Using CRISPR/Cas9 System To Create Stable New Germplasm Of High Oleic Acid Brassica Napus L.

On the basis of low-glucosinolate and low-erucic rapeseed（Brassica napus）,to obtain stable high-oleic rapeseed is one of the current important breeding objectives.In this study,CRISPR/cas9 system was used to improve the variety of XY15,selected by Hunan Agricultural University.Bna FAD2 and Bna FAE1 are two key genes for the conversion of oleic acid to unsaturated fatty acids and ultra-long-chain fatty acids.The CRISPR/Cas9system was introduced into XY15 by using high-efficiency rapeseed transformation technology,with Bna FAD2.A05,Bna FAD2.C05,Bna FAE1.A08 and Bna FAE1.C03simultaneously edited.Through target gene cloning and sequencing,it was found that no gene editing occurred in the 7 T₀transgenic plants.However,gene editing was found and detected in T₁plants.We tested and identified their editing types,compared the editing efficiency and editing preference of two CRISPR/cas9 gene editing systems,combined with the edited plant genotypes and their corresponding part of the fatty acid content and oil content,we classified and analyzed a variety of genotypes.The total gene editing efficiency of Bna FAD2.A05,Bna FAD2.C05,Bna FAE1.A08 and Bna FAE1.C03 were 38.89%,27.78%,22.22%,and 30.56%,respectively.In the T₂generation plants,6 mutant plants with no exogenous genes and 4 target genes edited were selected,named Cas9-1-12-4,Cas9-1-12-5,Cas9-2-7-11,Cas9-5-5-3 and Cas9-6-6-1,and their relative oleic acid content was 68.69%,80.16%,75.82%,75.97%and 74.37%,respectively,which were significantly higher than that of the wild type XY15（62.04%）.The relationship between the genotypes of Bna FAD2and Bna FAE1 to the oleic acid of Brassica napus was preliminary summarized.