Development of polymorphic EST-SSR markers and characterization of the autotetraploid genome of sainfoin (Onobrychis viciifolia)

Background. Sainfoin (Onobrychis viciifolia) is a highly nutritious, tannin-containing, and tetraploid forage legume. Due to the lack of detailed transcriptomic and genomic information on this species, genetic and breeding projects for sainfoin improvement have been significantly hindered. Methods. In this study, a total of 24,630,711 dean reads were generated from 14 different sainfoin tissues using Illumina paired-end sequencing technology and deposited in the NCBI SRA database (SRX3763386). From these dean reads, 77,764 unigene sequences were obtained and 6,752 EST-SSRs were identified using de novo assembly. A total of 2,469 primer pairs were designed, and 200 primer pairs were randomly selected to analyze the polymorphism in five sainfoin wild accessions. Results. Further analysis of 40 sainfoin individuals from the five wild populations using 61 EST-SSR loci showed that the number of alleles per locus ranged from 4 to 15, and the expected heterozygosity varied from 0.55 to 0.91. Additionally, by counting the EST-SSR band number and sequencing the three or four bands in one sainfoin individual, sainfoin was confirmed to be autotetraploid. This finding provides a high level of information about this plant. Discussion. Through this study, 61 EST-SSR markers were successfully developed and shown to be useful for genetic studies and investigations of population genetic structures and variabilities among different sainfoin accessions.