Summer Squash Identification by High-Resolution-Melting (HRM) Analysis Using Gene-Based EST-SSR Molecular Markers

Cucurbita pepo (squash, pumpkin, gourd), a worldwide cultivated vegetable of American origin, is extremely variable in fruit characteristics. Most of its widely grown commercial types are known as summer squashes and belong to the elongated forms of C. pepo ssp. pepo (Cocozelle, Vegetable marrow and Zucchini groups). Here, we have integrated the high-resolution-melting (HRM) analysis method with expressed sequence tags-simple sequence repeat (EST-SSR) marker genotyping, in order to facilitate the identification of 36 summer squash landraces originated from Greece. The six EST-SSR loci used were informative and generated a unique melting curve profile of EST-derived microsatellites for each accession allowing their comparison and classification. Moreover, HRM was highly informative, as by using only four microsatellite markers we were able to discriminate 36 summer squash landraces and by using six EST-SSRs. We were able to construct a highly informative and discriminative dendrogram where the 36 genotypes were classified in six distinct clusters. Furthermore, we acquired information about the genes containing the EST-SSRs using bioinformatics tools. We found that the EST-SSRs used in this study were hybridizing to genes involved in stress response to heavy metals and biotic stresses or the production of flavonoids or symporters of important nitrogen sources, like xanthine and uric acid amongst others. The results presented here suggest that the panel of EST-SSR markers used in combination with HRM analysis could be useful in a variety of applications, like squash biodiversity assessment but most importantly in managing squash germplasm to improve breeding programs.