Molecular Mapping Of Two Recessive Genic Male Sterility Genes In Brassica Napus L.

The utilization of heterosis in rapeseed is an efficient pathway to increase yield, to solve the problem between yield and quality, and to improve resistance/tolerance. The study and utilization of high efficient pollination control system is the key step in hybrid seed production. The genic male sterility (GMS) systems had great potential for hybrid seed production in virtue of the characteristics of complete and stable male sterility, no negative cytoplasmic effects on plant growth, and ease of transference of the male sterility gene(s) to diverse genetic backgrounds. However, the production of F1 hybrid seed requires the removal of 50% of segregating fertile plants in the sterile female line which limits its utilization for production of hybrid seed. In 1993, Chen et al. reported a recessive genic male sterility (RGMS) line with an epistatic inhibitor gene, a 100% sterile population can be obtained via temporary maintainer, which made three-line systems available in hybrid seed production, and set up condition for the widely utilization of RGMS lines.Now, the RGMS lines used in China included several RGMS lines (eg. 117A and S45A) and several RGMS lines with an epistatic inhibitor gene (eg. 9012A). The 117AB and S45AB are two RGMS lines in which the sterility is controlled by two duplicate recessive genes named ms1 and ms2, located at two separate loci. One or two of the dominant genes existed at any loci can restore their fertility. The Brassica napus oilseed rape line 9012AB is a recessive epistatic GMS two-type line in which the sterility is controlled by two pairs of recessive duplicate sterile genes (ms3 and ms4) interacting with one pair of a recessive epistatic inhibitor gene (rf). Homozygosity at the rf locus (rfrf) inhibits the expression of the recessive male sterility trait in homozygous ms3 ms3 ms4 ms4 plants. In order to make the RGMS system a wider application, it is necessary to initiate further study on them. In this research, RGMS two-type lines 117AB, S45AB and 9012AB were used to identify AFLP markers linked to the Ms genes of RGMS lines117A and 9012A, construct genetic linkage map surrounding the Ms loci, and initiate comparative mapping between Arabidopsis and Brassica napus surrounding the Ms locus towards map-based cloning of the Ms gene of 9012A. Main results of the present study are as following:1. Crosses were made between the fertile plants of 117AB and S45AB, then selfed the F1 plants, and harvested seeds from 10 F1 individuals. Fertility of the 10 F1-derived F2 populations showed that the dominant Ms loci in 117A and S45A were non-allelic.2. Sterile bulk (BS) and fertile bulk (BF) DNA samples prepared from 9 male sterile and 9 male fertile plants of the two-type line 117AB were subjected to AFLP analysis. From the survey of 512 AFLP primer combinations, six AFLP fragments (Y1, K1, K2, K3, K4 and K5) were identified as being tightly linked to the Ms locus. The genetic distances between the markers and the Ms locus were all less than 8 cM, among whichtwo fragments, designated as K2 and K3, co-segregated with the target gene in the tested population. The markers detected could be valuable in marker-assisted selection in the utilization of RGMS in B. napus. By the way, we sequenced the AFLP fragments of Yl, Kl, K2, and K3, and compared the sequences with the Arabidopsis data, then primarily mapped the Ms locus to the Arabidopsis chromosome I . And fragment K2 was successfully converted into a SCAR marker.3. Sterile bulk (BS) and fertile bulk (BF) DNA samples prepared from 10 male sterile and 10 male fertile plants of the homozygous two-type line 9012AB were subjected to AFLP analysis. A total of 256 primer combinations were used and seven markers tightly linked to one fertile gene (Ms) in the RGMS line were identified. Among them, six fragments co-segratated with the target gene in the tested population, and the other one had a genetic distance of 5.4 cM. Five of them were successfully converted into dominant SCAR markers, which would facilitate the selection and breeding of temporary maintainers.4. A NIL population including 1506 individuals of 9012AB was developed for further study of the 5 SCAR markers, and a fine linkage map surrounding the Ms locus was constructed. Among them, two markers (SEP5 and SEP8) were located at one side of the target locus with a genetic distance of 0.2 cM and 0.1 cM respectively; and three markers (SEP 10, SEP7 and SEP4) were located at the other side with a genetic distance of 1.4 cM, 0.7 cM and 0.4 cM respectively.5. Isolated flanking sequences of the AFLP markers by PCR walking were submitted to NCBI for BLAST analysis. Information about Arabidopsis of each marker was collected and submitted to the Arabidopsis genome database (TAIL), and a linkage map in Arabidopsis chromosome V were made by 5 out of the 7 AFLP markers in the same order as in B. napus. Then, we could map the Ms locus in Arabidopsis between EP4 and EP8, which spanned a physical distance of about 2.1 Mb.