Fine Mapping Of Dominant Male Sterility Gene And Restorer Gene In Brassic Napus L.

Rs1046AB is a dominant genic male sterility (DGMS) line derived from a spontaneous mutant of Yi-3A in Brassica napus. It has some advantages: such as complete and stable male sterility, widely spread of restorers and no negative cytoplasmic. The sterility of this mutant was previously regarded as to be conditioned by the interaction of a dominant male sterility gene Ms and its non-allelic dominant restorer gene Mf (or Rf in previous reports) (Li et al., 1985). Recent genetic analyses, however, indicated that Ms and Mf may be allelic (Song et al., 2006). Using an F₂ population (Rs1046A×195-14A) of 192 plants, Hong (2006) have constructed a primary linkage map of Ms/Mf genes. Based on these result, the present study emphasized on identifying DNA markers linked to Ms/Mf genes in the F_2:3 and sterile plants in F₂ populations constructed by crossing Rs1046A with a double haploid (DH) restorer line (19514A). Main results of the present study are as follows:1. Three AFLP markers E3M10, E1M13 and S5T5 (Hong, 2006) were converted into SCAR markers successfully (designated as SCD2, SCD7 and SCD8). Five SCAR markers, including SCE3 (Lu, 2003) and SCHDF (Hong, 2006) were used to analysis in F_2:3 population and all sterile plants in F₂ population, which including 708 individuals in former population and 987 individuals in latter population, respectively. As a result, four SCAR markers SCD2 , SCE3 , SCD7 and SCD8 all have been mapped in one side of the target genes (Ms/Mf), with the genetic distances of 2.0cM, 1.7cM, 1.5cM and 0.1 cM, respectively, another SCAR marker SCHDF has been mapped in the other side of the target genes, with the genetic distance of 2.3cM.2. AFLP technology combined with bulked segregant analysis (BSA) was used to identify the genetic markers more tightly linked to Ms/Mf genes. Additional 512 primers combinations were screened and four AFLP markers linked with the Ms/Mf genes were obtained. Among them, two markers (P1M1 and P1M4) were co-dominant markers, they have been mapped between the SCAR markers SCD7 and SCD8. Another two markers (P3M2 and P12M6) were dominant markers, have been mapped in two sides of the target gene (Ms/Mf), the former was between SCD7 and SCD8, and the latter was between SCHDF and target genes.3. Using comparative sequencing technology, two SCAR markers (SC6 and SC9) obtained by Song et al., 2006, which linked with the sterile gene Ms of 609AB in Brassica napus have been integrated in the linkage map of present study and converted into co-dominant markers, designated as SC6D and SC9H. SC6D was mapped between AFLP marker P12M6 and target genes, and SC9H was mapped between AFLP markers P3M2 and P1M1/P1M4, with the genetic distance of 1.0cM. Combining the above AFLP markers and SCAR markers, the target genes (Ms/Mf) have been finely mapped in a little genetic regions.4. Through comparing with the linkage map of DH (Tapidor×Ningyou7) population, the target genes {Ms/Mf) have been mapped on linkage group N8. One co-dominant SSR marker located on linkage group N8, HUA348, was confirm to linkage to the Ms/Mf genes.5. Based on the homologous region sequences of target genes (Ms/Mf) region and Arabidopsis homologous, 18 specific primers (DFN1-DFN18) have been designed to amplify Ms gene and Mf gene. As a result, 14 primers could have amplified Ms gene and Mf gene together, except for primers DFN3, DFN12, DFN14 and DFN18.6. Using the SCAR marker SCD8, linked with the restorer gene Mf as a probe screened the Tapidor BAC library by Southern hybridization, and 29 positive BAC clones were identified.7. Twenty-nine positive BAC clones were identified by four specific primers DFN1, DFN5, DFN8, DFN16 and SCAR marker SCD8, then, an overlap contigs maybe covered Ms/Mf genomic region were constructed by the 18 positive BAC clones in 29 positive BAC clones. 29 positive BAC clones were digested with HindIII, and a fingerprint of 29 positive BAC clones has been constructed. As a result, it is a further verification of the possible overlap contigs covered Ms/Mf genomic region were constructed by PCR amplification.