Identification of CaLAP2 as a candidate gene for the yellow-green mutation in bell pepper through fine mapping of quantitative trait loci with kompetitive allele-specific polymerase chain reaction markers

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Chili pepper (Capsicum annuum L.) is one of the most important vegetable crops worldwide, and leaf color mutants are valuable for studying chloroplast development and chlorophyll biosynthesis. This study used the yellow-green mutant 96-140YBM and its wild-type 96-140 as materials, aiming to fine-map candidate genes controlling the yellow leaf trait. On the basis of a preliminary mapping interval via bulked segregant analysis and bulked segregant RNA sequencing (BSA/BSR-seq) in the early stage, 21 single nucleotide polymorphism (SNP) markers were screened on chromosome 9 of pepper, which were developed into kompetitive allele-specific polymerase chain reaction (KASP) molecular markers. Genotyping analysis and quantitative trait locus (QTL) mapping were performed using 200 segregating F2 populations, and three intervals strongly associated with the yellow-green leaf trait were detected on chromosome 9 (690,139–760,388; 5,084,723–5,277,105; 5,281,991–5,516,629). Twenty-two genes were identified within the mapping intervals, four of which were related to chloroplast function. Capana09g000125 (named CaLAP2) was identified as the candidate gene. Sequence analysis revealed a base variation in its exon (GGA→GAA), resulting in a glycine (Gly) to glutamic acid (Glu) substitution. This mutation site was developed into a KASP marker for genotype–phenotype association analysis in the F2 population, and validation results showed that the association accuracy between this mutation site and the phenotype reached 96.5%. This study established a fine-mapping technical system combining BSA/BSR-seq with KASP markers, which significantly narrowed the mapping interval and rapidly located key genes for the yellow-green mutation. This work lays an important foundation for elucidating the molecular mechanisms of yellow-green mutants in pepper, functional verification, and their application in molecular marker-assisted selection breeding.