Gentic Mapping,Cloning And Functional Analysis Of Dwarf Genes In Brassica Napus L.

Plant height is an important component of plant architecture as well as an important foundational yield-related trait in crops.Utilization of semi-dwarf genes is crucial for the success of the first “green revolution”,which markedly increased the yield of rice and wheat by decreasing the plant height,improving lodging and fertilizer resistance and meanwhile increasing harvest index.As one of the important oil crops,current rapeseed varieties with tall plant type are easy to lodging,which leads to the losses in yield and quality of rapeseed.However,the dwarfed resources of rapeseed are fewer and the molecular mechanism of plant architecture regulation is less enoughly explored,which greatly restricts the progress of the dwarf breeding in rapeseed.Thus,it is important to develop dwarf resources and cloning the related genes as well as elucidate the mechanism underlying stem elongation for rapeseed dwarf breeding.In this study,the height-related gene DS-3 was genetically mapped using B.napus(AACC,2n = 38)dwarf mutant ds-3 as the research material.We finally cloned the dwarf gene Ds-3 encoding a DELLA protein negatively mediating the GA signaling and named as Bna C07.rga-ds.We further characterized the function of Bna C07.rga-ds and its homologs in B.napus.In addition,we fine mapped the dwarfing gene in dwarf mutant ds-4.Main results are described as follows: 1.Mapping,cloning and functional vertification of dwarfing gene DS-3The ds-3 mutant grew creepingly and the leaf color was dark green at the seedling stage.At maturity,number of node,internode length,main inflorescences length and height of the first primary branche in ds-3 significantly decreased,which together resulted in its reduced height.Besides,the elongation of the ds-3 hypocotyl showed a reduced response to exogenous GA3.F1 plants displayed the intermediate plant height between the mutant ds-3 and its original genotype “HS5”.The segregation of plant height in the F2 and BC1 population agreed with an expected ratio of 1:2:1 fand 1:1,respectively,which indicating that the dwarfism of ds-3 was controlled by single incompletly domidant gene.By conducting the bulked segregant analysis(BSA),the DS-3 dwarf locus was mapped to a 6.0 c M interval between Bo GMS4033 and Bo GMS4044 on C07 chromosome.The corresponding chromosomal region of the four linked SSR markers flanking DS-3 showed good collinearity to the flanking region of a previously identified plant height-related gene Bna A06.RGA on A06.An annoated gene model,Bna C07.RGA in the target region of C07 chromsome,is homologous to Bna A06.RGA.Thus,we taked Bna C07.RGA as the candidate gene of DS-3 for further characterization.Comparative sequencing identified a C-to-T transition at the +272 nucleotide in the coding region of mutant allele Bna C07.rga-ds in ds-3,which caused a substitution of conserved proline(P)to leucine(L)in the N-terminal VHYNP motif.Locus-specific marker analysis showed that this point mutation co-segregated with plant heights of individuals in the F2 and BC1 populations derived from the cross between ds-3 and HS5.The results of ectopic expression of Bna C07.rga-ds and wild-type Bna C07.RGA genes in wide-type Arabidopsis Columbia ecotype showed that plant height of Bna C07.RGA transgenic lines was similar to that of wild-type Arabidopsis plants,while the Bna C07.rga-ds transgenic lines displayed obviously dwarf stature.The results of yeast two-hybrid assays showed that Bna C07.RGA can interact with At GID1 a in a GA-dependent manner,while Bna C07.rga-ds cannot interact with At GID1 a with or without GA induction.2.Identification,evolutionary relationship and functional characterization of RGA homologs in B.napusReciprocal BLASTp searches were conducted using amino acid sequence of Arabidopsid RGA(At RGA)as a query to identify homologous genes in the genomes of B.rapa,B.oleracea and B.napus.Two Bra RGA gene models were identified in the B.rapa genome;two Bol RGA gene models were identified in the B.oleracea genome;four Bna RGA gene models were identified in the B.napus genome.The results of phylogenetic analysis based on the nucleotide sequence and identity analysis of amino acid sequence suggested that the four Bna RGA genes respectively evolved from the two Bra RGAs and the two Bol RGAs,which constituted four orthologous gene pairs.RT-PCR analysis indicated that each Bna RGA gene from ds-3 was expressed in leaves and stems.And GA can enhance the expression level of the four Bna RGAs while different Bna RGA showed different GA-induced expression pattern.Yeast two hybrid assays suggest that,all the four Bna RGAs are able to interact with GID1 a under the GA induction.Besides,it was shown that Bna C07.RGA exerts a weaker effect on plant height than Bna A06.RGA by comparing the effect of ds-1 and ds-3 dwarfing loci on plant height.3.Fine mapping of dwarf gene DS-4The leaves of the ds-4 mutant was a sharply down-curved and crinkled at the seedling stage,which enable ds-4 to grow lying along the ground;At maturation,plant height of the ds-4 mutant was considerably shorter than that of ZY821.In addition,lower seed germination rate,shorter hypocotyl and the upper stem of ds-4 showing slight positive gravitropism for a period of time before flowering also are the features of ds-4.But dark morphogenesis of ds-4 appear normal.F1?F2 and BC1 generations were generated from the cross between mutant ds-4 and wild-type ZY821.The heitht of F1 plants was considerably below the mid-parent value and more closer to that of ds-1.It is easy to classify the plants of F2 and BC1 population into two types as dwarf stature and normal stature.And the segregation of dwarf stature and normal stature obeyed an expected ratio of 3:1 for the F2 population and 1:1 for the BC1 population.By bulked segregant analysis(BSA),the DS-4 dwarf locus was mapped to a 25.6 c M interval between ID-147 and ID-162 on C05 chromosome.In the meantime on the basis of this mapping interval and its corresponding genome sequence of ds-4 and ZY821,four CAPS marker were developed and found to be linked to DS-4.By genotyping tall plants from F2 population to identify the recombinants with these linked CAPS markers,the DS-4 locus was further mapped to a 4.10 M interval between SNP2CAPS-1 and SNP2CAPS-4.Subsequently,several SNP markers within mapping region were developed.And recombination informations of the recombinants being recombined between SNP2CAPS-4 and SNP2CAPS-39 among these SNP loci were further obtained by using the SNPseq? genotyping technology.Finally,the mapping interval of DS-4 locus can be narrowed to a 475 kb in length between SNP32 and SNP40 in view of their positions on the C05 chromosome of B.napus cv.“Darmor-bzh” genome.