Phytoalexin deficient 4 is associated with the lesion mimic trait in watermelon clalm mutant (Citrullus lanatus)

In watermelon (Citrullus lanatus), lesion mimic 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, this trait 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. There are 6 genes with non-synonymous mutation SNP sites. The predicted target gene, ClCG04G001930, encodes a Phytoalexin deficient 4 (PAD4), a protein that plays an important regulatory role in leaf senescence in many plant species. According to quantitative real-time PCR analysis, the expression level of ClCG04G001930 was significantly higher in the clalm mutant than in normal watermelon. Twenty-five SNPs were identified in the ClCG04G001930 gene of F₂ individuals of the clalm mutant. Overexpression the ClCG04G001930 gene, designated as ClPAD4, yielded transgenic lines whose leaves gradually developed chlorotic lesions over 3 weeks. RNA interference of the ClPAD4 yielded transgenic lines whose cotyledon prone to diseased over 2 weeks. Our results suggest that ClPAD4 might be the candidate gene responsible for lesion mimic in the clalm mutant. Our findings may serve as a foundation for elucidating the mechanism underlying the molecular metabolism of programmed cell death and should be useful for marker-assisted selection breeding in watermelon.