Functional Analysis Of The Dwarf Gene BnaC04.BIL1 And Mapping Of The Up-curling Leaf Locus BnUC1 In Brassica Napus L.

Oilseed rape（Brassica napus L.）is one of the most important oil crops worldwide,and provides high-quality vegetable oil for human diets,protein-rich feed for animals,and raw materials for industrial processes.Plant type breeding plays an important role in elevating seed yield.Optimizing plant type and high photosynthetic efficiency can be a help to increase seed yield of B.napus.Plant height is an important architecture trait which is a fundamental yield-determining trait in crops.Variety with dwarf or semi-dwarf phenotype is a major objective in the breeders because dwarfing architecture can help to increase harvest index,increase planting density,enhance lodging resistance,and thus be suitable for mechanization harvest.The leaf morphological trait is an important part of the plant type since it has direct effects on photosynthesis,respiration and transpiration.The study on plant type in rapeseed is few till now,and futher mining germplasm with practical ideal plant type,and cloning gene related to ideal plant type,will be helpful to rapeseed breeding.In this study,a pure dwarf mutant Bndwarf2 from our rapeseed germplasm bank was used to identify its dwarfing gene by map-based cloning,and characterize the gene function.In addition,a locus controlling up-curling leaf trait from B.napus（rapeseed）line NJAU5734 was genetically dissected and fine-mapped by molecular marker technique.Main results are as follows:1.Map-based cloning and functional analysis of the dwarf gene Bna C04.BIL1With dwarf mutant Bndwarf2,the dwarf gene（Bna C04.BIL1）has been found,and characterized by molecular experiments.Inheritance analysis:The F₁ plants from the cross between Bndwarf2 and ZS11,exhibited dwarf and compact phenotype.The F₂ population shown gentic segregation of dwarf vs tall plant,which is in a Mendelian model of 3:1 tested by Chi-square test.The BC₁ population fitted an expected Mendelian inheritance ratio of 1:1.These results indicated that the dwarf trait of Bndwarf2 is controlled by a pair of dominant gene.Sensitivity experiment to BR:ZS11 and Bndwarf2 were tested to determine their The sensitivity to BR.Results shown that the Bndwarf2 was BR-insensitive and the root and hypocotyl lengths of ZS11 became significantly shorter than contred,implying that the mapping experiment parent ZS11 is BR-sensitve.Gene mapping and cloing:With 74 dwarf and 20 tall plants randomly from the F₂ population were used for single nucleotide polymorphism marker（SNP）genotyping.This generated a set of linkage map containing 7,457 SNP markers,and Bn DWARF2 was preliminarily mapped within the 787.88 kb SNP marker interval（1.98 c M）on C04chromosome.To fine map the dwarf locus,318 primer pairs of simple sequence repeat（SSR）were developed,uniformly covering the mapping interval.Among these SSR,15 markers were found polymorphic.By use of these polymorphic markers,scan of the F_2:3 populations derived from the same set of mapping parents,Bn DWARF2 locus was fine-mapped onto a34.62 kb region which containing a total of 5 annotated genes that are Bna C04g41640D,Bna C04g41650D,Bna C04g41660D,Bna C04g41670D,and Bna C04g41680D.Map-based cloning:Sequences of the mapping interval for the two parents used in the mapping,were cloned.The cloning experiments and bioinformatic analysis shown that only one causual mutation in Bna C04g41660D（Bna C04.BIL1）has been found,which was two amino acid residues substitutions（Thr187Ser and Gln399His）between ZS11 and Bndwarf2.Bna C04.BIL1 contains a 1233-bp open reading frame（ORF）with 11 introns in B.napus.Bna C04.BIL1 is a homologous gene of the Arabidopsis AT2G30980（At BIL1,BIN2-LIKE1）gene,which encodes a GSK3-like protein kinase,belonging to GSK3 II subfamily.Bioinformatic analysis indicated that Bna C04.BIL1 can also interact with transcription factor Bna BZR1 to regulate plant type,functioning similarly to BIN2,and Thr187Ser mutation in the conservative domain may affect the interaction with Bna BZR1.Expression analysis:The q RT-PCR analysis showed that the Bna C04.BIL1 geneexpressed in all tissues of oilseed rape.Subcellular localization experiment showed that Bna C04.BIL1 was localized to the nucleus in tobacco leaf cells,and localization signal was also found in the cytoplasm.Genetic transformation:Overexpression of Bna C04.BIL1 in ZS11 not only reduced plant height,but also manifested as compact plant architecture.Using CRISPR/Cas9 vector to knockout Bna C04.BIL1 gene of Bndwarf2,the plant height of the transgenic lines became significantly higher than that of the Bndwarf2.These results suggested that the Bndwarf2 was a gain-of-function of gene Bna C04.BIL1 leads to the dwarfing phenotype.Protein interaction assays:The yeast two-hybrid assays（Y2H）,bimolecular fluorescence complementarity（Bi FC）,and GST pull-down showed that Bna C04.BIL1^Mut could interact with Bna BZR1,whereas the interaction signal between Bna C04.BIL1^WT and Bna BZR1 was weak.These results clearly suggested that the Thr187Ser amino acid substitution residues in the conserved region affected the interaction between Bna C04.BIL1 with Bna BZR1.It can be concluded that the newly discovered mutant gene Bna C04.BIL1 negatively regulates plant height,and leads to a compact plant type.2.Fine mapping of an up-curling leaf locus（BnUC1）In this study,by use of the double-low oilseed B.napus（rape）line NJAU5734 with up-curling leaf,the up-curling leaf trait has been analyzed for its inheritance regulation,and for its locus fine-mapping.Inheritance analysis:Compared with ZS11,the NJAU5734 had an up-curling leaf phenotype at the seedling stage and the leaf development period.The F₁ plants from the cross NJAU5734 with ZS11,exhibited the up-curling leaves.The BC₁-BC₆ populations derived from the backcross F₁ to recurrent parent ZS11 shown gentic segregation of up-curling leaf vs flat leaf plants,which is in a Mendelian model of 1:1 tested by Chi-square test.The F₂ and BC₆F₂ populations fitted an expected Mendelian inheritance ratio of 3:1.These results indicated that the up-curling leaf trait is controlled by a pair of dominant gene（BnUC1）.Gene mapping:With 22 up-curling plants randomly from the BC₆ population together with the recurrent parent ZS11 were used for Illumina Brassica 60K Bead Chip Array genotyping.The BnUC1 locus was preliminarily maped within the 2.73 Mb interval on A05chromosome.To fine map the up-curling leaf locus,201 primer pairs of SSR markers were developed,uniformly covering the mapping interval.Among these SSR,16 markers were found polymorphic.By use of these polymorphic markers,scan of the BC₆F₂ populations derived from the same set of mapping parents,BnUC1 locus was fine-mapped onto a 54.8 kb interval（0.14 c M）on A05 chromosome.Candidate gene analysis:The mapping interval contains six annotated genes according to the B.napus reference genome,among which Bna A05g18250D and Bna A05g18290D may be regarded as candidate genes responsible for the up-curling leaf trait by gene cloning and gene expression analysis.Analysis of agronomic traits and photosynthetic efficiency:The effect of up-curling leaf locus on the agronomic traits was evaluated by investigating the agronomic traits of 30randomly individuals from the BC₆ population.The near-isogenic line of the up-curling leaf（ZS11-UC1）and ZS11 were constructed to evaluate the effect of BnUC1 on photosynthetic efficiency.The results indicated that the up-curling leaf trait locus was beneficial to improve the photosynthetic efficiency.This study provides the foundamental research for further elucidating the molecular mechanism of up-curling leaf trait,and provides new germplasm for breeding rapeseed seedling resistance to planting varieties of B.napus.