The Purity Identification Of Cucumber Hybrid With Rapd Markers

With the rapid development of Molecular Biology, RAPD (Random AmplifiedPolymorphic DNA) has been applied widely in Biological fields. This thesis focused onthe application of RAPD to the purity identification of Cucumber hybrid seeds andresearched into the optimal conditions of RAPD.It was analyzed that methods to extract DNA template and different tissues hadeffects on RAPD. The RAPD products with total DNA extracted by improved SDSmethod from dry seeds and by "one-step" method from the yellowing young plants werethe same as those with total DNA extracted by the traditional CTAB and SDS. Beingsimple and rapid, two methods could reduce the cost and make extraction of DNA veryeasily; thus they were suitable for the purity identification of Cucumber hybrid seeds.At the same time, It was shown that different tissues had no influence on RAPD.The paper also discussed the factors that influenced RAPD. The results showed thatthe best 2ⁱ for RAPD was 1.5-3.0 mmcl. L1 the best dNTPsconcentration was 0.1 5-0.2SmmoI. L1 suitable quantity of Taq polymerase 0.5-1 .5U;suitable template DNA quantity 20-1 OOng every reaction and the best concentration ofrandom primer 0.2-0.4 ⁱ mcI. V'. In addition, it was further found that Taq polymerasewith different factories and periods had effects on RAPD when the reaction system ofRAPD was optimized, and that there were effects of random primers with differentbases and periods on RAPD.The reaction procedure was improved and optimized. The reaction pre-denaturedDNA at 940Cfor 4mm, denatured DNA at 940C for 30sec, annealed primers at 370C for30sec, extended primers at 720C for 1mm, re-extended reaction at 720C for 7mm after35 cycles and then stored the products at 4 0C.The variety homogeneity was identified and the parental polymorphisms wereanalyzed. The paper identified the homogeneity of the parent seeds and the hybrid seedsand analyzed the polymorphisms of the parents before screening the primers. Theresults were that the three false seeds were found among 30 Cucumber hybrid seeds3E?1ⁱtZ 1tJ⁽Cucurnis sativus L. ^*PPflNi^J RAPD ^with three primers S 100, Si 133 and Si 151. The parental homogeneities were very goodbecause the fingerprints of RAPD were the same among 7 female seeds and 7 maleseeds. Therefore, there were no false seeds among them. It was shown that the parentshad abundant polymorphisms when their polymorphisms were identified with 6 primersS48, 5171, S443, 51019, 51062 and 51357.The primers, which were suitable for the purity identification of Cucumber hybridseeds, were screened and the fingerprints of RAPD were established among the parentsand their hybrid seeds in Cucumber. 32 primers were screened from 102 the ones thatcould be beneficial to the purity identification of Cucumber hybrid seeds. The resultsshowed that there were 13 primers of partial female type, 9 primers of partial male type,5 primers of complementary type, 2 primers of peculiar type and 3 primers of shortagetype. Hence, their percentages were 12.74%, 8.82%, 4.90%, 1.96% and 2.94%respectively. It was found there were 1 female seed, 2 male seeds and 6 false seedsamong 94 Cucumber hybrid seeds when the purity of Cucumber hybrid seeds wasidentified with 4 primers S287, 5293, S297 and S300. Therefore, the purity of theCucumber hybrid seeds was 90.04%. The fingerprints of RAPD were established forZhaoQing No.2 Female SystemT03, ZhaoQing No.2 Male System B35 and their hybridseeds in Cucumber.As a result, the paper shows that RAPD can be applied to purity identification of thecrop seeds and that RAPD has good reliability and accuracy after the reaction systemand the reaction procedure are established.