Development of Genomic SSR Markers in Cynodon dactylon.

Bermudagrass, Cynodon dactylon (L.) Pers., is economically important as a major turf and forage crop in the southern United States.  The grass is highly cross-pollinated.  Consequently, individuals are highly heterozygous. Microsatellite, or simple sequence repeats (SSR), DNA markers are co-dominant and therefore are useful in bermudagrass genetic and breeding research. However, genomic SSR markers are not currently available for bermudagrass research. The objectives of this study were to construct and sequence SSR-enriched genomic DNA libraries, to identify SSR containing sequences, to design SSR primer pairs, and to verify the designed SSR primer combinations. A genomic DNA sample was extracted from the bermudagrass genotype ‘Zebra’ using IBI Scientific Plant Genomic DNA Maxi Kit. Five libraries enriched in core SSR CA-, GA-, ATG-, AAC- and CAG-sequences were constructed using the pUC19 plasmid.  The SSR-enriched DNA inserts are being sequenced at the Oklahoma State University Core Facility. The software program ‘SSR Locator’ was used to identify SSR sequences, and to design SSR primers, which will be used in the polymerase chain reaction (PCR) to amplify and characterize SSR alleles in a panel of bermudagrass genotypes.  Initial sequence data for the –CA library (N=759) resulted in, 87% long reads (>600bp), 6% medium reads (300-600bp), and 7% showed short reads (<300bp).  ‘SSR locator’ found 96% of the sequences submitted contained an SSR of which 57% were perfect and 43% were compound.  Primers were designed for 82% of the sequences that contained an SSR.  A total of 65%, of the primers designed, were non-redundant and were used to screen genomic DNA from two bermudagrass species (Cynodon dactylon ‘Zebra’ and Cynodon transvaalensis ‘Uganda’).  The initial screening (N=96) of –CA library produced 82% reproducible and strong amplification products, 9% amplification products of the non-predicted size, and 7% resulted in no reactions.  The information obtained by this project will aid in the creation of a molecular map for this species.