Gene mapping and candidate gene analysis of a novel watermelon lesion mimic mutant clalm

Abstract The leaf is an extremely important plant organ exhibiting a broad range of phenotypic variation. In watermelon ( Citrullus lanatus ), leaf spotting is a rare, valuable trait that can be used by breeders for selection at early growth stages. In this study, we tested a seven-generation family to determine the inheritance and genetic basis of this trait. As revealed by analysis of the lesion mimic mutant clalm , leaf spotting is controlled by a single dominant gene. Whole genome resequencing–bulked segregant analysis demonstrated that this gene is located on chromosome 4 from 3,760,000 bp to 7,440,000 bp, a region corresponding to a physical distance of 3.68 Mb encompassing approximately 72 annotated genes and eight non-synonymous coding SNPs. According to quantitative real-time PCR analysis, the expression level of ClCG04G001930 was significantly lower in the clalm mutant than in normal watermelon. The predicted target gene, ClCG04G001930 , encodes a fatty acid amide hydrolase protein that regulates a variety of neurobehavioral processes in animals. Twelve-five SNPs were identified in the ClCG04G001930 gene of F 2 individuals of the clalm mutant . RNA interference of the ClCG04G001930 gene, designated as ClPAD4 , yielded transgenic lines whose leaves gradually developed chlorotic lesions over 3 weeks. Our results suggest that ClPAD4 is the gene responsible for leaf spotting in the clalm mutant. Our findings may serve as a foundation for elucidating the mechanism underlying the spotted leaf trait and should be useful for marker-assisted selection breeding in watermelon.