Construction Of Genetic Linkage Map And Identification Of Quantitative Trait Loci(QTL)for Days To Flowering In Spring Rapeseed(Brassica Napus L.)

Days to flowering(DTF)is an important trait in Brassica napus,when plants bloom at the appropriate time,it is closely related to crops maturity,yields and seed quality.Furthermore,plants flower at the right time that may be avoid the unfavorable natural factors and produce more and more important offspring.Brassica rapa varieties are planted in high altitude areas.Compared with the spring B.rapa varieties,the spring B.napus varieties exhibit more favorable characteristics,such as strong disease resistance,a high oil content and high seed yield,with double low quality.Although the quality of B.napus varieties is better than that of B.rapa varieties,B.napus varieties cannot mature in the high altitude areas.If we can breeding the early mature germplasms replace B.rape varieties,it will be useful for spring rapeseed improvements in high elevations regions.This study is mainly mapping and analysis of QTL for days to flowering,development the closely linked marker for major QTL loci,it can provide theoretical guidance for early flowering marker-assisted selection and gene pyramiding.The main contents are as follows:1.We analysis the survival rate and doubling rate of the haploid plants under different genotypes,doubled methods and panted environments through spore culture.The results show that the doubled rate is the highest,90.9%,through soaked root method,however,the survival rate as well as the doubled rate,is the highest,so it is suggesting that the medium doubled is the best way for haploid plants through weight the advantages and disadvantages.In addition,the doubled rate had diversity between different genotypes in embryo formation,embryos into seedlings(P<0.05).In aspect of haploid plants transplant environment,greenhouse to greenhouse is an optimal transplanting mode.2.The EAT is a critical factor in early flowering parent No.4512 and DH lines and the thermosensory pathway is important in spring B.napus.Gaussian distribution and transgressive segregations in NN DH population suggested a quantity genetic for DEF.DTF was controlled by major and minor QTL and was affected by the environment,genotype,and the genotype-by-environment interaction can not be ignored.3.Using SSR and AFLP constructed one genetic linkage map contains 495 markers for spring B.napus.The linkage map covered 2252.7cM of B.napus genomic with the length from 49.1cM to 201.8cM.The mean of genetic distance between loci was 4.5cM.4.A composite interval mapping method was conducted to mapping QTL for DTF.The results showed that there were detected 48 QTL related with flowering time in five environments,these QTL located on chromosome A2,A3,A5,A6,A7,A10,C2 and C8,explained phenotypic variation 3.2~46.5%,cqDTFA7 a,cqDTFC2a and cqDTFC2 c which located on the A7 and C2,respectively,these could repeatedly detected in five environments,cqDTFC8 located on C8 and stably detected t in four spring environments.A meta-analysis was conducted to integrate overlapping loci as a consensus QTL,and the results showed that there were three major QTL cqDTFA7 a,cqDTFC2a and cqDTFC8,explained 10.4%,23% and 10% of the phenotypic variation,respectively.These loci carried allele showed negative effect,means can shorten the days of flowering which belong to early flowering allele site.Epistasis analysis showed that there were eight pairs of significant epistatic interactions were detected.Seven of the eight interaction pairs were(additive × additive)× environment interaction effects,located on C8,C2,A7,A6,A3 and A2.Only one QTL,qDTF4,showed an additive × additive effect.The largest epistasis effects were shown by qDTF2×qDTF6 and qDTF5×qDTF7,with the contributions of these interactions accounting for 8.2% and 8.3% flowering variation.qDTF6(cqDTFC8)was a multiple epistatic interactions loci.The interaction loci collectively explained 40.7% of the flowering variation.All of the QTL × environments(QEs)interactions exhibited a negative effect in four spring environments.On the contrary,all QEs showed a positive effect in the winter environment.5.A homologous sequence strategy was applied to search the homologous region for adding markers and development closely linked markers,a closely linked SSR marker GI803 designed from B.rape BACs KBrB008(AC232449)for the major QTL cqDTFA7 a.A candidate gene cloning strategy was applied to clone the homologous gene of APETALA1(AP1)of Arabidopsis from the parents of the NN DH population,and identified BnAP1 gene was a candidate gene for major QTL cqDTFA7 a.We developed one closely linked SSR markers S034 for major QTL cqDTFC8,which sequences derived from the C8 genome physical location 5.5M to 5.1M of B.rape.Na12-C12 was mapped in qDTFC2 c peak value position.We added six SSR markers in the interval of the QTLcqDTFC2 a,the genetic distance between QTL loci and the nearest flanking marker reduced from 12.3cM to 5.9cM.6.Three closely linked markers G1803,S034 and Na12-C12 validated in NNDH groups and the DH lines were divided into two genotypes AA and BB.The group carried the allele from No.4512(genotype AA)with negative additive effects that the DTF was significantly shorter than the group with the No.5246 allele(genotype BB)in the four spring environments.For multiple locus effects,the DH lines were divided into eight groups(I—VIII)according to combinations of loci,and the group carrying the multiple allele with negative additive effects from No.4512 was found to be significantly shorter than that carrying the No.5246 allele(Group I and Group VIII)in DTF.The multiple allele with additive effects were directly related to DTF in the various genotype groups.To validate and estimate the accuracy and application value of the closer-linked markers in marker assisted selection of spring rapeseed,G1803 and Na12-C12 were validated in 93 B.napus breeding resources,the average flowering days with genotype AA was significantly shorter than BB genotype plants(P <0.05),while the effect of recombination site is more obvious.Three closely linked markers will be useful for MAS.