Induction Of Male Sterility By Targeted Mutation Of The RFL Gene With Crispr/Cas9-mediated Genome Editing In Brassica Napus L.

Brassica napus L.（canola,oilseed rape）is one of the world’s most important oilseed crops,supplying humans with oil products,nutrient feed for livestock,and natural resources for industrial applications.Due to immense population pressure,more seed production needs to be produced for human consumption due to its high quality of food products.So,for more yield,hybrid seed production is one of the standard methods in many countries.For hybrid seed production,cytoplasmic male sterility is most promising in rapeseed.Mitochondrial open reading frame（ORFs）conferring many CMS types in the Brassicaceae family has been discovered from the last four decades simultaneously their fertility restorer（Rf）genes.Most Rf genes encode PPR proteins,including mitochondrial transcription termination factor protein（m TERF）and ubiquitous super-family proteins（UBL）.Since 2011,restorers of fertility（RFL）genes have been excavated by many researchers due to their high similarity with Rf genes.The Rf gene（restorer of fertility）plays a vital role in fertility restoration in plants by interacting with genes that promote male sterility.In current study we find several male sterile lines in a single copy of the Bna A09g45590D（RFL11）gene knockout,we will focus on the RFL-CMS relationship.We generated mutants of BnaRFL11（bnarfl11）by using the CRISPR/Cas9-mediated genomic editing technique.This vector was successfully transformed in pure line Wester by Agrobacterium-mediated transformation.Among the resulted 108 independent lines,88（81.40%）carried T-DNA insertions.T₀ and T₁ generations’mutant plants’stable mutations were detected with an 81%average allelic mutation transmission rate.Furthermore,T-DNA free mutant lines across two successive generations were identified.The off-target effects analysis showed that 7 potential off-target sites displayed no off-target variations.The efficiency of designed sgRNA ranged from 0%to 65.10%among S1（64.81%）and S2（46.29）in the T₀ lines.The bnarfl11 with mutations in two target sites showed a dramatic male-sterile phenotype.The phenotypic observations of homozygous mutant plants’showed anther abortion stage similar to nap-CMS with Orf222,Orf139,Ap3,and nad5c gene upregulation.The paraffin section and q RT-PCR analysis of bnarfl11 suggest that BnaRFL11 knockout induces male sterility in the transgenic line because BnaRFL11 is present in chromosome A09 very close to the Rfn region.To functionally characterize the BnaRFL11,we overexpressed BnaRFL11 using Ca MV35S promotor（PS2300 vector）and transfer it into rapeseed through Agrobacterium-mediated transformation.Morphological and cytological analysis showed that all anthers are remarkably shortened,with minute pollen grain production having low viability.The q RT-PCR analysis of BnaRFL11-OE showed higher expression in the bud and leaf,suggesting PPR encoding genes are essential in the vegetative and reproductive growth of the plant.Our results demonstrated that the BnaRFL11 mutants have the potential to induce male sterility in rapeseed.