| This thesis deals with the RAPD analysis for the germplasm materials of "Kudingcha Tea" in Holly, putting the stress on Ilex kudingcha C.J.Tseng. The research results showed that the optimal PCR system for the RAPD analysis of Ilex kudingcha C.J.Tseng was that a 25l reaction mixture system contains: 20ng-80ng template DNA, 200mol/L-400mol/L dNTPs, 1. 5mmol/L-2.0mmol/LMg2+ and 5pmol arbitrary primer, 1.5U Taq DAN; and that the optimal amplification program is 45 cycles of 4 min at 94 ℃ for pre-melting, 30sec at 94 ℃ for melting, 30sec at36 ℃ for annealing, 2min at 72 ℃ for elongation; 7min at 72 ℃ for post-elongation and the ampli fied tubes are taken out of the TGRADIENT Thermal Cycler at 4℃. The RAPD analysis of all experimental materials used in this project was carried on under the optimal PCR experimental system and reaction program mentioned above. 22 germplasm materials of Kudingcha Tea, from 4 species in Holly, i.e. Ilex kudingcha C.J.Tseng, I. latifolia Thunb., I. pentagona S.K.Chen,Y X. Feng et C. F. Liang, I. cornuta Lindl. and a intermediate type (it lies between I. kudingcha and I.pentagona ) were analysis for PCR amplification. 50 10-bp random primers were used for the polymorphism test of RAPD, and 27 random primers with optimal polymorphism were selected from the 50 random primers. The 27 random primers were used for the amplification on 22 germplasm materials mentioned above, and 498 bands were obtained. 484 bands were polymorphic bands, which amounted to 97. 18%. Moreover, another 57 germplasm materials of /. kudingcha, 6 germplasm materials of I. pentagona and 6 germplasm materials of the intermediate type were used for PCR amplification. 22 random primers with ideal polymorphism were selected from 50 10-bp random primers. The 22 random primers were used for PCR amplification of the 69 germplasm materials above, and 298 bands were obtained. Of all the bands, 274 bandswore polymorphic hands, which amounted to 91.9%. UPGMA cluster analysis dendrograms showed that the conclusions of RAPD analysis could he considered as the basis of molecular taxonomy on species level and infra-specific classification, which can be used for studying the genetic difference, relationship and classification of "Kudingcha Tea " in Holly, among different species, or among the different germplasm materials of the same species. In fact, using RAPD technique, not only the 4 species and a intermediate type of "Kudingcha Tea " in Holly can be distinguished clearly, but the germplasm materials of the same species (I.kudingcha C.J.Tseng), from the different origin regions, or from the different population can also be distinguished clearly. The different germplasm materials in the same origin region had smaller genetic distance, while the germplasm materials from different origin regions had greater genetic distance. Especially, there was more significant genetic difference between the germplasm materials of I. kudingcha from Hainan Island and that from the different origin regions of the Main Land. Seeing that the morphological characteristics of the germplasm materials are significantly different from the germplasm materials from the different origin regions of the Main Land, the author thinks that the population on Hainan Island can be classed as an ecotype. The typical germplasm materials of I.pentagons clustered as a independent cluster, and the 6 germplasm materials of the intermediate type clustered as another independent clustered, which is simultaneous to the cluster of the typical germplasm materials of I. pentagons. The author, based on the morphological characteristics and the clustering analysis, thinks that the intermediate type can be classified as a varietas. The author inferred that this varietas was perhaps from the natural cross in the system development.
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