| Chen et al. firstly reported the genetic model of a rapeseed recessive genic male sterility line (REGMS)9012A controlled by duplicate recessive genes and recessive epistasis inhibitor gene, which made three-line systems available for hybrid seed production. The novel model was modified recently by ZU Feng (2010), Dong Faming (2010) and Ni Xiyuan (2011). They deemed that the male sterility of9012A is controlled only by two individual loci. In spite of REGMS in Brassica napus attracts great attention to rapeseed breeders due to its prominent advantages in stable and complete male sterility, extensive distribution of restorers and diverse cytoplasmic sources, however, since the sterility was controlled by the interaction between multiple-twin genes, it causes a complex segragation in offspring genotypes and makes it difficult to be efficitively used in hybrid breeding. Therefore, fine mapping these two target genes and developing co-dominant markers would be highly interesting. For this purposes, on the basis of previous research (Xu Xiaodong,2009), which had located Bnms3and Bnrf in280Kb and4Mb genomic region, respectively. We further developed larger number of mapping population and designed abundant locus-specific markers for fine mapping these two genes, to search for closely linked co-dominant markers to Bnms3and Bnrf. The potentials of marker assistant selection in hybrid breeding are well demonstrated in this research. Our experimental results and main conclusions are as follows:1. Fine mapping of recessive sterile the gene Bnms3and the finding of co-dominant linked markersOn the basis of previous research, we designed58location-specific primer pairs from Brassica rapa genome A10.16markers were screened out using BSA method and could be mapped in280kb region. Among these markers,8showed no recombinants in a segregation population licluding2490individual-plants (Sca08-6Ms3-66Ms3-81Ms3-82Ms3-89Ms3-93Ms3-95the Ms3-96) corresponding to five Arabidopsis thaliana genes (At5g16560, At5g16570, At5g16590, At5g16600, and At5g16640). The markers Ms3-74and Ms3-88were nearst flanking markers of Bnms3and produced only two and one recombinant plants in the detection of2490individuals, respectively. The physical distance between Ms3-74and Ms3-88is75Kb and contain nine ORFs. Compared to previous mapping results (280Kb) from Xu Xiaodong, Bnms3region was clearly reduced by200Kb. In addition, six co-dominant markers within the scope of0.05cM nearby Bnms3(Ms3-77Ms3-82the Ms3-88, Ms3-89, Ms3-93, Ms3-95) were screened out and they could be effectively used for marker-assisted selection in breeding program.2. Mapping of recessive epistasis gene Bnrf and the development of co-dominant flanking markersOn the basis of Xu Xiaodong (4Mb,2009), we further designed81pairs of primers from Brassica rapa genome A7together with4publised markers to check the polymorphisms by BSA method.10markers showing closely linked with Bnrf were determined. In the first step,576individuals from wy23-rf (9012A) population were used for preliminary localization and then mapped Bnrf in between of markers Sca17-5and Scal7-25. Further, we designed85pairs of locus-specific primers more from Brassica rapa genome A7, and expand the mapping population to1767individuals. The results indicated that16markers closely linked with Bnrf were observed. The nearest flanking markers were Seal7-(61-87)-13and Scal7-(61-87)-27, with physical distance of99.3kb. This consumedly shorten the distance from Xu Xiaodong reported (2009). In addition, our study screened out7co-dominant markers closely linked with Bnrf (MR166Sca17-5, Sca17-25, Sca17-61Sca17-75Scal7-87, Sca17-(61-87)-27. They provide an important tool for marker-assisted selection for breeding purpose.3. Exploration of recessive nuclear male sterile genetic model in Brassica napus L. and marker-assisted selection in breeding In the present study, twenty-two varieties/breeding lines of Brassica napus were test-crossed with sterile lines20118A/MSL-A, and their temporary maintainer20118A-TAM/MSL-TAM, respectively. Both traditional genetic analysis and molecular marker assistant technology were employed to confirm their genetic model of sterility with two genes control system, and to check the efficiency of marker-assisted selection (MAS). The results showed that the segregation proportion of male fertile to sterile plants in F2progenies from6varieties (lines) crossed with20118A/MSL-A line fitted mendelian segregation (3:1and13:3), and with20118A-TAM/MSL-TAM showed all male fertility, indicating that the sterility of both20118A and MSL are controlled by one recessive sterile gene interacting with a recessive epistatic suppression gene. In addition, a reverse validation approach based on Bnms3and Bnrf linked marker assistant selection was used to further confirm the two gene control system. From a total of925F2plants,63,64and110individuals carrying temporary maintainer (ms3ms3rfrf), homozygous stevile(ms3ms3RfRf)and fertile(Ms3ms3RfRf)marker genotypes were screened out, respectively, which also fitted the mendelian segregation proportions of two gene model (1/16,1/16and1/8). After test-crossing with known homozygous sterile or temporary maintainers or one another among marker genotypes, higher than95%of lines were approved to be the expected genotypes; According to the information from22testcross cultivars/lines, only two alleles of Rf and rf were identified in BnRf locus, implying the third allele naturally existed very few, if any. Therefore, for practical breeding purpose, a marker assistant strategy simply based on BnRf/rf and BnMs3/ms3linked co-dominant markers is proposed; Finally, the reciprocal testcross between20118A/20118A-TAM and MSL-A/MSL-TAM indicated that these two independently originated male sterile systems might be genetically identical. |
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