| Brassica oleracea L. is originated from Med seacoast, it includes a few variations,such as cabbage, savoy, purple cabbage, borecole, cauliflower, broccoli, Brussels sprouts, kohlrabi, and so on. Now, it is the main vegetable in many countries around the world. At the present time, the Molecular Marker technology has been widely used in the study of many crops, but in a beginning stage on Brassica oleracea L.. In this experiment, we did 3 aspects of work to research Molecular Marker technology and its application based on Brassica oleracea L.. First, using the genomic DNA of cabbage as template, RAPD & SRAP & ISSR technology systems of Brassica oleracea L. were optimized. Second, establishing DNA fingerprinting to identify the purity of cabbage hybrid Xiaguang & Zaoxia 16 by SSR & RAPD methods, and Zhengchun & Hanguang 2 by SRAP & RAPD methods, and then change the fingerprinting into digital form. Third, BSA method was employed to study the immature-bolting characteristic and find its linkage RAPD markers. The major results are as follows:1. Optimization of RAPD & SRAP & ISSR technology system and primary application on Brassica oleracea L.The optimal RAPD reaction system with total volume of 20μl for cabbage was 2μl of MgCl2(25mmol/L) + 2.0μl of PCR Buffer(10×) + 0.5μl of dNTP(10mmol/L) + 0.5μl of Taq E (5U/μl) + 1.5μl of Primer(20ng/μl) + 3μl of DNA(10ng/μl) + 10.5μl of H2O, and the appropriate amplification procedure was as follows: 94℃for 5 min; 94℃for 1 min, 37℃for 1 min, 72℃for 1.5 min, 40 cycles; 72℃for 10 min.The optimal SRAP reaction system with total volume of 20uL for cabbage was 2μl of MgCl2(25mmol/L) + 2.0μl of PCR Buffer(10×) + 0.4μl of dNTP(10mmol/L) + 0.2μl of Taq E (5U/μl) + 30 ng of forward primer + 30 ng of reverse primer + 25ng of DNA + H2O, and the appropriate amplification procedure was as follows: 94℃for 5 min; 94℃for 1 min, 35℃for 1 min, 72℃for 1.5 min, 5 cycles; 94℃for 1 min, 48℃for 1 min, 72℃for 1.5 min, 35 cycles; 72℃for 10 min. The optimal ISSR reaction system with total volume of 20uL for cabbage was 1.8μl of MgCl2(25mmol/L) + 2.0μl of PCR Buffer(10×) + 0.4μl of dNTP(10mmol/L) + 0.3μl of Taq E (5U/μl) + 30ng of Primer + 30ng of DNA + H2O, and the appropriate amplification procedure was as follows: 94℃for 5 min; 94℃for 1 min, 50℃for 1 min, 72℃for 1.5 min, 40 cycles; 72℃for 10 min.The optimal reaction systems and amplification procedure were applicable on the eight species of Brassica oleracea L., and all eight materials can be distinguished by three technology respectively based on RAPD primer of S18, SRAP primer pairs of M3-E5 and M4-E5, and ISSR primers of 844 and 888.2. Purity identification of cabbage hybrid by Molecular Marker methodThe genomic DNAs of 4 hybrids (Xiaguang, Zaoxia 16, Zhengchun, Hanguang 2) and its parents were used as group of template DNA respectively, in order to select efficacious RAPD primers that could identify the hybrid. In 4 groups, 3 efficacious primers were gained in each group, they were S15, S42, S147 and S42, S78, S88 and S42, S103, S193 and S42, S89, S151 respectively. The specific RAPD fingerprints in the 4 groups could be established with S42 primer and then transformed into digital fingerprinting.3. Study of Molecular Marker of cabbage immature-bolting characteristicThrough selecting of 260 RAPD primers, we gained 4 primers, which can amplicate markers linked to the immature-bolting gene of cabbage. There were 5 linked markers showed from the relationship analysis result of F2 population with the 4 primers, and the linkage distances of the markers are different, the closest one is 8.58 cM. Three markers that were closest to immature-bolting gene correspondingly were examined of sequence and named S97-392, U16-446, and U16-562 according to fragment size and primer source. The result of sequence comparison between the 3 markers showed that there was little comparability, and no same DNA fragment or gene in GeneBank. |
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