ClPS1 gene-mediated manipulation of 2n pollen formation enables the creation of triploid seedless watermelon.

Seedless watermelons are increasingly dominating the consumer market due to their convenience and high quality. However, traditional triploid watermelon breeding faces challenges such as long breeding cycles and low survival rates of triploid F progeny, severely hindering both breeding and production. In this study, we identified the ClPS1 gene as being associated with the formation of 2n gametes in watermelon. Expression analysis revealed that ClPS1 is highly expressed during meiosis and microsporogenesis. Using CRISPR/Cas9, we generated ClPS1-targeted mutants, which disrupted chromosome segregation at metaphase II. This led to the production of diploid male spores and abnormal division of male spores, ultimately generating diploid pollen grains, while female meiosis remained unaffected. Moreover, self-fertilization or crosses using these mutants as paternal parents yielded triploid and aneuploid watermelons. Our findings demonstrate, for the first time, the molecular manipulation of 2n gametes to create triploid seedless watermelons, offering new insights into polyploid breeding and evolutionary studies in the Cucurbitaceae family and other species.