Development of a reference panel as a way to harmonize data of genotyped Sclerotinia sclerotiorum

Abstract

Several population studies of S. sclerotiorum have been conducted and resulted in important publications furthering our knowledge of this pathogen. Genotype data from those studies has been published with the notion that this data could be re‐used by other scientists. However, amplicon sizes were known to vary across labs due to primer modifications for fluorophore labeling and the instrument used to perform fragment analysis. Thus, it is necessary to create a reference panel of DNA that represents allele sizes known to be present within the population, which is a technique used in human DNA forensics for the same type of genetic marker. Given recent completion of genotyping of more than 366 S. sclerotiorum isolates from across the U.S., development of a DNA Reference Panel that represents most alleles within S. sclerotiorum loci isis now possible. Here, we identified 22 S. sclerotiorum isolates that were collected and genotyped previously and represented all known possible alleles among the 366 S. sclerotiorum. Then, DNA was isolated from those 22 S. sclerotiorum strains and genotyping was performed using 11 previously developed SSR markers. PCR used a 3‐primer indirect fluorescent labeling system that was slightly different from the system used to genotype the 366 isolates, which used direct primer labeling. Amplicons were sized using capillary electrophoresis and raw chromatograms imported into R for sizing and analysis. This data was used to determine differences in allele sizes and create an R function to automatically re‐classify observed data so that it could be seamlessly integrated into our 366 genotype database. Using results of 22 isolates, harmonization of data from two previous studies that genotyped 95 isolates mainly from Brazil and 366 isolates from U.S. took place. From this data, estimation of population genetic statistics, such as genetic/genotypic diversity, evenness, and richness, and test for evidence of sexual recombination was done. Taken together, the DNA reference panel represents most alleles within S. sclerotiorum and allows for integration of previously published population genetic data of this plant pathogen.Support or Funding InformationSupported by Margarita Marroquin‐Guzman and Sydney E. Everhart. Funded by Undergraduate Creative Activities and Research Experience at UNLThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.