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Watermelon is an important horticultural crop in the Cucurbitaceae family, which exhibits a remarkable diversity of vegetative phenotypes. In this experimental study, two contrasted watermelon lines “natural mutant line (DW-2) with non-lobed leaves and the wild-type line (DG-2) with lobed leaves” were crossed to derive the biparental F2 mapping populations over two years. Genetic segregation analysis suggested that the non-lobed leaf ( nll ) phenotype is controlled by a single genetic locus with incomplete dominance. Bulk segregant sequencing analysis (BSA-seq) and preliminary linkage mapping with 183 F2 individuals identified the nll locus in a 0.7 Mbp region on chromosome 4, which was also verified in a genome-wide association study (GWAS) of 144 watermelon accessions with leaf variations. Fine genetic mapping with a large F2 population ( n = 1069) and screened recombinants exposed the delimited candidate region of 98.23 kb with eight functionally annotated genes. The sequence analysis in this region suggested that Cla97C04G076510 is the most likely candidate gene for regulating the nll , which encodes a homeobox leucine zipper (HD-Zip) transcription factor ( ClNll ), a homolog of the LATE MERISTEM IDENTITY1 ( LMI1 ) in Arabidopsis. A single nucleotide deletion in the second exon of nll was mainly responsible for the non-lobed leaf phenotype, which encoded a truncated protein. A genetic marker based on the 1-bp deletion showed co-segregation with leaf lobeness in the F2 population, exhibiting validation in the natural GWAS panel of 144 watermelon accessions. The phylogenetic associations of ClNll protein sequences with 11 homologous sequences from eight other plant species revealed an identical conserved function. Further, the expression level of the Cla97C04G076510 gene was noticed to be significantly lower in the non-lobed leaf of the DW-2 mutant. The identified results provide valuable insights for understanding the genetic regulation of leaf shape variation in watermelon and would be helpful for marker-assisted breeding aimed at the development of improved cultivars.
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