Development Of Molecular Markers Associated With Polima CMS Restorer Gene In Resynthesized Brassica Napus L.

Rapeseed oil is the main edible vegetable oil in China. Brassica campestris L. was eventually taken place by Brassica napus L. in semi winter type rapeseed cultivation region in China due to higher disease resistance and higher yield in B. napus. Strong heterosis has been observed by many breeders in B. napus. As a results, many breeders are focusing on the breeding of rapeseed varieties with high yield and quality by the aid of heterosis utilization in modern rapeseed breeding program.There are several methods for utilizing heterosis in rapeseed breeding, such as genic male sterile （GMS）, cytoplasmic male sterile （CMS）, genic and cytoplasmic male sterile （GCMS）, self-incompatibility （SI） and chemical hybridizing agents （CHA）. Among them, CMS has been widely used in hybrid production in China. Therefore, breeding of CMS sterile line and its restore line, the key components in CMS heterosis utilization method, was paid more attention by breeders. However, the genetic basis of B. napus in China is narrow since B. napus was originated in Europe and then introduced to China. This is adverse to the long-term heterosis utilization breeding program in China. Luckily, China was one of the origin center of B. campestris. Then, the genetic diversity of B. napus could be enlarged using the B. campestris germplasms by resynthesizing B. campestris with Brassica oleracea. Some new male sterility or restoration gene resources might be identified among the resynthesized B. napus lines. Which will be beneficial to both genetic and breeding researches of the heterosis utilization.In this study, genetic segregate populations were constructed using a pol CMS restorer line23-002-1, which was screened from resynthesized B. napus lines. Then conventional AFLP technology and bioinformatics technology were used together to develop molecular markers that tightly linked to the restoring gene（Rfp） in the resynthesized B. napus line. The results will promote the utilization of the restoring gene in rapeseed breeding program and the cloning of the pol CMS restoring gene. The main results of this study are as follows:1. Stepwise backcross population A and B that contain Rfp gene were constructed using resynthesized B. napus restoring line23-002-1, pol CMS line1141A and its maintainer line1141B and NPZ05. The ratio of fertility individuals and sterility individuals in both stepwise backcross populations were agreed with the expected ratio1:1. It was concluded that the parent line carried only one Rfp locus.2. Two DNA restriction endonucleases combinations, EcoRI/MseI and SacI/TaqI, were used to identify AFLP markers associated with Rfp locus by bulked segregant analysis （BSA） method. Two AFLP markers （EA08/MC10-180and EA08/MG05₂30） were identified linked to the Rfp locus from a total of1,280AFLP primer pairs. These two markers flanked on different sides of the Rfp locus with the minimum genetic distance of5.6cM between the closest marker and Rfp locus.3. EA08MC10-180and EA08MG05-230were cloned and sequenced. The size of these two fragments were162bp and215bp, respectively. One of the AFLP marker （EA08MG05₂30） was converted to SCAR marker （SCL2） successfully.4. A homologous Scaffold of Brassica rapa was identified based on the sequence information of the two AFLP markers. Then, this Scaffold was chosen to design microsatellite （SSR） and SCAR primer pairs. Sixty SSR primer pairs and110SCAR primer pairs were designed and12markers were identified strongly linked to the Rfp locus.5. It was concluded that the Rfp was located on the chromosome9of B. napus combining the information of5markers （BRSC01, BRIP14, BRIP47, BRIP33and OS36）, the homologous Scaffold of B. rapa and the map location of Rfp gene determined by the integrated DH genetic linkage map.6. To provide versatile molecular markers for marker aided selection of elite restorer line, the versatility of these12markers was validated with another two genetic segregated populations （a NILs and a DH） besides the two stepwise backcross populations. The results shown that OS36and BRIP14were the most versatile molecular markers, and BRIP33was the second.