Location Analysis And Developing Linked Markers For Glyphosate Resistance Gene In Brassica Napus L. Using Next Generation Sequencing

There is rich in edible,oil,feed,tourism and other values in Brassica napus L.The problem of weed damage has always existed,troublling the growers in the production and cultivation of Brassica napus L.The nutrient competition with weeds lead to low production efficiency.Selective herbicides is not complete,while glyphosate and glyphosate that have good weeding effect are not selective on rape.Therefore,if glyphosate resistant genetic engineering rape hybrid line with stable,high-yield,high-quality are obtained and applied,the problem of grass damage in rape would be solved in China.In actual production practice,after spraying glyphosate herbicide on glyphosate resistant rape plants,the plants will not wither,but their flower organs are seriously harmed,resulting in difficulties in cross breeding between glyphosate resistant materials and other materials.In order to solve this problem,the transgenic rape SWUWY-35 with glyphosate resistant CP4-EPSPS gene was crossed with the pure line L804 of yellow seeded rape that is sensitive to glyphosate to obtain F₁generation.The F₁generation was self crossed to get 1:1 ratio of F₂generation population with glyphosate resistant and glyphosate sensitive plants.Two pools of glyphosate resistant and glyphosate sensitive progeny will be constructed,based on the BSA analysis method.Then,the whole genome re-sequence will be done,and the candidate interval of CP4-EPSPS gene were located on the chromosome using Mut Map+method.SSR and In Del molecular markers closely linked to CP4-EPSPS gene located the candidate interval were developed by MISA and IGV softwares.This study provides new method for the selection of glyphosate resistant plants in Brassica napus L.The main research results are as follows:5.The genetic pattern observation of glyphosate resistance traits.SWUWY-35 being a glyphosate resistant plant with CP4-EPSPS gene and L804being a pure yellow-seeded rape plant that has not CP4-EPSPS gene were planted in Southwest University trial field.Two materials were crossed to produce F₁generation,then 86 plants from F₂generation segregating population were obtained.Glyphosate resistance traits were observed and recorded at the seedling stage after spraying these plants with glyphosate herbicide.37 glyphosate resistant plants and 49 glyphosate sensitive plants were tested at the seedling stage,and the glyphosate resistance segregation ratio was consistent with the expected theoretical value of 1:1 despite environmental effects.Segregation analysis showed that the glyphosate resistance trait was in accordance with Mendel’s law of genetic pattern and belonged to the quality trait attributing to single gene.6.Chromosomal localization of glyphosate resistance gene candidate intervals.Based BSA analysis,the F₂generation segregating population was constructed by the glyphosate resistant and glyphosate sensitive parents,which were extracted leaf DNA to construct a pool of glyphosate resistant offspring and a pool of glyphosate sensitive offspring,being resequenced at a depth of 30×.Darmor-bzh being as the reference genome,the resequencing data of the glyphosate resistant parents SWUWY-35,the glyphosate sensitive parents L804,glyphosate resistant offspring pools and glyphosate sensitive offspring pools were preprocessed,and the glyphosate resistant gene was located in the 19.5-20.5 Mb of A01 chromosome using Mutm Map+method in Brassica napus L.7.SSR molecular marker.MISA software was used to identify microsatellite and complex microsatellite sequences in the 19.5-20.5 Mb of A01 chromosome.In combination with the primer design software Primer3,a total of 50 pairs of SSR primers were designed rapidly and efficiently in the candidate interval.These SSR primers were used to amplify the DNA of glyphosate resistant plants and glyphosate sensitive plants by PCR respectively.After polyacrylamide gel electrophoresis and silver staining developed,five SSR markers closely linked to glyphosate resistance traits were obtained.8.In Del molecular marker.Visualize the In Del variation in the candidate interval between the parental pools and the resistant and susceptible offspring pools by IGV software,insertion sites and deletion sites larger than 5 bases were screened.The IGV software was applied to target the start and end positions of In Del in this interval and to design In Del primers.A total of 38 pairs of In Del primers were designed.These In Del primers were used to amplify the DNA of glyphosate resistant plants and glyphosate sensitive plants by PCR respectively.After polyacrylamide gel electrophoresis and silver staining had been finished,four In Del markers closely linked to glyphosate resistant traits were obtained.