Knock-out Of TERMINAL FLOWER 1 Genes Promoted To Alter Flowering Time And Plant Architecture In Brassica Napus

TERMINAL FLOWER 1(TFL1)is a member of phosphatidylethanolamine-binding protein(PEBP)family,which plays an important role in the determination of floral meristem identity and regulates flowering time in higher plants.In Arabidopsis,tfl1 mutant exhibits a shorter vegetative phase,produces fewer leaves,reduces the number of flower buds,branches and flowers and controls the conversion of inflorescence meristem to terminal flower.In contrast,overexpression of the TFL1 gene promotes secondary inflorescence production and delayed flowering.Similarly,mutation of CENTRORADIALIS(CEN),an Antirrhinum TFL1 ortholog,results in the conversion of the normally indeterminate inflorescence to a determinate inflorescence.In rapeseed,the genetic mechanism underlying the control of flowering time has not been fully understood yet.Thus,one of the objectives of rapeseed breeding was to look for new strategies to alter the flowering behavior.Introducing determinate type of inflorescence in the crop will result in shorter flowering time with earlier and consistent maturation which will greatly facilitate harvest in short time.In this study we aimed to evaluate the role of TFL1 genes in controlling the Brassica napus flowering time.A phylogenetic tree was constructed with twelve protein sequences of TFL1 genes from B.napus,B.rapa,B.oleracea,and Arabidopsis thaliana.They were divided into three clusters based on the evolutionary relationship.Phylogenetic analysis indicated that Bna C03.TFL1 was much closer to Arabidopsis TFL1.The phylogenetic analysis of TFL1 paralogous genes indicated that all the five TFL1 genes from B.napus contain the conservative PEBP motif.The expression of the Bna TFL1 s were confined to flower buds,flowers,seeds,siliques and stem tissues and displayed distinct expression profiles.In this study,we targeted mutagenesis of TFL1 gene in rapeseed(B.napus)and analyzed the gene editing status in rapeseed cotyledon and transgenic rapeseed plants using the CRISPR/Cas9 system.A total of 101 T0 transgenic plants were obtained for sg RNA1/2(Bna A02.TFL1,Bna A10.TFL1 and Bna C02.TFL1)and 130 T0 transgenic plants for sg RNA3/4(Bna C03.TFL1 and Bna C09.TFL1)TFL1 genes through Agrobacterium-mediated transformation.Among them,40 plants and 60 plants were found as positive plants at sg RNA1/2 and sg RNA3/4 target sites,respectively.Mutations occurred in these five TFL1 genes were screened from these Cas9-positive transgenic plants by ACT-PCR(Annealing at critical temperature PCR).Six T0 plants were identified to have mutations at sg RNA1 and/or sg RNA2 target sites and 12 T0 plants were identified to have mutations at sg RNA3 and/or sg RNA4 target sites by ACT-PCR.To investigate if the knockout of Bnatfl1 genes affected the flowering time in B.napus.In the T1 generation(2018),all the mutant lines of Bna A10.tfl1(L46 and L69),Bna C09.tfl1(K85)and Bna A02.tfl1(L98)had the same day number from sowing to flowering(DTF)as that of Westar,the wild-type transgene recipient.While,K70(130?3 days)for Bna C03.tfl1(bi-allele lines)and K67(133?3 days)for Bna C03.tfl1/Bna C09.tfl1(bi-allele lines)flowered much earlier than Westar.However,K105 and K112(Bna C03.TFL1/Bna C09.TFL1)had the same normal flowering time as that of Westar.K105 and K112 were chimeric mutations with one WT allele at both Bna C03.TFL1 and Bna C09.TFL1 locus.In the T2 generation(2019),the flowering time of these mutants was similar to that in the T1 generation.Especially,two K70(103?3 days)for Bna C03.tfl1(including homozygous and bi-allele lines)and K67(113?7 days)for Bna C03.tfl1/Bna C09.tfl1(including homozygous and bi-allele lines)T2 mutant lines exhibited earlier flowering than Westar.Therefore,the results indicated that the loss-offunction mutants of Bna C03.TFL1 exhibited early flowering in B.napus,suggesting that Bna C03.TFL1 played the dominant role in determining the floral transition,and other paralogues may have obtained new functions or function redundantly in the determination of floral transition.To investigated if the Bna TFL1 gene copies also regulate plant architecture in rapeseed including plant height(PH)and branch initiation height(BIN)of these single mutants and double mutants in the T1 and T2 generations(2018 and 2019,respectively).The knockout mutants of Bna A10.TFL1,Bna C03.TFL1 and Bna C09.TFL1 were significantly shorter than WT(150.2 ± 5 cm in 2018 and 149.1 ± 12 cm in 2019).Consequently,the branch initiation height of these knockout mutants were also significantly lower than that of the wild type.Of these knockout mutants,Bna C03.tfl1(K70)displayed the largest reduction in both plant height and branch initiation height.These results suggested that these four Bna TFL1 s are involved in the determination of plant height,with Bna C03.TFL1 having the strongest and Bna A02.TFL1 having the weakest effects on determining the plant height and the position of the first branch.To investigated four yield-related traits including branch number(BN),number of siliques on the main inflorescence(NSMI),siliques length(SL),and number of seeds per silique(NSS)of these mutants in the T1 and T2 generations.We noticed that Bna A10.tfl1(L69),Bna A02.tfl1(L98),Bna C03.tfl1(K70)and Bna C03.tfl1/Bna C09.tfl1(K67)had much less branches than the wild type,while Bna C09.tfl1 had a similar number of branches of the wild type.In addition,all the knockout mutants had less siliques on the main inflorescence(NSMI),shorter siliques and less seeds per silique than the wild type.These results indicated that the yield-related traits in all the Bnatfl1 s mutants were affected at different degrees,suggesting that these Bna TFL1 gene copies play roles in the determination of plant architecture.Conclusions,our results indicated that Bna C03.TFL1 negatively regulates flowering time in B.napus and Bna C03.TFL1 together with other paralogues was essential for controlling the plant architectures.Our findings may open a new window for the future research to the understand of the molecular basis of TFL1 genes’ function in B.napus.