Regulation Of Flowering Time By Targeted Mutation Of The SVP Gene With CRISPR/Cas9-mediated Gene Editing In Brassica Napus L.

Rapeseed?Brassica napus L.?is one of the essential oilseed crops.It is as important source of edible oil for humans,protein-rich feed for animals,as well as raw material for industrial use in the world.Throughout the process of growth and development in rapeseed,the flowering period is the stage crucial for productivity;thus,the regulation of flowering time is one of the key targets in rapeseed breeding.Exploration of effective approaches to the control of flowering time in rapeseed is of great interest in mordent breeding practice.The gene SVP?short vegetative phase?plays a vital role in controlling the flowering time in plants by interacting with multiple genes in the flowering regulation network and could be a potential target to manipulate in early maturity breeding.In the present study,we generated mutants that targeted the four copies of BnSVP,BnaA04g12990D,BnaA09g42480D,BnaC04g35060D and BnaC08g34920D through CRISPR/Cas9-mediated genomic editing technique.We used one binary construct of CRISPR/Cas9 with four sgRNAs,S1,S2,S3 and S4 driven by Pu3b,Pu3d,Pu6?1 and Pu6?29 promoter,respectively,to transform pure line J9707 by Agrobacterium-mediated transformation.Among the resulted 81 independent lines,67?82.71%?carried T-DNA insertions.Stable mutant lines with constant mutations in T₁ and T₂ generations were detected with 63.85%and 65.77%average transmission rate of allelic mutations,respectively.Furthermore,T-DNA free mutant lines across two successive generations were identified.To find out whether the off-target effect occurred in the present study,we examined at the putative off-target site in the B.napus.The off-targets for S1,S2,and S4 were 8,5,and 4,respectively,while no off-target for S3 was identified.It was observed that 17potential off-target sites in T₀mutated plants displayed no variations based on the high-throughput sequence of PCR products.The efficiency of mutagenesis ranged from 0%to 44.77%in the T₀ lines among S1-S4.These sgRNAs targeting the same gene showed various editing efficiencies,with14.92%at S1,44.77%at S2,0%at S3,and 10.44%at S4,indicating that for effective generation of targeted mutations in rapeseed,the appropriate selection of sgRNA is essential.The mutant lines with simultaneous mutations in four homologous copies of BnSVP showed a dramatic earlier flowering phenotype.Based on the observations on homozygous transgene-free mutant plants,the growth periods from sowing to the first flower were51.4±2.6 days in Gansu province and 71.9±2.6 days in Hubei province,much shorter than the respective wild type,86.5±2.1 days and 145.9±2.1days.Furthermore,leaf numbers in these homozygous mutant plants increased and plant height decreased significantly compared with the wild type.Our results demonstrated that the mutants have the potential to rapeseed breeding for early maturity.