Data_Sheet_1_Map-Based Cloning and Characterization of Br-dyp1, a Gene Conferring Dark Yellow Petal Color Trait in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).xlsx

Flower color is an important trait in Brassica species. However, genes responsible for the dark yellow flower trait in Chinese cabbage have not been reported. In this study, we identified a dark-yellow-flowered Chinese cabbage line SD369. Genetic analysis indicated that the dark yellow flower trait in SD369 was controlled by a single recessive locus, Br-dyp1 (dark yellow petal color 1 in Brassica rapa). Using bulked segregant RNA sequencing and kompetitive allele-specific PCR assays, Br-dyp1 was fine-mapped to an interval of 53.6 kb on chromosome A09. Functional annotation analysis, expression analysis, and sequence variation analysis revealed that Bra037130 (BraA09.ZEP), which encodes a zeaxanthin epoxidase, was the most likely candidate gene for Br-dyp1. Carotenoid profile analysis suggested that Bra037130 (BraA09.ZEP) might participate in the epoxidation from zeaxanthin to violaxanthin. The 679 bp insertion in dark yellow petal caused premature stop codon, thus caused the loss-of-function of the enzyme zeaxanthin epoxidase (ZEP), which disturbed the carotenoid metabolism, and caused the increased accumulation of total carotenoid, and finally converted the flower color from yellow to dark yellow. Comparative transcriptome analysis also showed that the “carotenoid biosynthesis” pathway was significantly enriched, and genes involved in carotenoid degradation and abscisic acid biosynthesis and metabolism were significantly downregulated. Furthermore, we developed and validated the functional marker Br-dyp1-InDel for Br-dyp1. Overall, these results provide insight into the molecular basis of carotenoid-based flower coloration in B. rapa and reveal valuable information for marker-assisted selection breeding in Chinese cabbage.

花颜色是芸苔属（Brassica）物种的重要性状。然而，目前尚未有关于大白菜深色黄花性状调控基因的报道。本研究鉴定得到一份深色黄花大白菜种质SD369。遗传分析表明，SD369的深色黄花性状由单个隐性位点Br-dyp1（白菜型油菜深色花瓣色1，Brassica rapa）控制。通过混合分离转录组测序（bulked segregant RNA sequencing, BSR-seq）与竞争性等位基因特异性PCR（kompetitive allele-specific PCR, KASP）分析，将Br-dyp1精细定位至A09染色体上53.6 kb的区间内。功能注释分析、表达分析及序列变异分析显示，编码玉米黄质环氧酶（zeaxanthin epoxidase, ZEP）的Bra037130（BraA09.ZEP）是Br-dyp1最可能的候选基因。类胡萝卜素组分分析表明，Bra037130（BraA09.ZEP）可能参与从玉米黄质到紫黄质的环氧化反应。深色花瓣中存在的679 bp插入片段导致提前终止密码子产生，使玉米黄质环氧酶（ZEP）功能丧失，扰乱了类胡萝卜素代谢途径，导致总类胡萝卜素积累量升高，最终使花色由黄色转变为深黄色。比较转录组分析同样显示，‘类胡萝卜素生物合成’通路显著富集，参与类胡萝卜素降解及脱落酸（abscisic acid, ABA）生物合成与代谢的基因显著下调表达。此外，本研究开发并验证了针对Br-dyp1的功能性标记Br-dyp1-InDel。综上，本研究结果为解析白菜型油菜类胡萝卜素介导的花色形成分子机制提供了新见解，也为大白菜的标记辅助选择育种提供了有价值的参考信息。