Generation Of Mutants Of Multilocular Silique By CRISPR/CAS9 In Brassica Napus L.

Targeted genome editing by CRISPR/Cas9 has been used to create new germplasm in many major crops.In Brassica napus,multilocular siliques can enhance the yield by the increased silique seed number.However,few mutants of multilocular siliques are discovered in the nature by now.According to the known genes involved in the formation of multilocular siliques in Arabidopsis thaliana,we choose Bna CLV1,Bna CLV2,Bna CLV3 and Bna RPK2 as candidates for the trait of multilocular siliques to perform gene editing and construct their CRISPR/Cas9 expression cassettes.With transformation mediated by Agrobacterium,we get mutants of these candidate genes.Based on these mutants,we could identify the function of these genes.On the other hand,the created germplasm can be used for high yield breeding.The main results are as follows:1.Bioinformatics analysis shows that the gene structures of Bna CLV1?Bna CLV2?Bna CLV3 and Bna RPK2 are basically similar to homologous genes from Arabidopsis thaliana.Bna CLV1 ?Bna CLV2 ? Bna CLV3 and Bna RPK2 individually have 2,2,2 and 3 copies.Based on these information,we design 2 to 4 sg RNA for each gene and construct CRISPR/Cas9 expression cassettes.After transformation mediated by Agrobacterium to hypocotyl of Brassica napus,we get199,40,319 and 108 transgenic plants and the positive frequencies are 84.9%,92.5%,85.3%and 84.9% respectively.47,22,35,and 38 plants are found edited by the verification of non-denaturing PAGE gel with the mutation efficiency of 12.9% to 59.5%.2.After further sequencing analysis of targets of edited plants of T0 generations,we find various types of insertions and deletions in the upper stream,3-4 bp from PAM of which insertions of single base happen most frequently.These mutations are heritable.3.We observe 2.9% to10.0% edited plants turn phenotype mutants of T0 generations of Bna CLV1?Bna CLV2 and Bna CLV3,which are probably homozygous mutations.To sum up,in this study,we create various mutants by targeted editing of multilocular siliques candidate genes-Bna CLV1,Bna CLV2,Bna CLV3 and Bna RPK2.And our research provides valuable materials for further identification of gene functions and breeding of multilocular siliques in Brassica napus.