High-throughput genotyping tools can effectively promote molecular breeding in crops. In this study, genotyping by target sequencing (GBTS) system was utilized to develop a genome-wide liquid SNP chip for facilitating genetics and breeding in melon (Cucumis melo L.), a globally cultivated economically important horticultural crop. Based on over eight million SNPs derived from 823 representative melon accessions, 16K, 8K, 4K, 2K, 1K, 500, 250 and 125 informative SNPs were screened and evaluated for their polymorphisms, conservation of flanking sequences, and distributions. The set of 2K SNPs was found to be optimal for representing the maximum diversity with the lowest number of SNPs, and it was selected to develop the liquid chip, named “Melon2K”. Using Melon2K, more than 1 500 SNPs were detected across 17 samples of five melon cultivars, and the phylogenetic relationships were clearly constructed. Within the same cultivar, genetic differences were also assessed between different samples. We evaluated the performance of Melon2K in genetic background selection during the breeding process, obtaining the introgression lines of interested trait with more than 97% genetic background of elite variety by only two rounds of backcrossing. These results suggest that Melon2K provides a cost-effective, efficient and reliable platform for genetic analysis and molecular breeding in melon.

Many biotic or abiotic factors such as CPPU (N-(2-chloro-pyridin-4-yl)-N'-phenylurea), a growth regulator of numerous crops, can induce bitterness in cucurbits. In melon, cucurbitacin B is the major compound leading to bitterness. However, the molecular mechanism underlying CuB biosynthesis in response to different conditions remains unclear. Here, we identified a set of genes involved in CPPU-induced CuB biosynthesis in melon fruit and proposed CmBr gene as the major regulator. Using CRISPR/Cas9 gene editing, we confirmed CmBr's role in regulating CuB biosynthesis under CPPU treatment. We further discovered a CPPU-induced MYB-related transcription factor, CmRSM1, which specifically binds to the Myb motif within the CmBr promoter and activates its expression. Moreover, we developed an introgression line by introducing the mutated Cmbr gene into an elite variety and eliminated CPPU-induced bitterness, demonstrating its potential application in breeding. This study offers a valuable tool for breeding high-quality non-bitter melon varieties and provides new insights into the regulation of secondary metabolites under environmental stresses.

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.

The basic helix-loop-helix (bHLH) family of transcription factors (TFs) participate in a variety of biological regulatory processes in plants, and have undergone significant expansion during land plant evolution by gene duplications. In cucurbit crops, several bHLH genes have been found to be responsible for agronomic traits such as bitterness. However, the characterization of bHLH genes across the genomes of cucurbit species has not been reported, and how they have evolved and diverged remains largely unanswered. Here we identified 1,160 bHLH genes in seven cucurbit crops and performed a comprehensive comparative genomics analysis. We determined orthologous and paralogous bHLH genes across cucurbit crops by syntenic analysis between or within species. Orthology and phylogenetic analysis of the tandem-duplicated bHLH genes in the Bt cluster, which regulate the biosynthesis of cucurbitacins, suggest that this cluster is derived from three ancestral genes after the cucurbit-common tetraploidization event. Interestingly, we identified a new conserved cluster paralogous to the Bt cluster that includes two tandem bHLH genes, and the evolutionary history and expression profiles of these two genes in the new cluster suggest the involvement of one gene (Brp) in the regulation of cucurbitacin biosynthesis in roots. Further biochemical and transgenic assays in melon hairy roots supported the function of Brp. This study provides useful information for further investigating the functions of bHLH TFs and novel insights into the regulation of cucurbitacin biosynthesis in cucurbit crops and other plants.