Evaluation of high resolution melting for rapid differentiation of Phytophthora hybrids and their parental species

Phytophthora species hybrids have been repeatedly reported as causing damaging diseases to cultivated and wild plants. Two known hybrids, P. andina and P. x pelgrandis, are pathogens of Solanaceae and ornamentals respectively, although the extent of their host ranges are unknown. P. andina emerged from hybridization of P. infestans and an unidentified related species, while P. x pelgrandis emerged from P. nicotianae and P. cactorum. Considering that hybrids and parental species can co-exist in the same regions and to distinguish them usually requires cloning or whole genome sequencing, we aimed to develop a rapid tool to distinguish them. Specifically, we used High Resolution Melting (HRM) assays to differentiate genotypes based on their amplicon melting profiles. We designed primers for P. x pelgrandis and parental species based on available sequences of P. nicotianae and P. cactorum nuclear genes containing polymorphisms between species. For P. andina, heterozygous sites from Illumina short reads were used for the same purpose. We identified multiple amplicons exhibiting differences in melting curves between parental species and hybrids. We propose HRM as a rapid method for differentiation of P. andina and P. x pelgrandis hybrids from parental species that could be employed to advance research on these pathogens.