Fine Mapping Of The Clubroot Resistance CRq Gene And Cloning In Chinese Cabbage

Clubroot,caused by the soil-borne protest Plasmodiophora brassicae,is considered as one of the most economically important diseases of Brassica crops worldwide.It is known as the "cancer" of cruciferous crops.The selection of disease resistant varieties is known as the most green,economical and effective method for the prevention and cure of the clubroot of Chinese cabbage.Therefore,it is of great significance for the study of the root swelling of Chinese cabbage to decipher the disease resistant genes and cultivate the disease resistant varieties.This study used the technique of microspore culture of Chinese cabbage to obtain the doubled haploid(DH)resistant line Y635-10 and susceptible line Y177-47 as parent materials.Construction of the F2 segregating population,use of BSA-Seq,RNA-Seq,gene cloning,transgenic technology,fine mapping and cloning of clubroot resistance gene CRq conducted.Moreover,we constructed the overexpression vector of CRq and the vector was transformed into Arabidopsis thaliana mediated by Agrobacterium.The main results of the paper can be summarized are follows:1.The experimental base of clubroot in Xinye County,Henan Academy of Agricultural Sciences,the resistance identification test of the constructed F2 population was carried out in spring and autumn.The results showed that the separation ratio of the F2 isolated population was about 3:1,which was in according with the Mendel's law of inheritance.It was preliminarily judged that the resistance of clubroot was controlled by single gene dominant inheritance.2.On the basis of phenotypic identification of F2 isolated population.Individuals with extreme resistance and phenotype were selected for BSA-Seq analysis.The resistance gene was initially located in the 23.80-28.10 Mb region(4.3Mb)of the A03 chromosome.After that,we designed 365 In Del markers and 298 SSR markers in the area,and further refined the candidate interval to 24.30 – 24.55 Mb(250Kb)by using QTL technology.3.Combined with the reference genome data of Chinese cabbage,the functional annotation of genes in the positioning interval was carried out.It was found that in the reference genome,there were 4 resistance genes in the region,and the 4 resistance gene sequences and the RNA-Seq De novo assembly sequence were analyzed by local Blastn.The CRq gene fragment of resistance clubroot of Chinese cabbage was found,and the total length of CRq gene c DNA was cloned by RACE technology.4.We used Gateway technology to construct the overexpression vector of CRq and the vector was transformed into Arabidopsis thaliana.The T1 generation of transgenic Arabidopsis thaliana seeds was harvested,and the resistance of transgenic Arabidopsis was identified by T2 generation.The results showed that the root of wild Arabidopsis was greatly expanded after the infection was treated with the transgenic plants,indicating that the transgenic Arabidopsis had some resistance.Therefore,it is speculated that the CRq gene plays a role in the process of clubroot disease resistance.