Transcriptome-Derived Amplicon Sequencing Markers Elucidate the U.S. Podosphaera macularis Population Structure Across Feral and Commercial Plantings of Humulus lupulus

Obligately biotrophic plant pathogens pose challenges in population genetic studies due to their genomic complexities and elaborate culturing requirements with limited biomass. Hop powdery mildew ( ) is an obligately biotrophic ascomycete that threatens sustainable hop production. populations of the Pacific Northwest (PNW) United States differ from those of the Midwest and Northeastern United States, lacking one of two mating types needed for sexual recombination and harboring two strains that are differentially aggressive on the cultivar Cascade and able to overcome the -gene ( ), respectively. To develop a high-throughput marker platform for tracking the flow of genotypes across the United States and internationally, we used an existing transcriptome of diverse isolates to design a multiplex of 54 amplicon sequencing markers, validated across a panel of 391 U.S. samples and 123 international samples. The results suggest that from U.S. commercial hop yards form one population closely related to of the United Kingdom, while from U.S. feral hop locations grouped with of Eastern Europe. Included in this multiplex was a marker that successfully tracked -virulence in 65 of 66 samples with a confirmed -phenotype. A new qPCR assay for high-throughput genotyping of mating type generated the highest resolution distribution map of mating type to date. Together, these genotyping strategies enable the high-throughput and inexpensive tracking of pathogen spread among geographical regions from single-colony samples and provide a roadmap to develop markers for other obligate biotrophs.