Cloning And Identification Of Early Bolting Mutant Genes In Chinese Cabbage(Brassica Rapa Ssp.pekinensis)

Chinese cabbage?Brassica rapa ssp.pekinensis?is an important vegetable crop originated in China,and it is various and nutritious,and is an important vegetable crop in China and East Asia.Flowering affects the formation of leafy-head in Chinese cabbage,which is an important agronomic trait of Chinese cabbage vegetables.In the production of Chinese cabbage,the yield and quality of leafy-head are seriously affected by premature bolting,and the breeding of resistant bolting varieties is an important breeding goal of Chinese cabbage.For the Chinese cabbage with the bolt as the product organ,it is also the key to the yield formation after the seedling stage.And it is very important to clone the key genes that affect bolting and flowering in Chinese cabbage.In this study,seven early boltin mutants were obtained by EMS treating DH line‘FT'seeds,which were stably inherited.We used three of these mutants as research materials,mutant ebm6,ebm5-1 and ebm5-2.Respectively,F₂isolated populations were constructed and candidate genes were predicted by the next generation sequencing technology.The early bolting mutant genes were fine mapped and identified.The main results are summarized as follows.1.The creation of early bolting mutants in Chinese cabbageSeven early bolting mutants were obtained by 0.8%EMS treating DH line‘FT'seeds,which were early bolting and stably inherited.For genetic analysis,seven mutants were all controlled by the single recessive nuclear gene,respectively.The bolting characteristics,DE5?DE10 and flowering time FT,were significantly earlier than those of wild type‘FT'.For the half wheel matching,the results showed that the hybrid progeny of two mutants displayed the same bolting phenotype as the parents,indicating that its mutant gene was allele.The above seven mutants were named ebm5-1?ebm5-2?ebm6?ebm7?ebm8?ebm9 and ebm10,respectively.Mutant ebm5-1 and ebm5-2 were allelic mutations.2.The identification of candidate gene Brebm6 using Mut Map techniqueThe F₂ mapping population was constructed by hybridizing the mutant ebm6 and wild type‘FT',and the candidate gene was identified by using Mut Map techniqu and KASP methods.The results showed that the candidate gene is Bra A02g003340.3C,which identified as the Arabidopsis homologous gene At FLC2,then named Br FLC2.Compared with wild type‘FT',there is a single base substitution C to T at 52bp of the first exon in mutant ebm6,which makes the codon CAA in the wild-type‘FT'mutate to stopgain codon TAA,leading to premature termination of translation.Br FLC2 is an inhibitor of flowering conversion,which encodes a MADS-box protein,we predict Br FLC2 as a candidate for mutant ebm63.Mapping of mutant ebm5-1 and ebm5-2 based on BSR-seqTo identify the bolting gene,we crossed ebm5-1 and ebm5-2?early bolting?with pakchoi K20?relatively late bolting?to create two F₂ mapping populations.According to BSR-seq,the allele mutant gene Brebm5-1 and Brebm5-2 were located in the overlapping regions of A07chromosomes.Then we use to map Brebm5-1 gene using its 1,741 F₂ mapping population with early bolting traits to shorten the mapping interval.The mutant gene Brebm5-1 was finally located between the tightly linked molecular marker Indel18 and Indel45 on the A07chromosome.The physical distance was 56.24 kb,a total of 10 candidate genes in this interval.4.The prediction of candidate gene Brebm5-1 by whole genome resequencingThe whole genome of mutant ebm5-1 and wild-type‘FT'were sequenced by the Next generation sequencing technique.The SNP between ebm5-1 and‘FT'were screened in the candidate interval,there is only one non-synonymous mutation occurred on the exon of Bra A07g040740.3C in the ebm5-1.According to sequence analysis,the gene Bra A07g040740.3C?Br SDG8?encoding histone methyltransferase was predicted to be a candidate gene for mutant ebm5-1.5.Validation of candidate gene Br SDG8 gene function using allele mutant ebm5-2Using the allele mutant ebm5-2 to clone Br SDG8 full length,the results showed that there was a SNP changed?base G deletion?on 12th exon in the allele mutant ebm5-2,which led to premature termination of translation.The reaults suggest that histone methyltransferase gene Br SDG8 regulates early bolting phenotype of allele mutant ebm5-1 and ebm5-2.In this study,steven new early bolting mutants of Chinese cabbage were creation,and the early bolting phenotypes were obvious,which has important practical and theoretical significance.This experiment identified and revealed two important genes regulating flowering time.One gene is the flowering conversion inhibitor Br FLC2,the other is Br SDG8,encoding histone methyltransferases.These two results will play an important role in the study of flowering regulation mechanism of Chinese cabbage.