Studies On Genetic Diversity Of Eucommia Ulmiodes Oliver And Active Constituents Of Eucommia Ulmiodes Leaves With Different Regions

Objective:In order to investigate the resource status of Eucommia ulmiodes Oliver. in China, and establish SSR-PCR (simple sequence repeat-polymerase chain reaction) reaction system for the study of genetic diversity and relationship of germplasm in Eucommia ulmoides using simple sequence repeat (SSR) markers. Explore differences in chemical constituents in the different origin of Eucommia ulmoides leaves that using high performance liquid chromatography (HPLC). It was in order to protect the biodiversity of Eucommia ulmoides, promote the sustainable development of Eucommia ulmoides resources and exploitation and utilization of traditional Chinese medicine to explore genetic differences and differences in chemical composition between Eucommia ulmoides leaves with different regions.Methods:(1) The main factors affecting amplication results were studied with single factor selection and L16(45) orthogonal design. The obtained data were analyzed by software SPSS.(2)29pairs of EST-SSR primers and20pairs published SSR primers were screened. The genetic diversity and relationships of125individuals for different areas were analyzed using highly polymorphic primers.(3) The main active ingredients of Eucommia ulmoides leaves were compared using an Agela Technologies Promosil C18(4.6mm×250mm,5μm) chromatographic column, with a gradient elution of acetonitrile-0.1%phosphate solution as mobile phase gradient elution, detection wavelength at210nm, to evaluate the main effective components between different regions of Eucommia ulmoides.Results:(1) The optimal reaction system was as follows:2μL10×PCR buffer,0.5U Taq DNA polymerase,1.25mmol·L-1Mg2+,0.2mmol·L-1dNTP,0.3μmol·L-1Primer and60ng DNA template in20μL reaction solution. The order of the influence was:Mg2+> Taq DNA polymerase> Primer> dNTP> DNA template.(2) The18pairs of highly polymorphic primers were screened and74clear bands were amplified totally. Percentage of polymorphic loci was95.95%～78.38%. Nei’s genetic diversity index (H) was0.3620～0.3114, Shannon’s information index polymorphism (I) was0.5285～0.4550. Gene differentiation coefficient (Gst) was0.1149. The coefficients are the largest in Shimen, second in Sangzhi, and the lowest in Lueyang. Eucommia ulmoides from Shimen showed a closer systematic relationship with Eucommia ulmoides from Sangzhi. The genetic distance was the largest between Chongqing and Lueyang.(3) Determination of active ingredient content of different regions and the result of cluster analysis showed that, the quality of Eucommla ulmoides leaves in Guizhou Zunyi and Chongqing Xiushan were the best, the quality of Shanxi Lueyang was the worst.Conclusion:The optimized SSR-PCR reaction system was suitable for the study of relationship and genetic diversity of Eucommia ulmoides among the different populations. This analysis of Eucommia ulmoides populations genetic relationship and genetic diversity reflected that Eucommia ulmoides populations had genetic diversity and low genetic diversity among different populations. The results showed that there is a larger gene flow between the populations. The difference between the content of active components of Eucommia ulmoides leaves reflected from the quality difference of Eucommia ulmoides from different areas, and provided scientific basis for the evaluation of the quality of medicines and breeding of superior species.