Map-based Cloning Of The Cleistogamy Locus BnC03CL And Fine Mapping Of The Up-curling Leaf Locus BnUC3 In Brassica Napus L.

Oilseed rape is one of the most important oil crop in the world as souces of vegetable oil,biodiesels and industrial compounds.Flower as a plant reproductive organ is associated with seed yield formation.Leaves are the primary photosynthetic organs in plants.Leaf size,shape and structure can affect plant dry-matter production.Plant scientists and breeders have focused much attention on researches of inheritance and molecular basis of flower and leaves.The present studies have investigated the cleistogamy trait by morphorlogical,genetic and molecular biological approaches to uncover mechanisms underlying the cleistogamy trait in Brassica napus.Also,present study has investigated the inheritance of an up-curling trait and fine-mapped the up-curling-leaf locus.The results have laid a foundation for uncovering cleistogamy and leaf shaping mechanism in B.napus.1.Map-based cloning of the cleistogamous locus BnC03CLRapeseed is an often cross-pollinated plant with chasmogamy.The clesitogamous rapeseed used in present research,unlikes the usual oilseed rape and has closed petals which maintain the petal-closing state untill petal dropping-off.Clesitogamous trait may be used to reduce breeding workload and maintain variety purity.Present study investigated a novel dominant cleistogamy germplasms by morphological,inheritance and molecular appraoch in B.napus.The main results are as follows:（1）Morphological and cytological observation The near-isogenic line with the cleistogamy trait（NIL-CL）and chasmogamous（NIL-CH）trait were constructed.NIL-CH had open flowers after stage 13,which is consistent with the developmental criteria of A.thaliana flowers,while NIL-CL maintained the petal-closed state till petal abscission from plants.Other flower organs grew normally,and the pollen viability was similar to the NIL-CH.Microscopic observation indicated that the adaxial epidermal cells of NIL-CH petals were expanded and enlarged at 14^th anthesis while the adaxial epidermal cells of NIL-CL petals did not change,resulting in closing petals.The cytological observation indicates the petal closure is due to lack of expansion of adaxial epidermal cells.（2）Inheritance of the cleistogamous trait The cleistogamous CP7130 was crossed with the chasmogamous FA135 to generate F₁.Then,the F₁hybrid plants were self-pollinated to produce F₂ and F_2:3 populations to investigate cleistogamous behavior inheritance.The chi-squared test for segregation ratio of the populations agreed with the theoretical ratio of one Mendelian factor,suggesting that the cleistogamous behavior is controlled by one dominant Mendel factor.（3）Mapping of the cleistogamy trait locus Seventy-one F₂ individuals derived from the CP7130×FA135 cross were genotyped using a Brassica napus 60K Illumina SNP array.Trait–SNP locus association analysis suggested that the locus for the cleistogamous behavior was located in the 465.3-kb interval between SNP markers M40525 and M40449.This cleistogamous locus was named BnC03CL.To further map the locus,simple sequence repeat（SSR）markers were developed using ZS11 genome sequence as reference.With 10polymorphic SSR markers,scan of the segregating populations composing of 2608individuals lead to a narrowed mapping interval between SSR marker Bna C03V2042 and Bna C03V2394.To narrow the mapping interval,the two NILs were re-sequenced to develop markers for fine-mapping BnC03CL.And these works uncovered 47 SNPs and 25 In Dels（Insertion/Deletion）in the mapping interval.Then,two polymorphic In Del markers,Bna C03In Del1 and Bna C03In Del2 developed help to fine-map the cleistigoumous locus to an interval of 85.4 kb in length.（4）Cloning of cleistogamy trait locus The genomic sequence of the fine-mapping interval was cloned from NIL-CL and NIL-CH by a chromosome walking approach.The cloned and assembled genomic sequences were aligned.The results revealed that there was a small chromosomal segment inversion of approximately 29.8 kb within the mapped interval of NIL-CL.Sequence alignment of the two NILs also revealed an inserted sequence of 327bp miniature inverted-repeat transposable element（BnDTH9）at each of the two inversion breakpoints of NIL-CL.The two molecular markers were designed based on the inversion breakpoints.These two markers co-segregated with the cleistogamous trait.The fine-mapping interval（85.4 kb）harbors 13 annotated genes in the B.napus cv.ZS11 genome.The coding sequences（CDSs）of these 13 genes were cloned from the two NILs using gene-specific primer pairs.The sequencings revealed that the 13 CDSs were identical in NIL-CL and NIL-CH,implying that the CDSs do not directly associate with the cleistogamous trait.These results suggest that the 29.8 kb inversion on chromosome C03 is responsible for the cleistogamous phenotype.（5）Expression pattern alteration of the two genes adjacent to the inversion breakpoint Sequence analyses showed that inversion event resulted in promoter exchange between Bna C03G0156800ZS（Bna C03.FBA）and Bna C03G0157400ZS（Bna C03.EFO1）.The semi-quantitative reverse transcriptional polymerase chain reaction（Semi-RT-PCR）experiments were performed to assess the expression levels of Bna C03.FBA and Bna C03.EFO1 in various tissues of NIL-CL and NIL-CH.The results shown that the gene expression pattern of Bna C03.FBA and Bna C03.EFO1 altered.These were probably resulted from the promoter exchange between the two genes located at the boundary of the inverted sequence.The expression levels of Bna C03.FBA and Bna C03.EFO1 in NIL-CL and NIL-CH petals at key stages of petal maturation（stages 13 and 14）were also quantitatively determined by q RT-PCR.These results indicate the expression level of Bna C03.FBA was much lower than that of Bna C03.EFO1.Meanwhile,in the NIL-CL petals,the expression level of Bna C03.FBA was much higher than that of Bna C03.EFO1.These results implied that these changes in gene expression are presumably responsible for the cleistogamous trait.（6）Genetic transformation To explore the mechanism underlying the clasgamous trait,Bna C03.FBA constructs（PFBA::Bna C03.FBA）were developed using the Bna C03.FBA promoter（PFBA）from NIL-CL.This vector was transformed into NIL-CH,generating five independent stable transgenic lines.Transformants exhibited cleistogamous flowers with completely or partially closed petals,confirming that the overexpression of Bna C03.FBA was responsible for the closed petal phenotype.RNAi silencing of the Bna C03.EFO1 gene was conducted in NIL-CH.This generated four independent transgenic lines.The transgenic plants with a silenced Bna C03.EFO1 gene exhibited flower opening similar to NIL-CH.This result suggested that reduced Bna C03.EFO1 expression did not affect flower morphology in B.napus.These results suggest that 29.8 kb inversion causes high expression of the Bna C03.FBA,resulting in cleistogamy.（7）Bna C03.FBA is involved in protein degradation The causal protein Bna C03.FBA for cleistogamous behavior contains an FBA domain and an F-box motif,may form an SCF complex that functions in the protein degradation pathway.To verify this putative mechanism,the yeast two-hybrid assays and bimolecular fluorescence complementarity aassays with Bna C03.FBA were completed.These assays demonstrated that Bna C03.FBA can interact with Bna C06.SKP13 to form an SCF complex.Bna C03.FBA presumably participates in ubiquitin-mediated protein targeting for degradation through the ubiquitin 26S-proteasome system,playing an important role in petal maturation.In summary,present works have provided insight on the cleisgamous trait formation mechanism.2.Fine mapping of an up-curling leaf locus（BnUC3）Moderate leaf rolling may help to improve crop yield by minimizing the shadowing by leaves.A novel leaf rolling trait in germplasm NJAU-M1295 have been investigated by morphology,inheritance and molecular approach in B.napus.The main results are as follows:（1）The up-curved leaf trait Compared with the mapping parent Zhongshuang 11（ZS11）with typical flat leaves,the NJAU-M1295 plant has up-curled leaves.The leaf chlorophyll content in the plants with up-rolling leaves did not differ from that of normal,flat leaves of the mapping parent ZS11 at 5-leaf seedling stage.However,the plants with up-rolling leaves has decreased photosynthetic efficiency at 5-leaf seedling stage in comparison with the leaf-flat parent.（2）Inheritance of the curved leaf trait The NJAU-M1295 with up-curled leaves was crossed to ZS11 to produce F₁,F₂,and backcross（BC₁）populations for genetic analysis.The results indicated that the up-curling behavior of leaves is controlled by a single pair of dominant locus,referred to as BnUC3.（3）Mapping of the up-curled leave traits locus BnUC3 locus was firstly located in the 1922 kb long interval on the A02 chromosome of B.napus by SNP-trait association analysis with 37 individuals from a backcross population（NJAU-M1295×ZS11）×ZS11.To further map BnUC3,SSR markers and In Del marker were developed.By these markers,the mapping interval was narrowed to 92.0 kb using 584 individuals from a（NJAU-M1295×NJAU-CP3756）F₂ population.（4）Candidate gene analysis for the up-curled leaf trait The mapping interval harbors11 annotated genes according to the ZS11 genome database.Among them,three genes may be listed as candidates based on gene annotation information.Sequence cloning and alignment showed a nucleotide mutation in the Bna A02T0157000ZS gene sequence of NJAU-M1295.This mutation in the plant with up-curled leaves may change the functional product of STMP（Secreted Trans-Membrane Peptides）at position 68（Arg）into His.Thus,Bna A02T0157000ZS is pabably the causual gene for the up-curled leaf trait formation.To conclude,the experiments with aid of the morphological,physiological and genetic approach with the novel germplasm with up-curling trait have fine-mapped the locus responsible for the up-curling trait and identified the candidate gene.These have provided a solid foundation for the leaf up-curling leaf trait genetics and breeding in B.napus.