Development and cross-species transferability of EST-SSR markers in Siberian wildrye (Elymus sibiricus L.) using Illumina sequencing

Abstract

Siberian wildrye (Elymus sibiricus L.) is a perennial, self-fertilizing grass that plays an important role in animal husbandry and environmental sustenance. However, the transcriptomic and genomic information on this species is very limited, which hinders genetic and breeding studies. In the present study, 76,686,804 clean reads were generated from 11 different tissue samples of E. sibiricus by Illumina paired-end sequencing, and the reads were deposited into the NCBI SRA database (SRX574376). A total of 8,769 EST-SSRs were identified from 94,458 unigene sequences, which were obtained by de novo assembly. Moreover, 1,078 primer pairs were successfully designed, and 500 pairs were randomly selected to assess polymorphisms in 15 E. sibiricus accessions. A total of 112 primer pairs were polymorphic, and the polymorphism information content (PIC) values ranged from 0.39 to 0.81, indicating a high level of informativeness. Furthermore, these 112 polymorphic primer pairs were used to evaluate the transferability to 13 other related species, and 55 EST-SSR markers were found to be polymorphic among these 13 Elymus species. This study collected the global sequence data for E. sibiricus, and the newly developed markers will prove valuable in facilitating genetic diversity in E. sibiricus and related Elymus species.

Figure 1. Representative tissues and samples used in this study.

(a) Callus cells (induced by young inflorescences). (b) Radicles (seven days after seed germination). (c) Young inflorescences (10 days before fertilization). (d) Tufted leaves in the tillering stage. (e) Flag leaves in the heading stage. (f) Inflorescences (five days before fertilization). (g) Old inflorescences (five days after fertilization). (h) Stems (less lignified stems, moderately lignified stems and highly lignified stems). (i) Whole seedlings (three weeks after seed germination).

Figure 2. >EST-SSR marker variations at the ES-7, ES-43, ES-50, and ES-282 loci of 15 E. sibiricus accessions.

Each accession includes three individual plants; the letter ‘M’ denotes the molecular markers, which are 200 bp, 150 bp, and 100 bp (top to bottom) in ES-7, ES-43, and ES-282 and 150 bp and 100 bp in ES-50 (top to bottom).

Figure 3. Comparative electropherogram analysis of three EST-SSR loci (ES-7, ES-21, and ES-192) among different populations of E. sibiricus.