| Rapeseed（Brassica napus L.）is the second largest oil crop in the world.Increasing yield is one of the main objectives of its breeding.Increasing seed size（thousand seed weight（TSW））is an important way to improve yield.However,the scarcity of large seed resources and the lack of cloned genes regulating seed size seriously limit the efficiency of TSW improvement.In Arabidopsis thaliana,which is the model organism of cruciferous family,a large number of functional genes related to seed size have been identified and a relatively perfect regulatory network for seed size has been established.These studies provide a good reference for genetic improvement of seed size in rapeseed.Here,we selected a rapeseed inbred line"Xiaoyun"as the receptor and three genes（EOD1,UBP15 and UPL3）that regulate seed size in the Arabidopsis thaliana ubiquitination pathway as target genes.A series of mutants edited by CRISPR/Cas9 system at BnaEOD1,BnaUBP15 and BnaUPL3 locus were obtained,which provided new germplasm resources for genetic improvement of seed size in rapeseed.The main results are as follows:1.Sequence analysis results showed that,there are 2 homologous copies of BnaEOD1,4homologous copies of BnaUBP15,and 2 homologous copies of BnaUPL3 in Xiaoyun genome.A double target CRISPR/Cas9 vector was constructed for the conserved sites of the above target gene families.118 strains（BnaEOD1）,51 strains（BnaUBP15）and 9 strains（BnaUPL3）T0 generation positive single plants were obtained by Agrobacterium mediated transformation of rape hypocotyl.After gene editing sequencing,59 strains（BnaEOD1）,22 strains（BnaUBP15）and 2 strains（BnaUPL3）of T0generation edited plants were obtained,with editing rates of 50.00%,43.14%and22.22%,respectively.Through screening and identification of T1 generation plants,homozygous edited plants with multiple copies were obtained in all three gene families.2.Analysis of the editing of T0,T1 and T2 generation of 3 gene families showed that,The probability of editing multiple copies at the same time for the vector targeting multiple copies is much higher than the probability of editing only one copy.The editing efficiency of target 1 is lower than that of target 2.In the T0 generation of BnaEOD1,the editing rates of target 1 are 75.00%and 83.22%,and those of target 2 are 97.33%and 100.00%.Target 1 had the highest probability of base insertion and target 2 had the highest probability of base deletion.Editing events occurr at 3-4bp upstream of PAM.Mutations can be passed on stably to future generations.Hi-tom sequencing was performed at12 off-targets on 81 T2 generation edited plants,and no off-target phenomenon occurred,indicating that the probability of miss phenomenon could be ignored when the target specificity was high.3.Examining homozygous mutant plants of three gene families of T2 the seeds of related traits and agronomic traits found that,TSW of BnaEOD1 mutants increased by 14.90%-16.12%compared with wild type,and the number of seeds per silique decreased,the length per silique became shorter.TSW of BnaUBP15 mutants decreased by 10.92%-27.81%compared with wild type,and the organs became smaller,the number of lateral branches increased.TSW of BnaUPL3 mutants increased by8.66%-15.54%compared with wild type,and the length per silique became shorter,the number of seeds per silique decreased in some edited plants.Through screening and identification,3 strains（BnaEOD1）,5 strains（BnaUBP15）and 4 strains（BnaUPL3）of T-DNA-free homozygous edited single plants were obtained in T2 generation.These genetic mutant materials can be a new germplasm for improving TSW of Brassica napus L..In conclusion,this study preliminarily demonstrated that members of the BnaEOD1,BnaUPL3and BnaUBP15 in Brassica napus L.are involved in the developmental regulation of seed size.These results indicated that the BnaEOD1 and BnaUPL3 negatively regulate seed size and affect the number of silique and silique length in Brassica napus L.,while BnaUBP15 positively regulate seed size and affect organ size and the number of lateral branches in Brassica napus L..And we created a batch of new materials that could stabilize the genetic variation of seed size in Brassica napus L.by CRISPR/Cas9 system.The results laid a theoretical foundation for the systematic analysis of the regulation network of seed size in Brassica napus L.and also created new resources for yield improvement. |
