Wax is an acellular structural substance attached to the surface of plant tissues. It forms a protective barrier on the epidermis of plants and plays an important role in resisting abiotic and biotic stresses. In this paper, nonheading Chinese cabbage varieties with and without wax powder were observed using scanning electron microscopy, and the surface of waxy plants was covered with a layer of densely arranged waxy crystals, thus differentiating them from the surface of waxless plants. A genetic analysis showed that wax powder formation in nonheading Chinese cabbage was controlled by a pair of dominant genes. A preliminary bulked segregant analysis sequencing (BSA-seq) assay showed that one gene was located at the end of chromosome A09. Within this interval, we identified BraA09000626, encoding an AP2 transcription factor homologous to Arabidopsis AtSHINE3, and we named it BrSHINE3. By comparing the CDS of the gene in the two parental plants, a 35 bp deletion in the BrSHINE3 gene of waxless plants resulted in a frameshift mutation. Tissue analysis showed that BrSHINE3 was expressed at significantly higher levels in waxy plant rosette stage petioles and bolting stage stems than in the tissues of waxless plants. We speculate that this deletion in BrSHINE3 bases in the waxless material may inhibit wax synthesis. The overexpression of BrSHINE3 in Arabidopsis induced the accumulation of wax on the stem surface, indicating that BrSHINE3 is a key gene that regulates the formation of wax powder in nonheading Chinese cabbage. The analysis of the subcellular localization showed that BrSHINE3 is mainly located in the nucleus and chloroplast of tobacco leaves, suggesting that the gene may function as a transcription factor. Subsequent transcriptome analysis of the homology of BrSHINE3 downstream genes in nonheading Chinese cabbage showed that these genes were downregulated in waxless materials. These findings provide a basis for a better understanding of the nonheading Chinese cabbage epidermal wax synthesis pathway and provide important information for the molecular-assisted breeding of nonheading Chinese cabbage.
Keywords: BSA-seq; BrSHINE3; epidermal waxiness; nonheading Chinese cabbage; preliminary positioning.