Identification and validation of polymorphic microsatellite loci for the analysis of Phytophthora nicotianae populations.

A large number of SSR loci were screened in the genomic assemblies of 14 different isolates of Phytophthora nicotianae and primers were developed for amplification of 17 markers distributed among different contigs. These loci were highly polymorphic and amplified from genetically distant isolates of the pathogen. Among these, nine were further validated using a multiplexed genotyping assay with differentially labeled primers (FAM or HEX) to allow for duplex PCR amplification. The use of reverse primers with a 5' PIG tail was important to increase the quality and reliability of the analyses. A total of 46 alleles were detected in 5 tester isolates of P. nicotianae representing the breadth of diversity in the species. Furthermore, a high incidence of heterozygosity was determined with two alleles detected in 67% of the primer/isolate combinations. Three different alleles where detected for a single locus/isolate combination, indicating variation in ploidy. These markers represent a valuable new tool for the characterization of populations of P. nicotianae.