Fine Mapping Of The Recessive Genic Male-sterile Restorer In Brassica Napus

The utilization of heterosis in rapeseed is an efficient pathway to increase yield and improve qulity. The recessive genic male sterility (RGMS) line with an epistatic inhibitor gene had been found by breeders in 1990s'. Studies by years revealed that the RGMS line could be restored by any other lines, and could generate a complete and stable pollination by a temporary maintainer, so when it was used to produce the F₁ hybrid seeds, it didn't require to remove 50% male fertile plants from the mother line. Therefore, it will be an important method to utilize the heterosis in rapeseed. The male sterile line 7-7365A, bred by our laboratory, had been proved to have the same genetic mechanism.In this research, we use two-type lines 7-7365AB as materials and carry out some works on identifying the male sterile restorer gene, BnMs₃, as follows:1. Thirteen DNA fragments obtained from AFLP markers was cloned and sequenced. Then we designed special PCR primers for each sequence. Results showed that five AFLP markers were successfully converted to SCAR markers, and they distributed both sides of the targeting gene.2. In April 2006, we sampled 1997 male sterile individuals from 7-7365AB lines, extracted their genome DNA and analyzed them with SCAR markers or AFLP markers in order to ascertain the accurate genetic distances between the markers and the targeting gene. In July 2006, we sampled 1861 male sterile individuals again and repeated above-mentioned experiments. The results revealed that the comparative position between the markers and the targeting gene was consistent with previous researches, the two nearest makers are SCAR11 (AFLP11) and AFLP16 both in two populations, interestingly, the precise genetic distances of SCAR11 (AFLP11) are 0.3cM and 0.1cM, the precise genetic distances of AFLP16 are 0.1 cM and 0.4cM.3. The results from BLAST analysis showed that the five sequences had homologous sequences in the genome of Arabidopsis, and they could be located on the same chromosome. We separated flanking sequences of the two nearest markers by the technology of PCR Walking, and the two new sequences could still be located on the original positions after prolongation.4. On base of the region of Arabidopsis locked by the two nearest markers, we searched some homologous sequences from the database of Brassica napus and designed fifteen pairs of primers for these sequences. Finally, one pair of primers showed polymorphism between the parents.5. We extracted, cloned, and sequenced some fragments generated from the primers which didn't show polymorphism. Some fragments didn't show differences between the male sterile and the male fertile plants, and then we designed PCR walking primers and separated the flanking sequences of both sides. The results elucidated that there were no variation of insertion, deletion, as well as restrictive bases between the parents, but the sequences which derived from the same parent showed homology after digested by different restrictive endonucleases, thus we hypothesized that it was the complexity of brassica genome that resulted in the difficulty of chromosome landing in Brassica napus.