Exploiting Illumina sequencing for the development of 95 novel polymorphic EST-SSR markers in common vetch (Vicia sativa subsp. sativa).

Zhipeng Liu,Dong Luo,Peng Liu,Yanrong Wang,Wenxian Liu

doi:10.3390/molecules19055777

Abstract

The common vetch (Vicia sativa subsp. sativa), a self-pollinating and diploid species, is one of the most important annual legumes in the world due to its short growth period, high nutritional value, and multiple usages as hay, grain, silage, and green manure. The available simple sequence repeat (SSR) markers for common vetch, however, are insufficient to meet the developing demand for genetic and molecular research on this important species. Here, we aimed to develop and characterise several polymorphic EST-SSR markers from the vetch Illumina transcriptome. A total number of 1,071 potential EST-SSR markers were identified from 1025 unigenes whose lengths were greater than 1,000 bp, and 450 primer pairs were then designed and synthesized. Finally, 95 polymorphic primer pairs were developed for the 10 common vetch accessions, which included 50 individuals. Among the 95 EST-SSR markers, the number of alleles ranged from three to 13, and the polymorphism information content values ranged from 0.09 to 0.98. The observed heterozygosity values ranged from 0.00 to 1.00, and the expected heterozygosity values ranged from 0.11 to 0.98. These 95 EST-SSR markers developed from the vetch Illumina transcriptome could greatly promote the development of genetic and molecular breeding studies pertaining to in this species.

Highlights

The common vetch (Vicia sativa subsp. sativa) is an important forage legume crop that is commonly used as green manure, pasture, silage, and hay
A total of 41 potential expressed sequence tag (EST)-simple sequence repeat (SSR) contained more than 13 repeat units, and all of the motifs were tri-nucleotide repeats
Previous researches have shown that SSR occurrence in coding regions seems to be limited by non-perturbation of the reading frame, and the tri- and hexa-nucleotide repeats are dominant in protein-coding exons of all taxa

Summary

Introduction

The common vetch (Vicia sativa subsp. sativa) is an important forage legume crop that is commonly used as green manure, pasture, silage, and hay. The vetch fixes atmospheric nitrogen through its symbiotic relationship with rhizobia Traditional approaches based on probe hybridizaiton (containing repeated motifs) against genomic or cDNA libraries followed by DNA sequencing for the development of SSR markers are time-consuming and resource-intensive [5]. Expressed sequence tag (EST) derived markers are more likely embedded in functional gene sequences, which make them act as “functional genetic markers” for rapidly establishing marker-trait linkages and to identify genes quantitative trait loci (QTLs) for traits of agricultural importance in crop plants [6]. EST-SSR markers are likely to be more highly conserved and may be more transferable between closely related species compared with the markers derived from genomic sequences. The development and characterisation of EST-SSR markers has become quite extensive in a wide range of plant species [1,9,10,11,12,13,14,15,16]

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