EST-SSR marker characterization based on RNA-sequencing of Lolium multiflorum and cross transferability to related species

Abstract

Simple sequence repeat (SSR) markers are considered very useful molecular markers for genetic and molecular breeding, specifically for non-model species. To develop a set of whole genome-wide distributed SSR marker for annual ryegrass (Lolium multiflorum L.), we obtained the first transcriptome dataset of annual ryegrass subjected to drought stress and identified a total of 11,254 EST-SSR. Fifty EST-SSR markers were selected to test the amplification and polymorphism rates in 23 accessions of Lolium and Festuca diploid species. The results showed that all of the 50 functionally relevant markers were polymorphic, with allele number and polymorphism content averaging 7.4 and 0.839, respectively, and fully transferable across 23 accessions of Lolium and Festuca species. Sequencing of PCR products showed that the nucleotide sequences of Lolium and Festuca species had a high level of conservation. Different types of tri-repeats as abundant SSR motifs were observed in repeat sequence regions. A number of EST-SSRs were mapped to genome sequences of Lolium perenne and chromosomes of Brachypodium distachyon. Among them, 973 and 1375 Lolium SSR markers were located on the five chromosomes of B. distachyon and L. perenne scaffolds, respectively. In addition, we confirmed that EST-SSRs as well as SRAP markers could clearly identify varieties of Lolium and Festuca species, and the two sets of molecular markers had a significant correlation (r = 0.74). This set of EST-SSR markers are valuable genetic tools for genetic diversity, evolutionary, and association mapping studies in Lolium, Festuca, and many other grass species.