| Chinese cabbage(Brassica rapa ssp.pekinensis)belongs to Brassica plants,and which is one of the important vegetable crops in China.As the main organ for assimilation,the leafy head of Chinese cabbage is rich in nutrition and dietary fiber with highly economic value.The leafy head formation of Chinese cabbage was affected by many factors,which is a complex biological process controlled by multiple genes.Now,the knowledge about the molecular mechanism of leafy head development is still very scarce.In recent years,multiple gene mapping methods have been continuously developed and updated with the development of high-throughput sequencing technology.Bulk segregant analysis(BSA)and bulked segregant RNA-Seq(BSR-Seq)are two methods used for gene mapping based on isolated populations(such as F2 generation).Because that these two methods only need to construct one isolated population with target trait,then the genomic sequence information and specific transcriptome sequence information will be quickly obtained.It has been widely used for gene mapping of multiple species due to the advantages of simple operation,low cost and wide application range.In this study,the crosstalk analysis of the high-throughput sequencing-based BSA(BSA-Seq)and BSR-Seq was performed to locate candidate regions that associated with the leafy head formation of Chinese cabbage.Additionally,VIGS was performed to analysis the gene function of BrYUCCA5.1 and BrGH3-5.1.The main results are as following:1.Firstly,the heading type cultivar‘W30’ was crossed with non-heading type cultivar‘082’,the former was the female parent,the latter was the male parent.The F2 segregation progeny was derived from the self-pollinate of outstanding F1 individual with overlapping leaves.Then,100 F2 individual plants were selected for the construction of F2 segregation bulks,with 50 F2 individual plants showing an extremely non-heading trait and 50 F2 individual plants with leafy head.Four candidate regions responsible for leafy head formation were located by BSA-Seq analysis,which includes 40 candidate genes.Eight candidate regions responsible for leafy head formation were located by BSR-Seq analysis,which includes 607 candidate genes.The conjunctive analysis of these two methods identified that Casein kinase gene BrCKL8(Bra035974)is the common candidate gene related with leafy head formation in Chinese cabbage,and which showed highly expression levels at the middle and basal part of heading type plant leaves of F2 segregation progeny.The differentially expression genes(DEGs)between two set pairs of cDNA sequencing bulks mainly contains two different types of signaling pathway-related genes,one category was related to five hormone signaling pathways(auxin,ethylene,abscisic acid,jasmonic acid,gibberellin),the other category was composed with genes that associate with calcium signaling pathway.Moreover,a series of transcription factors(TFs)were also identified by the association analysis of BSR-Seq data.The real-time quantitative PCR was performed to analyze the expression levels of DEGs and candidate genes that associated with leafy head formation,the results were consisted with the sequencing date,which demonstrated the accuracy of BSA-Seq and BSR-Seq.This research will promote us to reveal the molecular mechanism of leafy head formation of Chinese cabbage.2.This study was mainly focused on the functional analysis of candidate gene BrYUCCA5.1 obtained by the association analysis of BSR-Seq.The real-time quantitative PCR(RT-qPCR)was performed to verify the expression levels of BrYUCCA5.1 gene in the basal,middle and apical part of mature leaves of five individual plants,which showed that the expression levels of BrYUCCA5.1 gene in heading type plant leaves were higher than non-heading type plant.Multiple sequence alignment indicated that BrYUCCA5.1 protein is closely related to Arabidopsis thaliana,Brassica oleracea,Brassica napus,Raphanus sativus with homology of 84.11%,85.75%,85.08%,85.08%.The conserved domain analysis showed that BrYUCCA5.1 protein belongs to the CzcO superfamily.Additionally,the BrYUCCA5.1 gene was silenced by VIGS system,the BrYUCCA5.1-pTY plant showed curling and shrinking leaves,and the development of abaxial side of BrYUCCA5.1-pTY leaves was promoted comparing with wild type plant.3.This study was mainly focused on the functional analysis of BrGH3-5.1 gene obtained by the differentially expression gene analysis of BSR-Seq.The RT-qPCR analysis result showed that there are significantly difference between the expression levels of BrGH3-5.1 gene at the three parts of male parent(‘W30’)leaves and female parent(‘082’)leaves.Multiple sequence alignment and phylogenetic evolution indicated that BrGH3-5.1 protein was highly homologous with other GH3-5 proteins identified in Arabidopsis,Brassica oleracea,and Brassica napus,and showed lowest homologous to OsGH3-5 protein with homology of 35.93%.Motif analysis was performed by MEME software and showed that the protein structure of BrGH3-5.1 was highly consistent with the AtGH3-5 protein.The seedling formed narrow leaves in plant inoculated with BrGH3-5.1-pTY,and the blade area became smaller compared with the control seeding inoculated with pTY-S. |
