Identification Of Multilocular Genes And Analysis Of Molecular Regulation Mechanism Of Multilocular Character Formation In Rapeseed

The multilocular silique is one of the most important agronomic traits in rapeseed high-yield breeding.The research on the molecular mechanism of multilocular silique development and establishment of multilocular germplasm resources are the basis of genetic breeding and improvement.Previously,the multilocular gene of mutant ml4 in Brassica rapa had been studied by map-based cloning and functional identification.It was proved that a single nucleotide mutation of the Br CLV3gene resulted in a corresponding increase in shoot apical meristem(SAM)and carpel number respectively.In the present study,the cloning and sequencing of multilocular homologous genes in B.rapa were continued.The genetic and molecular mechanisms of multiloculus traits were determined by genetic analysis and functional identification of the multilocular candidate genes.In addition,Bn CLV1/2/3 multilocular mutants with stable mutation inheritance were successfully accomplished in B.napus through the use of transgenic technology and CRISPR/Cas9 gene editing technology.The results of this study will provide a theoretical basis for the application of multilocular traits in B.napus and also provide new genetic resources for high yield breeding.The results were described as follows:1.A novel natural single-nucleotide variation in Br CLV3 plays an important role in regulating multilocular silique in B.rapaPhenotypic analysis of multilocular mutant srb showed that the number of stamen and carpel increased compared to those in wild type with biloculus.The siliques of srb mutant which had 2-4 locules were significantly wider and shorter than wild type.We analyzed the genetic control of multilocular trait using reciprocal crosses between mutant srb and wild-type.The F1 siliques had two carpels like wild type,and a separation of accord at an approximated rate of 3:1(?2=0.27,p>0.05),which shows clearly that multilocular trait of mutant srb was controlled by a recessive nuclear gene.The allelism test between srb and mutant ml4 revealed that all F1 and F2 progeny display multilocular trait.All the results showed that multilocular mutant srb was most likely controlled by a recessive gene Br CLV3 which is allelic to ml4 locus in B.rapa var.srb.we carried out a comparative sequencing of the gene encoding region,2.3kb promoter region and 3'end of 2.5kb flanking sequence between mutant srb and wild type,it was observed that there were three variations between them.The C-to-G nucleotide substitution was the only mutation that led to the twelfth histidine in CLE motif substituting for aspartic acid.The mutation site was a novel natural single-nucleotide variation which differs from the mutation of Br CLV3 gene in ml4.The His-to-Asp substitution might affect the function of Br CLV3 protein,resulting in a lead to the multilocular phenotype.The heterotopic expression of Brclv3_Asp12 can partly restore the mutant phenotype of clv3-2 in A.thaliana.In vitro root inhibitory assays of synthetic dodecapeptides Br CLV3_ASP12 and Br CLV3 in A.thaliana,we found that Br CLV3_ASP12Peptides inhibited root growth and SAM enlargement,but the inhibitory effect of Br CLV3ASP12 is weaker than polypeptide Br CLV3,which further indicated that the histidine/aspartic acid mutation in Brclv3_Asp12 could cause partial loss of CLV3 polypeptide activity,making it unable to regulate the growth of SAM normally,leading to the production of multilocular trait2.Establishment of multiloculus resources with homologous co-suppressionTransgenic technology was performed to transfer multilocular gene Br CLV3_Leu9 to B.napus.in other to obtain multiloculus resources.A multilocular line 35S::Br CLV3-19(OE19)was discovered in the T₀ generation.After two generations of phenotypic screening,multilocular lines with 99%to 100%multilocular siliques per plant were obtained.The carpel increased to 4 locules,and the number of seeds per siliques also increased.The number of seeds per siliques increased with a simultaneous increase in the number of carpels.We detected the expression of endogenous and exogenous CLV3 genes and the genes related to CLV3-WUS regulation pathway in inflorescence meristem.The expression level of Br CLV3 was very low,and the expression of two copies of Bn CLV3gene was also down-regulated.The total expression was significantly lower than that of the wild type.It indicated that exogenous genes co-inhibited the expression of endogenous genes,which resulted in the weakening of the inhibition of Bn CLV3 on WUS gene,resulting in multi-compartment traits.3.Establishment of multiloculus resourcesWe cloned Bn CLV1/2/3 gene in B.napus L.and analyzed its tissue expression specificity.Southern hybridization showed that there were two copies of each CLV gene in B.napus.Several sg RNA targets were designed for Bn CLV1/2/3 gene editing,and CRISPR/Cas9 gene editing vector was constructed to transform B.napus.Each gene of targeted Bn CLV1/2/3 was successfully edited.The mutagenesis efficiency was variable in a wide range from 0%to 48.65%in T₀,indicating that the appropriate selection of sg RNA is important to effectively generate indels in rapeseed.The multilocular phenotype is recovered in the T₀ generation,shown stable transformation and stable transmission of the mutations across generations.A large number of single and double mutants with different genotypes were screened out from T1&T2 progenies.We found that all multilocular plants were homozygous double mutants with knock-out in any of the CLV homologues,suggesting that there was functional redundancy between Bn CLV1/2/3 gene copies.All single and double homozygous mutant in T2 lines of Bn CLV3 were further examined for phenotypic characterization.The results revealed that CRISPR/Cas9-induced double mutants of Bn CLV3 produces more leaves and multilocular siliques with significantly increased seed number per silique and seed weight compared with wild-type and single mutant plants,which could result to overall increase in seed production.Evaluation using high-throughput sequencing of PCR products of 57potential off-target loci detected no off-target mutations.We also assessed the efficiency to horizon transfer the Cas9/g RNA cassettes by pollination.Our notable findings typically provide the productive potential for plant breeding strategies to improve yield traits in the rapeseed varieties that are currently cultivated.