Studies On The Molecular Mechanism Of Advantageous Traits Produced During Hybridization And Polyploidization Between Brassica Juncea And B.oleracea

In this study,the heterotriploid and heterohexaploid obtained from tuber mustard(Brassica juncea)and red cabbage(B.oleracea)were used as experimental materials to study the gene expression and the molecular mechanisms of advantageous traits during the process of distant hybridization and polyploidization.Through the study of gene expression patterns,it was clarified that there are additive and non-additive gene expression patterns in the process of hybridization and polyploidization,and the key non-additive genes related to the advantageous traits such as waxy layer were screened,and the non-additive up-regulated genes related to DNA methylation process were identified;By investigating the changes of DNA methylation patterns,the correlation between DNA demethylation and the generation of advantageous traits in the process of hybridization and polyploidization was preliminarily resolved,and the function of the non-additive up-regulated DNA demethylase homolog gene AtDML3 was clarified;Meanwhile,it was found that miRNAs were expressed non-additively during the process of hybridization and polyploidization,and were involved in regulating the generation of advantageous traits such as TuMV resistance,further resolving the molecular mechanism of non-additively expressed miRNAs regulating the generation of advantageous traits;New alien addition lines of tuber mustard with strong disease resistance were obtained by backcrossing heterohexaploid with tuber mustard.The results of the study will provide a theoretical basis for the generation and utilization of advantageous traits in the process of distant hybridization and polyploidization.The main results obtained are as follows:(1)Expression profiling analysis of tuber mustard(T),red cabbage(C),heterotriploid(H₀)and heterohexaploid(S₀)revealed that gene expression differences occurred during distant hybridization and polyploidization,and the differences mainly originated from the hybridization.28.6%-29%of the genes were identified non-additive expression,with non-additive repression predominating.KEGG enrichment analysis of non-additively expressed genes showed that 8 non-additive up-regulated were found to be involved in glucosinolate biosynthesis pathway,consistent with the results of increased total glucosinolates content in heterohexaploids.And 58 non-additive down-regulated genes were found to be involved in the wax synthesis pathway,and the observation of wax layer was consistent with the results of qRT-PCR.In addition,4non-additive up-regulated expressed genes were involved in DNA methylation and demethylation processes,presumably leading to altered DNA methylation levels during hybridization and polyploidization.It was shown that extensive gene expression alterations occurred during distant hybridization and polyploidization of T and C,and non-additive expression of genes improves the nutritional and sensory quality of S₀leafy vegetables by regulating glucosinolates content and waxy traits.(2)To investigate whether non-additive up-regulated expression of DNA methylation process-related genes causes changes in DNA methylation levels and patterns in hybridization and polyploidization,MSAP analysis of T,C,H₀ and S₀revealed that a decrease in DNA methylation levels occurred in distant hybridization(T:42.1%,C:55.2%;H₀:22.7%),while it was largely maintained in heterohexaploids(22.8%).The frequency of DNA methylation patterns alteration in H₀ and S₀ was 2.40%(53/2205),and 19 differentially methylated fragments(DMF)with DNA demethylation were obtained by amplification.QRT-PCR analysis showed that DMF3,DMF6,DMF9and DMF18 were demethylated and gene expression was up-regulated in H₀ and S₀,including the ubiquitin ligase gene At ATL6 and the leaf senescence-related gene At NAC042.By homology analysis,2 non-additive up-regulated expression of genes BjuA003228 and BjuB045323 involved in DNA methylation and demethylation processes were found to homological with DNA demethylase AtDML3,and functional similarity was presumed to exist.It was shown that the non-additive expression of DNA demethylases was involved in triggering the decrease of DNA methylation level in the hybrids(H₀ and S₀),and the sites in which DNA demethylated occurred might be associated with traits such as delayed senescence.(3)Functional validation of AtDML3,a homologous gene of non-additively expressed DNA demethylase,was performed.It was found that dml3 was basically consistent with the growth and developmental status of wild-type plants except for the time of leaf senescence onset.DNA methylation patterns of dml3 in leaf development and senescence stages were analyzed by using whole-genome methylation sequencing,and it was found that dml3 was hypermethylated in CG context.634 differentially methylated genes(DMGs)in CG context are regulated by DML3,of which 16.2%(108/634)are activated during leaf senescence.Differential site methylation pattern validation and expression analysis of 12 senescence-associated DMGs by BSP and qRT-PCR revealed that DNA methylation levels were negatively correlated with expression level,while the opposite was found in dml3.It was shown that the biological function of AtDML3 is to regulate leaf development by participating in the DNA demethylation of a subset of senescence-associated genes.(4)To investigate the non-additive expression pattern of miRNAs during distant hybridization and polyploidization and the regulatory mechanism of gene expression,smallRNA sequencing analysis of T,C,H₀ and S₀ found that miRNA expression changes occurred during distant hybridization and polyploidization,and the differences also mainly originated from hybridization.15%-16%of miRNAs were non-additively expressed,mainly non-additively repressed,and about 40%of miRNAs were non-additively negatively correlated with their target genes.KEGG pathway enrichment analysis of the target genes of non-additive repressed miRNA revealed that they were involved in the pathways of oleuropein lactone synthesis,starch and sucrose metabolism,and it was hypothesized that the non-additive repressed miRNAs might increase biomass by regulating the target genes.In addition,bra-miR1885b,induced by turnip mosaic virus(turnip mosaic virus,TuMV)was non-additively repressed in both H₀ and S₀,while its disease resistance target genes BjuB038637 and BjuB029447 were expressed non-additively up-regulated.Functional analysis of bra-miR1885b revealed that the susceptibility of the tuber mustard overexpression line to TuMV was increased,while its susceptibility was reduced by STTM1885b inhibition,and the heterohexaploid showed low susceptibility.It was shown that the process of distant hybridization and polyploidization regulates the production of biomass,disease resistance traits through non-additive expression of miRNAs.(5)In order to utilize the advantageous traits of the heterohexaploid,S₀ was backcrossed with tuber mustard for 3 generations to obtain 1036 tuber mustard-red cabbage alien additional lines population TC_nA₂(‘T',‘C',‘n'and‘A'represent‘tuber mustard',‘red cabbage',‘the number of chromosome'and‘addition',respectively).The chromosome composition of the TC_nA₂ generation were identified by using a single EST-SSR(expressed sequence tag-derived simple sequence repeat)marker from red cabbage to tuber mustard,and more than 50%of the plants added 1-2 chromosomes.Field observations revealed that some of the leaves of the TC_nA₂ generation population were dark green,and the plants was weakened of TuMV susceptibility.On the one hand,it can form constitutive defense by raising the waxy layer,on the other hand,adding the red cabbage genome can trigger the non-additive suppression of bra-miR1885b to regulate disease resistance genes against TuMV.It was shown that using heterohexaploids obtained from distant hybridization and polyploidization as bridge parents can obtain high-quality disease-resistant alien additional lines with red cabbage chromosomes.