Simple sequence repeat marker development and diversity analysis in buffalograss

Buffalograss [Buchloe dactyloides (Nutt.) Engelm. syn. Bouteloua dactyloides (Nutt.) Columbus] is a stoloniferous low input turfgrass species native to the central Great Plains of the United States. Several molecular marker techniques have been used to compare relationships among elite buffalograss germplasm, but most rely on random amplification, are transferred from other species, or are developed from limited DNA sequence information. The objectives of this study were to develop buffalograss derived simple sequence repeat markers (SSRs) from buffalograss sequence data using an in-silico bioinformatics pipeline and use high resolution melt analysis to screen the SSRs based on their melt profiles on buffalograss cultivars and germplasm. Transcriptomes from buffalograss cultivars '378' and 'Prestige' were mined for SSRs using the MIcroSAtellite tool. There were 259 conserved SSRs that were polymorphic between 378 and Prestige. Of those, 96 were tested by HRM on a panel of eight genotypes and 24 were selected for producing melt profiles that could distinguish the genotypes. The selected reactions produced 356 distinct melt profiles when tested on a panel of 96 genotypes, including 88 buffalograss germplasm selections, six buffalograss cultivars, and two blue grama [Bouteloua gracilis (H. B. K.) Lag. ex Steud.] selections, with melt profile frequencies ranging from 0.01 to 0.97. A subset of 86 melt profiles segregating among the blue grama and elite buffalograss cultivars distinguished each genotype. The buffalograss-specific SSR markers presented here will be useful to study buffalograss genetic diversity, line purity maintenance, and for marker supported plant breeding strategies.