Utilization of Genomic Simple Sequence Repeat Markers to Study the Diversity and Reproductive Biology of Danthonia Spicata.

Danthonia spicata (Poaceae), commonly known as poverty oatgrass, is a perennial bunch-type grass native to North America. It is a very shade tolerant, drought tolerant and low maintenance grass, frequently found on unimproved turf sites typically under trees. However, it has some disadvantages such as poor seed yield and shattering of seed heads prior to harvest. D. spicata has dimorphic seed heads; the hypothesis is that terminal seed heads allow some level of outcrossing and axial seed heads are only self-fertilized. However, there is no genetic evidence for heterozygosity and outcrossing in the literature. Our objective was to study the diversity and reproductive biology of D. spicata using genomic simple sequence repeat markers (genomic-SSRs). Roche 454 sequenced randomly sheared genomic DNA reads of D. spicata were mined for SSRs using the MIcroSAtellite identification tool (MISA). A total of 66553 singlet sequences (approximately 37.5 Mbp) were examined, and 3454 SSRs were identified, including 1584 mono-, 997 di-, 791 tri-, 57 tetra-, 18 penta-, and seven hexa-nucleotide repeats. Trinucleotide motifs with greater than six repeats and possessing unique PCR priming sites within the genome, as determined by Primer-BLAST, were evaluated visually for heterozygosity and mutation consistent with stepwise evolution. A total of 63 candidate markers were selected for testing from the trinucleotide SSR sites meeting these in silico criteria. Ten primer pairs that amplified polymorphic loci in preliminary experiments were used to screen 91 individual plants composed of 3 to 5 plants from each of 23 different locations in Maryland, North Carolina, Virginia, and Washington, D.C. Fourteen multilocus genotypes were observed providing strong evidence for asexual reproduction in D. spicata. The presence of polymorphisms unique to individuals suggests somatic mutation may be generating variability. We will use these genomic-SSR primers to screen terminal and axial seed head-derived progeny to look for evidence of genetic segregation and further test the hypothesis of apomixis in D. spicata.