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Evaluation of High Resolution Melting Analysis to discriminate between parental and hybrid Phytophthora species

Erica Goss: Department of Plant Pathology, University of Florida

<div><em>Phytophthora</em> hybridization events have been reported repeatedly, resulting in new species that conserve some of their parental phenotypic traits but differ in genotypes and host range. <em>P. andina </em>and<em> P. x pelgrandis</em> are pathogens of Solanaceae and ornamentals respectively, although the extent of their host ranges and distributions are unknown. <em>P. andina</em> emerged from hybridization of <em>P. infestans </em>and an unidentified related species, <em>P. x pelgrandis</em> from <em>P. nicotianae </em>and <em>P. cactorum. </em>Considering that hybrids and parental species can co-exist in the same areas and identification of hybrids usually involves sequence analysis, we aimed to develop a rapid tool to discern hybrids from parental species. We used High Resolution Melting (HRM) assays to differentiate genotypes based on their amplicon melting profiles. We designed primers for <em>P. x pelgrandis</em> based on available sequences of nuclear genes of <em>P. nicotianae</em> and <em>P. cactorum</em> containing one or more polymorphisms. For <em>P. andina</em>, heterozygous sites from Illumina short reads mapped against <em>P. infestans</em> were used for the same purpose. <em> </em>We evaluated the discriminative potential of amplicons using uMelt prediction software; then we ran HRM experiments to obtain actual melting profiles. Significant differences in melting curves were detected for each species and hybrid. These assays could be used to identify <em>P. andina </em>and <em>P. x pelgrandis </em>hybrids, and potentially as a versatile diagnostic tool when suspected infections arise.</div>