Development And Utility Of The Genome Wide SCAR Markers In Brassica Napus L

Rapeseed (Brassica napus, AACC,2n=38) is one of the dominant oil crops (soybean, helianthus, rapeseed, peanut) in our country. More and more cultivars of rapeseed were obtained according to the effort of the breeders along with the improved science and technology. However, a heavy challenge that the deficiency of effective and precise system to identify the potential lines for agricultural industry. Compared with the morphological markers and biochemical methods, the DNA fingerprinting were widespread employed to the determinate the maize cultivars duo to its high accuracy and high throughput screening model. The utilization of heterosis was considered as one powerful approach in the improvement of crops such as rice, maize and rapeseed. Understanding the molecular mechanisms of heterosis could supply a definite direction for breeding, however, it’s almost unclear in rapeseed. Some findings as following were obtained in the present study, the developed SCAR markers and functional markers designed according to the yield-related genes would provide some groundwork for rapeseed improvement.1. Development and optimization of the cultivar-specific SCAR markers for B. napus.The genes might involve in the flowering time, biotic and abiotic resistant, and responses to phytohormones were selected for marker development. A total of149markers were designed according to the96coding sequences (CDS) obtained by searching in the genomic database of B.rapa. The markers were screened by using DNA of eight B.napus accessions with different level of oil content as the templates. The putative genes with different PCR products among the tested samples were used as the subjects to search the homologs in the genome of B.oleracea. Forty three sequences were obtained and138makers were developed. The markers were employed to amplify the8eight core collection of B.napus. At last,67cultivar-specific SCAR markers were obtained with clear, stable and polymorphic products. In addition, the system for these SCAR markers was optimized and the best reaction conditions were established.2. Evaluation of the role of SCAR markers in genotypical analyses.The fingerprints of172winter rapeseed lines, the potential accessions for national regional trial from2010to2011, were constructed by using the67SCAR markers obtained. The relationship among the tested lines could be calculated according to the clustered data, suggesting that the SCAR maker system developed here is an effective technological for distinguishing and classification of the accessions. Four internal controls were used in the present study to evaluate the system, which need an improvement due to two of them were not consistent.3. Isolation the putative genes involved in the regulation of circadian rhythms and seed size in B. nap us for functional marker development.Primers designed according to the reported genes related to the yield and circadian rhythms in Arabidopsis and Rice were used to isolate the homologues in B. napus. The genes such as GWD3, SEX4, PORB, POPA responsible for the regulation of circadian rhythms, and the ones might involve in the dry matters accumulation (eg. BnWRIl, BnLECl, BnCYp78A, BnUP2, BnNAPIN, BnBBMl, BnFADl, BnWOX9, BnCPl, BnWOX2) were obtained and confirmed by sequence analysis. Nine functional markers were developed for association analysis between the rapeseed lines with the yield. Of course, further studies should be performed to known whether the markers were available in molecular assistant breeding because the physiological roles of these sequences were unknown in B. napus at present.4. Preliminary characterization of the two DH (Double Haploid) populations obtained by microspore culture.Two DH (doubled haploid) populations derived from different hybridized combinations of rapeseed were constructed by using microspore culture method. There were340(11-09-5515),145(11-09-5509) regenerate lines, respectively. Colchicine was employed to get the doubled haploid lines and55.29%,71.03%of them were succeeded respectively. The progenies with variance on flowering time were observed in the lines from11-09-5509. In addition, some putative plants were generated with higher oil content, and better composition of fatty acid than control in both two populations. The lines were screened out for breading and study of the molecular mechanisms.