Genetic Mapping And Candidate Gene Identification Of The Feathered-leaved Trait In Ornamental Kale(Brassica Oleracea Var.acephala)

The ornamental kale?Brassica oleracea var.acephala?is a kind of biennial foliage plant in Brassica.Because of its wonderful leaf shape and colour,it became the important ornamental flower in fall,winter and early spring.Leaf shape was the major trait,especially the feathered-leaved trait is unique,however,the genetic inheritance and the key gene corresponding to the trait is unclear.In this study,the feathered-leaved inbred line F0819 and the smooth-leaved inbred line S0835 were used to construct genetic segregant population to analyze the inheritance of the feathered-leaved trait in detail.According to the whole genome resequencing BSA-seq and fine mapping to anchor the key gene in chromosome.Combining with functional annotation,expression and clone results to predict the key gene.The overexpression vector was constructed and transformed to ornamental kale in order to verify the gene function.The major results are as follow:1.Genetic analysis of the feathered-leaved trait in ornamental kaleThe phenotype F₁,RF₁,F₂,BC₁P₁,BC₁P₂and F_2:3population was observed by visual examination at the rosette stage.The trait of F₁and RF₁was both the semi-feathered leaf trait between the two parents.The segregant ratio of F₂population conformed to 1:2:1.The segregant ratio of BC₁P₁and BC₁P₂both conform to 1:1.The segregant ratio of F_2:3population which selfing from the semi-feathered-leaved F₂individual also conformed to 1:2:1.These results confrmed that the feathered-leaved trait is controlled by a single nuclear semidominant gene.2.Fine mapping of the feathered-leaved trait geneThe feathered-leaved trait gene was mapped between 50.5Mb and 54.67Mb by the whole genome resequencing BSA-seq.The InDel markers were designed from the initial region to make linkage analysis with the 3,651 F₂population.The Bo FL gene was mapped between marker INDEL940 and INDEL5443 with genetic distances of 0.370 cM and 0.411 cM.The gene was fine mapped to 54,059,854 bp and 54,434,386 bp by aligning the physical location of the flank markers to kale reference genome.3.Identifcation of the candidate geneThere were 38 genes in the candidate interval,the genes Bo9g183220 and Bo9184610were related to leaf growth according to the functional annotation.The gene Bo9184610 was the candidate one by expression analysis and clone.There were 2097bp and 2075bp in F0819 and S0835,respectively,containing 14 SNP and one InDel.There were 7 exons and 6 introns,and the coding sequence were both 1530bp.Among these variants,there were 3 SNP in the second exon containing two nonsynonymous SNPs and a sysnonymous one.Bo9g184610 gene encoded 509aa which belong to ALG10 superfamily,and was conserved among the 22 homologous proteins.Bo9g184610 was a hydrophobin protein which had 10 transmembrane domain.The promoter sequences in F0819 and S0835 were 2,197 bp and 2,196 bp,respectively,containing 26 SNPs and 7 InDels.There were 30 and 31 cis-acting regulatory elements in F0819 and S0835,respectively,and only 3 elements were different between the two promoters.4.Functional identification of the feathered-leaved leaf gene in ornamental kaleA cleaved amplified polymorphic sequence marker was developed according to the first nonsynonymous SNP which was co-segregate with the feathered leaf trait after screening in F₂ population.The recombined vector p CAMBIA3301-Bo9g184610 which containing the coding sequence of Bo9g184610 gene in S0835 was constructed to transform the gene to the feathered-leaved ornamental kale under Agrobacterium tumefaciens.The three transgenic strains were positive by phenotype observation.The two strains of them which grow well were confirm to be the positive transgenic one by analysis level of gene integration and expression.In this study,the inheritance of the feathered-leaved trait was illuminated by genetic analysis in detail.The key gene controlling the feathered-leaved leaf trait was discovered by the forward genetic mapping and prediction.A co-segregant CAPS marker with the feathered-leaved trait was construct by sequence difference between the two parents after the candidate gene clone.It will give a benefit to accelerate the leaf shape breeding.Identification of the gene function by overexpressing the candidate gene,and it will be the foundation for elucidating the molecular mechanism of regulating leaf shape changes of Bo9g184610.