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Development of New Polymorphic Microsatellite Markers for Three Closely Related Plant-Pathogenic Phytophthora Species Using 454-Pyrosequencing and Their Potential Applications

Phytophthora spp. (oomycetes) are causal agents of devastating diseases on a high number of crops, ornamentals, and native plants worldwide. Neutral molecular markers are increasingly being used to investigate the genetic population structure and possible pathways of spread of different plant pathogens, including Phytophthora spp. In this study, polymorphic microsatellite markers were developed for three species of the former Phytophthora citricola species complex—namely, P. multivora, P. plurivora, and P. pini (P. citricola I)—using the 454-pyrosequencing technique. In total, 35 polymorphic microsatellite loci were found and further characterized: 11 for P. plurivora, 16 for P. multivora, and 8 for P. pini. Microsatellites with dinucleotide motifs repeated 6 to 10 times were the most common for all three species. On average, 65 alleles per species and 5.3 alleles per locus were detected. Most loci were characterized by a low observed heterozygosity, which might be due to the homothallic mating system of the three Phytophthora spp. targeted. Cross amplification of the newly developed markers was tested on 17 Phytophthora spp. belonging to five different internal transcribed spacer clades. Transferability success was generally low and decreased with increasing genetic distance from the species to the three target species. A set of four loci was selected to easily discriminate P. plurivora, P. multivora, and P. pini on the basis of presence or absence of a polymerase chain reaction amplicon on an agarose gel.