Fine Mapping Of Ebm1, A Gene Referred To Early Bolting In Chinese Cabbage (Brassica Campestris L.ssp.Pekinensis)

Chinese cabbage(Brassica campestris ssp.pekinensis(Lour)Olsson)is the significant vegetable in East Asia.The genetic research of bolting will make significant sense for breeding because early bolting leads to the loss of merchandise value of Chinese cabbage.Since mutants are ideal meterials for detecting genetic mechanism,we utilized EMS mutagenesis and isolated microspore culture to obtain an early bolting mutant 1(ebm1)derived from a doubled-haploid(DH)line,'FT'.In our study,a linkage genetic map of mutant gene was constructed and a candidate gene was predicted mainly by molecular markers,gene mapping and gene cloning.The major results are as follows:1.ebm1 reveals significant differences of bolting compared with wild-type 'FT' by phenotype identification.Besides,ebm1 presents curly leaf during growth periods.It has been identified ebm1 is a diploid with normal pollen vigor by flow cytometry and TTC dyeing method.2.The mutant ebm1 and wild-type 'FT' were conducted as parents.Genetic analysis was performed in the F1,F2,and BC,generation.The results presented that the character of early bolting is controlled by a single recessive nuclear gene.3.The mutant ebm1 and a Pakchoi DH line,'13A516',were selected as parents to construct the mapping population.1502 individuals with recessive traits were derived from F2 plants for the linkage analysis between the SSR markers and ebm1.As a result,ebm1 was restricted to an interval between markers SSRh1-53 and SSRhl-61 at physical distance of approximately 73.4 kb.The target region contains 7 genes.4.Based on the clone of the 7 genes coding sequences,only gene Bra032169 showed differences between ebm1 and 'FT'.According to the gene notes,Bra032169 encodes a histoneme thyltransferases,CURLY LEAF(CLF),which can regulate the expression of genes(such as AG,FLC,AGL19,FT)related to flowering.Therefore,Bra032169 was predicted as a candidate gene.5.The clone and sequencing of Bra032169 cDNA of ebm1 performed that a 53-bp insertion appeared in the exon 12,leading to early termination of translation through analysis of amino acid sequence.It has been proven that the insertion occurred on Bra032169 of ebm1 doesn't influence its expression by analysis of expression pattern.This illustrates Bra032169 of ebm1 may produce the protein losing original founction of regulating its downstream genes.