Fine mapping of the major-effect QTL qPHS4.1 revealed a CsDOG1 gene controlling pre-harvest sprouting in cucumber (Cucumis sativus L.)

Pre-harvest sprouting (PHS) in cucumber (<i>Cucumis sativus</i> 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 <i>qPHS4.1</i> ^Q12 and <i>qPHS4.1</i> ^P60 were consistent with the respective Q12 and P60, confirming that the <i>qPHS4.1</i> locus has a major-effect on PHS in cucumber. Using BC₄F₂ and BC₄F₃ populations, the <i>qPHS4.1</i> was fine-mapped to a 69.34 kb region, explaining 38.8% phenotypic variation. Through map-based cloning, <i>CsaV3_4G032930</i> (designated <i>CsDOG1</i>) 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 <i>Csdog1</i>), leading to a single amino acid deletion. Expression profiling revealed that <i>CsDOG1</i> exhibits strict seed-specific expression, with minimal transcripts in vegetative tissues. Promoter analysis demonstrated identical promoter sequence between Q12 and P60. The <i>cis</i>-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 <i>CsDOG1</i> gene in the Q12 background resulted in mutants exhibiting extreme PHS susceptibility, confirming that <i>CsDOG1</i> is the causal gene responsible for PHS resistance underlying <i>qPHS4.1</i> 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.