Comparison And Identify Of Stemona Tuberosa Different Populations In Genetic Characteristics

Objective: In recent years,natural resources of Stemona tuberosa have been subject to excessive exploitation and utilization,as a result,finding a large natural population of this species becomes more and more difficult.The roots of this plant are used as a drug(Radix Stemonae Tuberosae),and unreasonable digging of them may result in regional extinction.Altogether,this study revealed S.tuberosa genetic characteristics and its classification at the molecular level,investigating affinity relationship between Stemona.spp.We hope these findings will provide useful information for subsequent introduction,cultivation,and utilization of medicinal plant resources,and for screening of specific alkaloids and their use in chemical classification,as well as for scientific selection of the genuine regional drug producing areas in the south of the Yangtze River Basin.Methods: The new plant genome extraction kit was adopted to extract Stemona.spp.genomic DNA.We used one-way ANOVA establish and optimize the ISSR-PCR reaction system response procedures.Using ISSR molecular marker and DNA barcoding technology analysis genetic diversity,sequence difference,cluster analysis and so on.Results: 1.Considering DNA purity,yield,test time and other factor,we chosed new plant genome extraction kit for plant material in this study.2.Through single factor method establish and optimize the ISSR-PCR reaction system response procedures,the optimal system was template DNA 30 ng,ISSR primers 0.4μmol/L,d NTP 0.15mmol/L,Mg2+ 2.5mmol/L,Taq DNA polymerase 1.5U,dd H2 O add to 25μL.3.7 ISSR primers which screenedform 100 ISSR primers(UBC)was used for the S.tuberosa by PCR.A total of 74 bands were obtain amplification reaction to 38 stemona spp material,among which 74 was found to be polymorphism.4.We also showed that the effective number of alleles(Ne)was 1.5347,the average Nei’s genetic diversity index(He)was 0.3189,the average Shannon information index(I)was 0.4841,the study revealed a high genetic diversity at the molecular level in different populations of S.tuberosa.On the basis of the matrix created from74 bands,the cluster analysis was performed using the UPGMA method and a dendrogram of 38 samples was established based on the SM genetic similarity coefficient.We can see that S.japonica,S.sessilifolia and S.parviflora were separated from S.tuberosa in the result,the population of S.tuberosa separated into three group,the first group included Guangxi Hechi,Baise,Yunnan Wenshan,Xichou,Guizhou Guanling,Xixiuqu;the second group included Guangxi Pingxiang,Longzhou,Daxin,Wuming,Dongxing,Fangchengqu,Hainan Lingshui;the third group included Guangdong,Hubei,Hunan,Fujian,Jiangxi,Sichuan,Chongqing,Guangxi Guilin,Hainan Baisha.5.Through the comparison of all sequence,the whole length ranged from 946 to 956bp.The difference in length of the sequence is mainly manifested in the absence and insertion of the base.The length of S.tuberosa was 946-951 bp,S.japonica was 946 bp,S.sessilifolia was 955 bp,S.parviflora was 950-961 bp.Variation sites was 56 bp account 5.66% in whole sequence,conservative sites was 914 bp account 92.42%,parsimony-informative sites was 20 bp account2.02%.6.We compute K2 P genetic distance between every two populations,the maximum distance was 0.031 between S.tuberosa and S.sessilifolia,the shortest distance was 0 in S.tuberosa species.The K2 P genetic distance in S.tuberosa species was 0.003,distance between S.tuberosa and S.japonica,S.sessilifolia,S.parviflora was 0.005,0.029,0.014 respectively.7.The Neighbor joining(NJ)and unweighted pair-group method with arithmeticmeans(UPGMA)method were used to construct phylogenetic trees,the results show that S.sessilifolia has big difference with others.S.parviflora can clustering alone.There are some classification in S.tuberosa species.Conclusion: This study revealed a high genetic diversity at the molecular level in different populations of S.tuberosa and indicated that the genetic relationship between these populations was correlated to geographical conditions at some extent.Cp DNA psb A-trn H region can identify different population of S.tuberosa well.These findings provide useful information for subsequent introduction,cultivation,and utilization of medicinal plant resources,and for screening of specific alkaloids and their use in chemical classification,as well as for scientific selection of the genuine regional drug producing areas in the south of the Yangtze River Basin.