Mapping Of The Ms/Mf Gene Of A Dominant Genic Male Sterility In Brassica Napus L

A new kind of genie male sterility (GMS) in Brassica napus was reported by Li et alin 1985. Due to its complete and stable sterility, and almost no negative cytoplasmiceffect, this mutant is considered as a promising alternative to CMS in heterosis utilization.A hypothesis of two dominant genes with interaction was subsequently proposed tointerpret it, that the sterility is conditioned by a dominant male sterility gene (Ms) and arestorer gene (Mf), and Ms can result in male sterility alone, but Mf can restore it. Basedon this model, a three-line hybrid production system just like CMS was then presented.However, after a systematic investigation on a natural dominant GMS mutant (609AB),Song et al. regarded that the dominant GMS is prone to be explained by one gene withmultiple alleles, which makes the mechanism of dominant GMS involved in a debate.Consequently, a further research on dominant GMS is fairly necessary.Towards this goal, our research was developed in three main fields. First, wedifferentiated the two genetic hypotheses of dominant GMS by cross, backcross and testcross, Rs1046AB used as a fundamental material. Then, a marker-assisted backcrossbreeding program to transfer the Ms gene to two elite inbred lines was carried out, usingtwo SCAR markers developed earlier. Finally, two mapping population were adopted tolocalize genes responsible for dominant GMS phenotype, and the results from differentpopulations were integrated together according to the same SCAR markers andcollinearity relation between Arabidopsis and Brassica napus. Main results of the presentresearch are as follow:1. By observing the fertility segregation of cross progenies, 10 temporary maintainers ofdominant GMS were identified from a total of 16 inbred lines. Other 5 inbred lines,which can restore the fertility of GMS, were selected as the restorers. Additional, oneinbred line which can restore the fertility of about a half progenies was concluded tocarry a heterozygous restorer gene.2. From the 1:1 segregation F1 population produced by crossing Rs1046B withmaintainers, fertile plants were selected to be selfed and baekcross to maintainers. Thesubsequent progenies were all fertile plants, suggesting that Ms and Mf werecompleted segregated in meiosis, so in Rs1046AB Mf is allelic to Ms. 20 sterile plantsfrom F2 progenies generated by crossing restorer 195A-14 with Rs1046A wererandomly selected to test crossing with maintainer 7-5, and no fertile individual wasobserved in test cross F1, indicating Mf from 195A-14 is allelic to Ms from Rs1046A,which confirmed the multiple allelism hypothesis again. Therefore, the genotypes of Rs1046A and Rs1046B are MsMs and MsMf and inbred line 195A-14 and 7-5 are ofgenotypes MfMf and msms, respectively.3. Two SCAR markers developed earlier were used to transfer Ms to two elite inbredlines, 7-5 and 195A-14, through a backcross breeding program. However, because nosterile plant was observed as expected in BC1 progenies, the program about 7-5 had tobe cancelled. By successive foreground selection and background selection for twotimes, Ms was successfully introgressed into 195A-14, and male sterile plants withsimilar appearance with 195A-14 were isolated, showing the MAS process is effective.Regretfully, due to the severely weak fecundity of female in sterile plants, thehomozygous dominant GMS line can't be obtained.4. Combining comparing NILs and BSA, 137 individuals from Rs1046AB were used formarker analysis. From the survey of 2, 048 pairs of AFLP primers, six fragments onthe same side were identified to be tightly linked to Mf, with a genetic distancevarying from 0.7 cM to 3.6 cM. Two of the four nearer AFLP fragments were thenconverted to SCAR markers directly. For the other two markers, after isolating theadjacent sequences by suppressed PCR, corresponding SCAR marker wassuccessfully obtained as well. All the SCAR markers have a same genetic distance toMf with original AFLP marker.5. By PCR analysis in a population of 1,017 individuals, three SCAR markerscosegregated with each other were differentiated, that SCHDF was mapped closer toMf than other two markers, with a distance of 1.1 cM.6. A local linkage map of Mf/Ms genomic region was constructed based on 192individuals of the F2 population from the Rs1046A/195A-14 cross. 20 AFLP markersand two SCAR markers, spanning a genetic distance of 10.4 cM, were identified to beassociated with the Mf/Ms locus. On this map, 14 markers and 8 markers reside oneach side of target gene. The Mf/Ms locus, cosegregated with marker S5T5-480, wasbracketed by E3M10-580 and ELM13-260, with a genetic distance of 0.1 cM and 1.0cM, respectively. Six markers with a nearer genetic distance were chosen for SCARconversion, and three of them were successfully converted, i.e. SCDG1 (E3M10-580),SCDG2 (S5T5-480) and SCDG3 (ELM13-260), which have a same genetic distanceto Mf/Ms with respect to original AFLP markers.7. BLASTn analysis of some markers' sequence (or flanking sequence) from above twopopulations on NCBI website can identify many homologues, most of whichdistribute on the short arm of Arabidopsis chromosomeâ… . Comparison of the markerloci in linkage map and the homologous loci in Arabidopsis indicated that there was a collinearity relation between the region flanking Ms/Mf and Arabidopsis homologues,though it was likely disrupted by some chromosome inversion and translocationevents. Integrating the BLASTn result of earlier researches, the homologous region inArabidopsis chromosomeâ… covered a physical distance of 3.4 Mb, betweenAT1g04950 and AT1g 14340.8. Utilization of the same SCAR markers or information of Arabidopsis homologues, weintegrated the mapping results of two earlier BC1 populations and two maps in thepresent research. Finally, the Ms/Mf gene can be positioned in an interval restricted bymarker E1M2-310 and E4M9-150, which contains 11 markers (including five SCARmarkers) and covered a genetic distance of 4.6 cM.