Pathogen enrichment sequencing (PenSeq) enables population genomic studies in oomycetes. PenSeq

• The oomycete pathogens Phytophthora infestans and P. capsici cause significant crop losses worldwide, threatening food security. In each case, pathogenicity factors called RXLR effectors contribute to virulence. Some RXLRs are perceived by resistance proteins to trigger host immunity but our understanding of the demographic processes and adaptive evolution of pathogen virulence remains poor. • Here we describe PenSeq, a highly efficient enrichment sequencing approach for genes encoding pathogenicity determinants that, as shown for the infamous potato blight pathogen Phytophthora infestans, make up less than 1% of the entire genome. • PenSeq facilitates the characterization of allelic diversity in pathogen effectors, enabling evolutionary and population genomic analyses of Phytophthora species. Furthermore, PenSeq enables the massively parallel identification of presence/absence variations and sequence polymorphisms in key pathogen genes, which is a prerequisite for the efficient deployment of host resistance genes. • PenSeq represents a cost-effective alternative to whole genome sequencing and addresses crucial limitations of current plant pathogen population studies, which are often based on selectively-neutral markers and consequently have limited utility in analyzing adaptive evolution. The approach can be adapted to diverse microbes and pathogens.