Development of SNP markers for Cucurbita species discrimination

There are five domesticated species in the genus Cucurbita. These are Cucurbita. pepo, Cucurbita maxima, Cucurbita moschata, Cucurbita ficifolia, and Cucurbita argyrosperma. Cucurbita has a large genetic diversity, with various growth forms, sizes, shapes, colors, compositions, tastes, and resistance to biotic and abiotic factors. Maintaining the biological diversity of genetic resources and managing the right historical information are important first steps in the development of new varieties with desirable traits. This study presents the classification of Cucurbita species using DNA barcodes and the development of powerful single nucleotide polymorphism (SNP) markers that can distinguish the five Cucurbita species. A total of 54 accessions from five Cucurbita species (C. pepo, C. maxima, C. moschata, C. ficifolia, and C. argyrosperma) were used for SNP marker development. The chloroplast DNA was extracted, and the three universal barcoding regions (matK, trnH-psbA, and rbcL) were sequenced. Neighbor-joining (NJ) trees were constructed using the nucleotide sequence of the cpDNA barcodes. The rbcL and trnH-psbA barcodes failed to differentiate between C. moschata and C. argyrosperma, whereas the matK region efficiently differentiated with 87% bootstrap support. Moreover, the matK region showed a species identification rate of 100%, making it an efficient marker for identifying the five domesticated Cucurbita species. Multiplex SNaPShot (mini sequencing) analysis of four SNPs in the matK region revealed four species-specific SNPs that effectively classify Cucurbita species into five groups. When all four markers are used together, they are effective. These four species specific SNP markers were validated using known Cucurbita germplasm (111 accessions) and applied for the species identification of unknown Cucurbita germplasm (370 accessions) available at the RDA gene bank in the Republic of Korea. These markers were 100% effective for identifying the five Cucurbita species. Therefore, these SNP markers can be used for effective management of Cucurbita genetic resources in genebanks around the world.