| Anhui Polygonatum plants in this article were analyzed using ISSR. The species mainly includes Polygonatum sibiricum, Polygonatum cyrtonema Hua, Polygonatum filipes Merr, Polygonatum zanlanscianense Pamp and Polygonatun odoratum (Mill.) Druce. By using ISSR molecular marker method, the researchers have analysed the genetic diversity of14kinds of Polygonatum which selected in14different places of Anhui. And this research is from the DNA level. At the same time, by using the UPGMA method, the researchers have constructed a cluster tree map to do dendrogram cluster analysis. This kind of research was based on specificity band. The following main results was achieved by the research:1.Taking polygonatum as an object of study, and using CTAB method, agarose gel electrophoresis detection and OD values show that the extracted DNA bands were at around23KB, OD260/OD280are between1.6to2.0, indicating that the DNA quality has met the requirements of molecular biology experiments. And then the researchers have confirmed the suitable way to extract Polygonatum’s plant leaves genomic DNA.2. Uing5factors and4levels of orthogonal experiment and effecting the selection of factors of PCR’s reaction (these factors including Taq DNA polymerase buffer (Mg2+), template DNA, dNTPs and primers), the researchers have built suitable reaction system of1SSR-PCR of Polygonatum plants, a reaction system of25μl containing1.0U of Taq DNA polymerase,3.5mmol/L buffer (Mg2+),80ng of template DNA,0.08mmol/L dNTP and0.16μmol/L primer.3. First,60primers for Polygonatum plant genome PCR amplification were selected from100UBC primers(UBC801-UBC899). Then, above system ware used to determine the optimum annealing temperature of each primer by gradient PCR reaction. At last,10effect optimal ISSR primers were selected.4.10best selected ISSR primers were used to detect the genetic variations in14species of polygonatum, which have gotten115clear, reproducible bands and114of them were polymorphic (99%). One primer detected a mean of10-13bands,of which the concentration was93%and the values was mainly in200-3000bp.5. The NTSYS-pc2.10e was used to caculate Jaccard’s genetic similarity coefficient of14polygonatum. By using the method of UPGMA, the researchers have constructed a clustering analysis map. In14polygonatum plant, the most closely related plants is Yuchu that mining from Jiuhua Mountain and Huangshan.The genetic similarity coefficient is0.6870and the genetic distance is0.3754. And these data have shown that the genetic variation has not been raised by the different distribution. The most farthest phylogenetic related plants is polygonatum that collected from Jiuhua Mountain, Huangshan and Hefei Academy of Forestry. The genetic similarity coefficient is0.4261and the genetic distance is0.8531. And these data have shown that the degree of genetic variation is much larger than that in other regions.6. The genetic diversity and population genetic structure of four subclasses have been analysed with POPGENE version1.32software, genetic parameters of this reasearch are as follows:the effective number of alleles (Ne) is1.5888, Nei’s gene diversity index (H) is0.3489, Shannon diversity index (I) is0.5229, the percentage of polymorphic loci (PPB/%) is99.13%.Coefficients of gene differentiation of4subgroup populations of polygonatum as follows:Ht (total gene diversity) is0.3500, Hs (gene diversity within the population) is0.1985, Gst (the coefficient of gene differentiation) is0.4328, Nm (the number of migrants per generation) is0.6553. |
