Transferability of Sorghum Genic Microsatellite Markers to Peanut

Currently development of new marker types has shifted from anonymous DNA fragments to gene-based markers. Simple 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 species 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 transferable 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.


Introduction
Peanut (Arachis hypogaea L.) is an important oilseed crop and it has acquired prominence because of its economic importance as well as its nutritional value.It is the third major oilseed crop in the world next only to soybean and cotton.A. hypogaea is believed to have originnated in the Southern Bolivia to Northern Argentina region of South America.The present day cultivated peanut 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 inheritance of important traits and lack of proper understanding 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 improvement has been relatively lagging behind chiefly owing to limited knowledge of genome and seldom molecular 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 systems 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].Previous 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 approaches, such as genomic library construction, enrichment and screening which are laborious and time consuming: this reduces the general utility of this marker system [11] and also dramatically discounts the advantages [12].The progress of development or discovery of new marker types has shifted from anonymous DNA fragments to gene-based markers, also called as functional markers.Gene based markers are more powerful 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.andtransferable 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 informative 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 transferability of markers has been attempted in several plant species across different taxa [14,15,19,[29][30][31][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 significant 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 advantage 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.

Materials and Methods
Total DNA from sorghum cultivar E36-1 and a set of four resistant and four rust and leaf spot diseases susceptible 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 required by them to produce sharp bands without much of spurious products.In the initial annealing steps, the annealing 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 MgCl 2 , 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 primers amplified during primer screening of peanut were also used for comparing amplification patterns (size and number of bands) to further confirm orthology or transferability 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 polymorphism 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 polymerphic banding pattern on 4% PAGE gel were considered as polymorphic markers.

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 produced a number of faint bands indicating non-specific amplifications.Out of 161 amplified primers 16 amplified 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 amplifiable markers implied that 39% of primer-binding sites were conserved between sorghum and peanut genomes.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 similar banding pattern for many of the SbEST-SSRs; however, 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.
Annotation for the common SbEST-SSRs was performed 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 membranes, stress and defense.The remaining SbESTs when searched for putative functions resulted in no hits (2.5%),Copyright © 2012 SciRes.AJPS   no significant homology (9.3%), or hypothetical proteins (37%) (Table 1).In major cases of ESTs putative functions matched both monocots (rice, maize) and dicots (Arabidopsis, soybean) indicating their common sharing.

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 theoretical 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 related species than SSR primer pair derived from genomic libraries.
In the present study 161 out of 411 SbEST-SSRs amplified in peanut.So, about 39% transferability or conservation of SSR motifs and flanking sequences found between sorghum (monocot) and peanut (dicot).The orthology was further confirmed by comparing amplifycation 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 regardless 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 polymorphism detection rate in this study (18%) is less compared 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 genomic 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% transferability from wheat (monocot system) to soybean (dicot system) was observed, but percent transferability or conserved EST-SSRs from wheat to rice, maize and soybean in the same study 43%.With regard to developing microsatellite markers, 3'-sequences yielded more polymorphic 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 distribution of SSR motifs, dinucleotides were found more common than tri, tetra or complex nucleotide repeats in transferred SbEST-SSRs or new gene based markers accounting 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 complex nucleotide (1.2%) repeat motifs.Dinucletotides were also found to detect higher polymorphism than others, 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 significant homology, could annotate putative functions for 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 membranes, 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) suggesting 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 similarity to genes with a range of functions could be used directly in determining putative traits.For example, ESTsequences 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 display significant conservation even after the long period of independent evolution.