Genetics Analysis Of Thousand Seed Weight And Fine Mapping Of Major QTL CqSw.A03-2 In Brassica Napus L.

Rapeseed(Brassica napus L.)is one of the most important oilseed crops in China.As an important factor determining seed yield of rapeseed,thousand-seed weight(TSW)is the primary target for breeding programs.Although previous studies have successfully identified numerous quantitative trait locus(QTL)for TSW,only two genes have been cloned.In this study,QTL mapping was conducted using a double haploid(DH)population derived from a high-TSW line ZY50 and a low-TSW line 7-5,and the genetic basis of TSW was dissected into six major QTLs.Moreover,a set of near-isogenic lines(NILs)for a major QTL,cq SW.A03-2,were developed.Consequently,cq SW.A03-2 was fine mapped and candidate gene prediction was conducted.The results are described as follows: 1.Linkage map construction for DH population.DH population derived from ZY50 and 7-5 was developed and a high-density genetic linkage map consisting of 2,237 SNP markers and 65 SSR markers was constructed.The linkage map consisted of 19 linkage groups and covers 1,951.5 c M of genetic distance with a mean of 0.85 c M between the adjacent markers.Comparative mapping between the linkage map and the physical map of B.napus revealed high collinearity between each linkage group and the corresponding chromosome.2.QTL analysis of TSW in the DH population.The phenotypic data of DH population was collected from Wuhan and Jingzhou across 2013-2014 and 2014-2015 growing seasons.QTL analysis detected a total of 29 QTLs for TSW,which were distributed on four linkage groups,and the percentage of phenotypic variation explained by individual QTL varied between 4.39% and 13.70%.Among the QTLs identified,24 QTLs were integrated into six consensus QTLs by meta-analysis.For all the QTLs except for those located on the C02 linkage group,the positive alleles were inherited from the parental line ZY50.3.Fine mapping of cq SW.A03-2.To uncover the genetic basis of cq SW.A03-2,a set of NILs were developed using ZY50 as the recurrent parent while 7-5 as the donor parent.Based on the progeny testing of the recombinants,the cq SW.A03-2 locus was restricted to a 61.6-kb physical region,in which 18 predicted coding sequences were identified.Candidate gene association analysis was performed in a panel of natural population consisted of 505 rapeseed lines and two significantly associated SNPs were identified.No significant difference was observed between NIL(ZY50)and NIL(7-5)for each of the five yield-related traits,suggesting that cq SW.A03-2 increases TSW of NIL(ZY50)without a negative influence on other yield-related traits,which represents a great potential of cq SW.A03-2 for yield improvement in rapeseed.5.Sequence analysis and expression analysis of the candidate gene underlying cq SW.A03-2.Comparison of the genomic sequence of Bna A03g37960 D between ZY50 and 7-5 revealed multiple variations in the coding and transcriptional regulatory regions.Expression analysis of Bna A03g37960 D in NILs revealed a higher transcript levels in early stages during ovule and seed development than other stages examined.Moreover,NIL(ZY50)consistently showed higher transcript levels than NIL(7-5)during ovule and seed development.The results suggested that Bna A03g37960 D denoted as Bna A.AHK2.a,is the candidate gene for cq SW.A03-2.In NIL lines,the individuals harboring the homozygous ZY50 allele showed a significantly higher expression level than the plants carrying chimeric genotype(promoter of ZY50 allele and coding region of 7-5 allele),suggesting that the expression of Bna A.AHK2.a may be controlled by the coding sequence or 3'-end transcriptional regulatory sequence.