Investigation And Genetic Diversity Analysis Of Paris Polyphylla Var. Chinensis

Paris polyphylla rhizome，a rare Chinese medicine, which possesses multi-pharmacological effects,such as clearing heat, detoxification, relieving swelling and pain, cooling the liver, calming frightened,anticancer, stoping bleeding, antibacterial and expectorant, relieving cough and asthma, sedation andanalgesia, resistance to cytotoxic, etc, has become the main raw material of many famous Chinesepatent medicines and has the broad development prospect. Paris polyphylla var. chinensis is one of thebase source plants for P. polyphylla rhizome recorded in “Chinese Pharmcopoeia”. Due to thedeterioration of their living environment and excessive human excavation, wild resources of P.polyphylla var. chinensis are diminishing gradually and even endangered in some places. Through thesystemically investigation，collection and genetic diversity research of P. polyphylla wild resources inZhejiang Province, this study try to understand the situitions of their distribution, living and geneticdiversity and establish their germplasm nursery to set a good foundation for their domestication,germplasm selection, reasonable utlization and effective protection. The mian results are listed asfollows:1. The total of124P. polyphylla var. chinensis wild germplasms was collected through theinvestigation of9representative areas in Zhejing Province including Lin’an, Pan’an, Kaihua andJingning, etc. The survey found that wild P. polyphylla var. chinensis resources had a wide distributionin Zhejing but with rare numbers and the most plants were scattered at the areas with an altitude of500-1500m, the annual average temperature of16℃and the annual precipitation of1400-2000mm.They grew under the moist shady areas such as the evergreen broad-leaved forests, bamboo forests,miscellaneous wood forests, bushes, forests by streams, slope of forests, etc, with the frequentlyassociated plants of lanceolata, sealwort, obtusiloba, lophatherum gracile, palm, nigatake, motherwort,araceae, ramie, lysimachia christinae, and houttuynia, etc..2.16botanical traits of48P. polyphylla var. chinensis germplasms from8provenances wereinvestgated and analyzed. The results showed there were certain degree of differences of each traitexisted between the different provenances and within different plants of the same provenance. Moreover,the variation extents were also different between16morphological traits. The average coefficient ofvariation was12.98%~42.64%with an rank from big to small of pedicel length> petiole length> petalwidth> plant height> stem diameter>petal length>leaf width>calyx width>leaf length>canopy>petalnumber>calyx number>stamen number>calyx length>stem number>leaf number. The overall averagecoefficient of variation on morphological traits from eight provenances are also different with adescending order of FH> JN> KH> SC> LS> TS> LQ> PA. According to the main component analysis,the results showed: both nutrient organs and reproductive organs had greater variations. In view ofnutrient organs’s traits influenced by eco-environments easily, the trait variations of reproductive organscould be inherited more stably. Based on morphological characteristcs P. polyphylla var. chinensisdisplay by UPGMA cluster analysis,48germplasms could be clustered into three categories. The firstcategory was samples from Kaihua, Pan’an and Taishun. The germplasms in Jingning, Suichang and Shunxi were clustered into the second category. The germplasms in Qingliangfeng, Lin’an and Fenghua,Ningbo were clustered into the third category.3. The optimum reaction system of ISSR-PCR was established for P. polyphylla var. chinensis: thereaction system was25μl, including0.2mmol/L dNTPs,0.75U TaqDNA polymerase,0.6μmol/Lprimers,2.0mmol/L Mg2+and40ng template. Reaction amplification program was:5min for95℃denaturation,40s for94℃denaturation,45s for52℃annealing,90s for72℃extension, whose cycleswas40, and7min for72℃extension.4.The selected13ISSR primers were applied in the amplification for48P. polyphylla var. chinensisgermplasms. A total of90bands were amplified with79polymorphic bands and a ratio of87.78%polymorphism. The polymorphic bands and polymorphism rates of8provenances were between26-46and28.89%-51.11%, the highest and lowest polymorphic provenances were samples from Lin’an andPan’an respectively; eight provenances Nei gene diversity index (H) between0.1133-0.2193, Shannonindex (I) between0.1654-0.3152, provenance Nei gene diversity index (H) and Shannon index (I) were0.2974and0.4475, indicating that P. polyphylla var. chinensis germplasms in Zhejiang possessed ahigh genetic diversity. The levels of genetic diversity of8provenances were ranked from big to small as:LQ> JN> LS> KH> FH> SC> TS> PA. According to the coefficient of genetic differentiation betweenprovenances (Gst) was0.4422, the conclusion could be drawn that the variations of genetic diversitywithin the same provenance were equvalent to those between different provenances. On the basis ofgenetic similarity coefficient, the UPGMA method was utilized to establish molecular level clusteringmaps for P. polyphylla var. chinensis germplasms. The48germplasms could be divided into twocategories, the first category was derived from Lin’an, Shunxi and Qingliangfeng germplasms, thesecond category included germplasms from Pan’an, Fenghua, Taishun, Suichang and Jingning, in whichcould be subdivided into two smaller categories, one with Pan’an’s and Fenghua’s germplasms, theother with Taishun’s, Jingning’s, Suichang’s germplasms. ISSR Cluster analysis results revealed thatthe genetic distance of wild P. polyphylla var. chinensis germplasms in Zhejiang presented certaingeographical features.