The leaf morphology is an important agronomic trait in crop production. Our study identified a maple leaf type (mlt) cucumber mutant and located the regulatory gene for leaf shape changes through BSA results. Hybrid F1 and F2 populations were generated by F1 self-crossing, and the candidate mlt genes were identified within the 2.8 Mb region of chromosome 2 using map cloning. Through the sequencing and expression analysis of genes within the bulk segregant analysis (BSA) region, we identified the target gene for leaf shape regulation as CsWOX4 (CsaV3_2G026510). The change from base C to T in the original sequence led to frameshift mutations and the premature termination of translation, resulting in shortened encoded proteins and conserved WUSCHEL (WUS) box sequence loss. The specific expression analysis of the CsWOX4/Cswox4 genes in the roots, stems, leaves and other tissue types of wild-type (WT) and mutant plants revealed that CsWOX4 was higher in the root, but Cswox4 (mutant gene) was significantly higher in the leaf. Subcellular localization analysis revealed that CsWOX4 was localized in the nucleus. RNA-seq analysis revealed that the differentially expressed genes were mainly enriched in the mitochondrial cell cycle phase transition, nucleosome and microtubule binding pathways. Simultaneously, the quantitative analysis of the expression trends of 25 typical genes regulating the leaf types revealed the significant upregulation of CsPIN3. In our study, we found that the conserved domain of CsWOX4 was missing in the mutant, and the transcriptome data revealed that the expression of some genes, such as CsPIN3, changed simultaneously, thereby jointly regulating changes in the cucumber leaf type.

Pre-harvest sprouting (PHS) in cucumber (Cucumis sativus L.) significantly reduces seed quality and yields in seed industry. The identification of PHS-associated quantitative trait loci (QTL) provides valuable genetic insights for improving PHS resistance in cucumber breeding practices. In this study, near-isogenic lines (NILs) targeting the major effect QTL locus were developed by the backcrossing of the highly PHS-sensitive donor parent P60 with the PHS-resistant recurrent parent Q12. PHS phenotypes of qPHS4.1^Q12 and qPHS4.1^P60 were consistent with the respective Q12 and P60, confirming that the qPHS4.1 locus has a major-effect on PHS in cucumber. Using BC₄F₂ and BC₄F₃ populations, the qPHS4.1 was fine-mapped to a 69.34 kb region, explaining 38.8% phenotypic variation. Through map-based cloning, CsaV3_4G032930 (designated CsDOG1) was identified as the candidate gene, which encodes a DOG1 (Delay of Germination 1) domain-containing protein. Sequence analysis revealed that the mutation in P60 was a 3-bp deletion in the second exon of this gene (designated Csdog1), leading to a single amino acid deletion. Expression profiling revealed that CsDOG1 exhibits strict seed-specific expression, with minimal transcripts in vegetative tissues. Promoter analysis demonstrated identical promoter sequence between Q12 and P60. The cis-acting regulatory elements, including several tissue-specific expression motifs, ABREs for ABA responsiveness and transcriptional promoter or enhancer elements were identified. Subcellular localization analysis revealed that the CsDOG1 protein was mainly localized in nucleus. Knockout of CsDOG1 gene in the Q12 background resulted in mutants exhibiting extreme PHS susceptibility, confirming that CsDOG1 is the causal gene responsible for PHS resistance underlying qPHS4.1 locus. This study establishes a crucial theoretical foundation for elucidating the genetic mechanisms controlling PHS and offers valuable genetic resources for cucumber PHS-resistance breeding.