Watermelon (Citrullus lanatus), one of the top five fruit crops based on the gross tonnage and cultivated area globally, holds major economic importance in agriculture and contributes substantially to farmers’ incomes. Watermelon cultivation relies on sexual reproduction, with meiosis playing a pivotal role in this process. However, our understanding of the meiotic mechanism in watermelon remains limited. In this study, we utilized CRISPR/Cas9 technology to target ClDYAD, a homolog of the meiosis-related genes AtDYAD and OsAM1, and conducted functional analysis to explore the initiation of meiosis in watermelon. ClDYAD was highly expressed in tender male flowers both before and during the early stages of meiosis. Mutations in ClDYAD led to meiotic arrest at the leptotene stage, impeding the normal enlargement of microspore mother cells and megasporocytes. This resulted in the absence of pollen in anthers and seed abortion. ClDYAD physically interacts with the protein encoded by Cla97C07G137480, which was identified as a switch-associated protein 70-like protein (ClSWAP-70). The expression pattern of ClSWAP-70 in tender male flowers of various sizes matched with the changes in ClDYAD mRNA levels. These findings shed light on the molecular mechanisms governing the initiation of meiosis in watermelon, offering valuable insights into male and female sterility in Cucurbitaceae plants and guiding future research.

The nuclear factor Y (NF-Y) gene family is a class of transcription factors that are widely distributed in eukaryotes and are involved in various biological processes. However, the NF-Y gene family members in watermelon, a valued and nutritious fruit, remain largely unknown and their functions have not been characterized. In the present study, 22 ClNF-Y genes in watermelon, 29 CsNF-Y genes in cucumber, and 24 CmNF-Y genes in melon were identified based on the whole-genome investigation and their protein properties, gene location, gene structure, motif composition, conserved domain, and evolutionary relationship were investigated. ClNF-YB9 from watermelon and its homologs in cucumber and melon were expressed specifically in seeds. Its expression remained low in the early stages of watermelon seed development, increased at 20 days after pollination (DAP), and peaked at 45–50 DAP. Moreover, the knockout mutant Clnf-yb9 exhibited abnormal leafy cotyledon phenotype, implying its critical role during seed formation. Finally, protein interaction assays showed that ClNF-YB9 interacts with all ClNF-YCs and the ClNF-YB9-YC4 heterodimer was able to recruit a ClNF-YA7 subunit to assemble a complete NF-Y complex, which may function in seed development. This study revealed the structure and evolutionary relationships of the NF-Y gene family in Cucurbitaceae and the novel function of ClNF-YB9 in regulating seed development in watermelon.