Transcriptome-derived amplicon sequencing (AmpSeq) markers elucidate the U.S. podosphaera macularis population structure across feral and commercial plantings of Humulus lupulus

Authors: WELDON, WILLIAM - Cornell University; KNAUS, BRIAN - Oregon State University; Grunwald, Niklaus - Nik; HAVILL, JOSHUA - University Of Minnesota; BLOCK, MARY - Oregon State University; Gent, David - Dave; Cadle-Davidson, Lance; GADOURY, DAVID - Cornell University

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/6/2020
Publication Date: 12/1/2020
Citation: Weldon, W.A., Knaus, B.J., Grunwald, N.J., Havill, J.M., Block, M.H., Gent, D.H., Cadle Davidson, L.E., Gadoury, D.M. 2020. Transcriptome-derived amplicon sequencing (AmpSeq) markers elucidate the U.S. podosphaera macularis population structure across feral and commercial plantings of Humulus lupulus. Phytopathology. 111:194-203. https://doi.org/10.1094/PHYTO-07-20-0299-FI.
DOI: https://doi.org/10.1094/PHYTO-07-20-0299-FI

Interpretive Summary: Tracking and understanding the diversity and structure of a pathogen's population is important for developing sound management approaches for plant diseases that mitigate disease spread and crop damage. Assessing population diversity and structure can be complicated, costly, and time consuming with certain organisms that have complex genomes and must be cultured on living tissue of their host. In this research, we use the hop powdery mildew system as a model to demonstrate how extant genetic markers can be adapted for a cost-effective, high-throughput sequencing to enable processing of hundreds of markers across hundreds of samples without the need for pathogen culture. We apply this technology to demonstrate that the hop powdery mildew fungus has distinct populations on wild versus cultivated plants in the eastern U.S., supporting recent findings that the pathogen population in the U.S. may be derived from Europe, and quantify distribution of virulent strains of the fungus. This research has immediate implications for the spread of the hop powdery mildew fungus, but more broadly the methods should be transferable to other organisms where genetic marker data are available yet potentially difficult to apply at scale.

Technical Abstract: 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. P. macularis populations of the Pacific Northwest (PNW) 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.