Finemapping And Functional Analysis Of A Major QTL,qDB.A09,for Branch Number In Brassica Napus

Rapeseed(Brassica napus)is one of the most important oilseed crops worldwide,providing 15%of the global supply of edible vegetable oil.At present,in order to meet the needs of China’s edible oil market,the main target of rapeseed breeding is further increase of yield.Yield is a complex trait controlled by many factors.The yield of rapeseed is determined by the number of siliques per plant(SP),the number of seeds per silique,and seed weight.Branching is the most important determinant of SP via its influence on(branch number)BN during vegetative development and(number of effective siliques on the main inflorescence)SMI during reproductive development,which in turn affects seed yield.However,the genetic architecture associated with branching remains unclear,and there have been no reports describing the fine mapping of branching-related traits in rapeseed.In the present study,a dense branching mutant,dense branching 1(db1),was crossed with T109,an inbred line with normal plant branching,and the resulting progeny were then backcrossed with db1 to produce a backcross population(BC1F1).We identified two major quantitative trait loci(QTLs)based on the BC1F1 population.Furthermore,fine mapped qDB.A09 to a DNA fragment of approximately 7.0 Kb in length.The branching mechanisms of db1 were researched based on haplotype analysis,genetic transformation,together with the sequence comparisons and expression analysis.The main results were as follows:1.Branching related QTL analysis based on Single nucleotide polymorphism(SNP)BeadChip Array-assisted BSA approach.The mutant db1 was crossed with the normal branching material T109,and the resulting Fiprogeny were then backcrossed with db1 to produce a(BC1F1)population.The phenotypic values of the length of the top four internodes(LFI)showed a significant deviation from a normal distribution in the BC1F1 population,which indicated that LFI might be mainly determined by major genes in this population.Four bulked-dense branching and four bulked-normal branching pools constructed from the BC1F1 population,and the two parents,i.e.,the mutant db1 and wildtype T109,were prepared and genotyped using the Brassica 60K SNP BeadChip array.Of the 52,157 SNPs on the SNP array,there were 346 polymorphic SNPs between "dense branching" bulks and "normal branching" bulks.Among these SNPs,247(71%)were mapped on chromosome A09 of B.napus,another 25 and 14 markers were located on chromosomes C06 and A07,respectively,these identified regions might harbor the major QTLs for branching.2.Confirmation of the QTLs and effect evaluation.The SSR and IP markers were developed based on the results of BSA,and partial linkage map of the chromosomal region spanning qDB.A09 and qDB.C06 were constructed.With the traditional QTL analysis,the QTL,qDB.A09,showed some effects on BN,LFI,AIL,SD,and SMI,and that was responsible for 9.1%,31.7%,17.7%,27.6%,and 7.6%of the phenotypic variation in these traits,respectively.We also identified the qDB.C06 locus,which explained 8.6%,29.1%,8.9%,24.3%,9.3%,and 6.3%of the phenotypic variation in BN,LFI,AIL,SD,SMI,and LMI,respectively.3.Fine mapping of qDB.A09.A BC1F3 population was developed via maker-assisted selection(MAS).The qDB.A09 locus was a single Mendelian factor and that the dense branching phenotype was controlled by a single recessive gene in the BC1F3 population.Based on 7785 plants from the BC1F3 population,qDB.A09 was delimitated to a 270Kb region between markers P2 and P19.To further narrow the genomic region,38,000 plants from a sib-mating population were selected for recombinant screening,and delimitated qDB.A09 to a 7.0Kb region between markers G8P4 and S65.4.Sequence analysis of the qDB.A09 candidate region.The 7.0Kb candidate region in B.napus genome contained 3 annotated genes,no reproducible DNA polymorphism between T109 and Darmor-bzh was observed for the three genes.In contrast,we identified several variations in the db1 sequence,several of which led to amino acid changes of three genes.By comparing sequence,it was found that T109 and Darmor-bzh were completely identical in the 7.0Kb sequence of the target segment,but there was one insertion in db1.Based on the sequence analysis of the Bacteria artificial chromosome clone from the ZS11 BAC library and the resequence data of db1,an additional insert fragment(10,734bp)in db1 and ZS11 was also identified.An F2 population(denoted Z-F2 population)developed from a ZS11 and dd(homozygous for the db1 allele)plants was genetically dissected for branching trait,and the result showed that the dense branching phenotype is controlled by was a single recessive gene.5.qDB.A09 candidate gene prediction.Through genetic transformation,haplotype analysis,and genetic analysis of the Z-F2 population,it was found that none of the three genes corresponding to the 7.0Kb segment of the Darmor-bzh reference genome were qDB.A09.Based on the transcriptome data,it was predicted that there were three genes in the 10,734bp insertion,of which ORF2 and BnaA09G0259200ZS were significantly differently expressed in the near-isogenic line.Among them,ORF2 had higher expressions in the shoot apical meristem(SAM)of dd plants than that in the DD(homozygous for the T109 allele)and heterozygous plants,which was consistent with the genetic model of qDB.A09 locus.Comparative sequencing revealed that there were only three single-base differences between db1 and ZS11 in the qDB.A09 locus.SNP1055,which was downstream of ORF2,was a unique variant for db1.Correspondingly,ORF2 had a higher expression level in dd than that in the ZS11 and other materials.Therefore,it was speculated that ORF2 might be the target gene of qDB.A09.6.ORF2 is a potential FAR1-RELATED SEQUENCE(FRS).ORF2 contains the core transposase domain of the FHY3 protein and the SWIM zinc finger domain at the Cterminus.Therefore,ORF2 may belong to the transposase-derived transcription factor of the FRS family,which directly binds to FHY3/FAR1-binding site of CLAVATA3(CLV3)and inhibits its transcription.Meanwhile,the SAM size of the genotype dd plants was larger than that of DD.CLV3 might be repressed by over expression of ORF2 in dd and in turn promote WUSCHE expression.As a result,the SAM size of dd plants was larger than that of DD,thus showed extremely dense branching in dd plants.In addition,RNA-seq data showed that ABA-INSENSITIVE5 and a series of ethylene response factors as well as WRKY transcription factors were highly expressed in dd plants,suggesting that qDB.A09 might be involved in plant stress response.