| Objective:To investigate the genetic diversities and variations of seven Aconitum L. species (including Aconitum soongaricum Stapf., Aconitum karakolicum Rapaics., Acomitum leucostomum Worosch., Aconitum leucostomum var. nalatiensis F. Zhang, Aconitum nemorum M. Pop., Aconitum apetalum B. Fedtsch., Aconitum anthoroideum DC., total forty-one provenances), to supply DNA characteristics for accurate identifying between above crude drugs. Methods:Plant genome extraction kit was applied to extract DNA, ultraviolet spectrophotometer and electrophoresis methods were used to detect the concentrations and purity of DNA. The impact of facts were optimized for ISSR-PCR reaction, then determine the reaction conditions. Thirteen primers were selected out from Sixty ISSR primers, to analyze the DNA of total forty-one provenances of seven Aconitum L.species for ISSR-PCR amplification. Amplification of band was statistics and then to establish data matrix. NTSYS-PC2.10software used to analyze the polymorphic bands obtained, SHAN and UPGMA methods were used to make up the systematic diagram of genetic relationship, two-dimensions scatter-graph and three-dimensions scatter-graph were constructed by using the principal coordinate analysis; POPGEN32software are used to calculate genetic similarity, genetic distance and related parameters of genetic diversity level of seven species Aconitum L; With the analysis of molecular variance (AMOVA analysis) statistic variance of genetic data to analysis intraspecific and interspecific genetic differences in size. Results:The concentration and purity of DNA of Aconitum L.samples are comply with the requirement of the experiment by tested. The optimal ISSR reaction system suitable for Aconitum L. herbs was founded, which was:20μL reaction volume,10μL the2×Taq PCR Master Mix;2μL(100ng) template DNA;2μL(1.00μmol/L) primers. Amplification program was:pre-denature at95℃for4min; denature at95℃for30s; annealing at46.0～56.0℃for45s; extension at72℃for90s,35 cycles; last extension at72℃for7min; preservation at4℃. According to cluster analysis result of ISSR the forty-one provenances of Aconitum L. were classified into seven groups, which accorded pretty with the classification of species. Thirteen primers were used for amplification and a total of163DNA bands were obtained, including151polymorphic bands, the percentage of polymorphic bands (PPB) was92.64%. From the population, Shannon information index (1) was0.5182, the genetic similarity coefficient (H) was0.3525, observed number of alleles was1.9264, effective number of alleles was1.6314, The genetic identity was from0.6119to0.8933, and the genetic distances were from0.1128to0.4913. Based on Nei’s analysis the gent diversity(Ht) of Aconituw L. is0.3590, the gene diversity(Hs) within species is0.1297, the gene flow(NM) among species is0.2828, the coefficient of genetic differentiation(Gst) among species is0.6387, shows that genetic variations of Aconitum L. major exist in different species, the genetic variations among species is63.87%in the total genetic variations, genetic variations within the species is only36.13%. AMOVA analysis showed that the interspecific genetic amounted to57.7%of total variance, and intraspecific variation is only42.3%, there were significant differences(P<0.001). AMOVA results were consistent with the POPGEN32, Aconitum L. have certain genetic differentiation between various groups, genetic variation mainly exists in species. Conclusion:Germplasm resources of Aconitum L. showed abundant polymorphism and higher genetic variation, which might supply molecular level basis, ISSR molecular maker technology is useful for identifying species of Aconitum L. plants, and provide possible for building DNA fingerprint. |
PDF Full Text Request