Functional Validation Of Restorer Gene (Bnams4~a) In Rapeseed Genic Male Sterile Line 7365A

The genic male sterility has become one of the important approaches to heterosis utilization in rapeseed in China.The fertility of genic male sterile material 7365 system is controlled by two loci,namely BnaMs3/Bnams3 and Bnams4~a/Bnams4~b/Bnams4~c.Previous research speculated that Bnams4~a may be responsible for restoring male sterility that caused by Bnams4~b through DNA methylation-related pathways.Based on previous research,the present study profiled the genome-wide DNA methylation of the 736512AB near-isogenic line that separated at the Bnams4 locus only,by expressing the invert-repeat sequence of the sRNA target site at the regulatory region of Bnams4~a,the hypothesis that the sRNAs regulate fertility through RNA directed DNA methylation(RdDM)was excluded.Further,through Single-molecule real-time(SMRT)sequencing of the BAC that contained Bnams4~a,genes that were closely linked to Bnams4~a was excluded to be restorer genes.And Genetic complementation in 7365A was accomplished by transformation with the full-length gDNA of Bnams4~a.The main results of this study are as follows:1.Two genome-wide bisulfite sequencing libraries of 736512AB near-isogenic lines were constructed,and the whole genome methylation profile in the buds of Brassica napus was completed.The overall methylation levels of male fertile and sterile libraries were 12.23%and 13.00%,respectively,with no significant differences.The DNA methylation levels at the different sequence contexts of the two libraries were CG>CHG>CHH.DNA methylation is not evenly distributed on each chromosome.Both the overall methylation level and that under different contexts of A_n subgenome were significantly lower than that of C_n subgenome.The average level of DNA methylation in flower buds is lower than that of roots and leaves.2.The methylation pattern of gene region differed from that of repeat region:the methylation level was highest in promoter,it increased rapidly once depart from the transcription start site(TSS),and significantly lower in gene body region.Among them,the methylation level of intron was higher than that of exon and UTR regions.The repeat region itself tended to be low in the middle and high in both flanks,and the methylation levels in the 1kb region of upstream and downstream were relatively low.3.There were local methylation differences between fertile and sterile lines:the number of hypo-methylated differentially methylated regions(DMRs)in 779differentially methylated regions was significantly more than that of hyper-methylated DMR.Eleven genes that may play roles in pollen development were identified among402 differentially methylated genes,of which 10 genes were expressed differently.Eight genes were randomly selected for validation by traditional bisulfite sequencing,and the results were in accordance with that of whole genome bisulfite sequencing.Both the DNA methylation level in the gene body region and expression level of Bnams4~a were significantly lower than that of Bnams4~b.4.Through the inverted-repeat constructs of the sRNA regulatory region,which was transformed into Arabidopsis plant that contained Bnams4~b,the DNA methylation level of the target region in Bnams4~b significantly increased,but the expression level did not change,and the male stertility had not been recovered.Over-expression of the gDNA of Bnams4~a and full-length Bnams4~a transformed into wild-type Arabidopsis resulted in male sterility.These results excluded the proposed hypothesis that Bnams4~a rescued the male fertility by the sRNAs in the regulatory region through RdDM.5.Through SMRT sequencing technology,the BACs contained Bnams4~a and Bnams4~b were completely assembled.The sequence comparison showed that two insertions were newly identifed at the position of about 5kb upstream of Bnams4~a,named806 and 1667 respectively.The fragment 806 was predicted to encode a protein with unknown function.The putative protein of fragment 1667 had a methyltransferase activity.Two complementary constructs contained the two fragments were used to transform into Arabidopsis that contained Bnams4~b,and neither of the two constructs could restore the fertility of Bnams4~b,which excluded the possibility that Bnams4~a is a closely linked gene of Bnams4~b.6.Genetic transformation of 7365A male sterile plants was performed with full-length gDNA of Bnams4~a.A total of 138 positive transformants were produced,of which81 plants had recovered the fertility.Both the DNA methylation level in the gene body region and expression level of Bnams4~a was significantly decreased.7.The Bnams4 promoter consists of three tandem repeats:TR1,TR2 and TR3.And by transformation of Arabidopsis with constructs,in which different number of tandem repeats drove the expression of Bnams4,we found that when 5'UTR drives Bnams4,the phenotype of the transformants was same to that of wild type;when Bnams4 was driven by TR3 only part of positive transformants showed male sterility;and when driven by two or more tandem repeats,all positive transformants were male sterile.These results indicated that tandem repeats in the Bnams4 promoter were indispensable for the expression of Bnams4.In this study,the whole genome methylation analysis of rapeseed buds was carried out,and the fertility changes caused by the difference in the promoter region of Bnams4~a were excluded.The fertility restorer genes closely linked to Bnams4~a were excluded,and the complementary transformation of Bnams4~a was successfully accomplished in rapeseed line 7359A.This study has improved the genome-wide methylation profiles in Brassica napus,and laid the foundation for the mechanism of fertility restoration and regulation of new chimeric genes.