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The plants in genus Bletilla Reichb. f. possess multiple medical uses in traditional Chinese medicine. However, most wild plants of this genus are critically endangered. In this paper, we searched and combed the origins of these medicinal plants, compared and identified the common adulterants. To explore the suitable soil, climate, and geographical conditions for the artificial cultivation of Bletilla Reichb. f. plants, we investigated and described the natural habitats in different spatial scales, collected and sorted out the information of geographical distribution for the wild plants. We also systematically summarized and prospected the technology of artificial breeding for genus Bletilla Reichb. f.. Based on the characteristics of the reproductive system and breeding features for Bletilla Reichb. f., the seed aseptic germination is considered to be the most appropriate breeding method. Therefore, the development of a new-generation technology for direct seeding germination and the breakthrough of the key cultivation technology will greatly improve the reproductive efficiency of Bletilla Reichb. f., resulting to change the mode of plant-resource supply. The formulation of more specific protective measures together with the acceleration of the research on artificial reproduction technology and the enhancement of the basic research on the resources and reproduction, will facilitate to improve the efficiency of the protection and utilization for the Bletilla Reichb. f. plants, which shows the essential significance for sustainable utilization of medicinal resources of Bletilla Reichb. f..
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OBJECTIVE: To analyze the genetic diversity of Lysimachia christinae Hance germplasm resources from 36 different places of origin with ISSR DNA markers. METHODS: Ten primers screened from 45 ISSR primers wrea mplified with PCR and the amplification products were examined by 1.5% agarose gel electrophoresis. POPGENE32 software was used for statistical analysis of genetic parameters and UPGMA method (NTSYS2.10 software) for cluster analysis and dendrogram constructing. RESULTS: A total of 91 sites were amplified from the 10 ISSR primers, of which 80 sites were polymorphic loci. The percentage of total polymorphic loci (PBB) was 87.91%. The number of observed alleles (Na) was 1.879 1 and the number of effective alleles was number (Ne) 1.381 7. The Nei's genetic diversity index (He) was 0.239 0 and the Shannon information index (I) was 0.374 5. The genetic similarity coefficient of the 36 germplasm resources varied from 0.69 to 0.97 and the 36 materials couldbe divided into six categories at the similarity coefficient of 0.76. Clustering results showed that the samples within small geographic range clustered together in the dendrogram, indicating the nearer genetic relationships of them. Meanwhile, the samples from different provinces or cities could not be differentiated from each other through cluster analysis. CONCLUSION: The germplasm resources of Lysimachia christinae have rich genetic diversities. The genetic diversities of Lysimachia christinae have no significant correlation with their geographic distribution.
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In order to establish the stable andreliable ISSR-PCR System of Lysimachia christinae, L16 (4(5)) orthogonal design, which based on 7 levels of single factor experiment, were used in this study. The variance analysis was carried out by SPSS 19.0, and 5 main factors affecting the reaction system were optimized in 4 levels. The best annealing temperature was selected by the optimized reaction system. And the stability and reliability of this system was tested by 23 samples from different origins. The results showed that the five factors (DNA template, primer, dNTP, Mg2+ and Taq enzyme) were the most impacts on the amplified results of ISSR-PCR of L. christinae. The order of the influence was: primer > Taq enzyme > DNA template > Mg2+ > dNTP. The optimal system, which was determined by multiple comparison on different levels of each factor, was total volume of 25 microL, including DNA template 60 ng, primer 0.3 micromol x L(-1), dNTP 0.2 mmol x L(-1), Mg2+ 1.8 mmol x L(-1), Taq enzyme 1.25 U. The optimal system was stable and reliable tested by 23 samples from different origins. This study lays the foundation for genetic diversity analysis, fine varieties selection and molecular identification of L. christinae, and provides reference for optimization on ISSR-PCR system of other speciesin future.