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AmpSeq based SNP marker development for population analysis of geographically diverse isolates of hop powdery mildew (Podosphaera macularis)

William Weldon: Cornell University

<div>The geographic distribution of powdery mildew of hop (<em>Podosphaera macularis</em>) spans all hop growing regions of the northern hemisphere. Notable differences in genetic diversity, mating type, and virulence have been observed across the continental US and Europe. Analysis of structure within populations, or across spatial distributions, typically requires genetic markers such as single nucleotide polymorphisms (SNPs) to characterize individual isolates. The <em>P. macularis</em> ‘HPM-663 reference’ transcriptome was recently sequenced and assembled <em>de novo</em>. RNA-seq libraries from 87 geographically diverse <em>P. macularis</em> isolates were mapped against this reference to generate an initial set of SNPs. Only 196 unique transcripts SNPs shared by at least two samples. Amplicon Sequencing (AmpSeq) primers were designed with the specifications: 1) the amplicon containing each variant should be 240 to 280 base pairs (bp) in length, ideally 260 bp; 2) the SNP site should be at least 30 bp from the end of the amplicon; 3) the amplicons may span multiple variants; and 4) the AmpSeq primers should bind sites with minimal homology to all other primer binding sites. Under these conditions, 166 transcripts (84.6%) were suitable for designing AmpSeq primers to genotype <em>P. macularis </em>populations. This concept of designing markers spanning divergent regions of transcripts provides a promising marker generation approach transferable across diverse species.</div>