Development of a SNP genetic marker system based on variation in microsatellite flanking regions of Phytophthora infestans

摘要

A single nucleotide polymorphism (SNP)-based molecular marker system for the oomycete pathogen causing late blight on solanaceous hosts, Phytophthora infestans, was developed by identifying sequence polymorphisms in microsatellite flanking regions (MFRs). MFRs were identified using the complete genome sequence for this pathogen and SNP rates were assessed by sequencing a total of 14 000 bases from 32 MFRs across a diverse international panel of 32 isolates. SNP rates were highly variable among loci, with nucleotide diversity ranging from a low of zero to a high of 0.013 (one change per 78 bp) and an average across all scorable loci (n = 28) of 0.0023 (one change per 426 bp). Nucleotide diversity estimates are highly conservative due to strict objective scoring rules used when calling SNPs. Overall, 102 SNPs were scored using objective criteria (versus 167 as scored by eye), and could distinguish all isolates in the panel, with pairwise per cent similarity between isolates varying from 58.5% to 99.4%. Observed and expected heterozygosities at most SNP sites were similar. The utility of a 'straincode' for strain typing based on up to 19 unlinked SNPs was investigated. The probability of identity was very low (PI = 3.5 x 10-6) when all 19 SNPs were included; however, to achieve probabilities of identity of about 1% and 0.1%, only four and seven loci were needed, respectively. The approach needs to be assessed with larger panels of isolates, but results suggest that MFRs and SNPs are a largely untapped source of useful sequence polymorphisms for strain typing of microbes and that their utility could extend to ecological and evolutionary research, especially with high throughput sequence-based technologies.