BrCYP71 mutation resulted in stay-green in pak choi (Brassica rapa L. ssp. chinensis).

BrCYP71 encoding multifunctional oxidase was mapped using BSA-Seq and linkage analysis, and its function in stay-green of pak choi was verified through Arabidopsis heterologous transgenic experiment. Stay-green refers to the phenomenon that plant leaves remain green during senescence and even after death, which is of great significance for improving the commerciality of leafy vegetables during storage or transportation and extending their shelf life. In this study, we identified a stay-green mutant of pak choi and named it nye2. Genetic analysis showed that the stay-green trait was controlled by a recessive nuclear gene. We obtained a 550 kb candidate region on chromosome A03 using BSA-Seq and linkage analysis. In this interval, BraA03g049920.3.5C, named BrCYP71, was identified as a candidate gene using sequence variation analysis. BrCYP71 is an ortholog of Arabidopsis AT4G13290, which encodes a multifunctional oxidase. A 4 bp insertion from T to TGATC in the first exon of BrCPY71 in the mutant led to the formation of a stop codon, TAA. Ectopic overexpression of BrCYP71 in Arabidopsis cyp71 could restored the wild-type phenotype. These results indicate that BrCYP71 contributes to the stay-green of nye2. The expression levels of chlorophyll catabolism-related genes in nye2 were significantly reduced compared to those in the wild-type, suggesting that BrCPY71 affected chlorophyll catabolism. Our achievement provides a novel genetic resource for breeding the stay-green varieties of Brassica rapa.