Development and Characterization of Microsatellite Markers for Sclerotinia homoeocarpa

A.I. Putman, I. Carbone, and L.P. Tredway

Sclerotinia homoeocarpa causes dollar spot, the most economically important disease of turfgrass worldwide.  Little is known about the diversity and structure of S. homoeocarpa populations infecting various turfgrass hosts worldwide.  The objective of this research is to develop and use microsatellite markers in population studies of S. homoeocarpa.  Microsatellites were initially isolated using a bead capture enrichment protocol, and additional repeats were identified in silico from an early draft genome assembly of S. homoeocarpa with the Tandem Repeat Database.  Microsatellites with sufficient flanking sequence to the end of the read or to adjacent repeats were deemed suitable for primer design.  To ensure usability in both cool- and warm-season isolates, primers were tested for amplification of single bands from a representative of each group in PCR reactions with an annealing temperature gradient.  Next, candidate loci were examined for polymorphisms by Sanger sequencing.  Candidates containing indels in the flanking region or exhibiting homoplasy due to compound polymorphisms were discarded as not usable.  From the genome data, 6,075 microsatellites were identified based on minimum thresholds of repeat number, copy number, and perfection of repeat units.  Three of 31 candidate loci from the enrichment protocol were selected as usable.  Of the 791 candidate loci identified in silico, 11 usable loci have been selected and 76 have been discarded.  Three to ten alleles per locus have been found among a select group of cool- and warm-season isolates from four continents.  Data collected to date indicate microsatellites may provide increased resolution in population studies when compared to DNA sequence genotyping.  Multiplex PCR protocols using fluorescent-tagged primers have been developed to enable rapid genotyping.  These microsatellites will be useful in determining the diversity and structure among worldwide populations of S. homoeocarpa.