Transposon-mediated structural haplotypes at CsMYB60 are associated with peel coloration in cucumber

A previously unrecognized MULE1+MULE2 structural haplotype at CsMYB60 was identified and associated with mature peel coloration in cucumber. Peel color is a major commercial and physiological trait in cucumber (Cucumis sativus L.), yet our understanding of the underlying molecular mechanisms remains incomplete. Previous work has shown that the R2R3-MYB transcription factor CsMYB60 regulates flavonoid and proanthocyanidin biosynthesis in cucumber fruit spines. More recent evidence further implicates this gene in peel color variation via intronic Mutator-like element (MULE) insertions or premature stop codons identified in diverse germplasm. In the present study, we utilized a pair of near-isogenic lines, a yellow-peel inbred line (L14) and its white-peel mutant (L19), to minimize genetic background effects and precisely dissect the genetic basis of peel color variation. Through bulked segregant analysis combined with high-resolution fine mapping in segregating populations derived from these lines, the peel color locus was narrowed to a 121-kb interval on chromosome 4 harboring the R2R3-MYB transcription factor CsMYB60. Sequence comparison revealed that the L14 allele carries both the previously-characterized MULE1 insertion and a second nearby MULE2 insertion within the second intron of CsMYB60, whereas L19 retains only MULE1. During the peel color-transition stage, the MULE1 + MULE2 haplotype showed higher CsMYB60 transcript abundance than the MULE1-only haplotype in the near-isogenic materials examined. CRISPR/Cas9-mediated knockout of CsMYB60 in the L14 background abolished yellow pigmentation, demonstrating that CsMYB60 is required for yellow mature peel formation in this background. Together, these results identify a previously unrecognized MULE1 + MULE2 structural haplotype associated with distinct CsMYB60 transcript abundance and mature peel coloration. The causal regulatory contribution of MULE2 itself remains to be determined. The associated marker may facilitate tracking of this structural haplotype in relevant cucumber breeding materials.