Efficient multiplex simple sequence repeat genotyping of the oomycete plant pathogen Phytophthora infestans

Abstract

Genotyping is fundamental to population analysis. To accommodate fast, accurate and cost-effective genotyping, a one-step multiplex PCR method employing twelve simple sequence repeat (SSR) markers was developed for high-throughput screening of Phytophthora infestans populations worldwide. The SSR markers reported for this species were evaluated and the twelve most informative and easily scored were selected. To accomplish a single step genotyping procedure, we optimized primers, fluorescent labels and PCR conditions to genotype using a capillary electrophoresis system with four fluorescent labels (FAM, NED, PET and VIC) and a labeled LIZ standard for sizing of the SSR fragments. The results obtained using commercially available multiplex PCR kits on a set of reference isolates were in agreement with that obtained using primer pairs in simplex reactions. In testing on many thousands of isolates, we have found the markers appropriate for resolving distinct multilocus genotypes (MLGs) of isolates of European and wider populations. Here we demonstrate the utility of the assay on a set of 19 reference isolates plus 77 others sampled from The Netherlands and Great Britain. In most isolates one to two alleles were observed at each locus but the presence of three alleles at a single locus in some isolates was consistent with increased ploidy. Methods are presented that are appropriate for the analysis of datasets comprising isolates of mixed ploidy levels. We also report on the direct P. infestans genotyping from infected field material to collect, store and extract pathogen DNA. A critical step in this multiplex method was the standardization of the protocol between two laboratories in The Netherlands and Great Britain. Reference isolates were exchanged and an allele nomenclature and scoring system agreed. Such co-operation is facilitating the genotyping of international collections of P. infestans isolates in wider networks of laboratories and providing the data required to expand an existing international database of pathogen diversity.