Identification of a non-redundant set of 202 in silico SSR markers and applicability of a select set in chickpea (Cicer arietinum L.)

Gaurav Agarwal,Mahendar Thudi,Vikas K Singh,Rajeev K Varshney,Murali M Sabbavarapu,S Sheelamary,Pooran M Gaur

doi:10.1007/s10681-015-1394-3

Abstract

Abstract The paucity of sequence information flanking the simple sequence repeat (SSR) motifs identified especially in the transcript sequences has been limiting factor in the development of SSR markers for plant genome analysis as well as breeding applications. To overcome this and enhance the genic SSR marker repertoire in chickpea, the draft genome sequence of kabuli chickpea (CDC Frontier) and publicly available transcript sequences consisting of in silico identified SSR motifs were deployed in the present study. In this direction, the 300 bp sequence flanking the SSR motifs were retrieved by aligning 566 SSR containing transcripts of ICCV 2 available in public domain on the reference chickpea genome. A set of 202 novel genic SSRs were developed from a set of 507 primer pairs designed, based on in silico amplification of single locus and having no similarity to the publicly available SSR markers. Further, 40 genic SSRs equally distributed on chickpea genome were validated on a select set of 44 chickpea genotypes (including 41 Cicer arietinum and 3 Cicer reticulatum), out of which 25 were reported to be polymorphic. The polymorphism information content (PIC) value of 25 polymorphic genic SSRs ranged from 0.11 to 0.77 and number of alleles varied from 2 to 9. Clear demarcation among founder lines of multi-parent advanced generation inter-cross (MAGIC) population developed at ICRISAT and near-isogenic nature of JG 11 and JG11 + demonstrates the usefulness of these markers in chickpea diversity analysis and breeding studies. Further, genic polymorphic SSRs reported between parental lines of 16 different mapping populations along with the novel SSRs can be deployed for trait mapping and breeding applications in chickpea.

Highlights

Chickpea (Cicer arietinum L.) is the second most important grain legume widely cultivated throughout tropical and sub-tropical regions of the world (Gaur et al 2012)
A total of 566 unique transcripts containing 623 in silico identified genic simple sequence repeat (SSR) were downloaded from Chickpea Transcriptome Database (CTDB v 1.0, http://www.nipgr.res.in/ctdb.html)
In order to determine the uniqueness and develop novel, robust genic SSR markers for applications in crop improvement the following in silico analysis was adopted: 1. All 507 primers designed were subjected to BlastN analysis against the reference chickpea genome to identify primer pairs that amplify single genomic locus

Summary

Introduction

Chickpea (Cicer arietinum L.) is the second most important grain legume widely cultivated throughout tropical and sub-tropical regions of the world (Gaur et al 2012). It is a self-pollinated crop with basic chromosome number eight (2n = 2x = 16) and genome size of 738 Mb (Varshney et al 2013b). It is cultivated on 11.55 million ha with an annual production of 10.46 million tons and average productivity of 905 kg/ha (FAOSTAT data 2013). The recent advances in genomics coupled with the availability of the genome sequence for several important crop plants (Michael and Jackson 2013) including chickpea (Varshney et al 2013b) provide an opportunity to translate the knowledge to breed superior variety with enhanced resistance/tolerance to biotic and abiotic stresses in several crop species including chickpea (Gaur et al 2012; Varshney and Tuberosa 2013a, b)

Methods

Results

Conclusion