Cloning Of A Female-sterile Mutant Gene Brfsm In Chinese Cabbage(Brassica Campestris Ssp. Pekinensis)

The genome sequencing results of the Chinese cabbage(Brassica campestris ssp.pekinensis)were released in 2011 and gradually improved through subsequent research.Revealing the function of each gene in the Chinese cabbage genome is an important task in the research of Chinese cabbage genome for a period of time to date.Chinese cabbage is a bisexual flower plant,female sterile mutant,which is an ideal material for screening and cloning genes related to pistil development.In this study,the Chinese cabbage double haploid pure line(DH line)'FT' was used as plant material,and a female sterile mutant fsm1(female-sterile mutant1)was created by combining microspore culture with EMS mutagenesis,its pollen vitality was normal.Genetic analysis showed that the mutant traits were recessively inherited by mononuclear genes.The female sterile mutant gene Brfsm of mutant fsm1 was cloned by the map cloning strategy.The molecular mechanism of the abortion of the pistil caused by the mutant gene Brfsm was discussed.The main results are as follows:1.Phenotypic and genetic traits of female sterile mutant fsm1The purified microspores isolated from Chinese cabbage were mutagenized by EMS aqueous solution with a concentration of 0.08%(v/v),and a female sterile mutant fsm1 was identified in the M1 population of the regenerated plants by conventional microspore culture.Its corolla is small,but normal pollen is released after the flowers are opened.Scanning electron microscopy showed that the pollen on the stigma of the mutant pistil can germinate after self-pollination,and the pollen tube can grow into the ovary to reach the ovule,indicating that the male gamete development is normal.Paraffin section observation showed that the intermediate membrane in the ovary of the mutant fsm1 was abnormally fused,the number of embryonic frame was reduced,and the structure of the embryo sac was incomplete.Genetic analysis showed that the pistil abortive trait of the mutant fsm1 was controlled by a single recessive nuclear gene,which was named Brfsm.2.Fine mapping of the female sterile mutant BrfsmThe F2 large-scale gene mapping population was constructed by crossing the Cabbage Sprouts DH line '701' with the AA genome of Chinese cabbage as the female parent and hybridizing with the female sterile mutant fsm1.3,108 invisible homozygous individuals with mutant phenotype were selected from the population.By screening SSR linkage markers,the mutation site was located on the A04 chromosome,near the cnu-m439 a marker.The SSR and In Del markers were further designed,and the mutant gene Brfsm was finally mapped between the two linked markers Indel-I2 and Indel-I7 with genetic distances of 0.05 c M and 0.06 c M,respectively.When mapping to the reference genome,the physical distance of this interval is 1376.69 kb,including 107 genes.After checking the information of the Chinese cabbage genome database(BRAD),it was found that the localization interval was just near the centromere of the A04 chromosome.Due to the complex domain and heterochromatin structure near the centromere of the chromosome,it is difficult to further narrow the localization interval by using the linkage marker analysis method.3.Whole-genome re-sequencing of mutant fsm1 to predicted candidate geneBy re-sequencing the wild-type 'FT' and the mutant fsm1,a total of 933,820 SNPs were detected in the female sterile mutant fsm,of which 12 SNPs were located in the target region and distributed over 6 genes.After PCR amplification and Sanger sequencing,it was found that only 1 of the above 12 SNPs were verified,which occurred on the gene Bra A04003010.In the wild type 'FT',the base was guanine(G),and in fsm1 it was cytosine(C),and the genetic code is mutated from TCG(tryptophan)to TAG(stop codon),and the CDS sequence of the amino acid encoding Bra A04003010 is terminated prematurely.It is speculated that Bra A04001030 is a candidate gene for female sterile mutant.4.Functional verification of the female sterile mutant gene BrfsmIn the laboratory,we also used the 'FT' as the mutagen material to construct another mutant library(containing more than 1,000 mutants)by EMS mutagenesis the germination seeds.Another female sterile mutant fsm2 was obtained and its phenotype was very similar to fsm1.Genetic analysis indicated that the female sterility trait of the mutant fsm2 was also regulated by a single recessive nuclear gene.In order to verify whether the two female sterile mutant fsm1 and fsm2 were caused by the same gene mutation,the F1 hybridization method was used for the allelic detection,and the progeny and the female sterile plants were found to have a 3:1 separation(137:32 and 147:45,respectively),indicating that the female sterility phenotype of fsm1 and fsm2 were controlled by the same gene.Using the Bra A04001030 gene cloning primer of the mutant fsm1,the corresponding gene of the mutant fsm2 was cloned,and it was found that one SNP on the Bra A04001030 gene of fsm2 changed from G to A,and the amino acid encoded by the CDS sequence was missensely mutated by GAA(Glutamate)becomes GGA(glycine),causing a change in the amino acid sequence of the protein.The Arabidopsis homologous gene AT5G35770 of the Chinese cabbage Bra A04001030 gene encodes the STERILE APETALA(SAP)transcription factor,which regulates the development of flower organs.5.Transcriptome analysis of wild type 'FT' and mutant fsm1The pistil of wild type 'FT' and the female sterile mutant fsm1 were selected for comparative transcriptome analysis.A total of 1,080 differentially expressed genes were obtained,and several genes related to floral organ development and ovule formation were identified,including ARF3/ETT,FIL,STM,ANT and KAN,etc.,significantly enriched GO terms are mainly related to the metabolic process of the organism.In KEGG metabolic pathway enrichment analysis,709 differentially expressed genes were aligned to 95 metabolic pathways,and significantly enriched metabolic pathways including important pathways such as metabolism and secondary metabolite synthesis.