BrKAO2 mutations disrupt leafy head formation in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

The role of BrKAO2 in leafy head formation was confirmed by using two allelic Chinese cabbage mutants. Chinese cabbage yield and quality are determined by leafy head formation. Cloning and characterising the key genes regulating leafy head formation are essential for its varietal improvement. We used an EMS-mutagenised population of the heading type 'FT' Chinese cabbage line and identified two allelic non-heading mutants, i.e. nhm3-1 and nhm3-2. Genetic analysis showed that the mutant trait was controlled by a single recessive gene. MutMap and Kompetitive Allele Specific PCR genotyping revealed that BraA05g012440.3C was the candidate gene, which encodes ent-kaurenoic acid oxidase 2 in gibberellin (GA) biosynthetic pathway. It was named BrKAO2. Two non-synonymous mutations in the second BrKAO2 exon, respectively, accounted for the mutant phenotypes of nhm3-1 and nhm3-2. BrKAO2 was expressed during all leaf development stages, and there were no significant differences between the wild type and mutants in terms of BrKAO2 expression. The mutant phenotypes were restored to the wild type via exogenous GA3 application. RNA-Seq was performed on wild-type 'FT', nhm3-1, and nhm3-1 + GA3 rosette leaves, and several key genes involved in GA biosynthesis, signal transduction, and leafy head development were identified. These findings indicate that BrKAO2 is responsible for the leafy head formation in nhm3 mutants.