Melon (Cucumis melo L.) is a globally important fruit crop, but progress in molecular breeding has been hampered by limited functional dissection of genes associated with agronomic traits. Therefore, we developed a comprehensive genome resource based on the C. melo ssp. agrestis accession 13C. This resource includes a complete telomere-to-telomere genome assembly, including accurate quantification of 45S rDNA copy number in melon, a transcriptome atlas from 31 tissue samples, a phenotypically diverse EMS-induced mutant library and a stable transformation system. By sequencing 1,125 M2 families, we identified about 660,000 variants, which cover 97.33% of the annotated gene space. Leveraging these integrated resources, we identified and functionally characterized several key genes, including CLAVATA3 INSENSITIVE RECEPTOR KINASES 2 (CmCIK2), which regulates carpel number; PARA-AMINOBENZOIC ACID SYNTHASE (CmACDS), a central regulator of folate biosynthesis; and a mutant allele of the known gynoecious gene WIP DOMAIN PROTEIN 1 (CmWIP1). In addition, we discovered a specific EMS-induced variant in the fruit ripening regulator CmNAC-NOR, and further validated its function by generating targeted mutants. The CmNAC-NOR mutants exhibited delayed fruit ripening, thus providing a valuable resource for improving ripening traits in agrestis accessions. To facilitate broader utilization, we developed the Melon Information Resource, available at https://zhanglab.qau.edu.cn/melon/index.php, an integrated platform housing 13C comprehensive genome resources and associated convenient analysis tools. This unified and accession-specific resource offers unprecedented opportunities to accelerate gene discovery and trait improvement in melon through functional genomics and molecular breeding.

Cucurbitacin B (CuB) is the primary compound responsible for bitterness in melon (Cucumis melo L.) and negatively affects fruit quality and consumer acceptance. Although CuB biosynthesis is regulated by multiple transcription factors, the regulatory mechanisms underlying CuB biosynthesis across different tissues remain unclear. Here, we generated two independent knockout lines (Cmbt-18 and Cmbt-19) of the basic helix-loop-helix (bHLH) transcription factor gene CmBt (Bitter fruit) using CRISPR-Cas9. Loss of CmBt reduces CuB levels in fruits and roots and produces non-bitter leaves, indicating that CmBt predominantly regulates CuB accumulation in leaves. By contrast, its paralog CmBr (Bitter root) predominantly controls CuB accumulation in fruits and roots. These results redefine CmBt as a leaf-predominant regulator and CmBr as a fruit/root-predominant regulator of CuB biosynthesis, revising previous findings. Furthermore, we demonstrate that CmBt physically interacts with CmBr. The CmbtCmbr double mutants exhibit a more severe reduction in CuB content in fruits, leaves, and roots, compared with either single mutant, confirming a synergistic interaction between CmBt and CmBr. Reverse transcription quantitative PCR (RT-qPCR) and tobacco dual-luciferase assays further show that CmBt and CmBr synergistically activate the CuB biosynthetic gene CmBi. Additionally, the CmbtCmbr double mutant completely prevents CPPU-induced fruit bitterness. Our findings reveal that CmBt and CmBr exert tissue-specific, partially redundant, and synergistic roles in activating CmBi and promoting CuB biosynthesis. These results identify the CmBt-CmBr locus as a promising target for breeding non-bitter melon cultivars.

Carpel number (CN) is an important trait affecting the fruit size and shape of melon, which plays a crucial role in determining the overall appearance and market value. A unique non-synonymous single nucleotide polymorphism (SNP) in CmCLAVATA3 (CmCLV3) is responsible for the variation of CN in C. melo ssp. agrestis (hereafter agrestis), but it has been unclear in C. melo ssp. melo (hereafter melo). In this study, one major locus controlling the polymorphism of 5-CN (multi-CN) and 3-CN (normal-CN) in melo was identified using bulked segregant analysis (BSA-seq). This locus was then fine-mapped to an interval of 1.8 Mb on chromosome 12 using a segregating population containing 1451 progeny. CmCLV3 is still present in the candidate region. A new allele of CmCLV3, which contains five other nucleotide polymorphisms, including a non-synonymous SNP in coding sequence (CDS), except the SNP reported in agrestis, was identified in melo. A cis-trans test confirmed that the candidate gene, CmCLV3, contributes to the variation of CNs in melo. The qRT-PCR results indicate that there is no significant difference in the expression level of CmCLV3 in the apical stem between the multi-CN plants and the normal-CN plants. Overall, this study provides a genetic resource for melon fruit development research and molecular breeding. Additionally, it suggests that melo has undergone similar genetic selection but evolved into an independent allele.

Trans-species polymorphisms (TSPs) are fundamental to preserving ancient genetic diversity, yet the evolutionary forces driving their long-term maintenance remain largely unexplored. Here, we investigate genome-wide TSPs in two Cucumis species, cucumber and melon, using whole-genome sequencing data from over 1200 accessions. A total of 5149 TSPs were identified, which predominantly located in genic and promoter regions. Coalescent analysis indicated that both gene flow and balancing selection have contributed to the persistence of these ancestral alleles. Moreover, among the 99 genes with shared coding-region polymorphisms, two genes that cluster by alleles rather than by species provide evidence of long-term balancing selection. These genes are involved in the immune and stress response processes with pleiotropic effects. Our findings elucidate the complex evolutionary forces driving TSPs in Cucumis, providing mechanistic insights into the maintenance of intraspecific genetic diversity in plants across deep evolutionary timescales.