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 F2 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.