Low temperature is an important abiotic stress that negatively affects morphological growth and fruit development in melon (Cucumis melo L.). Chilling stress at the seedling stage causes seedling injury and poor stand establishment, prolonging vegetative growth and delaying fruit harvest. In this study, association mapping was performed for chilling tolerance at the seedling stage on an expanded melon core collection containing 212 diverse accessions by 272 SSRs and 27 CAPSs. Chilling tolerance of the melon seedlings was evaluated by calculating the chilling injury index (CII) in 2016 and 2017. Genetic diversity analysis of the whole accession panel presented two main groups, which corresponded to the two subspecies of C. melo, melo, and agrestis. Both the subspecies were sensitive to chilling but with agrestis being more tolerant. Genome-wide association study (GWAS) was conducted, respectively, on the whole panel and the two subspecies, totally detecting 51 loci that contributed to 74 marker-trait associations. Of these associations, 35 were detected in the whole panel, 21 in melo, and 18 in agrestis. About half of the associations identified in the two subspecies were also observed in the whole panel, and seven associations were shared by both the subspecies. CMCT505_Chr.1 was repeatedly detected in different populations with high phenotypic contribution and could be a key locus controlling chilling tolerance in C. melo. Nine loci were selected for evaluation of the phenotypic effects related to their alleles, which identified 11 elite alleles contributing to seedling chilling tolerance. Four such alleles existed in both the subspecies and six in either of the two subspecies. Analysis of 20 parental combinations for their allelic status and phenotypic values showed that the elite alleles collectively contributed to enhancement of the chilling tolerance. Tagging the loci responsible for chilling tolerance may simultaneously favor dissecting the complex adaptability traits and elevate the efficiency to improve chilling tolerance using marker-assisted selection in melon.

Trade-offs between survival and growth are widely observed in plants. Melon is an annual, trailing herb that produces economically valuable fruits that are traditionally cultivated in early spring in China. Melon seedlings are sensitive to low temperatures, and thus usually suffer from cold stress during the early growth period. However, little is known about the mechanism behind the trade-offs between seedling cold tolerance and fruit quality in melon. In this study, a total of 31 primary metabolites were detected from the mature fruits of eight melon lines that differ with respect to seedling cold tolerance; these included 12 amino acids, 10 organic acids, and nine soluble sugars. Our results showed that concentrations of most of the primary metabolites in the cold-resistant melons were generally lower than in the cold-sensitive melons; the greatest difference in metabolite levels was observed between the cold-resistant line H581 and the moderately cold-resistant line HH09. The metabolite and transcriptome data for these two lines were then subjected to WGCNA, resulting in the identification of five key candidate genes underlying the balancing between seedling cold tolerance and fruit quality. Among these genes, CmEAF7 might play multiple roles in regulating chloroplast development, photosynthesis, and the ABA pathway. Furthermore, multi-method functional analysis showed that CmEAF7 can certainly improve both seedling cold tolerance and fruit quality in melon. Our study identified an agriculturally important gene, CmEAF7, and provides a new insight into breeding methods to develop melon cultivars with seedling cold tolerance and high fruit quality.

Seed size is an important agronomic trait in melons that directly affects the seed germination and subsequent seedling growth. However, the genetic mechanism underlying seed size in melon has remained unclear. In the present study, we employed Bulked-Segregant Analysis sequencing (BSA-seq) to identify a candidate region (∼1.35 Mb) on chromosome 6 that corresponds to seed size. This interval was confirmed by QTL mapping of three seed size-related traits from an F₂ population across three environments. This mapping represented nine QTLs that shared an overlapping region on chromosome 6, collectively referred to as qSS6.1. New InDel markers were developed in the qSS6.1 region, narrowing it down to a 68.35-kb interval that contains eight annotated genes. Sequence variation analysis of the eight genes identified a SNP with a C to T transition mutation in the promoter region of MELO3C014002, a leucine-rich repeat receptor-like kinase (LRR-RLK) gene. This mutation affected promoter activity of the MELO3C014002 gene and was successfully used to differentiate the large-seeded accessions (C-allele) from the small-seeded accessions (T-allele). qRT-PCR revealed differential expression of MELO3C014002 between the two parental lines. Its predicted protein has typical domains of LRR-RLK family, and phylogenetic analyses reveled its similarity with the homologs in several plant species. Altogether, these findings suggest MELO3C014002 as the most likely candidate gene involved in melon seed size regulation. Our results will be helpful for better understanding the genetic mechanism regulating seed size in melons and for genetically improving this important trait through molecular breeding pathways.

Simple sequence repeats (SSRs) are widely used in mapping constructions and comparative and genetic diversity analyses. Here, 103,056 SSR loci were found in Cucurbita species by in silico PCR. In general, the frequency of these SSRs decreased with the increase in the motif length, and di-nucleotide motifs were the most common type. For the same repeat types, the SSR frequency decreased sharply with the increase in the repeat number. The majority of the SSR loci were suitable for marker development (84.75% in Cucurbita moschata, 94.53% in Cucurbita maxima, and 95.09% in Cucurbita pepo). Using these markers, the cross-species transferable SSR markers between C. pepo and other Cucurbitaceae species were developed, and the complicated mosaic relationships among them were analyzed. Especially, the main syntenic relationships between C. pepo and C. moschata or C. maxima indicated that the chromosomes in the Cucurbita genomes were highly conserved during evolution. Furthermore, 66 core SSR markers were selected to measure the genetic diversity in 61 C. pepo germplasms, and they were divided into two groups by structure and unweighted pair group method with arithmetic analysis. These results will promote the utilization of SSRs in basic and applied research of Cucurbita species.