Germplasm Innovation And Study Of Bolting Tolerance In Chinese Cabbage(Brassica Rapa)

Spring Chinese cabbage is consumed during spring and summer,which is of greatsignificance to ensure the supply of vegetables in the off-season.However,premature bolting is the main limiting factor for spring production of Chinese cabbage.It is an urgent problem to select and breed Chinese cabbage varieties with strong bolting-resistant trait.The progress of conventional breeding is slowly,molecular breeding technology is urgent needed to improve the breeding efficiency.In view of the above problems,this study carried out the breeding of new Chinese cabbage varieties with bolting-resistant trait and explored the application of multiple molecular breeding techniques including:screening and validation of the SSR markers were performed on bolting-resistant and easy-bolting Chinese cabbage varieties.Candidate regions associating with bolting trait were identified by using the BSA-seq method.Clustering and functional analysis of differentially expressed genes were conducted with transcriptome sequencing,which further investigated the transcriptional changes of genes associating with the bolting trait.The differentially expressed MADS-box gene family was identified and analyzed,which explored the molecular regulation mechanism of the bolting-resistant trait of Chinese cabbage from the level of transcription factors.The main results were shown as below:1.Germplasm innovation of bolting-resistant spring Chinese cabbage.Due to the practical problem that premature bolting has become the main factor restricting the spring production efficiency of Chinese cabbage in China,new combination‘3-5-5-3-3×3-8-1-4-5'has been preliminarily bred by means of hybrid breeding,which exhibited better comprehensive characters including bolting-resistant,heading performance and resistance than those of'jujin'.A new fast-growing seedling-edible Chinese cabbage germplasm with bolting-resistant and hairless on leaf has been preliminarily obtained.2.Study on SSR molecular markers associated with bolting trait of Chinese cabbage.Molecular marker screening was performed on segregating population constructed with Chinese cabbage bolting-resistant mutants‘012'and its wild-type easy-bolting cultivar‘006'.A dominant SSR marker BRMS-026 was obtained through screening 62 pairs of SSR primers.Then,the marker BRMS-026 was validated in the parental lines and F₂population.The results showed that the marker BRMS-026 was linked wtih bolting trait.The genetic linkage distance between the marker and the gene associated with the easy-bolting trait was 13.51 c M.3.Study on BSA-seq analysis of bolting trait.By using the BSA-seq method,four candidate regions associated with bolting trait were detected,which located on chromosomes A01 and A08,respectively.The total length of candidate genomic regions was 2.97 Mb.A total of 576 genes were annotated in the candidate regions,among which 88 nonsynonymous mutated genes between their parentswere annotated.In Del marker was developed based on BSA-seq analysis,and polymorphism analysis of these In Del markers was conducted with two parental lines and two pools.The marker A08-8 on chromosome A08 showed polymorphism.4.Differentially expressed gene analysis based on RNA-seq.Transcriptome sequencing of the flower buds of easy-bolting Chinese cabbage'006'and its bolting-resistant mutant'012'was conducted.The regulation mechanism of bolting and flowering in Chinese cabbage was analyzed from transcriptome level.The results showed that there were 5,196 genes significantly differentially expressed between two parental lines.Compared with'006',2,521 genes were significantly up-regulated and 2,675 genes were significantly down-regulated in'012'.Through GO functional analysis and KEEG biological path way senrichment analysis,it was found that the differentially expressed genes were involved in numerous biological processes and signal transduction pathways,such as starch and sucrose metabolism,secondary metabolism,plant hormone signal transduction,oxidative phosphorylation and so on.Several genes that have been reported to be associated with bolting and flowering were also significantly differentially expressed between‘006'and‘012'.For example,AP3,SEP1,AP1and so on were up-regulated in‘012'.FLC was down-regulated in‘012'.It indicated that the bolting and flowering traits of Chinese cabbage were regulated by multiple pathways.5.Identification of the MADS-box gene family in Chinese cabbage.Thirty one members of the MADS-box gene family identified by transcriptome sequencing were analyzed.The most significant up-regulated gene Br MADS24 in the mutant belong to the A(AP1/FUL/CAL)subgroup,which had an inhibitory effect on bolting.Br MADS24 and the most significant down-regulated gene Br MADS28 were all located on the A09 chromosome.Br MADS28 was located in the SVP subgroup.Two more significant down-regulated genes Br MADS22 and Br MADS2 belong to the TM3-like(SOC1)subgroup and had a promoting effect on bolting.