Evaluation of Off-Type Grasses in Hybrid Bermudagrass (Cynodon dactylon (L.) Pers. x C. Transvaalensis Burtt-Davy) Putting Greens Using Genotyping-By-Sequencing.

Use of hybrid ultradwarf bermudagrasses (UDBG; Cynodon dactylon (L.) Pers. x C. transvaalensis Burtt-Davy) on golf course putting greens is increasing in the southern United States.  However, off-type grasses within many putting surfaces have been observed.  To explore the genetic variation among UDBG cultivars and off-type grasses, a genotyping-by-sequencing (GBS) approach was utilized.  Desirable UDBG cultivars and off-type grasses were selected from golf course putting greens in 2013 and cultured from single stolon transplants in a glasshouse at the University of Tennessee (Knoxville, TN).   All hybrid bermudagrass and off-type samples were confirmed as triploids (2n=3x=27) using flow cytometry.  A GBS protocol was then used to assess the genetic variation among UDBG cultivars, off-types, and progenitor species.  The ApeKI restriction enzyme was selected based on optimization trials for the GBS digestion in order to maximize the number of sampled genomic loci. Libraries were constructed from 96 DNA samples and multiplexed on a single lane of an Illumina HiSeq. Over 270 million reads were obtained. Data analysis was performed with the UNEAK pipeline designed for calling single nucleotide polymorphisms (SNPs) from GBS data without a reference genome. Using a minimum depth of 3 reads per sample, the analysis yielded 205,416 filtered SNPs (average read depth of 3.889).  The samples were run on a lane of an Illumina NextSeq to increase coverage. Analysis of the combined datasets yielded 272,039 filtered SNPs with an average depth of 4.718. A multidimensional scaling plot of the genetic variation among samples revealed clear clustering of the progenitor species (C. dactylon (L.) Pers. and C. transvaalensis Burtt-Davy) from UDBG cultivars and off-types. Of the 26 off-type samples, only 9 (35%) were genetically divergent from UBDG cultivars. Additional research is needed to determine the genetic origins of off-type grass phenotypes in UDBG putting greens.