Integrated Genetic Mapping, Transcriptomic, and Small RNA Analysis of Fruit Bitterness in Melon

Melon fruit bitterness, mainly caused by cucurbitacin B (CuB), is a major quality defect that reduces consumer acceptance and market value. However, the genetic and regulatory mechanisms underlying fruit bitterness remain largely unclear. In this study, two thin-skinned melon inbred lines, A54 (bitter leaves and fruit) and B15 (bitter leaves but non-bitter fruit), were used to dissect the genetic basis of fruit bitterness. Genetic analysis revealed that fruit bitterness in A54 is controlled by a single dominant locus. Through bulked-segregant analysis sequencing (BSA-seq) and fine mapping, the candidate region was delimited to a 133-kb interval on chromosome 9, which contains four bHLH genes. Among them, MELO3C005610 (formerly designated CmBr) exhibited 25.5-fold higher expression in A54 than in B15 and was identified as the most plausible positional candidate gene. We further developed a near-isogenic line with bitter fruit (B15^NIL) and performed integrated transcriptome, small RNA, and degradome sequencing. Twelve CuB biosynthetic genes were significantly up-regulated in B15^NIL fruits, indicating coordinated activation of the cucurbitacin biosynthetic pathway. In addition, small RNA and degradome analyses identified candidate miRNA-target interactions that may contribute to post-transcriptional regulation of CuB biosynthesis, although these interactions require further functional validation. Together, these findings provide a framework for understanding the genetic basis of fruit bitterness in melon and suggest candidate targets for future functional validation and breeding of non-bitter cultivars.