Transcriptome-derived amplicon sequencing (AmpSeq) 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 complexity and elaborate culturing requirements with limited biomass. Hop powdery mildew (Podosphaera macularis) is an obligately biotrophic ascomycete that threatens sustainable hop production. Podosphaera macularis populations of the Pacific Northwest United States (US) differ from those of the Midwest and Northeastern US, 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 Humulus lupulus R-gene R6 (V6), respectively. To develop a high-throughput marker platform for tracking the flow of genotypes across the US and internationally, we used an existing transcriptome of diverse P. macularis isolates to design a multiplex of 54 Amplicon Sequencing markers, validated across a panel of 391 US samples and 123 international samples. The results suggest that P. macularis from US commercial hop yards form one population closely related to P. macularis of the UK, while P. macularis from US feral hop locations grouped with P. macularis of Eastern Europe. Included in this multiplex was a marker that successfully tracked V6-virulence in 65 of 66 samples with a confirmed V6-phenotype. A new qPCR assay for high-throughput genotyping of P. macularis mating type generated the highest resolution distribution map of P. macularis 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.