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In this study, we identified a novel glossy mutant from Chinese cabbage, named SD369, and all wax monomers longer than 26 carbons were significantly decreased. Inheritance analysis revealed that the glossy trait of SD369 was controlled by a single recessive locus, BrWAX3. We fine-mapped the BrWAX3 locus to an interval of 161.82 kb on chromosome A09. According to the annotated genome of Brassica rapa, Bra024749 (BrCER60.A09), encoding a β-ketoacyl-CoA synthase, was identified as the candidate gene. Expression analysis showed that BrCER60.A09 was significantly downregulated in all aerial organs of glossy plants. Subcellular localization indicated that the BrCER60.A09 protein functions in the endoplasmic reticulum. A 5567-bp insertion was identified in exon 1 of BrCER60.A09 in SD369, which lead to a premature stop codon, thus causing a loss of function of the BrCER60.A09 enzyme. Moreover, comparative transcriptome analysis revealed that the ‘cutin, suberine, and wax biosynthesis’ pathway was significantly enriched, and genes involved in this pathway were almost upregulated in glossy plants. Further, two functional markers, BrWAX3-InDel and BrWAX3-KASP1, were developed and validated. Overall, these results provide a new information for the cuticular wax biosynthesis and provide applicable markers for marker-assisted selection (MAS)-based breeding of Brassica rapa.

In this study, we identified a novel glossy mutant from Chinese cabbage, named SD369, all wax monomers longer than 26 carbons were significantly decreased. Inheritance analysis revealed that the glossy trait of SD369 was controlled by a single recessive locus, BrWAX3. We fine-mapped the BrWAX3 locus to an interval of 161.82 kb on chromosome A09. According to the annotated genome of Brassica rapa, Bra024749 (BrCER60.A09), which encodes a β-ketoacyl-CoA synthase, was identified as the candidate gene. Expression analysis showed that BrCER60.A09 was significantly downregulated in all aerial organs of glossy plants. Subcellular localization indicated that the BrCER60.A09 protein functions in the endoplasmic reticulum. A 5,567-bp insertion was identified in exon 1 of BrCER60.A09 in SD369, which lead to a premature stop codon and thus causing a loss of function of the BrCER60.A09 enzyme. Moreover, comparative transcriptome analysis revealed that the 'cutin, suberine and wax biosynthesis' pathway was significantly enriched, and genes involved in this pathway were almost upregulated in glossy plants. Further, two functional markers, BrWAX3-InDel and BrWAX3-KASP1, were developed and validated. Overall, these results provide new information for the cuticular wax biosynthesis and provide applicable markers for marker-assisted selection (MAS)-based breeding of Brassica rapa.

Microspore culture has been implemented in breeding programs to produce doubled haploids (DH) and thus accelerate the breeding process. This study aimed to establish an efficient microspore culture protocol for Zengcheng flowering Chinese cabbage, a highly nutritious and valued vegetable, that would be suitable for heterosis breeding. Microspores of three genotypes, 17AY010, 17AY011, and 17AY012 were successfully induced to produce embryos in NLN-13 medium. Two genotypes, 17AY011 and 17AY012 were cultivated in NLN-13 medium supplemented with different concentrations (0, 0.2, 1, 5 or 25 μM) of L-ascorbic acid sodium salt (Vitamin C Sodium Salt, VcNa) to enhance microspore embryogenesis and plant regeneration without an intervening callus phase. In both genotypes, the addition of 1 μM and 0.2 μM VcNa significantly increased the frequency of embryogenesis, 6.55-fold and 10.33-fold as compared with the control, respectively. The optimum concentration of VcNa application that would improve the rates of direct plant regeneration in the two tested genotypes was 1 μM. Moreover, the doubled haploid rates of regenerated plants of the three genotypes were above 60%. Five DH lines of 17AY012 with high yield were obtained. According to the measurement of the affinity index, they were prospective self-incompatible lines for hybrid breeding.