Map-based cloning and functional validation of ClChlH governing yellow rind color in watermelon (Citrullus lanatus)

Map-based cloning and gene editing confirmed that ClChlH was the causal gene controlling yellow rind color in watermelon. Fruit rind color is a crucial agronomic trait influencing the commercial quality of watermelon (Citrullus lanatus). Although yellow rind is an important phenotype, its underlying molecular mechanisms remain poorly understood. In this study, genetic analysis using two mapping populations derived from crosses between a yellow rind line (W-21-4-2) and two green rind lines (W-21-301 and W-21-129) indicated that the yellow rind trait is controlled by a single dominant locus, ClYR. By combining BSA and KASP genotyping, we initially mapped ClYR to a 6.5 Mb region on chromosome 4. Subsequent fine mapping with 1345 F_2:3 individuals narrowed the candidate interval to 325.37 Kb, which contains 10 annotated genes. Among these, ClChlH (Cla97C04G068530) emerged as the most promising candidate gene due to multiple insertions/deletions (InDels) in its promoter region that distinguish the yellow and green rind lines. Expression analysis revealed significantly reduced ClChlH transcript levels in the yellow rind line W-21-4-2. Promoter activity assays further demonstrated that these structural variations suppress transcriptional activation of ClChlH. Haplotype analysis confirmed that these promoter InDels were correlated with yellow rind phenotype. Functional validation via CRISPR/Cas9-mediated mutagenesis generated mutants exhibiting yellow-green or sectored (half-green-half-yellow) pigmentation in both rind and leaf tissues. Collectively, our findings elucidate a key genetic regulator of rind coloration and provide valuable molecular resources for future watermelon breeding programs.