Improvement Of Male Sterile Line In Brassica Napus L. By Marker-assisted Selection

Rapeseed is one of the largest oil crops in China.Utilization of heterosis is an effective approach to improving the productivity and stability in the crop.The key to utilizing the heterosis in rapeseed is to develop effective male sterile pollination control systems.Based on genetic research of 9012AB,a recessive genetic male sterile germplasm,we introduced high oleic acid material and clubroot resistance genes into sterile line and temporary maintainer line in Brassica napus L.by means of marker-assisted selection.The main results are as follows:Based on the difference between Bnms3,BnC9.Tic40 and BnA10.Tic40sequences,allele-specific markers of M3C9-1F/1R and m3c9-4F/2R were developed,which can effectively distinguish two genotypes of Ms3/ms3,although in different genetic backgrounds might have effects on the detective results in some cases.Based on the differences between Bnms4~a and Bnms4~b sequences,we developed a co-dominant marker M4A1-5F/1R;Two markers were designed through analyzing the sequences of SSR markers of A7-24 and AT3G24240-1,which can be detected using agarose gel and thus simplified the detection process.The ms4~b and ms4~c genotypes were efficiently identified by our newly developed markers of C-1F/1R and M4A1-1F/b-1R.Based on the difference between the sequence of BnA5.fad2,BnA5.FAD2 and BnA5.FAD2,we also successfully developed a co-dominant Kasp marker and a single dominant marker of x2d-1-1F/1R for analysis of oleic acid related genotype.To understand the genetic relationship among the breeding materials,we have genotyped 118 pairs of two-type lines(A and B)and temporary maintainer lines(C)with a Brassica 6K SNP array followed by cluster analysis.The background recovery rate of both ZH5 and ZS9 with high oleic acid trait are higher than 86%.With the ZH5 and ZS9 high oleic acid male sterile materials,the genetic similarities of A-and B-lines and their respective C-line are higher than 90%,while the genetic similarity of A-and B-line within ZH5 and ZS9 is up to 96.8%and 95.7%.Moreover,the genetic similarity of AC in the two systems is up to 81.3%and 88.3%,respectively.That results thus provide valuable information to breed for AC material with higher genetic similarity.Using molecular marker-assisted selection,the Crb gene was successfully introduced into high oleic acid ZH5 and ZS9 three-line materials,and the background recovery rate up to 83.9%and 86.3%,respectively.The phenotype is similar with recurrent parent.The three-lines of ZH5 material with high-oleic acid and resistant clubroot traits has been obtained,and the high oleic acid ZS9 three-lines will be selected in the next generation.Based on the oleic acid content and plant fertility phenotype data,we found that the low oleic acid individuals in the ZH5 had lower death buds than the high oleic acid individuals,and found there have four linkage markers on the oleic acid site in the chromosome of A05,which may be one of reasons for the difference in phenotype,through genetic linkage analysis.By using molecular marker-assisted selection,alleles of Ms3 and ms4 were introduced into the elite recurrent parents of Y2009,15Miao15 and ZS11.After analyses of the genotypes and phenotypes in Y2009 and 15miao15 populations,we found that the dead of buds was significantly reduced,suggesting that pyramiding of multiple genes in breeding can be quickly realized through assisted selection by molecular markers.