Genetic Regulation of Chlorophyll Biosynthesis in Pepper Fruit: Roles of CaAPRR2 and CaGLK2

<b>Background:</b> Pepper (<i>Capsicum annuum</i> L.) is a widely cultivated vegetable crop worldwide, with its rich fruit colors providing unique visual traits and economic value. This study investigated the genetic basis of the immature green fruit color by constructing a F₂ segregating population derived from a cross between yellow fruit C20 and green fruit C62 parent lines. <b>Methods:</b> Bulked segregant analysis sequencing (BSA-seq) was performed to identify genomic regions associated with fruit color. Candidate genes were pinpointed through functional annotation and genetic variation analysis, supported by SNP markers, genotype analysis, and transcriptome profiling. <b>Results:</b> Two genomic regions associated with fruit color were identified on chromosomes 1 (14.55-20.85 Mb) and 10 (10.15-22.85 Mb), corresponding to previously reported loci <i>pc1</i> and <i>pc10.1</i>. Two chlorophyll synthesis-related genes, <i>CaAPRR2</i> and <i>CaGLK2</i>, were identified as candidate regulators of fruit color. Mutations in these genes include a premature stop codon in both <i>CaGLK2</i> and <i>CaAPRR2</i>. The mutation of <i>CaAPRR2</i> and <i>CaGLK2</i> jointly regulate the yellow fruit trait in pepper, with <i>CaGLK2</i> being the major gene and <i>CaAPRR2</i> being the minor gene. Transcriptome analysis showed that the expression levels of the two genes increased during the green ripening stage of the parent fruits, with higher expression levels of <i>CaGLK2</i>. <b>Conclusions</b>: This study identifies <i>CaGLK2</i> and <i>CaAPRR2</i> as key regulators of immature green fruit color in pepper, with <i>CaGLK2</i> playing a predominant role. These findings provide a theoretical foundation and data support for elucidating the molecular regulatory mechanisms of fruit color and advancing marker-assisted breeding in pepper.