanking sequences found
between sorghum (monocot) and peanut (dicot). The
orthology was further confirmed by comparing amplify-
cation pattern (number of amplification products and size)
in both sorghum and peanut genomes. Majority of them
had similar amplification pattern but a few showed extra
bands with common ones either in sorghum or peanut
which may be due to duplications, insertions or deletion
mutations during course of evolution which diverged 150
million years ago [46]. Similar bands amplified regard-
less of phylogenetic distances are an important feature of
EST-SSR markers which are transferred across species or
even genera [29]. But one of the concerns is alleles of
identical size with different numbers of repeats within
the SSR (size homoplasy)) observed in most of studies,
suggesting a need for caution when interpreting alleles of
identical size found using cross-amplified SSRs based on
band migration in the absence of DNA sequences. So
knowledge of DNA sequence is essential before SSR loci
can be meaningfully used to address applied and evolu-
tionary questions. Majority of SbEST-SSRs produced
multiple bands (range 1 - 16) which is a common feature
reported in most of the studies involving transferability
of EST-SSRs.
Transferability of SbEST-SSRs in the present study is
more (39%) compared to study of He et al. [47] using
soybean genomic SSRs (25%) in peanut. But the poly-
morphism detection rate in this study (18%) is less com-
pared to latter study (28%). This illustrate that EST-SSRs
are more transferable across species or distant taxa and
are less efficient in polymorphism detection than ge-
nomic SSRs as they are derived from transcribed regions
of genome which are conserved across species. However,
transferability (39%) in this study is less compared to the
study of Gao et al. (2003) [12]. In which 69% transfer-
ability from wheat (monocot system) to soybean (dicot
system) was observed, but percent transferability or con-
served EST-SSRs from wheat to rice, maize and soybean
in the same study 43%. With regard to developing mi-
crosatellite markers, 3’-sequences yielded more poly-
morphic markers (22.9%) than 5’-ESTs (13.4%) did.
This result is not unexpected as during the process of
cDNA generation (poly T priming) there is a preferential
selection of untranslated regions (UTR) within 3’-ESTs,
therefore are more variable than 5’-ESTs. In the distribu-
tion of SSR motifs, dinucleotides were found more
common than tri, tetra or complex nucleotide repeats in
transferred SbEST-SSRs or new gene based markers ac-
counting for 56%, 37.2%, 0.5% and 5% respectively and
also SbEST-SSRs with dinucleotide repeats detected
more polymerphism (18.7%) than tri (9.8%,) and com-
plex nucleotide (1.2%) repeat motifs. Dinucletotides
were also found to detect higher polymorphism than oth-
ers, which was observed in some the previous studies
[48]. This shows that there seems to be some correlation
between repeat number and polymorphism.
The transferable SbEST-SSRs subjected to BlastX
with an e-value more than or equal to 1E-5 as a signifi-
cant homology, could annotate putative functions for
Transferability of Sorghum Genic Microsatellite Markers to Peanut
52% of the common ESTs (Table 3). Most of annotated
SbESTs were related to basic functions of plant cells
such as metabolism, photosynthesis, signal transduction,
transcription, growth, and transportation across mem-
branes, stress and defense. The remaining SbESTs search
for putative functions resulted in poor hits (2.5%) no
significant homology (9.3%) or hypothetical proteins
(37%). These may represent transcriptomes which are yet
to be characterized for their putative functions. In major
cases of ESTs putative functions matched both monocots
(rice, maize) and dicots (Arabidopsis, soybean) suggest-
ing that these are highly conserved across plant species
mainly encoding for basic functions. Thus annotations of
transferred SbEST-SSRs help to explore the potential
utility of the EST-SSR loci for comparative mapping in
peanut. Functional EST-SSRs exhibiting sequence simi-
larity to genes with a range of functions could be used
directly in determining putative traits. For example, EST-
sequences of iabtgs366 and iabtgs269 showed a strong
homology to putative shrunken seed protein and EREBP
transcription factor, which is involved in stress tolerance
respectively. This potential will make them a valuable
source of new genic SSR markers so called “perfect”
genetic markers.
Thus, by using transferability technique it was possible
to develop a set of new gene based markers for peanut
crop using genomic resources of sorghum that will be
useful for different genetic studies in peanut. In this
study, we could demonstrate the feasibility of utilizing
EST-SSRs from monocots in dicots as plant genes dis-
play significant conservation even after the long period
of independent evolution.
5. Acknowledgements
Authors are thankful to the Indo-US Agricultural Know-
ledge Initiative and Department of Biotechnology (DBT)
of Government of India for supporting research in au-
thors’ (BF) laboratory.
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