G09-066 912.918

Identifying commercially relevant Echinacea
species by AFLP molecular markers
Luigi Russi, Chiaraluce Moretti, Lorenzo Raggi, Emidio Albertini, and Egizia
Falistocco
Abstract: 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 prop-
erties, 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 prepara-
tions 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 frag-
ments, 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 Echina-
cea.
Key words: Echinacea pallida, E. purpurea, E. angustifolia, AFLP, species identification, commercial frauds.
Re´sume´ : L'inte´reˆt croissant pour les plantes me´dicinales a attire´ l'attention de plusieurs chercheurs sur le genre Echina-
cea. Les espe ces E. angustifolia, E. pallida et E. purpurea sont les plus importantes pour leurs proprie´te´s immunologiques,
bien connues et largement utilise´es par les ame´rindiens. Les trois espe ces sont faciles a distinguer sur la base de leurs ca-
racte´ristiques morphologiques, mais il serait difficile, voire impossible, de les distinguer au sein de pre´parations commer-
ciales compose´es de parties se ches broye´es de l'E. purpurea (la plus prise´e des espe ces pour ses proprie´te´s
chimiothe´rapeutiques), meˆle´es avec celles provenant des deux autres espe ces. Des marqueurs mole´culaires permettant de
distinguer les espe ces seraient utiles dans ce cas. Dans le pre´sent travail, a partir de mate´riel frais d'e´chinace´es cultive´es,
des marqueurs AFLP ont e´te´ employe´s pour diffe´rencier les trois espe ces et pour de´tecter des amplicons spe´cifiques des
chacune. A
l'aide de 14 combinaisons d'amorces, 994 amplicons ont e´te´ de´tecte´s au total, dont 565 e´taient polymorphes.
Globalement, 89 amplicons e´taient uniques a l'E. purpurea, 32 a l'E. angustifolia et 26 a l'E. pallida. Les combinaisonsE+CAC/M+AAT et E+CAC/M+AGC ont permis, a elles seules, de fournir respectivement 13, 9 et 4 ou 7, 5 et 5 mar-queurs spe´cifiques des espe ces E. purpurea, E. angustifolia et E. pallida. Un travail de validation pour confirmer ces re´sul-tats a e´te´ mene´ sur des e´chantillons me´lange´s de 23 accessions couvrant l'essentiel de la diversite´ ge´ne´tique au sein de cestrois espe ces. Les re´sultats sont discute´s en fonction des applications pratiques dans le domaine des me´decines douces, dela de´tection des fraudes et des implications pour le genre Echinacea.
Mots-cle´s : Echinacea pallida, E. purpurea, E. angustifolia, AFLP, identification des espe ces, fraudes commerciales.
[Traduit par la Re´daction] past the genus was known as Brauneria and Rudbeckia,while the name Echinacea appeared for the first time in The genus Echinacea Moench (Compositae) is native to 1762. The classification of the taxa within the genus is con- the prairies of North America, from which it spreads from troversial. McGregor (1968) distinguished 9 species and 4 southern Canada to Texas and Georgia, but the greatest di- varieties, while Binns et al. (2002), on the basis of a mor- versity of species is found in Arkansas, Oklahoma, Missouri, phometric multivariate statistical analysis, supported 2 sub- and Kansas (McGregor 1968; Urbatsch et al. 2000). In the genera containing 4 species and 8 varieties.
Received 21 April 2009. Accepted 7 August 2009. Published on the NRC Research Press Web site at genome.nrc.ca on 21 October 2009.
Corresponding Editor: P. Gustafson.
L. Russi,1 L. Raggi, E. Albertini, and E. Falistocco. Dipartimento di Biologia applicata, Universita degli studi di Perugia, Borgo XX
Giugno 74, 06121 Perugia, Italy.
C. Moretti. Dipartimento di Scienze agrarie e ambientali, Universita degli studi di Perugia, Borgo XX Giugno 74, 06121 Perugia, Italy.
1Corresponding author (e-mail: [email protected]).
Genome 52: 912–918 (2009)
Published by NRC Research Press Russi et al.
Echinacea angustifolia D.C., Echinacea purpurea (L.) mer was used for selective PCR. The AFLP analysis was Moench, and Echinacea pallida Nutt. are the most wide- carried out on single plants, using 14 primer combinations spread and most known species because of their commercial as listed in Table 1.
importance due to immunological properties. The use ofthese species as medicinal plants dates back several centu- Trial 2: validation
ries; E. angustifolia was widely used by the indigenous pop- To validate the results of the first trial, seeds of 23 acces- ulations of North America for healing injuries, to treat sions (4, 11, and 8 for E. angustifolia, E. pallida, and snakebites, or to counter infectious diseases (Gilmore 1919).
E. purpurea, respectively) received from the USDA, ARS, In recent years many studies have been carried out to better National Plant Germplasm System (NPGS) were used. The explore the pharmacological activities of the most common accessions, all natural populations, were chosen randomly species of Echinacea and to identify the chemical com- from the NPGS germplasm collection list. The information pounds that confer such properties (Barrett 2003). However, available in the Germplasm Resources Information Network in the past, in popular medicine these species were probably (USDA, ARS, National Genetic Resources Program 2007) in used indifferently for the same medical treatments, either terms of collection sites, source history, and improvement because they have similar properties or because it was diffi- status ensured the sampling of sufficient genetic diversity cult to distinguish one from the other. Although the three for the validation trial. Seeds were germinated in Petri species have some of the same pharmacological activities dishes, and seedlings were transplanted in pots in the green- owing to the presence of active compounds that act synerg- house and grown in the same conditions described above. In istically, each species shows slight variations in the amount May 2008, similar leaf segments (of approximately 1 cm2) of individual active compounds (Percival 2000; Speroni et from 6 plants of each accession were sampled and bulked al. 2002). Efforts are being made to analyze the type and (Table 2). DNA extraction from bulked leaves was carried amount of the chemical compounds of each species, to opti- out using the GenElute Plant Genomic DNA Miniprep Kit mize their use for proper therapeutic application (Barrett (Sigma). Also, an equal amount of DNA from each bulk 2003). Following intense research activity on these species was used to prepare a single bulk for each Echinacea spe- in the medical and pharmacological fields, interest in their cies and, from these, a whole bulk was prepared with all genetic characteristics and phylogenesis has greatly in- species together. The AFLP analysis was carried out as de- creased in recent years. On the contrary, knowledge about scribed above, except that for the validation experiment only their cytology is scant, being limited to the chromosome one primer combination (E+CAC/M+AGC) was used. Am- number: E. angustifolia and E. purpurea are diploid, with plifications were performed in a 20 mL reaction mix contain- 2n = 22, while E. pallida is tetraploid, with 2n = 44 ing 1/100 of the pre-amplified DNA, 50 ng of 6-FAM- (McGregor 1968; Mechanda et al. 2004a; Qu et al. 2004).
labeled E+CAC primer, 50 ng of unlabeled M+AGC primer, In this study, AFLP (amplified fragment length polymor- 2 mL of 10 PCR buffer (Invitrogen), 0.2 mmol/L dNTPs, phism) molecular markers were used to characterize the and 0.4 U of Taq polymerase (Invitrogen). Samples were de- three species, investigate their genetic similarities, and gain natured and run on an ABI 3130xl genetic analyzer (Applied insight into their genetic relationships. Cytological investiga- tions were also carried out to confirm their chromosomenumber. The main objective of the study was to look at the possibility of distinguishing them using AFLP markers, a For determination of the chromosome number, actively field with potential practical applications and where litera- growing root tips were excised when they were about ture is scarce.
1.5 cm in length, pretreated in a saturated solution of a-bromonaphthalene for 3–4 h, and then fixed overnight in 3:1ethanol – acetic acid. Somatic chromosomes were stained Materials and methods
using the Feulgen procedure. Squashes were performed in Trial 1: plant material, DNA isolation, and marker
1.5% acetic orcein and attached to a cover slip with glycer- ine albumen.
The first trial was conducted in 2005 on seed samples of E. angustifolia, E. purpurea, and E. pallida provided by Aboca S.p.A. (Arezzo, Italy), a company growing, process- AFLP fragments were scored as dominant markers, and ing, and marketing medicinal plants. The seed lots of the individual profiles were considered as phenotypes (Me- three species were originally imported by Aboca in 1999 chanda et al. 2004b). In trial 1, fragments that could be un- from the USA in a commercial quantity. Seeds were germi- equivocally scored (1 for presence and 0 for absence) across nated in Petri dishes, and some of the seedlings were uti- all individuals were included in the analysis; the scores were lized for cytological preparations; the remainder were used to prepare a data matrix based on 30 individuals (10 transplanted in Jiffy pots, transferred to a greenhouse, and plants per species). The binary data were analysed by tabu- grown at a constant temperature of 21 8C and a day length lation and frequency procedures using SAS software (SAS of 16 h. The molecular analysis was carried out on 10 plants Institute Inc. 1999) to inspect for the presence of specific per species. Young, newly formed leaves were collected for fragments and polymorphism within and between species.
total genomic DNA extraction based on the protocol de- The binary data were also used to prepare a matrix of ge- scribed by Dellaporta et al. (1983). AFLP marker analysis netic similarity by using the coefficient of Jaccard (1908).
was carried out according to Vos et al. (1995), as modified The similarity matrix was used in clustering the individu- by Cnops et al. (1996). A fluorescently labeled E+CAC pri- als by the unweighted pair group method with arithmetic Published by NRC Research Press Genome Vol. 52, 2009 Table 1. Number of total, polymorphic, and species-specific AFLP fragments, scored over 14 EcoRI/MseI
primer combinations, in three Echinacea species from trial 1.
Primer combination* E. angustifolia E. pallida E. purpurea Table 2. Accession number from the USDA, ARS, NPGS catalogue, geo-
graphic coordinates, and state of the collection sites of three species of
Echinacea used in the validation trial.
Accession No.
E. angustifolia
PI 631268
E. pallida
Ames 26351
E. purpurea
Ames 27338
mean (UPGMA) (Sneath and Sokal 1973). Cophenetic ma- matrix. The results of the cluster analysis were also vali- trices derived from the dendrograms were compared with dated by principal coordinate analysis (Gower 1966) by dou- the original similarity matrix by the test of Mantel (1967) to estimate the goodness-of-fit of the clustering to the data eigenvalues and eigenvectors, and displaying the relation- Published by NRC Research Press Russi et al.
ships among individuals in 3 dimensions. Estimation of ge- M+AAT, able to provide 13, 9, and 4 specific fragments for netic distances, cluster analysis, the Mantel test, and princi- E. purpurea, E. angustifolia, and E. pallida, respectively.
pal coordinate analysis were carried out by NTSYS-pc The UPGMA dendrogram in Fig. 1 indicates a high simi- software (Rohlf 1993).
larity within each species and clear differences among spe- AFLP fragments in the validation trial were scored by cies. The goodness-of-fit of the analysis was validated by GeneMapper 4.0 software (Applied Biosystems). An AFLP the high and significant correlation coefficient between the locus in the bulked samples was considered to be polymor- similarity and cophenetic matrices (r = 0.997, Mantel t = phic if the amplified band was present in some accessions 20.557, P < 0.001). All individuals of E. purpurea are and absent in others, species-specific if the band was shared grouped together, apart from the rest of the individuals, at a between all accessions belonging to one species and absent similarity value of 0.623. The rest of the individuals are then in all other accessions, and monomorphic if the band was split into 2 other groups at a similarity value of 0.728, present in all samples. To avoid an underestimation of the clearly distinguishing E. angustifolia and E. pallida and con- genetic similarities, all loci, polymorphic or not, were con- firming that they are phylogenetically closer to one another than to E. purpurea. Although the three species share a largepart of their genome, as shown by the high number ofmonomorphic fragments, peculiarities do exist, thus allow- ing distinctiveness. This is confirmed by the pattern of clus- ters, with no individuals being mis-classified into different The analysis confirmed the chromosome number typical species. A further statistical validation of these results is of each species: E. angustifolia and E. purpurea are diploid, provided by principal coordinate analysis, where the projec- with 2n = 22, while E. pallida is tetraploid, with 2n = 44.
tion of individuals plotted against the axis representing themost significant eigenvectors shows low within-species vari-ability and consistent variability between species (Fig. 2).
Trial 1: AFLP analysis of cultivated Echinacea spp.
Individuals of each species were tightly grouped together, The scoring of AFLP gels showed the presence of 994 confirming the clustering pattern. The first 3 eigenvalues fragments. Of these, 429 (43%) were monomorphic, found were able to explain as much as 0.82 of the total variation in all plants of all three species, while 565 (57%) were poly- (0.51, 0.28, and 0.02, respectively), with the first 2 able to morphic (Table 1). Of the latter group, 147 fragments were significantly explain more variation than expected under the discriminant, that is, unique to all plants of a single species.
broken stick model (Joliffe 1986).
The analysis showed that 89 fragments were unique toE. purpurea, 32 were unique to E. angustifolia, and 26 Trial 2: validation
were unique to E. pallida. However, discrimination among The primer combination E+CAC/M+AGC, used for vali- species was also possible by using combinations of frag- dation analysis, produced a total of 76 amplification prod- ments. For instance, E. angustifolia and E. purpurea were ucts, a value very close to the 74 found on cultivated found to share 28 fragments that were absent in all individu- Echinacea spp. in trial 1; 31 of these amplicons (41%) were als of E. pallida. Echinacea angustifolia and E. purpurea polymorphic. Of these, 10 amplicons were species-specific could, in turn, be distinguished from one another by 103 (3, 2, and 5 for E. angustifolia, E. pallida, and E. purpurea, bands, 69 present in the former and absent in the latter and respectively), while 4 amplicons were common between 34 absent in the former and present in the latter (data not E. angustifolia and E. pallida and absent in E. purpurea.
The remaining polymorphic fragments were specific to Discrimination properties shown by each primer combina- some accessions within the same species rather than to the tion are of particular interest. For instance, the primer com- whole species; in particular, 3 amplicons were found in E. angustifolia, 3 in E. pallida, and 11 in E. purpurea. The M+ACG, E+CAC/M+AGA, and E+CAC/M+AGT did not species-specific amplicons were also easily found in the produce any AFLP fragment specific to E. angustifolia, bulked samples of all accessions of the same species and in while E+CAC/M+AAT alone was able to generate 9 specific the bulk comprising all 23 accessions. A duplicate lane of fragments. In E. pallida the best primer combinations able one sample confirmed the correctness of the profile.
to generate specific fragments were E+CAC/M+ACG (6 The numbers of species-specific AFLP fragments detected fragments) and E+CAC/M+AGC (5 fragments), while for by the E+CAC/M+AGC primer combination in the valida- E. purpurea the best primer combinations were E+CAC/ tion trial were lower than those found in the cultivated sam- M+AAT and E+CAC/M+AGT, with 13 and 12 fragments, ples (3, 2, and 5 vs. 5, 5, and 7 in E. angustifolia, E. pallida, and E. purpurea, respectively), and this could be due to the The primer combination that gave the highest number of high number of accessions used in the validation test. Most fragments was E+CAC/M+AAG, while E+CAC/M+ACC likely, some of the bands scored as species-specific in trial 1 gave the lowest. It is interesting that the former also gave might belong to those classified as accession-specific in the the highest number of polymorphic fragments, but was not validation trial (i.e., bands present in only some of the ac- the most effective in terms of species discrimination. Five cessions of a given species). The sizes of the species- primer combinations were able to detect fragments specific to each of the three species, namely E+CAC/M+AAA, E+CAC/M+AGC are as follows: 75, 160, and 200 bp for E. angustifolia; 127 and 212 bp for E. pallida; and 38, E+CAC/M+AGG. Of these, the most powerful was E+CAC/ 53, 73, 153, and 155 bp for E. purpurea.
Published by NRC Research Press Genome Vol. 52, 2009 Fig. 1. Dendrogram of individual plants belonging to E. angustifolia, E. pallida, and E. purpurea, generated by UPGMA cluster analysis of
Jaccard's similarity coefficients calculated from AFLP marker data.
Fig. 2. Principal coordinate analysis of individual plants belonging to E. angustifolia, E. pallida, and E. purpurea, based on AFLP markers.
tion and highlight several differences with few primer com-binations. Moreover, results from AFLP data sets have Echinacea purpurea, E. angustifolia, and E. pallida are proved to be concordant with those from other molecular the most known species of the whole genus, especially for markers (Powell et al. 1996). Most, but not all, AFLP frag- their immunological properties. Echinacea purpurea in par- ments of a specific size can be considered to represent the ticular is the most significant for its medicinal use (Tyler1997; Percival 2000; Oomah et al. 2006). The objective of same loci (Cervera et al. 2001; Peters et al. 2001), although the present study was to investigate the genetic similarities this likelihood apparently decreases with an increase in ge- of the three species and look at the possibility of distin- netic distance between species (Still et al. 2005). Large data guishing them using AFLP markers. Characterization of ge- sets can offset the assumption of orthology simply by in- nomes using molecular markers has developed rapidly and a creasing the number of independent loci sampled across a vast literature is available. Useful applications of molecular genome and establishing ‘‘correct'' phylogenetic relation- markers include genetic control of plant reproduction, ge- ships among species (Rokas et al. 2003). Still et al. (2005) netic mapping, marker-assisted selection, cultivar character- reported that AFLP methodology largely fulfills this require- ization, estimates of genetic distances, analysis of gene ment and any non-orthologous fragments detected among expression, and cloning. In the last decade the fields that populations should be overcome by the much higher number have benefited the most from extensive use of molecular of orthologous fragments.
markers are those connected with phylogenetic studies, cul- AFLP analysis has been found to be useful in predicting tivar characterization, and marker-assisted selection.
The use of AFLP technology for these purposes has be- E. purpurea germplasm (Baum et al. 2001). Twenty-four come very popular because of its unique ability to detect AFLP primer combinations were used to construct a genetic polymorphisms within the genome without requiring prior map of this species via individual pollen DNA fingerprinting sequencing information (Still et al. 2005). This technology (Aziz and Sauve 2008), with 104 scorable markers in 11 can faithfully generate many fragments per primer combina- linkage groups. RAPD markers were successfully used by Published by NRC Research Press Russi et al.
Kapteyn et al. (2002) to investigate the genetic relationships using only a single combination of primers, as confirmed and diversity of the same three Echinacea species studied in by the validation trial based on E+CAC/M+AGC.
the present work; they found 17 diagnostic markers useful A sequence characterized amplified region assay carried for distinguishing among taxa. They did not find any spe- out on DNA from the three Echinacea species of the same cies-specific marker, and postulated as discriminant those origin as in the present study showed that the expected found with a frequency of at least 0.95 in a taxon and absent band (330 bp) was present in E. purpurea and not in the in other taxa, or present with a frequency of less than 0.05.
other two species (Adinolfi et al. 2007). The same approach The results of the present research show that in Echinacea, applied to our species-specific fragments and verified exper- AFLP markers seem to be much more effective than RAPD imentally would provide a simple tool able to distinguish markers in distinguishing among species, firstly because of each of the three species.
the higher number of polymorphisms detected (Table 1) and The present study focussed on the most commercially im- secondly because many of the fragments were found to be portant species and has no contribution to make in the dis- specific to a single species. AFLP analysis was also success- pute on the origin of the tetraploid E. pallida. However, the fully employed by Kim et al. (2004) and Mechanda et al.
results indicate a closer genetic similarity between E. pallida (2004a) in studying the genetic diversity of the whole Echi- and E. angustifolia than between E. pallida and E. purpurea, nacea genus, and the former pointed out the superiority of in line with most of the published literature based on molec- AFLP versus RAPD markers in phylogenetic studies, in ular markers. McGregor (1968) supposed E. pallida to de- terms of the number of polymorphic fragments per primer rive from a chromosome doubling of the hybrid between pair. In the present study the average number of polymor- E. simulata and E. sanguinea. On the basis of morphometric phic fragments per primer pair was 40, significantly lower analysis based on 74 characters, Binns et al. (2002) revised than the 66 fragments reported by Kim et al. (2004), 62 by the genus Echinacea and included 4 species rather than 9.
Mechanda et al. (2004a), and 82 by Still et al. (2005), but According to Binns et al. (2002), 5 botanical varieties consistently higher than the 4.6 fragments obtained by (E. pallida var. pallida, var. angustifolia, var. tennesseensis, RAPD analysis. Independently of the type of marker used, var. simulata, and var. sanguinea) belong to E. pallida, but the results from cluster analysis are in perfect agreement the tetraploidy of E. pallida var. pallida and the diploidy of with those presented by Kapteyn et al. (2002), who analyzed var. angustifolia and/or the other 3 varieties would be in natural and cultivated populations from several sources and contrast with the accepted definition of a species (a group found low variability within species. Kim et al. (2004) also of individuals able to interbreed and give rise to fertile prog- reported similarities within species of the same size as those enies). The work of Binns et al. (2002), while confirming in the present study. The explanation suggested by Kapteyn the similarities of E. pallida and E. angustifolia, also shows et al. (2002) is that the high genetic uniformity among pop- high similarities of these species with E. sanguinea and ulations within species is the result of a continuous gene E. simulata, sensu McGregor (1968), thus giving support to flow, particularly in E. purpurea, the most widespread spe- cies, and in E. pallida. The differences found among popula- tions of E. angustifolia were ascribed to a discontinuousdistribution and a consequent difficulty of gene flow. How- Adinolfi, B., Chicca, A., Martinotti, E., Breschi, M.C., and Nieri, P.
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