A Gypsy LTR retrotransposon insertion in BrNYC1-A04 causes the stay-green phenotype by inhibiting chlorophyll b degradation in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

A Gypsy LTR retrotransposon insertion in BrNYC1-A04 causes the stay-green phenotype of Chinese cabbage via bulked segregant analysis sequencing, fine-mapping, and gene silencing analysis. The stay-green trait positively influences the postharvest quality of Chinese cabbage (Brassica rapa L. ssp. pekinensis), a leafy vegetable crop in the Brassicaceae family. This trait is typically caused by defects in chlorophyll degradation. In this study, we identified BraA04g008820.3C as a candidate gene associated with the stay-green phenotype in Chinese cabbage through BSA-seq and fine-mapping analyses. The gene was subsequently named BrNYC1-A04 due to its high homology to Arabidopsis thaliana AtNYC1 (At4G13250). Sequence analysis revealed that a Gypsy long terminal repeat (LTR) retrotransposon (RT) insertion in the first intron of BrNYC1-A04 disrupts normal splicing. This produces a premature termination codon in the mRNA of BrNYC1-A04 in the 5BTL (stay-green) line, resulting in a loss-of-function mutation. The BrNYC1-A04 mutation affects both chlorophyll b degradation and chloroplast structure by modulating chlorophyll b reductase activity. Under dark-induced senescence, the transcriptional expression of BrNYC1-A04 is regulated by BrORE1. Virus-induced gene silencing (VIGS) of BrNYC1-A04 in the wild-type line HN19-G significantly delayed chlorophyll b degradation. Based on the identification and functional verification of the Gypsy LTR-RT insertion in BrNYC1-A04, we propose a model for how the BrNYC1-A04 mutation inhibits chlorophyll b degradation and confers the stay-green trait in Chinese cabbage.