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Investigation of Erysiphe necator Population Structure using Amplicon Sequencing (AmpSeq) without Clonal Isolation

Breanne Kisselstein: Cornell University

<div>Studies of aerially-dispersed fungal pathogens are often limited by: (i) subculturing samples of uncertain purity to achieve clonal lines, and (ii) conducting follow-up bioassays to quantify fungicide resistance or other traits. Amplicon Sequencing (AmpSeq) has the potential to circumvent subculturing by detecting clonality, for targeted bioassay-based validation of known genotypes. In a proof-of-principle study of the haploid fungus <em>Erysiphe necator</em>, from 2015 to 2017 we used 1-cm pieces of tape to collect over 800 samples of conidia from single colonies on grapevine foliage from 5 vineyards. AmpSeq was used to compare heterozygosity (an indicator of paralogy or mixed infection) and allele frequencies for over 200 molecular markers. For the first two years, the median heterozygosity rate was 3%, but the <em>CYP51</em> gene (with known paralogs) had sites with 6% and 28% heterozygosity, and two other genes had heterozygosity above 70%. Based on markers with low heterozygosity, at least 5.9% of samples represented mixed infections, suggesting that subculturing to achieve clonality may be unnecessary for some applications. The DMI fungicide resistance allele<em> EnCYP51 </em>A495T<em> </em>was consistently observed in approximately half of samples across all commercial vineyard sites for both years. This approach of genotyping many loci, including genes under selection, enables numerous analyses without clonal isolation.</div>