Microsatellite Markers Development From EST Sequences of Bermudagrass (Cynodon spp.).

Bermudagrass is an important warm-season, perennial and rhizomatous grass, widely used for turf and forage in the southern United States and other warmer temperate and tropical regions in the world. Microsatellites, or simple sequence repeat (SSR) markers are highly useful in genetic studies and breeding, but are not currently available in bermudagrass. The objective of this study was to develop SSR markers from EST sequences of bermudagrass. A total of 20,237 EST sequences from National Center for Biotechnological Information (NCBI) database were screened with ‘SSR Locator’ program. Analysis revealed 1,303 sequences containing one or more SSRs, and consequently 918 primer pairs were designed to amplify SSR markers. Genomic DNA samples from four bermudagrass accessions, C. dactylon var. aridus (2n=2x=18), C. transvaalensis PI 290812 (2n=2x=18), C. dactylon ‘Zebra’ (2n=4x=36), and C. dactylon cv. Tifton 10 (2n=6x=54) were extracted with Zymo Research, Plant/Seed DNA Extraction Kit. In each SSR primer pair combination, Polymerase Chain Reaction (PCR) amplification was performed for each DNA sample of the four bermudagrass genotypes and was amplified in two replications. Amplicons were separated and visualized using a Li-Cor 4300 DNA Analyzer and data were collected for highly scoreable and reproducible bands.  Of 304 primer pairs that amplified discrete bands in the four genotypes, 41% amplified bands for the var. aridus genotype, 45% for PI 290812, 31% for Tifton 10, and 36% for Zebra. Research is ongoing to screen additional primer pairs with more results to be updated.