Genetic Diversity Of Epimedium Plants In Chongqing Area

There are about 60 species of Epimedium in Berberidaceae worldwide,distributed in Europe and East Asia.China is the modern geographic distribution and differentiation center of this genus,with about 50 species.Epimedium plants are evergreen,with peculiar flower shapes and high ornamental value.In addition to being a source of nectar plants,some species are also the source of Epimedium,traditional Chinese medicinal materials,with great development potential.In this paper,through the investigation the wild resources in Chongqing,morphological research,component content analysis and ISSR molecular marker research,the genetic diversity of Epimedium plants in the area was understood,which provides an important theoretical basis for scientic protection and rational utilization of Epimedium plant resources in this area.Through field surveys and planting management of wild populations of Epimedium in Chongqing,we recorded the wild environment of Epimedium in detail,and the resource garden of Epimedium in Chongqing was established in Chongqing Medicinal Botanical Garden,and 47 living resources of wild Epimedium in Chongqing were collected and preserved,Eleven species have been identified,There were Epimedium acuminatum(sixteen populations),Epimedium sagittatum(seven populations),Epimedium leptorrhizum(three populations),Epimedium borealiguizhouense(five populations),Epimedium sutchuenense(two populations),Epimedium myrianthum(two populations),Epimedium epsteinii(one populations),Epimedium dolichostemon(two populations),Epimedium wushanense(five populations),Epimedium fargesii(three populations),Epimedium glandulosopilosum(one populations)。Morphological comparison of 11 species of Epimedium in Chongqing showed that the length of the spacing,sepal size,anther color,inflorescence and rhizome state were relatively stable in flower morphological characteristics,which could be used as morphological classification basis.The instability of plant coat or white powder,the number of leaves on the stem and the color of flowers in some species can be used as a reference for morphological classification.Epimedium acuminatum,Epimedium leptorrhizum,Epimedium sutchuenense,Epimedium epsteinii,Epimedium dolichostemon,Epimedium wushanense,Epimedium fargesii,Epimedium glandulosopilosum belong to the large flower group in 11 species.The Epimedium sagittatum,Epimedium borealiguizhouense and Epimedium myrianthum belong to the small flower group.Composition analysis showed that 39 of the 48 wild Epimedium crude drug samples were tested for total ash content in accordance with the standards of the 2015 Chinese Pharmacopoeia,with the qualified rate of 81.25%.For the content of dilute ethanol cold extracts,41 items met the pharmacopoeia standards,with a pass rate of85.42%.Three of the icariin content items tested met the pharmacopoeia standard,with a qualified rate of 6.25%.Nineteen of the total flavonoids content items met the pharmacopoeia standard,with a qualified rate of 39.58%.The comprehensive statistical results of five index components including moisture,total ash,dilute ethanol extract,icariin,and total flavonoids content show that there is only one sample of wild Epimedium in Chongqing that meets the pharmacopoeia standards.Morphological observation was conducted on 47 living plants of the population collected and preserved in Chongqing Medicinal Botanical Garden.Morphological characteristics of 20 character with obvious differences,such as root morphology,number of flower stems and leaves,and type of inflorescence,were evaluated,and cluster analysis was conducted by UPGMA method.The results showed that Epimedium plants in Chongqing can be divided into two branches by five morphological indexes,namely flower diameter,petal shape,petal length/inner sepal length,pedicel and inflorescence axis.Morphological characters and contents of total flavones,icariin and other contents were extracted by principal component analysis with the characteristic value greater than 1 as the standard.A total of 7 principal components were obtained,and the cumulative variance contribution rate was 79.464%.Based on the results of cluster analysis and principal component analysis,flower diameter,petal shape,petal length/inner sepal length,inflorescence type and number of flowers are of great value in the classification of Epimedium plants in Chongqing.The genetic diversity of Epimedium plants in Chongqing area was analyzed by ISSR molecular marker technology.A total of 12 primers were screened and 210 DNA bands were amplified,of which 197 were polymorphic bands,accounting for 93.83% of the amplified bands.The results show that the similarity coefficient between different species is high,with the similarity coefficient above 0.56.Among all the tested materials,the highest similarity coefficients were NC-001,NC-002 and NC-002 as well as NC-002 and NC-003,whose similarity coefficients were all 0.871.The results of cluster analysis showed that the 47 materials could be divided into 3 groups with the threshold value of 0.668.The first group was YY-001 of Epimedium borealiguizhouense from Youyang.The second group mainly includes four species of Epimedium myrianthum、Epimedium dolichostemon、Epimedium epsteinii 、Epimedium glandulosopilosum,and part of Epimedium sagittatum、Epimedium leptorrhizum、Epimedium wushanense、Epimedium sutchuenense.The third group mainly includes three species of Epimedium fargesii、Epimedium acuminatum、Epimedium borealiguizhouense.and part of Epimedium sagittatum 、 Epimedium leptorrhizum 、 Epimedium wushanense 、Epimedium sutchuenense.The results of the clustering were consistent with the results of plant taxonomy and geographical distribution,which combined the diversity of species and the difference of geographical location in taxonomy.