Development And Application Of SCAR Markers Linked To Clubroot Disease In Brassica Oleracea L.

Cabbage belongs to Brassica oleracea in the cruciferous plants.Cabbage is one of main vegetable crops in China,and it is cultivated throughout the country.Clubroot is a worldwide soil-borne disease,and it is infected by Plasmodiophora brassicae Woronin.Any susceptible cruciferious variety can be infected by this disease.In recent years,clubroot disease has developed rapidly,and resulted in a significant loss in the quality and yield of cabbage.Breeding disease-resistant cultivars is one of the effective ways to control clubroot disease.Resistance identification is the basis of disease-resistance breeding,but it is easily affected by the environment.It is beneficial to provide accurate and reliable genetic markers forDNA molecular markers,and is also important for the study of the genetic diversity of clubroot disease.The resistance identification of high quality varietiesby molecular marker assisted selection can speed up the process of disease resistance breeding.In recent years,RAPD marker technology is simple and convenient,however,its reaction isaffected easily by conditions,and its repeatability and stability are poor.The SCAR marker derived from RAPD marker technology has simple operation,low cost and more reproducible than other molecular markers.The development and utilization of SCAR marker improved the efficiency of marker assisted selection breeding,and facilitated the rapid analysis of a large number of samples at the same time.In this study,firstly,we studied the resistance identification system of cabbage clone using a susceptible cultivar Jingfeng No.1.Secondly,using resistant resouce“GZ87”from Syngenta company as the material,and being based on the published RAPD markers,the SCAR markers were developed.Thirdly,A three-cross population containing 110 lines was constructed from F₁ cultivar“GZ87”andinbred line“263”.Then BC₁ population was constructed by backcrossing with the inbred line“263”.BC₁combining A DNA-BSA pool was constructed by the artificial inoculation identification of BC₁ population and SCAR molecular marker-assisted selection,and association analysis was conducted.The main research results are as follows:?1?Using the susceptible cultivar Jingfeng No.1 as materials,the clubroot resistance identification system of cabbage clones was established through water culture inoculation and soil culture inoculation.The clones of the Jingfeng No.1 were obtained through tissue culture,and were transplanted to water culture and soil culture.Water culture inoculation way:At seventh days after transplanting,the artificial inoculation in nutrient solution was made with 2×10⁸ CFU/mL concentration of the resting spores in water,and irrigated the nutrient solution regularly.Soil culture inoculation way:After seedlings recovering,the clones were transplanted into the matrix made up of peat,vermiculite and perlite in the proportion of 2:1:1?volume ratio?.The nutrient pot and matrix were sterilized two times by high temperature.The concentration of the spore suspension was adjusted to 2×10⁷ CFU/mL,andthen each plant was poured into20mL spores solution by irrigating root inoculation method.One day before inoculation and one week after inoculation,the solution of tray maintained a depth of 1cm,and irrigated the nutrient solution regularly.The culturing environment of two inoculation methods was as follows:day and night temperature was 26/20?day/night?,and illumination intensity was 5000 Lx,and photoperiod was 14 h,and Hoagland solution and distilled water was watered regularly.After 6 weeks of inoculation,the root of cabbage was washed and cleaned for 1 time in sterile water,then the incidence of plants was investigated.Under the condition of water culture inoculation,clones did not have a clubroot disease phenotype in the roots of the plants,but after the inoculation of soil culture,the incidence of the plants were obvious.The inoculation method of soil culture was more suitable for the clubroot disease identification of Cabbage clones.?2?Based on two RAPD markers reported by Syngenta for clubroot disease resistance,“GZ87”was used as test material.Through PCR amplification,specific fragment recovery,cloning,and sequencing,the SCAR markers were successfully designed according to the sequencing results of sequencing fragments.SCAR markers were used to amplify PCR from 110 three-cross population genotypes,of which 58 lines could amplify specific bands.The accuracy of SCAR markers was confirmed by PCR amplification,sequencing and comparing with RAPD marker sequences using other resistant cabbage cultivars of Syngenta company.?3?SCAR markers were used to amplify PCR from 110 three-cross population genotypes,of which 58 lines could amplify specific bands.76 BC₁ lines of the BC₁population from three-cross population and recurrent parent“263”was inoculated by artificial inoculation.The DNA-BSA mixed pool was constructed by selecting 17 lines of extreme resistance and susceptibility respectively.Through re-sequencing,the high-quality and reliable SNP loci and InDel loci were 1338555 and 3381999respectively.Using ED method and SNP-index method to correlate the results respectively,the resulting intersection-associated region is C7:39590000-44050000with a distance of 4.46 Mb,a total of 737 genes.The results were correlated using the ED method and the InDel-index method.The intersections of the two regions were C7:39400000-43840000 and C7:43880000-43880000,and the distances were 4.46Mb and 0Mb,respectively,and there were 737 genes and 1 gene respectively.Depth annotations of multiple databases of candidate genes within candidate regions were performed by the BLAST software.A total of 700 genes were rapidly screened and annotated in the candidate region.Among them,there were 542 non-synonymous genes in the parents,and 233 were observed in the frameshift mutation genes.