| Chen et al (1993,1998) reported that the sterility of RGMS was controlled by two recessive genes (Bnms3 and Bnms4) and one recessive epistatic suppression gene (Bnrf) interactively. This sterile system has several remarkable advantages, a stable, complete male sterility, which makes sure for yielding high rate F1 hybrid seeds; a complete open flower petal, which benefits for getting a good seeds set. Of them, the most important one is that almost all the normal rapeseed cultivars are its restorers, which provides possibility to produce huge number of F1 combinations for breeding purpose. Therefore, this RGMS was regarded as one of the most potential sterile system in oilseed rape nowadays. However, due to the polygene control system, resulting in complex genotypes in segregation generations, the improvement for sterile lines becomes difficult if without advanced technique support in practical breeding procedures. So it is necessary to develop a set of specific markers, which linked either to sterile or epistatic genes for creating or improving a two-type lines and temporary maintainer lines, and thereby to get geneticly stable full sterile lines finally. To achieve this goals, firstly we used the DNA samples from sterile and fertile plants of two-type lineand also DNA's from temporary maintainer line and full sterility lines provided by Shan Xi province for marker screening. Secondly, the linked markers were confirmed and fine mapping for Bnms3 and Bnrf genes with large segregation population generated from sib-mating (9012A×9012B) and by full self-mating (9012B×9012B) of fertile plants, which from Huyouza 1 hao (F1 hybrid) originally. The locus specific PCR primers were developed on the basis of the syntenic region of the Arabidopsis genome, the Brassica BAC sequences and the published reference markers. The main results and conclusions are as following1. Fine mapping of the Bnms3 gene1) Screening markers linked to the Bnms3 gene:Based on published reports, we obtained 7 markers linkaged with the Bnms3 gene (SC1,SC5,SEP4B,SEP7, SEP8, AR23 and AR48), Of them, SEP7 was also mapped to SG (Sollux×Gaoyou) map in linkage group 10 (A10) Further, two markers nearby SEP7were proved to be also linked to Bnms3 (PBI and ZAAS133), PBI was a co-dominant marker. Thus, according to our result, Bnms3 gene was located on A10 of B. rape instead of C9 of B. oleracea as previously published. Finaly we choose 4 markers (PBI SEP7,AR23 and AR48) for future mapping Bnms3 gene in large population of 1896 plants.The Bnms3 gene was genetically mapped in a flanking region of 1.4 cM.Bnms3 gene was 0.4 cM from marker AR23 in one side and 1.0 cM from marker AR48 in another side.2) Fine mapping of the Bnms3 gene:According to the 4 marker mapping result of Bnms3 gene, we future developed 45 genome region specific primers from the syntenic search of the BAC clone in Brassica database between marker AR23 and AR48 and found 15 of them showing clear polymorphism between fertile and sterile plants. At last,6 markers (Ms3-6,Ms3-9,Ms3-10,Ms3-20,Ms3-32 and Ms3-44) showing clear co-segregation with the Bnms3 gene were selected for fine mapping in population of 1896 plants. The results future narrowed Bnms3 gene in a flanking region of 0.5 cM, with 0.1 cM from marker Ms3-32 and 0.4 cM from marker Ms3-6 in respective sides. Syntenic analysiswith Arabidopsis genome, a physical distance of 280 Kb between Ms3-6 and Ms3-32 was identified, which was 190 Kb less than published interval between flanking markers of Bnms3 gene.3) Co-dominant marker:Two of six co-segregation markers (Ms3-9 and Ms3-32) with Bnms3 showed co-dominant nature, with 0.4 cM and 0.1 cM to Bnms3 respectively. So far, all the published markers were dominant in nature.4) Identification of Bnms3 alleles in normal varieties:Using co-dominant marker PBI and Ms3-32,72 representative rapeseed cultivars worldwide were analyzed for allelic detection. The results indicated that all the checked cultivars were homozygosis locus for Bnms3 gene of BnMS3MS3. This result explained the reason for why almost all the normal rapeseed cultivars can be used as restorers.2. Fine mapping of the Bnrf geneInitially, more than 200 markers evenly distributed on SG-map were screened with sterile and fertile pools from temporary maintainer line and full sterility lines (1:1), searching for Bnrf linked markers. As a result, three markers (MR 166, R118 and sR0282) from A7 showed tightly linked with Bnrf gene. These markers together with another three published dominant markers (ESPSC1,ESPSC2 and XSC5) were used for co-segregation analysis in a F2 population of 864 plants. The result indicated that Bnrf gene was located on linkage group 7 (A7) of B. napus, which agreed with previously published result. The genetic distance between Bnrf gene to flanking markers MR166 and ESPSC1 were 3.2 and 6.0, respectively. One co-dominant marker sR0282 was detected, which located 11.9 cM away from Bnrf gene.All the co-dominant markers obtained from this research are positively contributed in present hybrid breeding.
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