Construction And Screening Of EMS Mutant Library In Chinese Caggage

Chinese cabbage (Brasscia campestris ssp. pekinensis L.) is an important species of Brassica vegetable crops, which belongs to Cruciferae family and has highly homologous genome with model organism Arabidopsis. A number of information about gene functions in Arabidopsis is clarified, providing a useful reference for genomics research in Chinese cabbage. With the successfully finalizing of genome sequencing and its corresponding BRAD database in Chinese cabbage, the research on functional genomics of Chinese cabbage has become more attractive. One of the most effective methods of rationally utilizing those resources and researching functional genes is to construct a saturated mutant library and to create a certain scale of TIILING population.In this study, elite Chinese cabbage inbred lines was used as the experimental materials. We researched the related aspects of constructing mutant library including selection of wild genotype, the implementation of mutagen dosage and mutagenesis method, ananlysis of phenotypic variation, and screening and evaluation of mutation frequency. TILLING and HRM technologies were combined in creating and screening the mutant library in Chinese cabbage, thus we integrated artificial mutagenesis with high efficiency, DNA mixed pool and high-throughput detection techniques in this research. The assessment of mutant library could be performed and realized in orientation at gene level and in a large scale system at high technology platform. We aimed to broaden the genetic background of Chinese cabbage, to provide basic materials for studying functional genomics and breeding new varieties in Chinese cabbage. The results are listed as follows:1) In the choice of wild type material, the self-compatibility, embryo survival rate from microspore culture, and comprehensive phenotypic traits were analyzed statistically. One optimum wild genotype inbred line A03was determined to be used for constructing mutant library, which was self compatible, easy microspore embryogenesis and good comprehensive characters.2) In the seed treatment of EMS mutagenesis, two treatment ways including EMS "soaking+interval concussion" and "soaking+continued concussion" were analyzed. The results indicated that "soaking+interval concussion" was the appropriate way because of processing repeatability and less mechanical damage. In EMS mutagen dose, treatment time and effective mutation, using M1and M2generations with different treatments, seeds germination rate, survival rate, vitality index, seedling growth, peroxidase activity such as POD, SOD and CAT enzyme, and MDA content were investigated. And the self pollination ability of M1and survival percentage of M2plants were also analyzed. The appropriate mutation scheme was determined as soaking seeds by0.4%EMS with16h after2h soaking in water. At the same time, it could effectively improve the Chinese cabbage mutation frequency by continuous seed treatments with two times of mutation of seeds.3) By the way of one time seeds treatment with EMS, two times of seeds treatment with EMS, and buds treatment with EMS combined with microspore culture, the Chinese cabbage mutant library contained4253M1families and their selfing seeds of M2was constructed. One M2population including7756individuals derived from4004M1family was constructed and will be used for screening.4) For M2individuals derived from4004M1families in the mutant library, phenotypic variations in the vegetative growth stage including seedling, rosette, heading and harvest stage, in different periods of reproductive growth stage and in multiplying seeds stage were investigated. Within6404M2, seedlings,2409mutants were detected, with variations ratio of37.61%;363mutants were detected in1677M2individuals at heading stage, with variations ratio of21.65%;121mutants were detected in544M2individuals when cutting heading, with variations ratio of22.24%;382mutants with cold tolerance were detected in6490M2individuals, with variations ratio of5.89%;230mutants with whole plant or part of plant were detected in960M2individuals at reproductive growth stage, with variations ratio of23.96%; some mutants with seeds color and shape were detected in M2seeds.5) DNAs of4224plants randomly selected from7756M2population were extracted,8times of mixed pool technology was used to construct48elementary DNA pools and6DNA mixed pool with uniform concentration. Sixteen available specific primers were obtained by designing and screening based on three genes related to auxin and heading, which were used to amplify the DNA mixed and elementary pools. Using HRM technology to screen PCR amplified products, single mutant plant DNA with different solubility curve were amplified and sequenced. Within screening mutants of AX gene, in total83point mutants were found. In general, the mutation density of the mutant library was one mutant point per41Kb averagely each plant. The population mutation density for EMS seeds mutagenesis once is1mutant per58Kb each plant, for EMS continuous mutation of two times it was40each plant, for small scale mutation population obtained by bud mutation combined with microspore culture it was39each plant.