| The clustering relations of medicinal plants in Asteraceae, the optimization of the RAPD (Random Amplified polymorphic DNA) protocol, the analysis of genetic diversity and convertion of the RAPD marker into SCAR (sequence characterized Amplified Region) marker in Coptis were studied in this paper. The main results were as follows:1. Seven species of medicinal plants within Asteraceae were analyzed using random amplified polymorphic DNA (RAPD) markers 50 primers were screened by RAPD, while 10 selected primers were able to produce 337 clear polymorphic bands which were all polymorphic. The results were generated by UPGMA algorithm to cluster genetic distance caculated from RAPD data. The analysis revealed that the genetic results based on RAPD markers were consistant with that of the traditional classification to some extent.2. The genomic DNA were extracted from the Coptis, including the C. chinensis Franch, C. omeinensis C. Y. Cheng and C. deltoidea C. Y. Cheng. The genomic DNA of C. omeinensis C. Y. Cheng was used to optimize the RAPD reaction protocol in Coptis, which contained screening the DNA polymerases. Furthermore the advantage of introducing TD-PCR (Touch Down PCR) was studied through the combinations of three primers/one template and one primer/two templates. It was showed that in order to obtain the specific and reproducible marker, in the 25μl RAPD-PCR, the reaction system could be described as the proper concentration of primer, template and dNTPs was 0.32μmol/L,0.40 mg/L and 0.25 mmol/L respectively, and the best DNA polymerase was PyrobestTM DNA polymerase. The optimized reaction process also was listed in the following,2 min for a predenaturation step at 94℃; 50 s at 94℃, and 1 min at 72℃for denaturation and extension, in the first 7 cycles, the annealing temperature was initiated from 39℃, and then 0.5℃lower per cycle, in the remaining cycles, the annealing temperature was constantly kept at 36℃and a total of 40 cycles; 5 min at 72℃for a final extension step. In our assay, the TD-PCR played important role in enhancing the specific amplified fragments, and based on certain target to screen the DNA polymerase could be favored to gain the scientific result.3. Based on the optimization of the RAPD protocol,34 selected primers were able to produce 528 clear polymorphic bands in all 10 materials of Coptis and 325 in C. omeinensis C. Y. Cheng, the percent of polymorphic bands was 86.36% and 34.15% respectively. In the RAPD fingerprints of 10 Coptis plants using S55, the three species including C. chinensis Franch, C. omeinensis C. Y. Cheng and C. deltoidea C. Y. Cheng could be characterized. By clustering the 10 plants among Coptis by RAPD, it was shown that all the C. omeinensis C. Y. Cheng were clustering together, C. deltoidea C. Y. Cheng collected from different ecological environment also had high similarity. C. chinensis Franch had higher similiraty with C. deltoidea C. Y. Cheng than C. omeinensis C. Y. Cheng. Meanwhile, the clustering result of C. omeinensis C. Y. Cheng was consistent with their ecological environment.4. The RAPD marker which was observed in the amplified fingerprints of the C. omeinensis C. Y. Cheng with phoenix-tail type (Sandaohe), C. omeinensis C. Y. Cheng with double-wings type (Sandaohe) and C. omeinensis C. Y. Cheng with phoenix-tail type (Huangwanchadi) was cloned, and the size of the sequence was 954 bp. Then the RAPD marker was converted into SCAR marker by designing the specific primers. The favorable annealing temperature was 68.5℃.
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