This study aims to enhance the coloration of pepper fruit by identifying valuable genetic resources through the analysis of single nucleotide polymorphism (SNP). markers and candidate genes associated with fruit pigmentation. Utilizing 197 natural populations of both hot and sweet peppers, we employed specific-locus amplified fragment sequencing (SLAF-seq) to examine 1496 high-quality SNP markers, thereby identifying significant loci contributing to fruit color variation. Our genome-wide association study pinpointed 30 significant SNP sites located on chromosome 6. Further analysis using kompetitive allele-specific PCR(KASP) and phenotypic correlation with fruit color led to the identification of the CA.PGAv.1.6.scaffold919.44 gene, which is implicated in anthocyanin synthesisregulation via the NAC domain, thereby influencing pepper fruit coloration. These findings offer a valuable reference for the advancement of molecular-assisted breeding strategies aimed at improving the fruit color of both sweet and hot peppers.To improve the fruit color of sweet peppers, this study aimed to identify single nucleotide polymorphism (SNP) loci and candidate genes significantly associated with fruit color. A natural population of 197 sweet pepper accessions was used as the material. SLAF-seq was conducted with 1496 high-quality SNP markers to mine excellent variant loci and predict candidate genes. Through Manhattan plot analysis and association analysis with the best linear unbiased prediction (BLUP) values of fruit color, 30 significant loci were detected on chromosome 6. Combining KASP genotyping technology with field phenotypes, the gene CAPGAv.1.6.scaffold919.44 was identified as a candidate gene regulating mature fruit color. It is related to the NAC domain and is hypothesized to alter fruit color by regulating anthocyanin biosynthesis. This study lays the foundation for molecular-assisted breeding of sweet peppers related to fruit color.

To investigate the molecular mechanisms underlying ascorbic acid (AsA) accumulation in pepper fruits and to identify key genes involved in its biosynthesis, we performed integrated metabolomic and transcriptomic analyses on two pepper cultivars—T41 (sweet pepper) and 22-5 (chili pepper)—at three developmental stages, including young fruit, green ripe, and color change stages. The results show that AsA content in both cultivars was significantly higher at the green ripe stage than the young fruit stage, with T41 exhibiting significantly higher AsA levels than 22-5 at both the young fruit and green ripe stages. Transcriptomic analysis identified a total of 24,433 differentially expressed genes (DEGs). KEGG pathway enrichment analysis revealed that genes associated with AsA biosynthesis were mainly enriched in the “ascorbate and aldarate metabolism” pathway. Follow-up validation confirmed APX3 as the most likely candidate gene responsible for the difference in AsA content between the two pepper fruit types, with its expression pattern negatively correlating with AsA accumulation. This study unveils the molecular regulatory mechanisms underlying AsA biosynthesis and provides a theoretical foundation for breeding pepper cultivars with elevated AsA levels. This study provides valuable insights into the molecular regulation of AsA biosynthesis and lays a theoretical foundation for breeding high-AsA pepper varieties.

Yellow leaf phenotypes represent an important trait in pepper and enrich genetic germplasm resources. Previous studies have primarily examined leaf yellowing caused by defects in chlorophyll biosynthesis, whereas the functions of chlorophyll degradation-related genes remain less well characterized. In this study, a yellow leaf phenotype (NY) was identified. Transmission electron microscopy and RNA-seq showed that differentially expressed genes were concentrated in photosynthesis- and chlorophyll-related pathways. CaCLH1 was then demonstrated to promote chlorophyll degradation. Yeast one-hybrid analysis identified the CabHLH18-like gene as a direct interactor with the CaCLH1 promoter and as a positive regulator of CaCLH1 transcription. The CabHLH18-like gene was highly expressed in yellow leaf pepper. The CabHLH18-like gene was subsequently isolated and characterized, revealing nuclear localization and transcription factor activity. Functional analysis showed that silencing the CabHLH18-like gene increased chlorophyll content, whereas its overexpression reduced chlorophyll content. These findings indicate that CaCLH1 is a direct downstream target of the CabHLH18-like gene and contributes to CabHLH18-like-gene-mediated regulation of leaf yellowing in pepper. This study refines mechanistic understanding of leaf yellowing and provides a foundation for future research and breeding applications in pepper.