Study On Mutant Creation Using Microspore Culture Combined With Mutagenesis And Functional Genomics In Chinese Cabbage

Chinese cabbage (Brassica campestris ssp. pekinensis (Lour) Olsson) belongs to the A genome class of the Brassica of Cruciferae family, and is an important vegetable crop that is widely cultivated in China. Since the release of the Chinese cabbage genome sequence, and it has since guided research on functional genomics. Mutants are ideal materials for studying the functional genomics in plants. In this study, a doubled haploid (DH) line of the Chinese cabbage variety ’FT’ was employed as the experimental material. In order to quickly obtain homozygous mutants, a new method was constructed using the isolated microspore cultures combined with mutagenesis methods. And the studies on functional genomics of these mutants were carried out, mainly including molecular marker, gene mapping and transcriptome sequencing. The main research results are as follows:1. Five homozygous mutants were quickly obtained using the isolated microspore cultures combined with 60Co γ-ray mutagenesis in Chinese cabbageThe flower buds containing late uninucleate spores were irradiated with 60Co γ-rays at doses of 20,40 and 60 Gy, then the microspores from the treated flower buds were isolated and cultured. A total of 467 putative mutants were isolated from 1,483 regenerated plants. Of these, five M1 mutants were verified, and the mutant frequency was 0.34%. Comparing with the wild-type ’FT’, the mutants which exhibited stable inheriting traits, mainly including petal degeneration, developmental retardation, male sterility, chlorophyll-deficient leaf and non-heading.2. Seven homozygous mutants were quickly obtained using the isolated microspore cultures combined with EMS (ethyl methanesulfonate) mutagenesis in Chinese cabbageThe isolated microspores were treated with different concentrations of EMS solution (0.04%,0.08% and 0.12%), and mutagenic microspores were cultured. After verification in the Mo and M1 generation, a total of seven mutants were selected, which were accounted for 0.46% of the 1,304 regenerated plants. Comparing with the wild-type’FT’, the mutants exhibited stable inheriting traits, mainly including female sterility, early bolting, slow growth, chlorophyll-deficient leaf and vertical leafy head.3. Fine mapping and candidate gene prediction of the mutant gene pdm were performed using molecular markers and RNA-Seq technologyPetals of the petal degeneration mutant (pdm) were observed to be shriveled and not fully expanded. Genetic analysis indicated that the phenotype of pdm was controlled by a single recessive nuclear gene. The F2 segregation population was constructed, and a total of 1,419 recessive homozygous individuals with the pdm phenotype were investigated as the mapping population. Results showed that the mutant gene pdm was located between the two markers Indelhsn26 and SSRhsnl23 at a genetic distance of 0.04 cM and 0.04 cM, respectively, on chromosome A01. Physical distance was estimated to be approximately 285.2 kb. Comparing with the Chinese cabbage genome sequence, twenty-eight genes were predicted in the target region. Based on comparative transcriptome analyses between’FT’and pdm, Bra040093 was predicted to be the candidate gene for pdm. Bra040093 encodes a peroxisomal acyl-coenzyme A oxidase 1 (ACX1), which has a role in the biosynthesis of jasmonic acid (JA). Comparison of the gene sequences in ’FT’ and pdm revealed two single-nucleotide polymorphisms (SNPs) in pdm. qRT-PCR analysis showed that the expression level of Bra040093 was dramatically higher in’FT’than in pdm.4. The DEGs related to the phenotype of drm were analyzed using RNA-Seq technologyThe developmentally retarded mutant (drm) exhibited slow growth and development, leading to the production of a tiny, leafy head and slightly chlorophyll-deficient leaves, especially in seedlings. Genetic analysis indicated that the phenotype of drm was controlled by a single recessive nuclear gene. Compared with the wild-type’FT’, the drm’s chlorophyll content was significantly reduced and its chloroplast structure was abnormal. Moreover, its photosynthetic efficiency and chlorophyll fluorescence parameters were significantly decreased. Comparative transcriptome analyses on the leaves of’FT’and drm 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 involved in chlorophyll degradation and photosynthesis were identified, such as chlorophyllase and Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The expression patterns of chlorophyll degradation-and photosynthesis-related genes were analyzed using qRT-PCR. The presence of the mutant gene drm likely impeded chlorophyll biosynthesis and chloroplast development, which would further influenced the growth and development of plant.5. The DEGs related to the phenotype of fsm were analyzed using RNA-Seq technologyThe female-sterile mutant (fsm) exhibited pistil abortion, however, the fertility of stamens was normal. Also other floral organs exhibited abnormal morphology. Genetic analysis indicated that the phenotype of fsm was controlled by a single recessive nuclear gene. Compared with the wild-type’FT’, the fsm’s ovules were abnormal. Comparative transcriptome analyses on the flower buds of’FT’and fsm using RNA-Seq revealed a total of 1,872 DEGs between’FT’and fsm. According to GO and KEGG pathway analysis, a number of DEGs involved in ovule development were identified, such as PRETTY FEW SEEDS 2 (PFS2) and temperature-induced lipocalin (TIL). And various DEGs related to floral development and flowering were also found. The expression patterns of 18 DEGs, including two ovule development-related genes and sixteen floral development and flowering-related genes, were analyzed using qRT-PCR. The presence of the mutant gene fsm likely influenced ovule development, and thus the fertilization process can not be accomplished.