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 intraspecific 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- traspecific 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 Amplified 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 Amplified 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, flatulence, 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 (Guo 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 intraspecific level. Therefore, taxonomic evaluati- on, identification of species and systematic classification has been a difficult task for decades. With regard to species identification 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 leaflets and rayflorets (rauchensteiner et al., 2002), and pollen morphology (akyalcin et al., 2011) have been defined 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; Daniheka and rotreklová, 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 flavonoids may have taxonomic importance, as well (valant, 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 firm 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 identifica- tion and systematics (orth, 2000; rauchensteiner at al., 2002; raDulovic et al., 2007). The use of molecular markers in the systematic studies of yarrow was introduced from the 1990th. wallner et al. (1996) proved the applicability of RFLP and PCR based fingerprinting 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 (Guo et al., 2004), although this method could not assure a well established separation of A. millefolium aggr. In the work of Guo 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; Farajpour et al., 2011; Gharibi et al., 2011) and AFLP (rahimmalek 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 official 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 intraspecific taxa of A. collina, the most frequently collected and cultivated species. According to our knowledge, molecular genetic study of intraspecific 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 officially 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. filipendu- lina of the A. Millefolium agg. (Guo 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 flow 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 (kraFt and säll, 1999) from 10-15 plants/accession was carried out for PCR trials as well as checking of chromosome numbers by flow cy- tometry (Fig. 1) according to the modified method of Galbraith et al. (1983). After 2 months, the seedlings were planted into open field plots in three replicates at our experimental station in Budapest. At flowering 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. filipendulina 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- plification 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 amplification 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 specific annealing temperature, 1 min at 72 °C and a final extension for 7 min at 72 °C. Amplified 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 specific annealing temperature, 45 s at 72 °C and final 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. Amplified 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 (yeh and boyle, 1997) was used to estimate number of polymorphic bands, percentage of polymorphic bands, nei’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 (hammer 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 fluorescence intensity of octoploid (A. panno- nica), hexaploid (A. millefolium), tetraploid (A. collina) and diploid (A. filipendulina) 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 significance 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. filipendulina. Taking into account only the A. collina accessions, the differences are much lower; significant 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 significant (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 significant 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 significantly 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 significantly 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 difficult to compare with the present data, significant 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 (Farajpour 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. filipendulina 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. (Guo 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 reflect 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 intraspecific 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 (Farajpour 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 coefficient analysis of the studied samples shows a clear separation of A. ptarmica (Fig. 3) which reflects 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 defined 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 reflect 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 first of all for the separation of A. collina accessions while the relationships of other species inside the section are less specific. 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. filipendulina (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 profiles of these two species were also hardly distinguishable from each other (Guo 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 coefficients 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) reflect 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 intraspecific 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 specific 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 reflect 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 first 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 findings of Iranian authors in case of some other yar- row species (Farajpour 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 fin- 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. 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