Development Of The Germplasm With Novel Fatty Acid Profiles Using Genome Editing Techniques

Rapeseed is one of the important oil crops in China.Increase of oleic acid and decrease of linolenic acid plays an important role in the breeding of quality in rapeseed.It is the basis for quality breeding to develop high oleic and low linolenic acid germplasm.The study was aimed to precisely mutate the FAD2 and FAD3 genes by using CRISPR/Cas9 technique to obtain the new germplasm with high oleic acid and low linolenic acid Through self-bred and backcross of transgenic progeny,we separated the insertion site of T-DNA from the CRISPR editing site,and obtained the mutant plants without transgenic components.The main research results are as follows:1.After bioinformatics analysis of published FAD2 and FAD3 gene in Brassica napus,we used the online software to screen gene editing targets,and constructed a hygromycin resistance marker gene and CRISPR/Cas9 plant expression vectors which carry both FAD2 and FAD3 of Brassica napus with the specific targets.Brassica napus J9707 was used as the material for this study.After the hypocotyls segments transformated by Agrobacterium in Brassica napus,143 and 169 transgenic plants of FAD2 and FAD3 were obtained.2.All transgenic positive plants were detected by non-denaturing PAGE gel,and selected for TA cloning and sequencing.In this study,the editing efficiency of FAD2 was 16%,while the editing efficiency of FAD3 was 5%.The sequence alignment between FAD2 mutant and target gene shows that single nucleotide insertion mutation is the main type,almost above 50%.A variety of 2 to 13 bp deletion are also found.Partial FAD3 mutants are detected G-A base substitutions and 2bp AC insertion.3.Comparative analysis of the efficiency of FAD2 gene mutation at 2 target sites showed that for the first target,the mutant efficiency of Bna A.FAD2.a and Bna C.FAD2.a were 53% and 29%;although there are differences in the target sequence of the first base of Bna A.FAD2.b and Bna C.FAD2.b copy,the mutation rate was 18%,and no mutations of Bna C.FAD2.b copy were detected.The results also show that PAM sequence has a great influence on mutation efficiency.At second target,the Bna A.FAD2.a copy of the PAM sequence was-GGG,with a mutation efficiency of 71%;however,no other mutations in the PAM sequence of-NGA were detected.4.The genotype analysis of mutant inbred and hybrids lines showed that the ratio of the genotype of each offspring of heterozygous mutation was in accordance with Mendel’s genetic rule.The mutant without T-DNA was detected in T0 generation,but also observed some detected mutations of T0 generation do not exist in next generations;transgenic components can be removed through backcrossing and self-bred.5.We analyzed the mutation of FAD2 target gene,the contents of various fatty acids in the seeds of T1,T2 and T3.The oleic acid content in mutant seeds increased significantly,with the highest exceeding 80%(wild type average 66.43%),while linolenic acid content decreased significantly.The oleic acid content in the leaves of the mutant was positively correlated with the oleic acid content in the seed.The mutation of FAD3 target gene alone decreased the linolenic acid content in the seeds of T2 generation.