American Journal of Plant Sciences, 2012, 3, 1169-1180 http://dx.doi.org/10.4236/ajps.2012.39142 Published Online September 2012 (http://www.SciRP.org/journal/ajps) 1169 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. Email: bfakrudin@gmail.com Received April 14th, 2012; revised May 12th, 2012; accepted May 20th, 2012 ABSTRACT 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. Copyright © 2012 SciRes. AJPS
Transferability of Sorghum Genic Microsatellite Markers to Peanut 1170 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 markers. 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 SbEST-SSRs 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 4). 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 Copyright © 2012 SciRes. AJPS
Transferability of Sorghum Genic Microsatellite Markers to Peanut 1172 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. Sl No GI No PRIMER PUTATIVE FUNCTION E-value Repeat Motif P/M Temp Condn 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 Copyright © 2012 SciRes. AJPS
Transferability of Sorghum Genic Microsatellite Markers to Peanut 1173 Continued 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 Copyright © 2012 SciRes. AJPS
Transferability of Sorghum Genic Microsatellite Markers to Peanut 1174 Continued 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 TRANSPORTER 2) (TG)n (GT)n P T-II 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 Continued 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 1176 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 1177 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 libraries. 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 1178 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. 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