Phenotypic characterization of a dominant glossy mutant and fine mapping of qCWA9.1 for controlling cuticular wax biosynthesis in rapeseed (Brassica napus L.)
Introduction Plant cuticular wax has gained increasing attention owing to its critical role in biotic and abiotic stress tolerance. Identifying key genes regulating cuticular wax biosynthesis and understanding their mechanisms are important for rapeseed resistance breeding. However, the candidate genes and mechanisms involved in cuticular wax biosynthesis in rapeseed remain poorly understood. Methods and results In this study, a dominant glossy mutant 39J7H was identified in rapeseed through genetic analysis. Two stable and pure sister lines ‘4074’ (glaucous) and ‘4075’ (glossy) were developed from the glossy mutant 39J7H via successive self-crosses. The leaves of ‘4075’ exhibited significantly increased cuticle permeability as indicated by water loss rate and toluidine blue (TB) staining. Conversely, scanning electron microscopy (SEM) and GC-MS analysis demonstrated a dramatic reduction in wax crystals density, total wax content, and chemical components in ‘4075’ leaves compared to ‘4074’. Gene mapping analysis using F2 populations from the ‘4074’ × ‘4075’ cross identified the qCWA9.1 locus within a 2.3 Mb region on chromosome A09 of rapeseed through BSA-seq. Subsequent linkage analysis in the F2 population, along with phenotypic data from the F2:3 generation, refined the qCWA9.1 region to a 49.0-kb interval between the SSR markers 9AS337 and 9AS339. This interval contains nine genes, among which BnaA09G0695600ZS, an orthologue of the Arabidopsis gene AT1G01600 (AtCYP86A4), encoding a fatty acid omega-hydroxylase CYP86A4, was identified as a potential candidate gene for qCWA9.1 associated with the glossy phenotype. Sequence analysis uncovered SNP variations in the first exon and promoter region of BnaA09G0695600ZS in the glossy line ‘4075’. Additionally, qRT-PCR analysis indicated that a significant upregulated in BnaA09G0695600ZS expression in this line. Discussion Based on these results, we hypothesize that BnaA09G0695600ZS emerged as one of candidate genes for cuticular wax synthesis. The absence of two key cis-regulatory elements, I-box and TATC-box, in the promoter region of BnaA09G0695600ZS in the glossy line ‘4075’ may potentially alter cuticular wax metabolism in rapeseed leaves. These findings enhance our understanding of cuticular wax biosynthesis in rapeseed and provide valuable genetic resources for the development of high-stress-resistant rapeseed germplasms.