Capsicum chinense (habanero pepper) exhibits substantial variation in fruit pungency, color, and flavor due to its rich secondary metabolite composition, including capsaicinoids, carotenoids, and volatile organic compounds (VOCs). To dissect the genetic and regulatory basis of these traits, we conducted an integrative analysis across 244 diverse accessions using metabolite profiling, genome-wide association studies (GWAS), and transcriptome-wide association studies (TWAS). GWAS identified 507 SNPs for capsaicinoids, 304 for carotenoids, and 1176 for VOCs, while TWAS linked gene expression to metabolite levels, highlighting biosynthetic and regulatory genes in phenylpropanoid, fatty acid, and terpenoid pathways. Segmental RNA sequencing across fruit tissues of contrasting accessions revealed 7034 differentially expressed genes, including MYB31, 3-ketoacyl-CoA synthase, phytoene synthase, and ABC transporters. Notably, AP2 transcription factors and Pentatrichopeptide repeat (PPR) emerged as central regulators, co-expressed with carotenoid and VOC biosynthetic genes. High-resolution spatial transcriptomics (Stereo-seq) identified 74 genes with tissue-specific expression that overlap with GWAS and TWAS loci, reinforcing their regulatory relevance. To validate these candidates, we employed CRISPR/Cas9 to knock out AP2 and PPR genes in tomato. Widely targeted metabolomics and carotenoid profiling revealed major metabolic shifts: AP2 mutants accumulated higher levels of β-carotene and lycopene. In contrast, PPR mutants altered xanthophyll ester and apocarotenoid levels, supporting their roles in carotenoid flux and remodeling. This study provides the first integrative GWAS–TWAS–spatial transcriptomics in C. chinense, revealing key regulators of fruit quality traits. These findings lay the groundwork for precision breeding and metabolic engineering to enhance nutritional and sensory attributes in peppers.

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