Phenotypic and Genotypic Differences Between Bahiagrass Hybrids in Georgia.

Bahiagrass biotypes were collected from 12 counties throughout the state of Georgia between July 3, 2012 and July 20, 2012. Approximately 12 bahiagrass biotypes were obtained from 4 locations within each county. Seedhead branch number was used to identify possible bahiagrass hybrid biotypes. Biotypes with seedhead branch numbers ranging from 2 to 8 were collected. Each biotype was excavated from the site and transplanted into the greenhouse in Athens, GA. One month later, each pot was destructively harvested and approximately 20 to 30 rhizome cuttings, 2.5 cm in length, were replanted into pots. Each transplanted rhizome was considered a distinct bahiagrass biotype. Biotypes were grown to maturity in the greenhouse (not all survived). Morphological data were collected from regenerated bahiagrass biotypes (n = 1002) between November 11, 2012 and December 9, 2012. Collected data included leaf width at leaf base (mm), leaf width midway to leaf apex (mm), leaf length (cm), ligule description (membranous, hairy, or absent), ligule length (mm), seedhead branch number, seedhead length (cm), and flowering culm length (cm). Bahiagrass biotypes with 2, 3, and 4 seedhead branches were collected from all 12 counties. Biotypes with 5 seedhead branches were collected in 9 of 12 counties, while biotypes with 6, 7, and 8 branches were collected from 6, 3, and 1 counties, respectively. Both membranous- and hairy-ligules were observed on all bahiagrass biotypes regardless of location or original seedhead branch number. DNA was extracted from 30 potential bahiagrass hybrids (representative of the vast differences in morphology): GAR2-2, GAR7-1, GAR10-1, GAJ7-8, GAJ12-6, GAL4-15, GAL9-33, GAL11-17, GASU2-6, GASU4-4, GASU4-5, GASU9-2, GASU11-1, GASU11-2, GASU11-6, GAC8-4, GAC12-6, GAC12-8, GALC9-10, GACL13-10, GACL14-4, GAG1-10, GAW1-4, GAW1-5, GAW4-4, GAB11-15, GACH5-2, GACLCH1-7, GACLCH1-11, and GALCH4-9, using a PureLink Genomic Plant DNA Purification Kit. The universal primers that amplify the barcoding region of the gene were used on the 30 potential bahiagrass hybrids. Amplicons were cleaned with a PCR Clean-up column and sequenced bi-directionally by Eurofins MWG Operon. Sequences were imported into Sequencher version 4.9, trimmed, and aligned with the matK barcoding region of Paspalum dilatatum (Genbank accession: HM850547), P. distichum (FN908063), P. notatum (HM850548), P. urvillei (HM850549), and P. vaginatum (HM850550). The sequence of the matK marker for 30 accessions was identical to only the P. notatum sequence (HM850548). Thus the maternal parent for the 30 potential bahiagrass hybrids is bahiagrass. Thirty three P. vaginatum SSR markers were used to generate marker data for 80 fragments. The resulting dendrogram revealed three major groups: P. dilatatum, P. urvillei, and P. notatum. The P. notatum group can be further divided into 2x and 4x bahiagrass. ‘Argentine’, GACLCH1-7, GACLCH1-11, GACLCH4-9, and GAL4-15 were all identified as 4x bahiagrass samples, while all other potential bahiagrass hybrid biotypes were identified as 2x bahiagrass.