Analysing Genetic Diversity Of Panax Notoginsengbased On CpDNA Molecular Markers

Based on NCBI,this study has developed the molecular markers using the published chloroplast genomes data of Panax notoginseng and analyzed the genetic diversity of 89 samples from 9 populations.We choosed Wenshan as the center,and nine samples' places were Wenshan,Yanshan,Malipo,Maguan,Qiubei,Guangnan,Jianshui and Mengzi.We extracted DNA from the fresh leaves by using the CTAB methodand kit.Molecular markers are made up of three types,including chloroplast genome Simple Sequence Repeat(cpSSR),Chloroplast genome(cpDNA)polymorphism and the matK gene.The number of cpSSR is 90.There are seven successful primers,besides,the primer of cpSSR-5 showed obvious stripe difference.The type of three base repeating units is most than other while two base repeating units is least in cpSSR.The cpSSR distribution location in the coding gene area is higher than intergenic region.Based on the type,frequency and number of repetitions of repeating units,we focused on the areas are psbJ-psbL,rpl14-rpl16 and ycf1.Based on the sequence alignment,we have obtained thirty polymorphic sites,including 14 SNPs and 16 InDels.Then,we got the eleven primers base on the polymorphic sites.Contrary with cpSSR,the distribution location in the coding gene area is less than intergenic region.According to the number of polymorphic sites in the region,we identified key areas were trnH(GUG)-psbA,psbM-trnD(GUC),rpl14-rpl16,atpF-atpH,psbZ and ycf1.It's worth noting that the area of rpl14-rpl16 belonged to cpSSR and cpDNA polymorphism.Consider the differentiation of DNA gel electrophoresis,the study will focus on cp InDel,and the primer of pgcp019 and pncp08 can be used for the differentiation of Panax notoginseng population.The gene matK is the control group,because of no polymorphic sites in the region.The results of gel electrophoresis did not observe the obvious difference of bands.So,it is necessary to further verify polymorphism by sequencing.The gene ycf1 was similar to rpl14-rpl16 that belonged to cpSSR and cpDNA polymorphism.Although the difference was obvious from the gel results,there were non-specific bands,and no further studies were conducted.Through the above analysis,the four primers(cpSSR-5,pgcp019,pncp08 and pncp-M)amplification were sequenced.Base on the sequence of cpSSR-5,we acquired the two and ten kinds of haplotypes base on SNP and InDel,respectively.We coulde distinguish four types population by Combination cpSSR-5 haplotype.Using the same method,pgcp019 got five and seven kinds of haplotypes,respectively,and distinguished five types groups.pncp08 obtained four kinds of haplotypes,and distinguished two types groups.pncp-M got nine and fourteen kinds of haplotypes,respectively,and distinguished six types groups.The primer pgcp019 contains polymorphic locus,but the matK gene has no polymorphic locus.However,the primer pgcp019 and matK gene had some degree of differentiation.This indicates that the polymorphic region has a certain reference value.It also indicated that the polymorphic region was determined based on the four cpDNA of Panax notoginseng,and it could not contain all the polymorphic regions.Based on the sequencing of the primer pgcp019 and matK gene,the NJ phylogenetic tree was constructed,we got four and six group of classes,respectively.Use multiple indicators(length of sequence,nucleotide diversity and average number of nucleotide differences),we determine pgcp019 is the best primer to distinguish between groups.Combination all kinds of haplotypes,we finally gained 39 kinds of haplotypes,can distinguish all kinds of groups.Based on the number of haplotypes within populations,the highest diversity of population 3(Malipo)was determined.