Mutations in BrMYB31 lead to a glossy phenotype caused by a deficiency in epidermal wax crystals in Chinese cabbage.

key messageMutations in BrMYB31 were responsible for glossy phenotype, which was verified in two allelic mutants and gene silencing analysis. BrMYB31 regulated wax biosynthesis by modulating BrCER4 expression in Chinese cabbage. Plant cuticular wax plays a crucial role in resisting both biotic and abiotic stresses, but its deficiency is beneficial for improving the commercial properties of certain leafy vegetables. The glossy appearance resulting from the absence of epidermal wax crystals is a striking product feature of Chinese cabbage (Brassica rapa L. ssp. pekinensis). In this study, we identified two allelic mutants with wax crystal deficiency (wdm2 and wdm5) derived from an ethyl methane sulfonate (EMS)-mutagenized population of Chinese cabbage. Genetic analysis indicated that a recessive nuclear gene is responsible for the glossy phenotype. Based on MutMap sequencing combined with the kompetitive allele-specific PCR (KASP) genotyping approach, BraA02g022420.3C, a gene belonging to MYB transcription factor family, was predicted as the candidate gene and designated it as BrMYB31. Allelic mutant sanger sequencing analysis revealed a single-nucleotide polymorphism (SNP) (G/A) in the 2nd exon of BrMYB31 in wdm2, resulting in the substitution of lysine (K) with arginine (R), and also a SNP (G/A) in the 3rd exon of BrMYB31 in wdm5, causing tryptophan (W) to be converted into a terminator (X). Further virus-induced gene silencing analysis demonstrated that suppression of BrMYB31 exhibited a glossy phenotype. Transcriptome analysis revealed that BrMYB31 likely regulates glossy leaf characteristics probably by impacting wax biosynthesis-related genes expression. Transcriptional activity assays confirmed that BrMYB31 functions as a transcriptional activator. Yeast one-hybrid assays, luciferase reporter assays, and GUS activity analyses indicated that BrMYB31 could directly bind to BrCER4 promoter. This study illustrates the effectiveness of integrating MutMap and KASP in mining candidate genes associated with glossy leaf traits in EMS mutants and provides new insights into the regulatory network of cuticle wax biosynthesis in Chinese cabbage.