BrWAX2 plays an essential role in cuticular wax biosynthesis in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

Abstract

Key message Map-based cloning was used to identify the BrWAX2 gene, which was involved in the cuticular wax biosynthesis. The malfunction of BrWAX2 together with other reduced expression of genes in alkane-forming pathway caused the glossy phenotype. Abstract Cuticular wax covering the outer plant surface plays various roles in protecting against biotic and abiotic stresses. Wax-less mutant shows glossy in stem and leaf surface and plays important roles in enriching Chinese cabbage germplasm resources for breeding brilliant green varieties. However, genes responsible for the glossy trait in Chinese cabbage are rarely reported. In this study, we identified a glossy Chinese cabbage line Y1211-1. Genetic analysis indicated that the glossy trait in Y1211-1 was controlled by a single recessive locus, BrWAX2 (Brassica rapa WAX 2). Using bulked segregant sequencing (BSA-Seq) and kompetitive allele-specific PCR (KASP) assays, BrWAX2 was fine-mapped to an interval of 100.78 kb. Functional annotation analysis, expression analysis, and sequence variation analysis revealed that Bra032670, homologous to CER1 in Arabidopsis, was the most likely candidate gene for BrWAX2. The gene Bra032670 was absent in glossy mutant. Cuticular wax composition analysis and RNA-Seq analysis suggested that the absence of BrWAX2 together with the decreased expression of other genes in alkane-forming pathway reduced the wax amount and caused the glossy phenotype. Furthermore, we developed and validated the functional marker BrWAX2-sp for BrWAX2. Overall, these results provide insight into the molecular mechanism underlying cuticular wax biosynthesis and reveal valuable information for marker-assisted selection (MAS) breeding in Chinese cabbage.

BrWAX3, encoding a β-ketoacyl-CoA synthase, participates in cuticular wax biosynthesis in Chinese cabbage

“…In total, three biological replicates of W-bulk and G-bulk were constructed. The cuticular wax composition and components in W-bulk and G-bulk were assessed via GC-MS at Shanghai Jiao Tong University, which was performed as previously described by Yang et al (2022a).…”

“…The SAMtools software (V1.3.1) (Li et al 2009) was used to detect the single-nucleotide polymorphism (SNP) and insertion/deletion (InDel) variants between the W-pool and G-pool. Then, we calculated the SNP-index and △(SNP-index) for all genomic positions in the W-pool and G-pool, which was performed as our previous studies (Yang et al 2022a;Yang et al 2022b). Finally, the absolute value of △(SNP-index) was calculated for sliding window analysis, with a 1-Mb window width and a 50-kb sliding window step.…”

“…The analysis of gene relative expression data was performed using the 2 −ΔΔCt method (Livak and Schmittgen 2001). BrGAPDH was employed as the reference gene (Yang et al 2022a;Yang et al 2022b). The primers were listed in Table S2.…”

“…Our qRT-PCR analysis of wax metabolism-related genes was consistent with the RNA-seq results (Figure S8). Cuticular waxes contain not only VLCFAs and their derivatives, but also other secondary metabolites, such as terpenoids, sterols, and avonoids (Bernard and Joubès 2013; Samuels et al 2008;Yang et al 2022a). As expected, pathways of ' avonoid biosynthesis', 'phenylalanine metabolism', 'glucosinolate biosynthesis', 'stilbenoid, diarylheptanoid and gingerol biosynthesis' were signi cantly enriched (Fig.…”

“…For economically important leafy vegetables in Brassica species, such as Brassica rapa (A genome) and Brassica oleracea (C genome), the waxless and glossy phenotype is an important commodity trait in breeding (Ji et al 2021;Yang et al 2022a). Therefore, identifying genes responsible for the glossy phenotype facilitates the breeding of new varieties with a glossy green phenotype.…”