Conjunctive BSA-Seq and BSR-Seq to Map the Genes of Yellow Leaf Mutations in Hot Peppers (Capsicum annuum L.)

Yellow leaf mutations have been widely used to study the chloroplast structures, the pigment synthesis, the photosynthesis mechanisms and the chlorophyll biosynthesis pathways across various species. For this study, a spontaneous mutant with the yellow leaf color named 96-140YBM was employed to explore the primary genetic elements that lead to the variations in the leaf color of hot peppers. To identify the pathways and genes associated with yellow leaf phenotypes, we applied sequencing-based Bulked Segregant Analysis (BSA-Seq) combined with BSR-Seq. We identified 4167 differentially expressed genes (DEGs) in the mutant pool compared with the wild-type pool. The results indicated that DEGs were involved in zeatin biosynthesis, plant hormone signal transduction, signal transduction mechanisms, post-translational modification and protein turnover. A total of 437 candidates were identified by the BSA-Seq, while the BSR-Seq pinpointed four candidate regions in chromosomes 8 and 9, containing 222 candidate genes. Additionally, the combination of BSA-Seq and BSR-Seq showed that there were 113 overlapping candidate genes between the two methods, among which 8 common candidates have been previously reported to be related to the development of chloroplasts, the photomorphogenesis and chlorophyll formation of plant chloroplasts and chlorophyll biogenesis. qRT-PCR analysis of the 8 common candidates showed higher expression levels in the mutant pool compared with the wild-type pool. Among the overlapping candidates, the DEG analysis showed that the CaKAS2 and CaMPH2 genes were down-regulated in the mutant pool compared to the wild type, suggesting that these genes may be key contributors to the yellow leaf phenotype of 96-140YBM. This research will deepen our understanding of the genetic basis of leaf color formation and provide valuable information for the breeding of hot peppers with diverse leaf colors.