Fine Mapping And Identification Of Genes Controlling Branching In Flowering Chinese Cabbage (Brassica Campestris L. Ssp. Chinensis Var. Utilis Tsen Et Lee)

Flowering Chinese cabbage is a kind of cabbage subspecies of Brassica genus of the Brassicaceae family,whose edible organs is flower stalk.It is a characteristic vegetable of the Guangdong region of China and is widely cultivated.The main inflorescences of common flowering Chinese cabbage has significant growth advantages,and each plant usually only harvested single flowering stalk;the Zengcheng variety has more axillary buds produced on rosette stem,which can further form multiple branches and be harvested many times in one planting.Breeding varieties with multiple branches can increase the yield of flowering Chinese cabbage,save seed and planting labor,and have broad prospects for industrial development.In this study,the F₂population was constructed using the less branching common flowering Chinese cabbage DH line‘CX010’and the multiple branching Zengcheng flowering Chinese cabbage DH line‘CX020’as parents,the gene regulating the branching was located by Graded Pool-seq technology and KASP genotyping technology,and the candidate genes were cloned and initially identified.The main results of this study are as follows:（1）Genetic characteristics of branches in flowering Chinese cabbage.The number of branching of 1050 F₂plants was counted when the branching phenotype was significant.The results showed that the branching number of F₂generation plants was obviously separated and showed a normal distribution trend.This suggests that the branching of flowering Chinese cabbage were a quantitative trait.（2）Preliminary mapping of branching genes in flowering Chinese cabbage.A total of 50 F₂plants with multiple branching,less branching and middle branching phenotypes were selected to construct 3 DNA mixing pools.The flowering Chinese cabbage branching gene was localised by the Graded Pool-Seq localisation method to 28.0～28.9 Mb on chromosome A07,an interval of 900 kb in length,which contains a total of 178 genes.（3）Fine mapping of branching genes in flowering Chinese cabbage.Used parent plants and 150 randomly selected plants from the F₂populations as material,plant genotypes were determined using the KASP genotyping technique.Used QTL Ici Mapping software and combined genotype and phenotype data,the candidate interval for the branching gene was finally narrowed down to between two markers A0716 and A0717,with a interval of approximately 24.6 kb,which included five candidate genes.（4）Cloning and identification of candidate genesThe candidate genes were annotated by reference to Chinese cabbage database and Arabidopsis database.The tandem repeats genes Bra A07g041560.3C and Bra A07g041570.3C correspond to the same Arabidopsis gene AT1G78440,encoding GA2ox1,which regulates rice tiller.Therefore,Bra A07g041560.3C and Bra A07g041570.3C were predicted to be candidate genes for branching of flowering Chinese cabbage.Full-length gene sequence cloning results show that there was no difference between Bra A07g041560.3C in parents,while there was a mutation in the first exon of Bra A07g041570.3C,but it was synonymous mutation.Promoter sequence cloning and element prediction results are displayed,besides the31 bp deletion of Bra A07g041560.3C in the promoter of‘CX020’,there were 16 SNPs difference between‘CX020’and‘CX010’,the promoter element in‘CX020’lacked an ERE cis-regulatory element compared to‘CX010’.Also,the position of the TATA-boxes were different;The promoter sequence of the Bra A07g041570.3C gene of‘CX020’has a SNP（G→C）at a position 103 bp upstream of the transcription start site compared to‘CX010’,an MYB cis-regulatory element was absent in‘CX020’.This element has been shown to play a role in the development of axillary meristematic tissues and branching in Arabidopsis.q RT-PCR results showed that the relative expression level of Bra A07g041560.3C and Bra A07g041570.3C were the significantly expressed between the two parents in rosette stem.After GA3 treatment of‘CX020’.