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Mutation of BrKAO2, encoding ent-kaurenoic acid oxidase, mediates leafy head formation by affecting GA biosynthesis in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

ABSTRACT: Leafy head is the main product of Chinese cabbage, and also is the primary character to determine its yield and quality. Cloning and characterizing key genes involved in leafy head formation is imperative for varietal improvement in Chinese cabbage. From an EMS mutagenesis population of a heading wild-type ‘FT’ of Chinese cabbage, we identified two allelic non-heading mutants, nhm3-1 and nhm3-2. Genetic analysis showed that the mutant character was controlled by a single recessive gene. MutMap and Kompetitive Allele Specific PCR genotyping results revealed that BraA05g012440.3C encoding an ent-kaurenoic acid oxidase 2 which functions in the GA biosynthetic pathway, was the causal gene for leafy-head formation, and we named it as BrKAO2. Two kinds of non-synonymous mutations in the second exon of BrKAO2 were responsible for the nhm3-1 and nhm3-2 mutant phenotype, respectively. BrKAO2 was expressed at all stages of leaf development, and there was no significant difference between the wild-type ‘FT’ and the mutants nhm3-1 and nhm3-2. The mutant phenotype was restored to the wild-type through the application of exogenous GA3. RNA-Seq was performed on the rosette leaves of wild-type ‘FT’, nhm3-1 and nhm3-1+GA3 plants, and a number of key genes involving in GA biosynthesis, signal transduction and leafy head development were identified. These findings indicated that BrKAO2 is responsible for leafy head formation of nhm3, and a new mechanism of the leafy head formation in Chinese cabbage was proposed.

ORGANISM(S): Brassica rapa

PROVIDER: GSE183651 | GEO | 2021/09/12

REPOSITORIES: GEO

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Project description:The leaf of Chinese cabbage is the major place of photosynthesis, the mutation of leaf may directly affect the rate of plant growth and development and the formation of leafy head, and ultimately influence the yield and quality of Chinese cabbage. We identified a developmentally retarded mutant (drm) exhibiting stable inheritance, which was derived from Chinese cabbage DH line ‘FT’ using a combination of isolated microspore culture and radiation treatment (60Co γ-rays). The drm exhibited slow growth and development at the seedling and heading stages, leading to the production of a tiny, leafy head, as well as chlorophyll-deficient leaves, especially in seedlings. Genetic analysis indicated that the phenotype of drm was controlled by a single recessive nuclear gene. Compared with wild-type line ‘FT’, the drm’s chlorophyll content was significantly reduced and its chloroplast structure was abnormal. Moreover, the photosynthetic efficiency and chlorophyll fluorescence parameters were significantly decreased. The changes in leaf color, combined with these altered physiological characters may influence the growth and development of plant, ultimately resulting in the developmentally retarded phenotype of drm. To further understand the molecular regulatory mechanisms of phenotypic differences between ‘FT’ and drm, comparative transcriptome analysis were performed using RNA-Seq, a total of 338 differentially expressed genes (DEGs) were detected between ‘FT’ and drm. According to GO and KEGG pathway analysis, a number of DEGs which involved in the chlorophyll degradation and photosynthesis were identified, such as chlorophyllase and ribulose-1,5-bisphosphate carboxylase/oxygenase. In addition, the expression patterns of 12 DEGs, including three chlorophyll degradation- and photosynthesis-related genes and nine randomly selected genes, were confirmed by qRT-PCR. Numerous single nucleotide polymorphisms were also identified, providing a valuable resource for research and molecular marker-assistant breeding in Chinese cabbage. These results contribute to our understanding of the molecular regulatory mechanisms underlying growth and development and lay the foundation for future genetic and functional genomics studies in Chinese cabbage.

Project description:The leaf of Chinese cabbage is the major place of photosynthesis, the mutation of leaf may directly affect the rate of plant growth and development and the formation of leafy head, and ultimately influence the yield and quality of Chinese cabbage. We identified a developmentally retarded mutant (drm) exhibiting stable inheritance, which was derived from Chinese cabbage DH line âFTâ using a combination of isolated microspore culture and radiation treatment (60Co Î³-rays). The drm exhibited slow growth and development at the seedling and heading stages, leading to the production of a tiny, leafy head, as well as chlorophyll-deficient leaves, especially in seedlings. Genetic analysis indicated that the phenotype of drm was controlled by a single recessive nuclear gene. Compared with wild-type line âFTâ, the drmâs chlorophyll content was significantly reduced and its chloroplast structure was abnormal. Moreover, the photosynthetic efficiency and chlorophyll fluorescence parameters were significantly decreased. The changes in leaf color, combined with these altered physiological characters may influence the growth and development of plant, ultimately resulting in the developmentally retarded phenotype of drm. To further understand the molecular regulatory mechanisms of phenotypic differences between âFTâ and drm, comparative transcriptome analysis were performed using RNA-Seq, a total of 338 differentially expressed genes (DEGs) were detected between âFTâ and drm. According to GO and KEGG pathway analysis, a number of DEGs which involved in the chlorophyll degradation and photosynthesis were identified, such as chlorophyllase and ribulose-1,5-bisphosphate carboxylase/oxygenase. In addition, the expression patterns of 12 DEGs, including three chlorophyll degradation- and photosynthesis-related genes and nine randomly selected genes, were confirmed by qRT-PCR. Numerous single nucleotide polymorphisms were also identified, providing a valuable resource for research and molecular marker-assistant breeding in Chinese cabbage. These results contribute to our understanding of the molecular regulatory mechanisms underlying growth and development and lay the foundation for future genetic and functional genomics studies in Chinese cabbage. The RNA from the third true leaves (day 15 to day 24 after the appearance of the third true leaves) of a developmentally retarded mutant (drm) and its wild type âFTâ in Chinese cabbage were sequenced by RNA-Seq, in triplicate.

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Mutation of BrKAO2, encoding ent-kaurenoic acid oxidase, mediates leafy head formation by affecting GA biosynthesis in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

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