The mottled rind is an important fruit external appearance trait that influences consumer preferences. Previous studies reported that CmMt1 and CmMt2 regulate rind mottling in melon, yet CmMt2 has not been cloned. In this study, we developed near-isogenic lines (NILs) using the nonmottled rind ‘13C’ as the recurrent parent and mottled ‘P114’ as the donor parent, and screened a mottled rind mutant ‘S249’ by ethyl methanesulfonate mutagenesis of ‘13C’. Combined with these genetic materials, CmMt2 was delimited to a 44-kb region on chromosome 2. Within this genetic interval, a CACTA-type TIR transposon insertion was detected in all nonmottled rind lines, and this insertion may lead to impaired nuclear localization and dimerization capability of CmKNAT2-like2 encoding a homeobox protein through the loss of conserved ELK and Homeodomain. Further, CRISPR/Cas9-mediated knockout of CmKNAT2-like2 confirmed its pivotal role in regulating mottled rind phenotype. In addition, transcriptome analysis suggested that the transposon insertion in CmKNAT2-like2 results in nonmottled rind by disrupting chloroplast development and altering the expression of chlorophyll biosynthesis-related genes, and population analysis revealed that the transposon associated with CmKNAT2-like2 has undergone selection in cultivated melons. Collectively, these results demonstrate that CmKNAT2-like2 is the causal gene underlying CmMt2, which regulates mottled rind in melon.

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