The use of RAPDs for assessment of identity, diversity, and quality of Echinacea (original) (raw)
Related papers
Identifying commercially relevant Echinacea species by AFLP molecular markers
Genome, 2009
The rising interest in medicinal plants has brought several species of the genus Echinacea to the attention of many scientists. Echinacea angustifolia, E. pallida, and E. purpurea are the most important for their immunological properties, well known and widely used by the native Americans. The three species are easily distinguishable on the basis of their morphological characteristics, but it would be difficult, if not impossible, to distinguish them in commercial preparations of ground, dry plant parts of E. purpurea (the most valuable species for chemotherapeutic properties) mixed with the other two species. Species-specific molecular markers could be useful to address this issue. In the present work, using fresh material collected from cultivated Echinacea spp., AFLP analysis was used to discriminate the three species and to detect species-specific DNA fragments. By using 14 primer combinations it was possible to detect a total of 994 fragments, of which 565 were polymorphic. Overall, 89 fragments were unique to E. purpurea, 32 to E. angustifolia, and 26 to E. pallida. E+CAC/M+AAT or E+CAC/M+AGC alone provided 13, 9, and 4 or 7, 5, and 5 specific fragments for E. purpurea, E. angustifolia, and E. pallida, respectively. A validation trial to confirm the results was carried out on bulked samples of 23 accessions covering most of the genetic diversity of the three species. The results are discussed in terms of practical applications in the field of popular medicine, detecting frauds, and implications for the genus Echinacea.
Plants
The Echinacea genus includes a number of species that are commercially employed for the preparation of herbal products. Echinacea angustifolia DC. is one of these and is widely used, mainly for its immunomodulating properties, as it contains a wide range of compounds that belong to different chemical classes. In particular, echinacoside, cynarin and lipophylic alkylamides are the main specialized metabolites of the roots and can be considered to be marker compounds. In this work, 65 E. angustifolia accessions have been compared in a field trial in Italy, with the aim of investigating the variability/stability of the weight and chemical composition of their roots in order to identify the accessions that are most promising for future genetic-improvement programs. The morphological characteristics of the aerial parts have also been investigated. Seventeen samples were discarded due to germination or plantlet-development issues. Seven of the remaining accessions were identified as being...
Fitoterapia, 2007
In our previous study, RAPD (Random Amplified Polymorphic DNA) analysis revealed species-specific markers for three medicinal Echinacea species (Asteraceae): E. angustifolia DC., E. pallida (Nutt.) Nutt. and E. purpurea (L.) Moench. In the present work, we have converted a RAPD marker (750 bp) for E. purpurea into a SCAR (Sequence Characterized Amplified Region) marker. SCAR-PCR, in fact, revealed the expected amplicon (330 bp) only in E. purpurea and not in the other two species, giving further evidence for differences in medicinal Echinacea spp. genome and confirming a greater similarity between E. pallida and angustifolia.
The Study of Several Genotypes of Echinacea Purpurea (L.) Moench
Research aimed at the evaluation and selection of biological material, genetically valuable for several genotypes of Echinacea purpurea (L.) Moench. The experiment, that took place during the years 2008 şi 2009, aimed the phenological study and the content of active principles in several genotypes of Echinacea purpurea (L.) Moench received from 8 different locations in Europe and of a population from Cluj (acclimated since 1982 in our university). All the tested plants have been multiplied by means of seedlings. Among the investigated genotypes during both consecutive years, under the climatic conditions from Cluj – Napoca, the earliest (regarding the vegetation period) were: Porrentry (Switzerland) and the population from Cluj, related to the control variants (the average of the experimental plot).The aerial parts had a major contribution to the whole plant weight. It was noticed that, during each year of experience, the genotypes Warsaw-Poland, Műnchen-Germany, Siena-Italy, and Je...
Phytochemical variation within populations of Echinacea angustifolia (Asteraceae)
Biochemical Systematics and Ecology, 2002
Quantitative evaluation of phytochemical diversity in Echinacea angustifolia DC. populations from different natural geographic areas supports the existence of distinct natural chemotypes within the species. Consumers, growers and manufacturers of phytomedicines are interested in chemotype identification for prediction of phytochemical content in cultivar development. Six month old E. angustifolia roots, grown from nine different wild seed sources in a controlled environment, were extracted into 70% ethanol and 28 reported phytochemicals were measured by HPLC separation. Two-way ANOVA between the nine populations revealed quantitative differences (pϽ0.05) in the caffeic acid derivatives 2,3-O-dicaffeoyl tartaric acid (cichoric acid), 2-O-caffeoyl tartaric acid (caftaric acid), 1,3-dicaffeoyl-quinic acid (cynarin), echinacoside and ten reported alkamides. Canonical discriminant analysis determined the phytochemical variables which contributed the most towards chemotype distinction for five of the nine populations: undeca-2E,4Z-diene-8,10-diynoic acid-2-methylbutylamide * , dodeca-2E,4Edienoic acid isobutylamide * , dodeca-2E-ene-8,10-diynoic acid isobutylamide * * , hexadeca-2E,9Z-diene-12,14-diynoic acid isobutylamide * , cichoric acid * * , caftaric acid * , and echinacoside * * (* pϽ0.0001, * * pϽ0.05). Five of those compounds were also significantly associated with latitudinal variation by regression analyses (pϽ0.05).
2005
Using a micromanipulator (Narishige, East Meadow, NY), pollen grains of E. purpurea 'Magnus' were individually collected in separate PCR (polymerase chain reaction) tubes that contained 5µl of pollen germination medium. The pollen germination medium consisted of 0.29M sucrose, 1.62mM H3BO3 and 1.27mM Ca(NO3)2, (pH 5.7). After 1-2 hours of incubation at room temperature, the DNA was amplifi ed via MasterAmp ™ Extra-Long PCR kit (EPICENTRE ® , Madison, WI) using Zhang et al. (11) 15-mer primers (Operon Technologies, Alameda, CA). After staining with ethidium-bromide, Primer-Extension Pre-amplifi ed (PEP) DNA products were visualized by 2% agarose gel and photographed using an AlphaImager 2000 System (Alpha Innotech, San Leandro, CA). AFLP markers were generated from PEP products by DNA amplifi cation via PCR using the AFLP System-Analysis Kit of GibcoBRL (Rockville, MD). Agarose gel analyses were conducted to check restriction digestion, pre-amplifi cation and amplifi cation of the DNA samples. AFLP profi les (DNA fi ngerprints) were scored by separating the selective amplifi cation products through denaturing 6.5% polyacrylamide gel electrophoresis (9). All AFLP profi les were recorded with an automated DNA analyzer (Global IR 2 DNA Analyzer and Sequencer, LI-COR). Saga MX Generation 2-AFLP ® Analysis Software Version 3.1 (Li-Cor Inc., Lincoln, Nebraska) was ® Analysis Software Version 3.1 (Li-Cor Inc., Lincoln, Nebraska) was ® used to score AFLP profi le images and subsequently to add the marker data to an Oracle ® database. ® database. ® Results and Discussion: AFLP assays of individual conefl ower pollen grains were possible only after development of innovative methods described in this report. Because there is only a very small amount of DNA in a pollen grain, the primer extension pre-amplifi cation (PEP) protocol of Zhang et al. (11) was used to increase the amount of genomic DNA in each sample. The PEP procedure was modifi ed by using MasterAmp ™ Extra-Long PCR kit (EPICENTRE ® , Madison, WI) which contained the PCR enhancer betaine and Taq polymerase with 3'-5' exonuclease activity along with an array of continuous random 15-mer primers. These enhancers have been reported (3) to increase the fi delity and accuracy of the PCR products, therefore, PreMix 3 and PreMix 9 were used for pollen DNA amplifi cations via PEP procedure. The amplifi cation of pollen DNA by PEP resulted in genome distributed in fragments of varied lengths. AFLP analysis of the parental tissue revealed that 24 primer pairs were suitable for Echinacea fi ngerprinting and these were selected for subsequent pollen analyses. Restriction digestion was conducted on PEP products and PCR based
Industrial Crops and Products, 2010
Echinacea angustifolia var. angustifolia (Asteraceae) is widely used in the nutraceutical, cosmetic and pharmaceutical markets. Echinacoside, a phenol glycoside, is actually considered the marker compound and it is used for the assessment of quality of the roots. The plant material containing at least 1% of echinacoside is required by the market for the production of standardized extracts. The selection of highproducing secondary metabolites plants is necessary for an advantageous cultivation of this medicinal plant. Two Canadian accessions of E. angustifolia were cultivated and analyzed for their content in secondary metabolites showing high content of echinacoside (1.29% and 1.65%, respectively) resulting as interesting candidates for a profitable cultivation and a further selection work.
Industrial Crops and Products, 2019
Echinacea purpurea (L.) Moench is a plant species important for the phytopharmaceutical industry and in horticulture. Currently, there is lack of standardized plant material with an increased content of secondary metabolites in purple coneflower. The following research meets the expectations of the industry, as new selected lines of purple coneflower were micropropagated by somatic embryogenesis. The plant lines were analyzed both in terms of the content of main secondary metabolites by High Performance Liquid Chromatography (HPLC), as well as the genetic stability within the line and the genetic distance between lines using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) genetic markers. Significant differences were found in the relative percentage composition of individual phenolic acids in the tested plant material. Among six selected lines of Echinacea purpurea, three were characterized by a higher content of cichoric acid in relation to the other lines studied. A higher mean polymorphism rate (> 90%) was found with the RAPD technique, with a total of 1427 scorable bands produced (142.7 products per one primer). Unlike the RAPD analysis, ISSRs detected mostly monomorphic loci (63.4%), followed by polymorphic ones (36.6%), while there were no specific loci present. Cluster analysis of both marker systems showed that the tested genotypes were grouped according to their respective lines.
Clonal propagation and production of cichoric acid in three species of Echinaceae
In Vitro Cellular & Developmental Biology - Plant, 2012
In vitro tissue culture protocols were tested for propagation of Echinacea purpurea, Echinacea pallida and Echinacea angustifolia in order to obtain biomass for the production of cichoric acid, which is the major active compound in the Echinacea extracts. The in vitro culture process was initiated by seed germination on half-strength Murashige and Skoog (MS) medium. Multiplication was achieved on MS medium supplemented with naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 2-iso-pentenyladenine (2iP), and N 6 -benzyladenine (BA) in different concentrations. Shoot explants produced the highest number of shootlets on MS medium, which was supplemented with 0.1 mg/l 2iP and 0.1 mg/l IBA. RAPD markers revealed genetic polymorphism in some instances between in vitro generated plantlets such as for E. purpurea plantlets analyzed with the OPO-8 primer. RAPD markers generated with the primer 4A-29 revealed low levels of genetic variation between in vitro plantlets for all three species of Echinacea, while remaining RAPD markers revealed no variation. Content of cichoric acid in leaves, shoots, and callus was analyzed by high-performance liquid chromatography/MS and was identified in all studied samples, independent of species or tissue type. Highest levels (0.39-0.73 mg/g dw) were observed in shoots and leaves.