Evaluation of yarrow (Achillea) accessions by phytochemical and molecular genetic tools

Abstract

Yarrow (Achillea) species are known and utilized worldwide. In the recent study our primarily goal was to get information about the intraspecific diversity of A. collina in the Carpathian Basin. Five cultivated genotypes and six populations of wild origin were compared involving seven other species as control. Essential oil (EO) and proazulene (PA) contents were determined and the DNA samples were evaluated by RAPD (11 primers) and ISSR (12 primers) methods. The EO content varied between 0.010 (A. distans) and 0.365 (A. collina) ml/100 g DW, the PA content was found between 0.021 and 0.173% DW. The used RAPD markers provided 140 bands (97.14% polymorphic). They distinguished primarily among species and less characteristically among the A. collina populations. With ISSR primers we detected 188 bands (97.34% polymorphic). ISSR markers and combined RAPD and ISSR method enabled an informative intraspecific evaluation of A. collina accessions. The largest genetic distances were found between A. ptarmica and the members of sect. Achillea (genetic distances 0.52 - 0.72). Similarity is highest (genetic distance 0.27) among the populations of lower geographical distances. Nei's genetic distances of cultivated populations are also relatively low (0.23 - 0.36). Some wild accessions may represent valuable biological resources for breeding.

Full text

Journal of Applied Botany and Food Quality 89, 105 - 112 (2016), DOI:10.5073/JABFQ.2016.089.013
Department of Medicinal and Aromatic Plants, Corvinus University of Budapest, Budapest, Hungary
Evaluation of yarrow (Achillea) accessions by phytochemical and molecular genetic tools
Katalin Inotai, Zsuzsanna György, Sára Kindlovits, György Várady, Éva Németh-Zámbori*
(Received November 11, 2015)
* Corresponding author
Summary
Yarrow (Achillea) species are known and utilized worldwide. In the
recent study our primarily goal was to get information about the
intraspecic diversity of A. collina in the Carpathian Basin. Five
cultivated genotypes and six populations of wild origin were com-
pared involving seven other species as control. Essential oil (EO)
and proazulene (PA) contents were determined and the DNA sam-
ples were evaluated by RAPD (11 primers) and ISSR (12 primers)
methods.
The EO content varied between 0.010 (A. distans) and 0.365 (A. col-
lina) ml/100 g DW, the PA content was found between 0.021 and
0.173% DW. The used RAPD markers provided 140 bands (97.14%
polymorphic). They distinguished primarily among species and less
characteristically among the A. collina populations. With ISSR pri-
mers we detected 188 bands (97.34 % polymorphic). ISSR markers
and combined RAPD and ISSR method enabled an informative in-
traspecic evaluation of A. collina accessions. The largest genetic
distances were found between A. ptarmica and the members of sect.
Achillea (genetic distances 0.52 - 0.72). Similarity is highest (ge-
netic distance 0.27) among the populations of lower geographical
distances. Nei’s genetic distances of cultivated populations are also
relatively low (0.23 - 0.36). Some wild accessions may represent
valuable biological resources for breeding.
Abbreviations
AFLP Amplied Fragment Length Polymorphism; DNA Deoxy-
ribonucleic Acid; DW Dry Weight; EO Essential OIl; ISSR Inter-
Simple Sequence Repeat; PA Proazulene; PCA Principal Coordinate
Analysis; RAPD Random Amplied Polymorphic DNA; UPGMA
Unweighted Pair Group Method with Arithmetic averages
Introduction
Achillea species are well known medicinal plants having an im-
portant role both in folk medicine and in the modern phytotherapy.
Main indications include loss of appetite, bloating, atulence, mi-
nor menstrual spasms and wound healing (Final community herbal
monograph, 2011). The majority of the drug is still obtained from
wild collection.
The genus Achillea consists of six sections and appr. 140 species
(Gu o et al., 2005), which are allogamous, herbaceous perennials
in the Northern hemisphere. The presence of spontaneous hybrids,
allo- and autopolyploids, aneuploids, and phenocopies results in a
big cytological, morphological and chemical variability not only at
species but also at intraspecic level. Therefore, taxonomic evaluati-
on, identication of species and systematic classication has been a
difcult task for decades. With regard to species identication earlier
studies were obviously focusing on morphological traits. Although
the majority of characteristics proved to be extremely variable, some
of them like fruit size (Dabrowska, 1977), shape of leaets and
rayorets (rauchensteiner et al., 2002), and pollen morphology
(ak ya l c i n et al., 2011) have been dened as stable ones at least in
the investigated taxa. Cytological studies ascertained that in some
species different caryotypes are present which, however, do not ne-
cessarily show connection with morphological traits (Dabrowska,
1977; Da n i h e k a and ro t r e k l o v á , 2001).
Phytochemical parameters represent a valuable part of taxonomic
evaluation of yarrow species. Based on the pharmacopoeial require-
ments, most frequently, the presence of chamazulene in the essen-
tial oil has been in the focus of investigations (tétényi et al., 1962;
oswiecimska, 1962; michler et al., 1992) although avonoids
may have taxonomic importance, as well (va l a n t , 1978). It is wide-
ly accepted now that chamazulene is a characteristic of the mem-
bers of Millefolium group; however, the presence of azulenes is
not a universal phenomenon for all of these species. Earlier, a rm
connection between chromosome number of the species and the po-
tential for accumulation of chamazulene was supposed. Recently, it
has been accepted that the production of proazulenes in polyploids
may depend on the chemism of the parent/original diploid species
(kästner et al., 1992; ma et al., 2010). By the development of ana-
lytical methods evaluation of a wider range of oil components and
their enantiomers provided a more complex approach in identica-
tion and systematics (or t h , 2000; rauchensteiner at al., 2002;
ra D u l o v i c et al., 2007).
The use of molecular markers in the systematic studies of yarrow
was introduced from the 1990th. wa l l n e r et al. (1996) proved the
applicability of RFLP and PCR based ngerprinting methods for
characterisation of micropropagated Achillea clones. Investigations
by nrITS and plastid trnL-F DNA sequences revealed phylogene-
tic connections: differentiation patterns of Achillea s.l. in time and
space (Gu o et al., 2004), although this method could not assure a
well established separation of A. millefolium aggr. In the work of
Gu o et al. (2005) characterization was realized by AFLP markers
and more recently, ma et al. (2010) demonstrated the ongoing in-
trogression of diploid progenitor and tetraploid progenies in the A.
millefolium complex by analysing single copy nuclear genes and
AFLP markers.
Besides taxonomic studies, several publications appeared in the last
years about RAPD, ISSR (ebrahimi et al., 2012; Fa r a j p o u r et al.,
2011; Gharibi et al., 2011) and AFLP (rah i m m a l e k et al., 2009) as-
sessment of yarrow species. Characteristically, the authors compared
populations from different geographical locations for conservation
purposes without describing the phytochemical values of the plant
material. These studies are focusing on indigenous species (A. santo-
lina, A. tenuifolia, A. eriophora, etc.), not found at the international
market and ofcial therapies.
It can be established that practice-oriented investigations on econo-
mically important taxa of yarrow are scarce up to now. The goal of
our investigations was to look for reliable, relatively simple methods
for differentiation/determination of intraspecic taxa of A. collina,
the most frequently collected and cultivated species. According to
our knowledge, molecular genetic study of intraspecic diversity of
this species has not been published till now.

106 K. Inotai, Z. György, S. Kindlovits, G. Várady, É. Németh-Zámbori
Materials and methods
Plant material
Eleven Achillea collina Becker accessions have been included into
the investigation. Three of them are ofcially registered cultivars,
two ones are cultivated commercial materials without special de-
nomination and six accessions originate from wild populations
(Tab. 1). For comparative studies, seven other species of different
ploidy level were used as control. Among them A. ptarmica belongs
to Section Ptarmica (DC.) W. Koch. All other species are members
of the Section Achillea and except A. crithmifolia and A. lipendu-
lina of the A. Millefolium agg. (Gu o et al., 2004).
Seeds of accessions nr. 1-2 were obtained from the maintainers of
cultivars, nr. 3 derived from own maintenance breeding, nr. 4-5 were
obtained from farmers, 6-11 were collected from wild populations of
Hungary and accessions nr. 12-18 were obtained from the genebank
of the National Botanical Garden, Vácrátót. The species identity has
been controlled according to the morphological traits and through
checking the ploidy level by ow cytometry.
Plants were grown from seeds in climatic chambers in 2014. In 5-6
weeks they reached a 4-5 leafy stadium when bulk sampling (kr a F t
and sä l l , 1999) from 10-15 plants/accession was carried out for
PCR trials as well as checking of chromosome numbers by ow cy-
tometry (Fig. 1) according to the modied method of Galbraith
et al. (1983).
After 2 months, the seedlings were planted into open eld plots in
three replicates at our experimental station in Budapest. At owering
stage, representative bulk samples with max. 20 cm of shoots were
taken from each plot for essential oil extraction. The plant material
was dried at room temperature and stored at +4 °C until distillation.
Tab. 1: List of the investigated Achillea accessions
Accession Species Origin of population
Nr. sign /chromosome nr.*/
Sect. Achillea
Agg. A. millefolium s.l.
1 C1 A. collina Becker (4x) German variety ‘Proa’
2 C2 A. collina Becker (4x) Slovakian variety ‘Alba’
3 C3 A. collina Becker (4x) Hungarian variety ‘Azulenka’
4 C4 A. collina Becker (4x) Cultivated commercial plant material, Gyula 46° 38’ 50.2” N/ 21° 16’ 42.3” E
5 C5 A. collina Becker (4x) Cultivated commercial plant material, Földes 47° 17’ 22.8” N/ 21° 21’ 47.8” E
6 CW1 A. collina Becker (4x) Wild collected population, Aszód 47° 39’ 12.1” N/ 19° 29’ 3.6” E
7 CW2 A. collina Becker (4x) Wild collected population, Csörötnek 46° 56’ 59.3” N/ 16° 22’ 14.7” E
8 CW3 A. collina Becker (4x) Wild collected population, Diósd 47° 24’ 29.8” N/ 18° 56’ 36.5” E
9 CW4 A. collina Becker (4x) Wild collected population, Mikóújfalu 46° 3’ 13.5” N/ 25° 50’ 6.7” E
10 CW5 A. collina Becker (4x) Wild collected population, Nagymaros 47° 47’ 16.9” N/ 18° 57’ 14.9” E
11 CW6 A. collina Becker (4x) Wild collected population, Remeteszőlős 47° 33’ 23” N/ 18° 55’ 44.6” E
12 ASP A. asplenifolia Vent. (2x) Genebank of Vácrátót Bot.Garden
14 DIS A. distans Walds. et Kit. (6x) Genebank of Vácrátót Bot.Garden
16 MIL A. millefolium L. s.s. (6x) Genebank of Vácrátót Bot.Garden
17 PAN A. pannonica Scheele (8x) Genebank of Vácrátót Bot.Garden
Excl. agg. A. millefolium
13 CRI A. crithmifolia Walds. et Kit. (2x) Genebank of Corvinus University
15 FIL A. lipendulina Lam. (2x) Genebank of Vácrátót Bot.Garden
Section Ptarmica
18 PTR A. ptarmica L. (2x) Genebank of Vácrátót Bot.Garden
The accessions are maintained as living genebank collection at the
Research Station of the Faculty of Horticulture, Corvinus University,
Budapest.
Essential oil extraction and analysis
The essential oil content was measured from the dried drug in tripli-
cate, by hydrodistillation in a Clevenger-type apparatus according to
the Hungarian Pharmacopoeia VII (Millefolii herba). After 3 hours
of distillation, n-hexane was added to take up the essential oil. Af-
ter evaporation of the hexane, the collected extract was stored in a
cool place. The essential oil content was calculated as ml/100 g dried
plant material. Water content of the drug was determined by heating
4 g of the drug at 105 °C for 3 hours.
The proazulene content in the essential oil samples was determined
by spectrophotometric method at 608 nm as described in the Euro-
pean Pharmacopoeia VII (Millefolii herba) in triplicate and calcula-
ted as a percentage of the dry weight expressed as chamazulene.
DNA isolation
One young leaf from each of 10-15 plants of each accessions was
collected and ground together with liquid nitrogen. Genomic DNA
was extracted from these bulk samples of fresh young leaves by
DNeasy Plant Mini Kit (Qiagen, BioScience, Hungary). DNA con-
centration and quality was assessed using NanoDrop (BioScience,
Hungary) and visually checked on 1% agarose gel.
Out of the primarily screened 17 RAPD and 13 ISSR primers only
11 RAPD and 12 ISSR primers produced clear, reproducible and
scorable bands, thus, the investigations have been carried out by
these ones.

Evaluation of Achillea accessions 107
RAPD Analysis
11 RAPD primers have been used, the optimum annealing tem-
perature was determined for each one individually (Tab. 2). Am-
plication reactions were performed in 12 μl volume containing
15-25 ng genomic DNA, 1 μM primer, 6 μl of 2× GoTaq Hot Start
Green Master Mix (Promega), 3 mM MgCl2 and nuclease free water.
PCR amplication was performed in a SuperCycler SC-200 ther-
mocycler (Kyratec) under the following conditions: 2 min at 95 °C,
followed by 35 cycles of 30 s at 94 °C, 1 min at specic annealing
temperature, 1 min at 72 °C and a nal extension for 7 min at 72 °C.
Amplied DNA fragments were separated in a 1.5% agarose gel
(SeaKem LE Agarose, Lonza) at 100 V for 90-120 min in 1× Tris-
Acetate EDTA (TAE) buffer (pH 8.0) and stained by 1 % (w/v) ethi-
dium bromide. The PCR products were visualized under UV light
by AlphaImager EP Imaging System (Cell Bioscience). The 100 bp
ladder (Promega) was used as a molecular weight size marker.
ISSR Analysis
ISSR analysis has been performed with 12 primers (Tab. 2), the op-
timum annealing temperature was determined for each one individu-
ally. PCR reactions were carried out in a volume of 12 μl containing
15-25 ng genomic DNA, 2 μM of primer, 6 μl of 2× GoTaq Hot Start
Green Master Mix (Promega), 3 mM MgCl2 and nuclease free water.
The SuperCycler SC-200 (Kyratec) was programmed as follows: an
initial cycle of 3 min at 95 °C, followed by 35 cycles each consis-
ting of 30 s at 94 °C, 45 s at specic annealing temperature, 45 s at
72 °C and nal extension of 7 min at 72 °C. Fragment separation and
visualization was performed as above.
Statistical analysis
The results of essential oil and proazulene analysis were evaluated
by one-way ANOVA using the IBM SPSS Statistics 22 program. The
pairwise comparisons of the variances were made by the Tukey Post
Hoc test.
Amplied DNA fragments were scored visually for presence (1) or
absence (0) of homologous bands and the results were summarized in
Microsoft Excel table. Popgene version 1.32 (ye h and bo y l e , 1997)
was used to estimate number of polymorphic bands, percentage of
polymorphic bands, ne i ’s (1973) gene diversity (h) and Shannon’s
Information Index (I) (lewontin, 1972) for dominant marker data.
Genetic relatedness among genotypes was studied by UPGMA (Un-
weighted Pair Group Method with Arithmetic averages) cluster ana-
lysis and principal coordinate analysis (PCA) using Past software
(ha m m e r et al., 2001).
Results and discussion
Essential oil and proazulene content
The essential oil content of the examined accessions varied between
0.002 (A. distans) and 0.365 (A. collina CW4) ml/100 g (Tab. 3). The
Fig. 1: Histograms of relative uorescence intensity of octoploid (A. panno-
nica), hexaploid (A. millefolium), tetraploid (A. collina) and diploid
(A. lipendulina) Achillea species (from left to right), (codes of ac-
cessions as in Tab. 1).
Tab. 2: The tested RAPD and ISSR primers
RAPD Sequence Annealing No. of
primer name temp. (°C) bands
OPA-20 5’-GTTGCGATCC-3’ 39 10
OPG-18 5’-GGCTCATGTG-3’ 38 14
OPB-11 5’-GTAGACCCGT-3’ 38 14
OPA-02 5’-TGCCGAGCTG-3’ 35 13
OPG-13 5’-CTCTCCGCCA-3’ 43 13
m2 5’-ACAACGCCTC-3’ 41 13
g11 5’-TGCCCGTCGT-3’ 48 14
seg1 5’-AGGGGTCTTG-3’ 35 15
seq2 5’-GGGTTTAGGG-3’ 35 9
seq3 5’-GACAGACAGG-3’ 35 15
seq4 5’-CGAAGCTACC-3’ 35 10
Total no. of bands (RAPD) 140
Number of polymorphic loci 136
Percentage of polymorphic loci 97.14%
ISSR Sequence Annealing No. of
primer name temp. (°C) bands
818 5’-CCCCCCCAAAAAAAG-3’ 47 10
825 5’-AAAAAAAACCCCCCCCT-3’ 49 14
849 5’-GGGGGGGGTTTTTTTTC-3’ 49 14
CAg5 5’-CCCCCAAAAAGGGGG-3’ 49 15
ctc4rc 5’-CCCCCCCCCTTTTR-3’ * 50 18
issr1 5’-CACACACACACACACAGT-3’ 51 21
issr2 5’-GAGAGAGAGAGAGAGAG-3’ 49 10
issr3 5’-GTGTGTGTGTGTGTGTC-3’ 49 12
issr4 5’-ACACACACACACACACTG-3’ 51 21
issr5 5’-AGTGAGTGAGTGAGTG-3’ 45 18
issr6 5’-GATAGATAGATAGATAGATA-3’ 47 14
issr7 5’-TCTTCTTCTTCTTCTTCT-3’ 45 15
Total no. of bands (ISSR) 188
Number of polymorphic loci 183
Percentage of polymorphic loci 97.34%
Total no. of bands (RAPD+ISSR) 328
Number of polymorphic loci 319
Percentage of polymorphic loci 97.26%

108 K. Inotai, Z. György, S. Kindlovits, G. Várady, É. Németh-Zámbori
accumulation levels of each species are in the range of data men-
tioned in other investigations (németh, 2005).
Evaluating the studied populations with giving consideration to the
accumulation level of the essential oil, the Tukey test distinguished
5 subsets at p=0.05 signicance level. Among them both A. dis-
tans having the lowest content (0.01 ml/100 g) and A. crithmifolia
having the highest one (0.42 ml/100 g) represent distinct subsets.
On the other hand, the largest homogenous subset includes all of
the A. collina accessions, besides A. crithmifolia, A. pannonica and
A. lipendulina. Taking into account only the A. collina accessions,
the differences are much lower; signicant differences were proven
only for A. collina wild growing population CW2 and another wild
growing population CW4 compared to all of the other ones. These
accessions show the two extreme values of the essential oil con-
tent: CW4 (Mikóújfalu, Transylvania) produced the highest content
(0.327 ml/100 g) and the genotype CW2 (Csörötnek, West-Hungary)
the lowest one (0.135 ml/100 g). In other wild collected accessions
(in Central Hungary) concentrations between these extreme values
were detected. However, each A. collina sample could surpass the
requirements of the European Pharmacopoeia. In our previous study
on 23 Hungarian wild populations of this species we detected also
signicant (up to four-fold) differences among the samples, however,
no connection between the geographical location and the level of the
essential oil content could be ascertained (németh et al., 2007). The
essential oil content of the selected cultivars and the cultivated geno-
types was much more similar to each other, within a range of 0.2 and
0.3 ml/100 g without signicant difference among them.
According to the recent chemotaxonomic conception, among the in-
vestigated species, proazulenes are only accumulating in A. collina
and A. asplenifolia (kastner et al., 1992; rauchensteiner et al.,
2002). This has been ascertained by our results. The distilled oil of
the other species had a yellowish colour indicating the lack of azu-
lenes, while the samples of the mentioned two species each showed
a blue colour of different intensity. The concentration of proazule-
nes prescribed by the European Pharmacopoeia VIII is 0.1% which,
however, was exceeded only by half of the samples. Besides the
sample of A. asplenifolia it was the case in three wild growing ac-
cessions and in two selected cultivars of A. collina (Tab. 3).
The lowest proazulene content (0.020%) was detected in a wild
growing A. collina population (CW2), while signicantly the high-
est values (0.135% and 0.148%) were also measured in accessions
of wild origin (CW3 and CW5, respectively). Among the cultivated
genotypes, the registered cultivars ‘Proa’ (C1) and ‘Azulenka’ (C3)
showed signicantly the highest proazulene contents (0,110% and
0.133%, respectively). Both cultivated populations (C4 and C5),
furthermore the cultivar ‘Alba’ (C2) and the sample from Remetes-
zőlős (CW6) have each statistically similar proazulene contents
(0.074-0.079%). In previous Hungarian investigations the proazu-
lene content of the wild growing populations was between 30% and
67% (németh et al., 2007). Although these are area percentages de-
tected by GC method, therefore difcult to compare with the present
data, signicant differences among accessions were detected in both
studies. Other trials on European wild yarrow populations A. collina
has been rarely evaluated. In Germany, michler et al. (1992) de-
scribed large differences among populations concerning the presence
of proazulenes.
Genetic markers
In the RAPD analysis 140 bands were detected of which 97.14%
was polymorphic. The numbers of obtained bands were between 9
and 15, the average was 12.36 polymorphic bands/primer. It is high-
er than obtained with A. santolina, A. tenuifolia (ebrahimi et al.,
2012) and with A. millefolium (Fa r a j p o u r et al., 2011), (Tab. 2).
Based on the PCA of RAPD markers, segregation of the taxonomi-
cally most distant A. ptarmica from the accessions of A. collina is
obvious while samples of A. crithmifolia and A. lipendulina are
between them (Fig. 2). These latter diploid species both belong to
sect. Achillea, however, are less closely related to the polyploid
members of the A. millefolium agg. (Gu o et al., 2004). A. collina
genotypes C3 (Hungarian cultivar ‘Azulenka’) and CW6 (wild col-
lected genepool from Remeteszőlős, central Hungary) show the
largest distances from the other ones while genotypes CW1, CW3
and C5 show the closest linkage to each other. The mentioned pat-
tern, however, may not reect geographical or genetic connection.
In case of the studied genotypes, it could be established, that RAPD
markers tend to distinguish primarily among species and less charac-
teristically among intraspecic populations of A. collina.
Using the ISSR markers, the total number of detected bands was
188, about 30% more than in case of RAPD primers. The percentage
of the polymorphic bands reached 97.34% exceeding the values of
other related studies with A. millefolium (Fa r a j p o u r et al., 2012;
Gharibi et al., 2011). The mean number of polymorphic bands/pri-
mer was 15.25, ranging from 10 to 21, also higher than in RAPD
analysis (Tab. 2).
Principal coefcient analysis of the studied samples shows a clear
separation of A. ptarmica (Fig. 3) which reects well the fact that
it is a member of Sect. Ptarmica and taxonomically the less related
species with all the other ones. This is a similar result as that in the
analysis with RAPD markers. A well dened group is formed by four
of the accessions of A. collina of wild origin and another one by the
cultivated accessions of this species. CW2 which is a population of
wild origin shows a larger separation from all of the other A. collina
accessions and especially from the other wild growing ones. A single
Tab. 3: Essential oil and proazulene content of the examined accessions
(codes of accessions as in Tab. 1)
Accession Essential oil content Proazulene content
code (ml/100 g DW) (% D.W.)
Mean Standard Mean Standard
deviation deviation
C1 0.248 c,d 0.082 0.105 b,c 0.029
C2 0.273 c,d 0.097 0.075 b 0.035
C3 0.290 c,d 0.051 0.173 b,c 0.052
C4 0.236 c,d 0.940 0.078 b 0.039
C5 0.248 c,d 0.108 0.074 b 0.043
CW1 0.202 b,c 0.145 0.061 a,b 0.018
CW2 0.135 a,b,c 0.088 0.021 a 0.005
CW3 0.235 c,d 0.141 0.135 c 0.057
CW4 0.365 d,e 0.183 0.106 b,c 0.038
CW5 0.317 c,d 0.212 0.148 c 0.086
CW6 0.199 c,d 0.103 0.079 b 0.075
ASP 0.249 c,d 0,038 0.136 c 0.014
DIS 0.485 e 0,044
MIL 0.005 a 0,001
PAN 0.198 b,c,d 0,026
CRI 0.159 a,b,c 0,062
FIL 0.189 a,b,c 0,009
PTR 0.059 a,b 0,011
Different letters represent statistically different subsets according to the Tukey
test at p=0.05

Evaluation of Achillea accessions 109
Fig. 2: Patterns of relationships among the investigated Achillea accessions revealed by principal component analysis based on RAPD data (codes of acces-
sions as in Tab. 1)
Fig. 3: Patterns of relationships among the investigated Achillea accessions revealed by principal component analysis based on ISSR data (codes of acces-
sions as in Tab. 1)
wild growing accession of A. collina (CW4) and other species of the
sect. Achillea form a further, larger group. In this pattern, species of
the A. millefolium agg. do not reect a close relationship with each
other. The most characteristically separated wild growing populati-
on CW2 (Western Hungary) and CW4 (Transylvania) are located to
larger distances (200-400 km) from the central populations (CW1, 3,
5 and 6). According to the results, ISSR markers proved to be appro-
priate rst of all for the separation of A. collina accessions while the
relationships of other species inside the section are less specic.
A joint evaluation of RAPD and ISSR analysis revealed a good se-
paration of Achillea species (Fig. 4.). Similarly to the results of both
RAPD and ISSR markers separately, A. ptarmica and the members

110 K. Inotai, Z. György, S. Kindlovits, G. Várady, É. Németh-Zámbori
Tab. 4: Genetic distances matrix of the investigated Achillea accessions based on RAPD and ISSR data (codes of accessions as in Tab. 1)
CW2 C3 C2 C1 C4 C5 CW6 CW1 CW5 CW3 CW4 DIS PAN FIL CRI ASP MIL PTR
CW2 1.00
C3 0.40 1.00
C2 0.38 0.30 1.00
C1 0.44 0.36 0.30 1.00
C4 0.46 0.28 0.28 0.29 1.00
C5 0.42 0.35 0.26 0.30 0.23 1.00
CW6 0.45 0.44 0.36 0.36 0.34 0.26 1.00
CW1 0.47 0.41 0.35 0.36 0.33 0.30 0.27 1.00
CW5 0.47 0.45 0.38 0.43 0.32 0.33 0.36 0.30 1.00
CW3 0.46 0.39 0.32 0.39 0.27 0.30 0.30 0.27 0.28 1.00
CW4 0.44 0.42 0.45 0.44 0.38 0.38 0.38 0.36 0.39 0.32 1.00
DIS 0.50 0.46 0.44 0.43 0.46 0.47 0.46 0.42 0.43 0.42 0.39 1.00
PAN 0.48 0.46 0.45 0.46 0.49 0.49 0.48 0.48 0.50 0.50 0.40 0.25 1.00
FIL 0.62 0.57 0.61 0.65 0.65 0.68 0.68 0.63 0.66 0.60 0.55 0.55 0.59 1.00
CRI 0.65 0.64 0.64 0.62 0.69 0.64 0.57 0.54 0.58 0.62 0.64 0.56 0.61 0.54 1.00
ASP 0.47 0.46 0.48 0.43 0.48 0.50 0.49 0.50 0.52 0.41 0.47 0.44 0.45 0.45 0.55 1.00
MIL 0.50 0.47 0.49 0.53 0.51 0.59 0.54 0.49 0.50 0.46 0.45 0.38 0.46 0.53 0.51 0.37 1.00
PTR 0.63 0.63 0.59 0.56 0.63 0.62 0.56 0.59 0.72 0.62 0.59 0.57 0.59 0.57 0.60 0.52 0.53 1.00
Fig. 4: Patterns of relationships among the investigated Achillea accessions revealed by principal component analysis based on RAPD and ISSR data (codes
of accessions as in Tab. 1)
of sect. Achillea are the most characteristically distinguishable from
each other. The accessions of the two related species A. crithmifolia
and A. lipendulina (Section Achillea, exc. A. millefolium agg.) are
situated closer to each other in the PCA analysis than to any of the
accessions in agg. A. millefolium. The studied genotypes of A. dis-
tans and A. millefolium seem to be closely linked with each other.
AFLP proles of these two species were also hardly distinguishable
from each other (Gu o et al., 2005) being two related polyploids of
the A. millefolium agg. The species A. pannonica, A. distans and
A. millefolium gave overlapping patterns and could not be clearly
separated based on essential oil markers, either (rauchensteiner
et al., 2002).
Accessions of A. collina represent another group. By the joint evalu-
ation of RAPD and ISSR markers, cultivated and wild growing ac-

Evaluation of Achillea accessions 111
cessions do not seem to separate characteristically from each other,
except the geographically more distant CW2 and CW4 which are
situated in the PCA coordinate system characteristically far away.
In coincidence with the above mentioned, the largest genetic distance
coefcients were proven between A. ptarmica and the members of
sect. Achillea (genetic distances between 0.52 and 0.72), (Tab. 4).
Distances of the species inside the Section Achillea are in most cases
below 0.5. The genetic distances of CW2 originating from Western
Hungary are the largest to any of the other wild originated accessi-
ons exceeding 0.4. On the other side, similarity is highest (genetic
distance 0.27 both) between the populations CW1-CW3 and CW1-
CW5 where geographical distances of the original locations are
52-55 km. Nei’s genetic distances among the cultivars are also rela-
tively low, between 0.23 and 0.36.
The values of the Shannon diversity index (H) (Lewontin, 1972)
reect similar results. This value calculated for all of the accessions
in this study is 0.5209 while taking into account only the A. collina
accessions it shows a lower value: 0.4098. The intraspecic diversity
seems to be higher among the wild growing accessions (H=0.3793)
than among the cultivars (H=0.3028).
Based on the investigated markers, it can be established, that the
studied RAPD provided a more specic approach for distinction of
species while the used ISSR and combined RAPD and ISSR primer
evaluation enabled an informative evaluation among A. collina ac-
cessions, as well. Nevertheless, the separation of populations based
on the investigated molecular markers does not reect any connec-
tion with their essential oil and proazulene contents.
According to the results of this study, a common origin of the cul-
tivated populations might be anticipated. ‘Proa’ is the rst selected
cultivar of A. collina. It is a German cultivar, which has been on the
market since 1973. Compared to this, ‘Alba’, a Slovakian cultivar
was registered almost twenty years later in 1992. As no relevant in-
formation on the original genetic background of these selections is
available, a relationship cannot be excluded. Besides, even in case
of their different origins, during the decades of their cultivation in
Central Europe, a stepwise reduction of the genetic divergence might
be hypothesized. It is supported by the fact that the two accessions
from commercial cultivation seem to be closely related to them, as
well. ‘Azulenka’, however, a recently (2013) registered Hungarian
cultivar which has been selected from a wild population of central
Hungary shows the lowest similarity with the formerly mentioned
taxa.
Genetic relationships of the accessions collected from the wild
show a connection with their original geographical habitats. This is
similar to the ndings of Iranian authors in case of some other yar-
row species (Fa r a j p o u r et al., 2011; Gharibi et al., 2011) although
the geographic distances of the studied populations were much lar-
ger than in our study. In our case the most distant populations (200-
600 km from Central Hungary) proved to have the smallest similari-
ty with each other and with the central ones. The populations within
a 50-60 km range of distance show much higher similarity. This n-
ding may be in connection with the fact that A. collina is a common
weed species in these area having the potential to be transported and
distributed easily around resulting in a decreased genetic diversity
inside the mentioned central region. As since the start of our work,
SSR markers are also available, a continuation of this work is for-
seen inclusing further A. collina accessions from the region.
Conict of interest
The authors declare that they have no conict of interest.
References
ak ya l c i n , h., arabaci, t., yil D iz , b., 2011: Pollen morphology of six
Achillea L. sect. Achillea (Asteraceae) species in Turkey. Turk. J. Bot.
35, 183-201.
Dabrowska, j., 1977: Observations of the fruit size of nine taxons from the
genus Achillea L. representing natural di-, tetra-, hexa- and octaploids.
Herba Pol. 28, 55-66.
Danihelka, j., ro t r e k l o v á , o., 2001: Chromosome numbers within the
Achillea millefolium and A. distans groups in the Czech republic and
Slovakia. Folia Geobot. 36, 163-191.
ebrahimi, m., Far a j p o u r , m., ra h i m m a l e k , m., 2012: Inter- and intra-
specic genetic diversity of Iranian yarrow species Achillea santolina
and A. tenuifolia based on ISSR and RAPD markers. Genet. Mol. Res.
11, 2855-2861.
Fa r a j p o u r , m., ebrahimi, m., am i r i , r., no o r i , s.a.s., sa n j a r i , s.,
Go l z a r i , r., 2011: Study of genetic variation in yarrow using inter-
simple sequence repeat (ISSR) and random amplied polymorphic DNA
(RAPD) markers. Afr. J. Biotechnol. 10, 11137-11141.
Final community herbal monograph on Achillea millefolium L., herba, EMA/
HMPC/290284/2009
http://www.ema.europa.eu/docs/en_GB/document_library/Herbal_-_
Community_herbal_monograph/2011/09/WC500115470.pdf (accessed:
23.02.2015)
Galbraith, D.w., ha r k i n s , k.r., maDDox, j.m., ay r e s , n.m., sh a r m a ,
D.p., Fi r o o z a b a D y , e., 1983: Rapid ow cytometric analysis of the cell
cycle in intact plant tissues. Science 220, 1049-1051.
Gharibi, s., ra h i m m a l e k , m., mirlohi, a., majiDi, m.m., ta b a ta b a e i ,
b.e.s., 2011: Assessment of genetic diversity in Achillea millefolium
subsp. millefolium and Achillea millefolium subsp. elbursensis using
morphological and ISSR markers. J. Med. Plants. Res. 5, 2413-2423.
Gu o , y.p., eh r e n D o r F e r , F., sa m u e l , r., 2004: Phylogeny and systematics
of Achillea (Ateraceae-Anthelmideae) inferred from nrITS and plastid
trnL-F DNA sequences. TAXON 53, 657-672.
Gu o , y.p., sa u k e l , j., mittelmayr, r., ehr e n F o r F e r , F., 2005: AFLP
analyses demonstrate genetic divergence, hybridization and multiple
polyploidization in the evolution of Achillea (Asteraceae-Anthemideae).
New Phytologist 166, 273-290.
Ha m m e r , Ř., Harper, D.a.T., ry a n , n.p.D., 2001: PAST: Paleontological
statistics software package for education and data analysis. Palaeontol.
Electron. 4, 9.
kaFt, t., sä l l , t., 1999: An evaluation of the use of pooled samples in stu-
dies of genetic variation. Heredity 82, 488-494.
kästner, u., sa u k e l , j., zitterl-eG l s e e r , k., la n G e r , r., reznicek, G.,
jurenitsch, j., ku b e l k a , w., 1992: Ätherisches Öl − ein zusätzliches
Merkmal für Charakterisierung der mitteleuropaischen Taxa der Achillea
millefolium-Gruppe. Sci. Pharm. 60, 87-99.
lewontin, r., 1972: „The Apportionment of Human Diversity“. Evol. Biol.
6, 391-398.
ma, j.x., li, y.n., voGl, c., eh r e n D o r F e r , F., Gu o , y.p., 2010: Allopoly-
ploid speciation and ongoing backcrossing between diploid progenitor
and tetraploid progeny lineages in the Achillea millefolium species com-
plex: analyses of single-copy nuclear genes and genomic AFLP. BMC
Evol. Biol. 10, 100. doi: 10.1186/1471-2148-10-100
michler, b., pr e i t s c h o p F , a., er h a r D , p., arn o l D , c., 1992: Achillea
millefolium: Zusammenhänge zwischen Standortfaktoren, Ploidiegrad,
Vorkommen von Proazulenen und Gehalt an Chamazulen im ätherischen
Öl. PZ-Wissenschaft 137, 23-29.
ne i , m., 1972: Genetic distance between populations. Am. Nat. 106, 83-
292.
németh, é., 2005: Essential oil composition of species in the genus Achillea.
J. Essent. Oil. Res. 17, 501-512.
németh, é., be r n á t h , j., ta r j á n , G., 2007: Quantitative and qualitative
studies of essential oils of Hungarian Achillea populations. J. Herbs.
Spices. Med. Plants, 13, 57-69.
or t h , m., juchelka, D., mo s a n D l , a., czyGan, F., 2000: Enantiomere
Monoterpene im ätherischem Öl von Achillea millefolium s.l. – eine zu-
satzliche taxonomische Bestimmungshilfe? Pharmazie 55, 456-459.
oswiecimska, m., 1962: Variable content of volatile oil and azulenes in

112 K. Inotai, Z. György, S. Kindlovits, G. Várady, É. Németh-Zámbori
herbs milfoil. Part I. variability independent of habitat. Dissertationes
Pharmaceuticae 14, 539-545.
ra D u l o v i c , n., zl a t k o v i c , z., pa l i c , r., st o j a n o v i c , G., 2007: Chemo-
taxonomic signicance of the Balkan Achillea volatiles. Nat. Prod. Com-
mun. 2, 453-74.
ra h i m m a l e k , m., ta b at a b a e i , b.e.s., arz a n i , a., et e m a D i , n., 2009: As-
sessment of genetic diversity among and within Achillea species using
amplied fragment length polymorphism (AFLP), Biochem. Syst. Ecol.
37, 354-361. doi: 10.1016/j.bse.2009.06.2002
rauchensteiner, F., ne j at i , s., werner, i., Gl a s l , s., sa u k e l , j., ju-
renits, j., ku b e l k a , w., 2002: Determination of taxa of the Achillea
millefolium group and Achillea crithmifolia by morphological and phy-
tochemical methods I. Characterisation of Central European taxa. Sci.
Pharm. 70, 199-230.
tétényi, p., ty i h á k , e., má t h é , i., sv á b , j., 1962: Untersuchungen über
die Azulenverbindungen der Achillea-Arten. 1. Mikrochemische Unter-
suchungsmethoden. Die Pharmazie 17, 463-466.
va l a n t , k., 1978: Characteristische Flavonoidglykoside und verwandt-
schaftliche Gliederung der Gattung Achillea. Naturwissenschaften 65,
437-438.
wa l l n e r , e., we i si n G, k., ro m p F , r., ka h l , G., ko p p , b., 1996: Oligo-
nucleotide ngerprinting and RAPD analysis of Achillea species: Cha-
racterisation and long-term monitoring of micropropagated clones. Plant.
Cell Rep. 15, 647-652.
ye h , F.c., ya n G , r.c., bo y l e , t.b.j., ye, z.h., ma o , j.x., 1997: POPGE-
NE, the user-friendly shareware for population genetic analysis. Molecu-
lar Biology and Biotechnology Centre, University of Alberta, Canada.
Addresses of the authors:
Éva Németh-Zámbori, Katalin Inotai, Sára Kindlovits, Department of Me-
dicinal and Aromatic Plants, Szent István University, H-1118, Budapest,
Villányi Str. 29-43., Hungary
E-mail: zamborine.nemeth.eva@kertk.szie.hu
E-mail: inotai.katalin@kert.szie.hu
E-mail: kindlovits.sara@kertk.szie.hu
Zsuzsanna György, Department of Genetics and Breeding, Szent István Uni-
versity, H-1118, Budapest, Ménesi Str. 44., Hungary
E-mail: gyorgy.zsuzsanna@kertk.szie.hu
György Várady, Institute of Enzymology, Research Centre for Natural Sci-
ences, Hungarian Academy of Sciences, 1117 Budapest, Magyar tudósok
körútja 2., Hungary
E-mail: varady.gyorgy@ttk.mta.hu
© The Author(s) 2016.
This is an Open Access article distributed under the terms
of the Creative Commons Attribution Share-Alike License (http://creative-
commons.org/licenses/by-sa/4.0/).