Creation Of Brassica Napus Germplasm With Resistance To Herbicide And Clubroot Disease

Brassica napus L.is one of the most important oil crops and the main source of domestic edible vegetable oil in China.In recent years,the frequent occurrence of grass pests and clubroot diseases have seriously affected the safe production of rapeseed in China,but there is a shortage of excellent rapeseed varieties resistant to herbicides and clubroot diseases,so it is of great significance to bred a new germplasm of B.napus rapeseed with resistant to herbicides and clubroot disease to ensure the security of edible oil supply in China.In this study,on the one hand,the method of molecular marker-assisted backcross Breeding was used to bred new B.napus germplasm that was resistant to herbicides and clubroot.On the other hand,through transgenic technology,the resistance gene of clubroot disease was transformed into herbicide resistant B.napus material to bred the transgenic germplasm of B.napus rapeseed with resistant to herbicides and clubroot disease.The main findings are as follows:1.The F₁ generation was obtained by hybridization between the hybrid clubroot resistant material T13 and homozygous sulfonylurea resistant herbicide material DS3,Spraying herbicides and inoculating Plasmodiophora brassicae during the three to five leaf stage indicates that all F₁ plants have herbicide resistance,but there is a separation of clubroot disease resistance;Using herbicide resistance markers to select,16 F₁ generation single plants with robust growth and resistance to both herbicides and clubroot were obtained,and then backcrossed with the clubroot resistant parent T13 to obtain BC₁F₁generation.2.Identification of herbicide and clubroot phenotypes in BC₁F₁seedlings,18 BC₁F₁single plants with resistance to both herbicides and clubroot were obtained.Using herbicide resistance marker-assisted selection,12 BC₁F₁ single plants with both herbicide and clubroot resistance were obtained.Continuing with SSR marker-assisted background selection,five single plants with herbicide resistance markers and genetic background recovery rates between 74.08%and 87.04%were obtained.They were backcrossed with the resistant parents T13 to obtain BC₂F₁ generation.3.Bna A02g15190D（Bna A02.MLP）,Bna A02g07380D,and Bna A02g32980D were obtained as candidate genes for resistance to clubroot disease through previous study,After inoculating P.brassicae in the resistant T13 materials for analysis,we found that the relative expression level of Bna A02.MLP gene was significantly up-regulated,and the data analysis showed that there was a significant difference in the relative expression level of Bna A02.MLP gene between resistant and sensitive clubroot disease materials under P.brassicae stress.Therefore,Bna A02.MLP gene was selected as the candidate gene for resistance to clubroot.4.Bna A02.MLP gene contains two exons,and the promoter contains low-temperature,drought,salicylic acid and abscisic acid responsive elements.It is mainly expressed in the root,similar to the infection site of clubroot.The encoded latex protein amino acid is 229,isoelectric point（p I）is 5.17 belonging to acidic protein,grand average of hydropathicity is-0.297 belonging to hydrophilic protein,Bet＿v＿1 is the main conservative structural domain.The members of the latex protein gene family in B.napus are remarkably similar in gene structure,protein structure,physical and chemical properties,etc,at the same time,the cis acting elements and tissue expression analysis indicate that this family may play a role in regulation of growth and development and response to stress in plants.Study has proved that the expression of At MLP28 in Arabidopsis thaliana,a homologous gene of Bna A02.MLP,is significantly up-regulated by P.brassicae stress.Based on the above results,Bna A02.MLP gene may have the function of resisting clubroot disease.5.The coding sequence of Bna A02.MLP gene was cloned in the clubroot disease resistant material T13,and an overexpression vector of 35S::Bna A02.MLP was constructed.Furthermore,the overexpression recombinant vector was transformed into herbicide resistant rapeseed material DS3 through Agrobacterium tumefaciens mediated hypocotyl transformation,and eight T₀ generation plants have been obtained.This study used marker-assisted selection and transgenic technology respectively to create germplasm with both herbicide resistance and clubroot disease resistance,will provide B.napus materials basis for herbicide resistance and clubroot resistance in China.