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In silico mining of EST-SSRs in Arachis hypogaea L. and their utilization for genetic structure and diversity analysis in cultivars/breeding lines in Odisha, India

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Abstract

A total of 26,685 unutilized public domain expressed sequence tags (ESTs) of Arachis hypogaea L. were analyzed to give a total of 4442 EST-SSRs, in which 517 ESTs contained more than one simple sequence repeat (SSR). Of these EST-SSRs, 2542 were mononucleotide repeats (MNRs), 803 were dinucleotide repeats (DNRs), 1043 were trinucleotide repeats (TNRs), 40 were tetranucleotide repeats (TtNRs), six were pentanucleotide repeats (PNRs) and eight were hexanucleotide repeats (HNRs). Out of these 4442 EST-SSRs, only 1160 were found to be successful in non-redundant primer design; 1060 were simple SSRs, while the remaining 100 were compound forms. Among all the motifs, MNRs were abundant, followed by TNRs and DNRs. The AAG/CTT motif was the most abundant (~33 %) TNR, while AG/CT was the most abundant DNR. For redundancy and novelty, a stringent criterion deploying three different strategies was used and a total of 782 novel EST-SSRs were added to the public domain of peanut. These novel EST-SSR markers will be useful for qualitative and quantitative trait mapping, marker-assisted selection and genetic diversity studies in cultivated peanut as well as related Arachis species. A subset of 30 novel EST-SSRs was further randomly selected for validation and genotyping studies with eight well-known cultivars and 32 advanced breeding lines (ADBX lines, ADBY lines and ADBZ lines) from Odisha state, India. The number of polymorphic markers among accessions of A. hypogaea was low; however, a set of informative EST-SSR markers detected considerable levels of genetic variability in peanut cultivars and uncharacterized breeding lines collected from Odisha. The 30 newly developed EST-SSRs from Arachis spp. showed ~97 % amplification in Cicer arientinum and 93 % in pigeon pea. Thus, the EST-SSRs developed in this study will be a very useful asset for genetic analysis, comparative genome mapping, population genetic structure and phylogenetic inferences among wild and allied species of Arachis.

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Acknowledgments

We thank the Vice Chancellor, OUAT, Bhubaneswar, Odisha, for providing facilities to carry out this work; the SSR analysis bioinformatics team of CSIR-National Botanical Research Institute, Lucknow, for their rapid support; Vice Chancellor, Utkal University, Odisha, for his support extended to our collaborators.

Author contributions

Sushree Shivani Sardar was involved in collections of plant materials and breeding lines, DNA isolation and SSR genotyping. Kedareswar Pradhan coordinated collections of plant materials and breeding lines, DNA isolation, SSR genotyping and manuscript drafting. Ravi Prakash Shukla designed EST-SSRs primers and their comparison with existing public domain database. Ribha Saraswat was involved in peanut public domain data search and analysis for unutilized ESTs, designing of EST-SSRs and their primers. Anukool Srivastava was involved in repeat motif analysis and their frequency and comparison of EST–SSRs with existing public domain database and novelty of EST-SSRs. Satya Narayan Jena coordinated designing EST-SSRs, their primers, redundancy check, analysis for genetic relationship and population structuring pattern among breeding line and cultivars with SSRs and manuscript writing. Anath Bandhu Das was involved in amplification of EST-SSRs and their sequencing for number of repeat motif analysis, novel EST-SSRs annotation and cross-transferability analysis.

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Authors and Affiliations

  1. Department of Genetics and Plant Breeding, Orissa University of Agriculture and Technology, Bhubaneswar, Odisha, 751003, India

    Sushree Sibanee Sardar & Kedareswar Pradhan

  2. Plant Molecular Biology Laboratory, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow, 226001, India

    Ravi Prakash Shukla, Ribha Saraswat, Anukool Srivastava & Satya Narayan Jena

  3. Department of Botany, Utkal University, Vani Vihar, Bhubaneswar, Odisha, 751004, India

    Anath Bandhu Das

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Supplemental table S1

Details of number of public domain ESTs, utilized ESTs and studied ESTs (DOCX 14 kb)

Supplemental table S2

Frequencies of different subclasses of EST-SSRs measured in repeat number of count (DOCX 22 kb)

Supplemental figure S3

Frequencies of different subclasses of EST-SSRs found in 26,685 unutilized ESTs of Arachis (PPTX 74 kb)

Supplemental table S4

Details of protein homology search with NR database (XLSX 41 kb)

Supplemental table S5

Details of protein homology search with TAIR database (XLSX 63 kb)

Supplemental table S6

Unmatched SSR IDs with references to whole sequence, primer sequence and flanking region searches (XLSX 86 kb)

Supplemental table S7

Venn diagram of novel search of 1160 EST-SSRs with reference to whole sequence, primer sequence and 50-bp 3′ and 5′ flanking regions with public domain database (PPTX 94 kb)

Supplemental table S8

Details of non-redundant EST-SSRs, their primer pairs, EST sources, GenBank ID, repeat motif, Tm and expected size (XLSX 123 kb)

Supplemental table S9

Details of validation of 30 EST-SSRs with eight parental genotypes of Arachis of Odisha state (XLSX 18 kb)

Supplemental table S10

Allele-size data of 30 validated EST-SSRs in 40 genotypes/advanced breeding lines of peanut (XLSX 25 kb)

Supplemental table S11

Multiple alignment of size-variant fragments amplified from two peanut parents and advanced breeding lines for EST-SSR marker, NBRI RS 492, showing the presence of repeat motif (CT)n with seven units in both parents and ADBZ1, but six units in ADBY9, ADBY12, ADBY15, ADBY16, ADBZ3. The flanking region of EST-SSR revealed the presence of SNPs (PPTX 105 kb)

Supplemental table S12

Detailed pair-wise genetic distance of 40 cultivars/breeding lines of Arachis collected from Odisha state. (XLSX 52 kb)

Supplemental table S13

Population structure of 40 genotypes/advanced breeding lines estimated from 30 EST-SSRs using STRUCTURE program at K = 2 (PPTX 89 kb)

Supplemental table S14

Various values of ln′(K), |ln″(K)| and delta-K at different presumed K values in Evanno test (XLSX 11 kb)

Supplemental table S15

Principal coordinate analysis of 30 EST-SSRs profiles to resolve genetic relationships among the eight parents and 32 advanced breeding lines (PPTX 48 kb)

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Sardar, S.S., Pradhan, K., Shukla, R.P. et al. In silico mining of EST-SSRs in Arachis hypogaea L. and their utilization for genetic structure and diversity analysis in cultivars/breeding lines in Odisha, India. Mol Breeding 36, 49 (2016). https://doi.org/10.1007/s11032-016-0466-y

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