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Genetic diversity of roseroot (Rhodiola rosea) in North-Norway
Biochemical Systematics and Ecology 50 (2013) 361–367
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Genetic diversity of roseroot (Rhodiola rosea) in North-Norway Zsuzsanna György a, *, Erling Fjelldal b, Márta Ladányi c, Paul Eric Aspholm b, Andrzej Pedryc a a
Department of Genetics and Plant Breeding, Corvinus University of Budapest, P.O. Box 53, H-1518 Budapest, Hungary Bioforsk, Norwegian Institute for Agriculture and Environmental Research, Soil and Environment Svanhovd, Norway c Department of Mathematics and Informatics, Corvinus University of Budapest, P.O. Box 53, H-1518 Budapest, Hungary b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 March 2013 Accepted 18 May 2013 Available online
Roseroot (Rhodiola rosea L.), an adaptogenic herb, has received increased attention in the last years. Genetic diversity of roseroot in Northern-Norway was studied with ISSR markers. Plants were collected in Finnmark County, Norway at 10 habitats. Using 8 ISSR primers 53 DNA fragments were generated and 92.45% of those were found to be polymorphic, indicating high genetic variability at the species level (Shannon index ¼ 0,4122). Lower level of diversity was detected at the population level (Shannon-index ranged from 0.21 to 0.36). Generated UPGMA dendrogram revealed 2 groups. An attempt was made to connect molecular marker data to the pharmacologically important glycoside content of the plants. The habitat with the lowest glycoside content separated from the others on the dendrogram, but no ISSR marker could be assigned to this trait. AMOVA showed that molecular variance has no effect on the glycoside content, it is only effected by environmental factors. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Rhodiola rosea Molecular markers ISSR Genetic diversity Rosavins Salidroside
1. Introduction Rhodiola rosea L., commonly known as golden root, arctic root or roseroot has been used in the traditional medicine of Russia and Scandinavia for centuries. People have used it as general immune-stimulant and the ultimate to overcome fatigue. This traditional adaptogen medicinal plant is receiving increased attention in the last years. Roseroot belongs to the family Crassulaceae. It is a herbaceous plant with thick rhizome, which contains pharmacologically important secondary metabolites (Brown et al., 2002). The species displays a circumpolar distribution in the higher latitudes and elevations of the Northern hemisphere mainly in Asia and Europe. According to Hegi (1963), its distribution in Europe extends from Iceland and the British Isles across Scandinavia as far south as the Pyrenees, the Alps, the Carpathian Mountains and other mountainous Balkan regions. Roseroot is highly variable both in phytochemical (Kurkin et al., 1988; Wiedenfeld et al., 2007) and in morphological aspect (Ohba, 1981, 1989, Asdal et al., 2006). Nowadays several commercially available products exist based on the extracts of its rhizome, of which raw material mostly comes from harvesting from wild populations. A key to the successful cultivation is the stable high value cultivars achieved through breeding. Establishing a fruitful breeding work starts with the assessment and evaluation of the natural populations.
* Corresponding author. Tel.: þ36 14826530. E-mail addresses: [email protected] (Z. György), [email protected] (E. Fjelldal), [email protected] (M. Ladányi), [email protected] (P.E. Aspholm), [email protected] (A. Pedryc). 0305-1978/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2013.05.009
362
Z. György et al. / Biochemical Systematics and Ecology 50 (2013) 361–367
Roseroot is widely spread in Norway. In 2006 ca. 200 plants from 10 geographic regions distributed along the coast of Finnmark County in Northern-Norway were collected (Fjelldal et al., 2010). One mixed root sample (including several individuals) from each habitat was analysed for glycoside content. The results showed large geographical variations in the content of the studied metabolites. Total content of rosavin varied between 0.067 % and 2.7%, with a mean value of 1.54% for the 10 studied regions. Studies concerning the genetic diversity of roseroot have been conducted with different methods. Elameen et al. (2008) investigated the genetic diversity in a Norwegian germplasm collection by AFLP. Finnish Rhodiola rosea populations were analysed by György et al. (2012) with ISSR markers. In 2009 Zini et al. published eight microsatellite sequences (simple sequence repeats, SSR) and flanking primer pairs. These primers were tested on two Rhodiola rosea populations from the Trentino Alps. Four out of these primers were also used by Kylin (2010) for evaluating genetic diversity of roseroot plants collected in Sweden, Greenland and Faroe Islands. Recently Kozyrenko et al. (2011) analysed the genetic structure of Rhodiola rosea mostly of Russian origin using ISSR polymorphisms. The aim of the present work was to characterize genetic diversity among roseroot individuals from habitats in NorthernNorway using ISSR markers and to examine if ISSR polymorphism is able to evince the big alterations among the habitats or individuals that were earlier detected in course of studying the chemical composition of the investigated plant material. 2. Materials and methods 2.1. Plant material Rhodiola rosea plants were collected in Finnmark County, Northern-Norway at 10 habitats along the coast (Fig. 1, Table 1). All of the habitats were close to the sea (less than 200 m), in an altitude approximately 10–50 m above sea level. The collected plants were further cultivated in the experimental field of Bioforsk, Svanhovd. From each habitat 5–6 plants were included in the study, all together 58 plants. The plant material was frozen in liquid nitrogen and was stored in 80 C until used. DNA was extracted from the frozen leaves according to a CTAB-based protocol (Pirttilä et al., 2001). DNA concentration and quality was assessed using NanoDrop (BioScience, Hungary) and on 1% agarose gel. 2.2. PCR amplification of ISSR markers PCR was performed in 25 ml reaction volume containing 20–80 ng DNA, 10X PCR reaction buffer, 2.5 mM MgCl2, 0.02 mM dNTP mix, 2.5 mmol of primers, 1 unit of Taq DNA polymerase (Fermentas, Szeged, Hungary) and sterile distilled water. Eight primers from the University of British Columbia, Canada set 9 of ISSR primers (http://www.michaelsmith.ubc.ca) (BC809, BC840, BC841, BC857, BC873 BC885, BC887 BC888) were chosen based on preliminary experiments. For the amplification of ISSR fragments the following program was used: initial denaturation at 94 C for 4 min; followed by 40 cycles of 94 C for 60 s, 49 C for 90 s, 72 C for 90 s; and a final synthesis at 72 C for 7 min. The PCR products were applied on a 1% (w/v) ethidium bromide-stained agarose gel in 1xTBE buffer with xylencyanol loading buffer. PCR products were separated for 2 h at 120 V. Amplified fragments were scored visually for presence (1) or absence (0) of homologous bands and the results were summarised in MS Excel table. 2.3. Data analysis 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
Fig. 1. Map of Northern-Norway, showing the 10 locations of the examined roseroot habitats.
Z. György et al. / Biochemical Systematics and Ecology 50 (2013) 361–367
363
Table 1 Description and geographic coordinates of the 10 habitats where Rhodiola rosea plants were collected. Habitat
Coordinates
Description of the spot
Zone Zone Zone Zone
1 2 3 4
36w04147/utm77437 35w05874/utm77690 36w03996/utm77834 36w04192/utm78161
Zone Zone Zone Zone Zone Zone
5 6 7 8 9 10
35w05844/utm7855 35w05319/utm78143 36w05002/utm78334 35w0485/utm78357 35w04151/utm7875 36w05982/utm78349
Slope leaning north. Both rich in humus and mixture of humus/gravel. Slope leaning north. Thin layer of humus over solid rock. Hardly any soil at all, growing in cracks in rocks/slate. Leaning south-east. Close to sea. Growing in cracked rocks and sand/gravel. Leaning north, close to rough sea. Tall plants, often growing in the shade from rocks/slate. Growing in cracked rocks and sand/gravel. Leaning north, close to rough sea. Open terrain. Steep hill, leaning east, quite high above sea level. Growing in humus/gravel (slate). Leaning east. Growing in sand/gravel/rocks. Leaning east. Growing in sand/gravel/rocks (slate). Leaning east Growing on humus over solid granite.
data. Genetic relatedness among habitats and genotypes was studied by UPGMA (Unweighted Pair Group Method with Arithmetic averages) cluster analysis using Popgene. The results of chemical analysis were earlier published (Fjelldal et al., 2010). Based on the standardized results clusters were formed with Ward-linkage according to squared Euclidean distance. To confirm these clusters, canonical discriminant analysis was performed. The binominal ISSR matrix was used for the analysis of molecular variance (AMOVA) implemented in Genalex 6.5 (Peakall and Smouse, 2012). AMOVA was used to estimate the partition of the genetic variation within and among the clusters, which were created based on the glycoside contents. The significance of the variance components was determined with a permutation test (999 replicates). 3. Results Using 8 ISSR primers analysis of the genetic diversity of 58 roseroot plants from the coast of Northern-Norway was conducted. The selected 8 ISSR primers generated 53 bands, ranging from 5 to 9 bands, corresponding to an average of 6.63 bands per primer. At the population level the Shannon-index was 0.41 with a standard deviation of 0.22 and the percentage of polymorphic loci (PPL) was 92.45%. However genetic variability was rather low at the habitats as indicated in Table 2. The highest level of variability occurred in habitat 3 (PPL ¼ 64.15%; h ¼ 0.24; I ¼ 0.36), whereas the lowest level in habitat 4 (PPL ¼ 35.85%; h ¼ 0.15; I ¼ 0.21). Genetic relationships among the studied habitats were calculated (Table 3) and an UPGMA-based dendrogram obtained is shown in Fig. 2A. The 10 habitats of roseroot were grouped into two subgroups. One clade included Habitat 1 and 2, while the second clade included all remaining habitats. In the second subgroup Habitat 10 was divergent from the other habitats. The closely situated habitats had highest genetic similarity (Habitat 1 and 2; 3 and 4; 6 and 7; 8 and 9). These habitats are always located on the same peninsula, while habitat 10 is located on an island. Fig. 3 shows the salidroside, rosavins and total rosavins content of the 10 habitats (Fjelldal et al., 2010). The graph shows large geographical variations in the content of these metabolites. The content of rosavins and total rosavins show strong correlation since rosavins is one of the three compounds called the “rosavins”. Some habitats have high salidroside content and lower rosavins content (HS_LR) like habitat 2, 3, 4. Some has lower level of salidroside and higher rosavins content (LS_ HR) like habitat 7 and 9. Some has about the same (middle) levels of salidroside and rosavins (MS_MR) like habitat 1, 5, 6, 8. Habitat 10 has outstandingly low levels of each compound (OLC). Fig. 2B shows a dendrogram using Ward-linkage hierarchical cluster analysis where the observed 4 clusters with different compound amounts are supported. Canonical discriminant analysis (Fig. 4) also confirmed these 4 clusters (Wilk’s lambda: 0.26 and 0.29 with Chi-square(9) ¼ 20.03 and Chi-square(4) ¼ 6.79; p < 0.05 and p ¼ 0.148 for the first two canonical discriminant functions, respectively). The group membership was predicted 100% successfully. Table 2 Genetic variability measures of the 10 roseroot habitats based on 8 ISSR marker. Habitat
Number of polymorphic bands
% of polymorphic bands
Gene diversity (h)
St. dev.
Shannon-index (SI)
St. dev.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. All
32 34 34 19 31 25 24 30 28 26 49
60.38 64.15 64.15 35.85 58.49 47.17 45.28 56.60 52.83 49.00 92.45
0.2093 0.2218 0.2415 0.1478 0.1941 0.1542 0.1428 0.2057 0.1591 0.1842 0.2666
0.2029 0.1966 0.2064 0.2100 0.1975 0.1857 0.1806 0.1999 0.1841 0.2093 0.1641
0.3137 0.3339 0.3567 0.2140 0.2939 0.2356 0.2196 0.3073 0.2462 0.2719 0.4122
0.2867 0.2792 0.2919 0.2985 0.2806 0.2706 0.2644 0.2882 0.2652 0.2982 0.2197
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Table 3 Nei’s genetic identity of the roseroot individuals of the 10 North-Norwegian habitats based on 8 ISSR markers. Habitat
1
2
3
4
5
6
7
8
9
10
1 2 3 4 5 6 7 8 9 10
X
0.90 X
0.90 0.87 X
0.87 0.85 0.94 X
0.83 0.80 0.90 0.90 X
0.85 0.86 0.89 0.93 0.94 X
0.85 0.88 0.90 0.92 0.91 0.95 X
0.84 0.84 0.90 0.90 0.93 0.93 0.92 X
0.86 0.84 0.92 0.92 0.93 0.95 0.92 0.97 X
0.88 0.85 0.89 0.87 0.91 0.90 0.88 0.88 0.89 X
Fig. 2. A: Dendrogram of the 10 roseroot habitats assayed in this study generated by UPGMA cluster analysis of the similarity matrix obtained using Nei’s genetic distance based on ISSR data (Nei, 1973) B: Dendrogram using Ward linkage hierarchical cluster analysis with squared Euclidean distance based on the salidroside and rosavin content of the 10 habitats.
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30
mg/g dried material
25
20 salidroside 15
rosavine tot.rosavin
10
5
0 1
2
3
4
5
6
7
8
9
10
Habitat
Fig. 3. Salidroside, rosavin and total rosavins content of the 10 studied North-Norwegian habitats.
AMOVA (Table 4) of the 10 habitats and the 4 glycoside groups showed that the most molecular variation, 85% was found within habitats, and only 16% among habitats. When the habitats were analysed in four groups based on their glycoside content, the molecular variance due to the groups was 0.00%. 4. Discussion The aim of the present study was to estimate the genetic diversity of Rhodiola rosea with ISSR markers in 10 habitats in Northern-Norway. ISSR markers were sensitive enough and revealed polymorphism. Genetic variability within the populations detected with the ISSR method was somewhat higher than in the study of Kozyrenko et al. (2011). Shannon index for the 10 North-Norwegian habitats ranged between 0.21 and 0.36 while in the study of Kozyrenko et al. (2011) it ranged between 0.14 and 0.26. However, since only half of the primers used are the same in the two studies comparison of the results is not fully appropriate. The genetic diversity among the habitats seems to be much
Fig. 4. Classification plot of the discriminant scores of the first two canonical discriminant functions from the discriminant function analysis of the 10 habitats (P1-P10). Different symbols denote groups with different amount of compounds such as high salidroside-low rosavins (HS_LR), higher rosavins-low salidroside (LS_HR); middle salidroside and rosavins (MS_MR) and outstandingly low compound (OLC).
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Z. György et al. / Biochemical Systematics and Ecology 50 (2013) 361–367
Table 4 Results of molecular variance (AMOVA) for 10 Rhodiola rosea habitats based on ISSR markers and 4 groups based on the glycoside contents. Source of variation
Degrees of freedom
Sum of squares
Estimated variance
% of total variance
Fixation index (V)
Significance (P)
Among groups Among populations Within population Total
3 6 48 57
41.43 82.37 314.93 438.72
0 1.25 6.56 7.81
0 16 84 100
0.00a 0.16b 0.16c
0.534 0.001 0.001
a, b, c Fixation indices are calculated as the correlations. a among groups. b among populations. c within population relative to the total.
higher in the present study. Shannon index was 0.41 while in the study of Kozyrenko et al. (2011) it was just 0.29. On the other hand this would be very strange, considering that in the present study the sampling area was far much smaller, than in the study of Kozyrenko et al. (2011). This phenomenon can be due to the subjective evaluation of the ISSR gels. Earlier two Northern-Finnish roseroot populations were compared with ISSR method (György et al., 2012) and Shannonindexes of 0.31 and 0.23 were calculated within the two populations, while 0.34 among the populations. These are comparable values to the present results. Generally all these Shannon index values are similar to those of Rhodiola crenulata from the Hengduan Mountains (0.17–0.33, Lei et al., 2006) and somewhat higher than of Rhodiola alsia (0.07–0.22, Xia et al., 2005) and Rhodiola chrysanthemifoila (0.08–0.24, Xia et al., 2007) in the Tibetian Plateau, indicating rather low genetic diversity in the studied populations. Results of chemical analysis of these 10 habitats showed also big differences. Salidroside content varied between 0.46 and 2.61% and the content of total rosavins varied between 0.67 and 2.7% comparing the 10 habitats. Both the lowest salidroside level and also the lowest level of rosavins were detected in Habitat 10 (Fjeldal et al., 2010). This habitat was also found to be separated based on its genetic background in the present study. This separation is approved by the geographics, as this habitat is located on an island. 4 groups were formed based on the glycoside content. AMOVA was performed to see if the molecular variance is in correlation with the glycoside content. The results showed that molecular variance has no effect on the glycoside content, it is only effected by environmental factors. In conclusion, using ISSR markers we were able to assess genetic diversity of roseroot of 10 habitats from NorthernNorway. Even though the habitat with the lowest glycoside content showed some separation from the others no molecular marker could be assigned to the low glycoside content and molecular variance of these ISSR markers has no effect on the glycoside content. Acknowledgement This study was financed by Hungarian Scientific Research Fund (OTKA 83728), and the National Development Agency (TÁMOP-4.2.1/B-09/1/KMR-2010-0005 and TÁMOP-4.2.2/B-10/1-2010-0023). References Asdal, A., Galambosi, B., Olsson, K., Wedelsback Bladh, K., Porvaldsdóttir, E., 2006. Spice-and medicinal plants in the Nordic and Baltic countries. In: Asdal, A., Olsson, K. (Eds.), Spice-and Medicinal Plants in the Nordic and Baltic Countries: Conservation of Genetic Resources: Report from a Project Group at the Nordic Gene Bank. Alnarp Nordic Gene Bank, pp. 94–104. Brown, R.P., Gerbarg, P.L., Ramazanov, Z., 2002. Rhodiola rosea, a phytomedicinal overview. Herbal Gram 56, 40–52. Elameen, A., Klemsdal, S.S., Dragland, S., Fjellheim, S., Rognli, O.A., 2008. Genetic diversity in a germplasm collection of roseroot (Rhodiola rosea) in Norway studied by AFLP. Biochem. Syst. Ecol. 36, 706–715. Fjelldal, E., Svenske, M., Martinussan, I., Volodin, V., Galambosi, B., 2010. Geographic variation in chemical composition in roseroot (Rhodiola rosea) in Finnmark County. In: The 7th Circumpolar Agricultural Conference, Alta, Finnmark, Norway September 6–8, 2010, p. 35. Book of Abstract. György, Z., Derzsó, E., Galambosi, B., Pedryc, A., 2012. Genetic diversity of Finnish Rhodiola rosea populations based on SSR and ISSR analysis, Acta Horticulturae. In: Proceedings of the I. International Symposium on Medicinal, Aromatic and Nutraceutical Plants from Mountainous Areas, 2011. 07. 06–09, vol. 955. Saas-Fee, Switzerland, pp. 197–202. Hegi, G., 1963. Rhodiola, Rosenwurz. Liefering 2/3. In: . In: Hegi, G. (Ed.), Illustrierte Flora von Mitteleuropa, Zweite völlig neubearbeitete edn, Band IV/2, pp. 99–102. Hamburg/Berlin. Kozyrenko, M.M., Gontcharova, S.B., Gontcharov, A.A., 2011. Analysis of the genetic structure of Rhodiola rosea (Crassulaceae) using inter-simple sequence repeat (ISSR) polymorphisms. Flora 206, 691–696. Kurkin, V.A., Zapesochanaya, G.G., Nukhimovskii, E.L., Klimakhin, G.I., 1988. Chemical composition of rhizomes of Mongolian Rhodiola rosea L. population introduced into districts near Moscow. Khim. Farm. Zhþ 22, 324–326. Kylin, M., 2010. Genetic Diversity of Roseroot (Rhodiola rosea L.) from Sweden, Greenland and Faroe Islands (Diploma thesis). Swedish University of Agricultural Sciences, Alnarp, Sweden. Lei, Y., Gao, H., Tsering, T., Shi, S., Zhong, Y., 2006. Determination of genetic variation in Rhodiola crenulata from the Hengduan Mountains Region, China using inter-simple sequence repeats. Genet. Mol. Biol. 29, 339–344. Lewontin, R.C., 1972. The apportionment on human diversity. Evol. Biol. 6, 381–398. Nei, M., 1973. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89, 583–590. Ohba, H., 1981. A revision of Asiatic species of Sedoideae (Crassulaceae). Part 2. Rhodiola (subgen. Rhodiola, sect. Rhodiola). J. Fac. Sci. Univ. Tokyo 13, 65–119. Ohba, H., 1989. Biogeography of the genus Rhodiola (Crassulaceae), with special reference to the floristic interaction between the Himalaya and Arctic region. In: Ohba, H. (Ed.), 1989. Current Aspects of Biogeography in West Pacific and East Asian Regions, vol. 1. University of Tokyo, Tokyo, pp. 115–133.
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Peakall, R., Smouse, P.E., 2012. GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research-an update. Bioinformatics 28, 2537–2539. Pirttilä, A.M., Hirsikorpi, M., Kämäräinen, T., Jaakola, L., Hohtola, A., 2001. DNA isolation methods for medicinal and aromatic plants. Plant Mol. Biol. Rep. 19, 273 (a-f). Wiedenfeld, H., Dumaa, M., Malinowski, M., Furmanowa, M., Narantuya, S., 2007. Phytochemical and analytical studies of extracts from Rhodiola rosea and Rhodiola quadrifida. Pharmazie 62, 308–311. Xia, T., Shilong, C., Shengyun, C., 2005. Genetic variation within and among population of Rhodiola alsia (Crassulaceae) native to the Tibetan Plateau as detected by ISSR markers. Biochem. Genet. 43, 87–99. Xia, T., Shilong, C., Shegyun, C., Defang, Z, Dejun, Z, Qingbo, G, Xuejun, G, 2007. ISSR analysis of genetic diversity of the Qinghai-Tibet Plateau endemic Rhodiola chrysanthemifolia (Crassulaceae). Biochem. Syst. Ecol. 35, 209–214. Yeh, F.C., Boyle, T.J.B., 1997. Population genetic analysis of co-dominant and dominant markers and quantitative traits. Belg. J. Bot. 129, 157. Zini, E., Clamer, M., Passerotti, S., Vender, C., Vendramin, G.G., Komjanc, M., 2009. Eight novel microsatellite DNA markers in Rhodiola rosea L. Conserv. Genet. 10, 1397–1399.
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Genetic diversity of roseroot (Rhodiola rosea) in North-Norway Zsuzsanna György a, *, Erling Fjelldal b, Márta Ladányi c, Paul Eric Aspholm b, Andrzej Pedryc a a
Department of Genetics and Plant Breeding, Corvinus University of Budapest, P.O. Box 53, H-1518 Budapest, Hungary Bioforsk, Norwegian Institute for Agriculture and Environmental Research, Soil and Environment Svanhovd, Norway c Department of Mathematics and Informatics, Corvinus University of Budapest, P.O. Box 53, H-1518 Budapest, Hungary b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 March 2013 Accepted 18 May 2013 Available online
Roseroot (Rhodiola rosea L.), an adaptogenic herb, has received increased attention in the last years. Genetic diversity of roseroot in Northern-Norway was studied with ISSR markers. Plants were collected in Finnmark County, Norway at 10 habitats. Using 8 ISSR primers 53 DNA fragments were generated and 92.45% of those were found to be polymorphic, indicating high genetic variability at the species level (Shannon index ¼ 0,4122). Lower level of diversity was detected at the population level (Shannon-index ranged from 0.21 to 0.36). Generated UPGMA dendrogram revealed 2 groups. An attempt was made to connect molecular marker data to the pharmacologically important glycoside content of the plants. The habitat with the lowest glycoside content separated from the others on the dendrogram, but no ISSR marker could be assigned to this trait. AMOVA showed that molecular variance has no effect on the glycoside content, it is only effected by environmental factors. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Rhodiola rosea Molecular markers ISSR Genetic diversity Rosavins Salidroside
1. Introduction Rhodiola rosea L., commonly known as golden root, arctic root or roseroot has been used in the traditional medicine of Russia and Scandinavia for centuries. People have used it as general immune-stimulant and the ultimate to overcome fatigue. This traditional adaptogen medicinal plant is receiving increased attention in the last years. Roseroot belongs to the family Crassulaceae. It is a herbaceous plant with thick rhizome, which contains pharmacologically important secondary metabolites (Brown et al., 2002). The species displays a circumpolar distribution in the higher latitudes and elevations of the Northern hemisphere mainly in Asia and Europe. According to Hegi (1963), its distribution in Europe extends from Iceland and the British Isles across Scandinavia as far south as the Pyrenees, the Alps, the Carpathian Mountains and other mountainous Balkan regions. Roseroot is highly variable both in phytochemical (Kurkin et al., 1988; Wiedenfeld et al., 2007) and in morphological aspect (Ohba, 1981, 1989, Asdal et al., 2006). Nowadays several commercially available products exist based on the extracts of its rhizome, of which raw material mostly comes from harvesting from wild populations. A key to the successful cultivation is the stable high value cultivars achieved through breeding. Establishing a fruitful breeding work starts with the assessment and evaluation of the natural populations.
* Corresponding author. Tel.: þ36 14826530. E-mail addresses: [email protected] (Z. György), [email protected] (E. Fjelldal), [email protected] (M. Ladányi), [email protected] (P.E. Aspholm), [email protected] (A. Pedryc). 0305-1978/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2013.05.009
362
Z. György et al. / Biochemical Systematics and Ecology 50 (2013) 361–367
Roseroot is widely spread in Norway. In 2006 ca. 200 plants from 10 geographic regions distributed along the coast of Finnmark County in Northern-Norway were collected (Fjelldal et al., 2010). One mixed root sample (including several individuals) from each habitat was analysed for glycoside content. The results showed large geographical variations in the content of the studied metabolites. Total content of rosavin varied between 0.067 % and 2.7%, with a mean value of 1.54% for the 10 studied regions. Studies concerning the genetic diversity of roseroot have been conducted with different methods. Elameen et al. (2008) investigated the genetic diversity in a Norwegian germplasm collection by AFLP. Finnish Rhodiola rosea populations were analysed by György et al. (2012) with ISSR markers. In 2009 Zini et al. published eight microsatellite sequences (simple sequence repeats, SSR) and flanking primer pairs. These primers were tested on two Rhodiola rosea populations from the Trentino Alps. Four out of these primers were also used by Kylin (2010) for evaluating genetic diversity of roseroot plants collected in Sweden, Greenland and Faroe Islands. Recently Kozyrenko et al. (2011) analysed the genetic structure of Rhodiola rosea mostly of Russian origin using ISSR polymorphisms. The aim of the present work was to characterize genetic diversity among roseroot individuals from habitats in NorthernNorway using ISSR markers and to examine if ISSR polymorphism is able to evince the big alterations among the habitats or individuals that were earlier detected in course of studying the chemical composition of the investigated plant material. 2. Materials and methods 2.1. Plant material Rhodiola rosea plants were collected in Finnmark County, Northern-Norway at 10 habitats along the coast (Fig. 1, Table 1). All of the habitats were close to the sea (less than 200 m), in an altitude approximately 10–50 m above sea level. The collected plants were further cultivated in the experimental field of Bioforsk, Svanhovd. From each habitat 5–6 plants were included in the study, all together 58 plants. The plant material was frozen in liquid nitrogen and was stored in 80 C until used. DNA was extracted from the frozen leaves according to a CTAB-based protocol (Pirttilä et al., 2001). DNA concentration and quality was assessed using NanoDrop (BioScience, Hungary) and on 1% agarose gel. 2.2. PCR amplification of ISSR markers PCR was performed in 25 ml reaction volume containing 20–80 ng DNA, 10X PCR reaction buffer, 2.5 mM MgCl2, 0.02 mM dNTP mix, 2.5 mmol of primers, 1 unit of Taq DNA polymerase (Fermentas, Szeged, Hungary) and sterile distilled water. Eight primers from the University of British Columbia, Canada set 9 of ISSR primers (http://www.michaelsmith.ubc.ca) (BC809, BC840, BC841, BC857, BC873 BC885, BC887 BC888) were chosen based on preliminary experiments. For the amplification of ISSR fragments the following program was used: initial denaturation at 94 C for 4 min; followed by 40 cycles of 94 C for 60 s, 49 C for 90 s, 72 C for 90 s; and a final synthesis at 72 C for 7 min. The PCR products were applied on a 1% (w/v) ethidium bromide-stained agarose gel in 1xTBE buffer with xylencyanol loading buffer. PCR products were separated for 2 h at 120 V. Amplified fragments were scored visually for presence (1) or absence (0) of homologous bands and the results were summarised in MS Excel table. 2.3. Data analysis 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
Fig. 1. Map of Northern-Norway, showing the 10 locations of the examined roseroot habitats.
Z. György et al. / Biochemical Systematics and Ecology 50 (2013) 361–367
363
Table 1 Description and geographic coordinates of the 10 habitats where Rhodiola rosea plants were collected. Habitat
Coordinates
Description of the spot
Zone Zone Zone Zone
1 2 3 4
36w04147/utm77437 35w05874/utm77690 36w03996/utm77834 36w04192/utm78161
Zone Zone Zone Zone Zone Zone
5 6 7 8 9 10
35w05844/utm7855 35w05319/utm78143 36w05002/utm78334 35w0485/utm78357 35w04151/utm7875 36w05982/utm78349
Slope leaning north. Both rich in humus and mixture of humus/gravel. Slope leaning north. Thin layer of humus over solid rock. Hardly any soil at all, growing in cracks in rocks/slate. Leaning south-east. Close to sea. Growing in cracked rocks and sand/gravel. Leaning north, close to rough sea. Tall plants, often growing in the shade from rocks/slate. Growing in cracked rocks and sand/gravel. Leaning north, close to rough sea. Open terrain. Steep hill, leaning east, quite high above sea level. Growing in humus/gravel (slate). Leaning east. Growing in sand/gravel/rocks. Leaning east. Growing in sand/gravel/rocks (slate). Leaning east Growing on humus over solid granite.
data. Genetic relatedness among habitats and genotypes was studied by UPGMA (Unweighted Pair Group Method with Arithmetic averages) cluster analysis using Popgene. The results of chemical analysis were earlier published (Fjelldal et al., 2010). Based on the standardized results clusters were formed with Ward-linkage according to squared Euclidean distance. To confirm these clusters, canonical discriminant analysis was performed. The binominal ISSR matrix was used for the analysis of molecular variance (AMOVA) implemented in Genalex 6.5 (Peakall and Smouse, 2012). AMOVA was used to estimate the partition of the genetic variation within and among the clusters, which were created based on the glycoside contents. The significance of the variance components was determined with a permutation test (999 replicates). 3. Results Using 8 ISSR primers analysis of the genetic diversity of 58 roseroot plants from the coast of Northern-Norway was conducted. The selected 8 ISSR primers generated 53 bands, ranging from 5 to 9 bands, corresponding to an average of 6.63 bands per primer. At the population level the Shannon-index was 0.41 with a standard deviation of 0.22 and the percentage of polymorphic loci (PPL) was 92.45%. However genetic variability was rather low at the habitats as indicated in Table 2. The highest level of variability occurred in habitat 3 (PPL ¼ 64.15%; h ¼ 0.24; I ¼ 0.36), whereas the lowest level in habitat 4 (PPL ¼ 35.85%; h ¼ 0.15; I ¼ 0.21). Genetic relationships among the studied habitats were calculated (Table 3) and an UPGMA-based dendrogram obtained is shown in Fig. 2A. The 10 habitats of roseroot were grouped into two subgroups. One clade included Habitat 1 and 2, while the second clade included all remaining habitats. In the second subgroup Habitat 10 was divergent from the other habitats. The closely situated habitats had highest genetic similarity (Habitat 1 and 2; 3 and 4; 6 and 7; 8 and 9). These habitats are always located on the same peninsula, while habitat 10 is located on an island. Fig. 3 shows the salidroside, rosavins and total rosavins content of the 10 habitats (Fjelldal et al., 2010). The graph shows large geographical variations in the content of these metabolites. The content of rosavins and total rosavins show strong correlation since rosavins is one of the three compounds called the “rosavins”. Some habitats have high salidroside content and lower rosavins content (HS_LR) like habitat 2, 3, 4. Some has lower level of salidroside and higher rosavins content (LS_ HR) like habitat 7 and 9. Some has about the same (middle) levels of salidroside and rosavins (MS_MR) like habitat 1, 5, 6, 8. Habitat 10 has outstandingly low levels of each compound (OLC). Fig. 2B shows a dendrogram using Ward-linkage hierarchical cluster analysis where the observed 4 clusters with different compound amounts are supported. Canonical discriminant analysis (Fig. 4) also confirmed these 4 clusters (Wilk’s lambda: 0.26 and 0.29 with Chi-square(9) ¼ 20.03 and Chi-square(4) ¼ 6.79; p < 0.05 and p ¼ 0.148 for the first two canonical discriminant functions, respectively). The group membership was predicted 100% successfully. Table 2 Genetic variability measures of the 10 roseroot habitats based on 8 ISSR marker. Habitat
Number of polymorphic bands
% of polymorphic bands
Gene diversity (h)
St. dev.
Shannon-index (SI)
St. dev.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. All
32 34 34 19 31 25 24 30 28 26 49
60.38 64.15 64.15 35.85 58.49 47.17 45.28 56.60 52.83 49.00 92.45
0.2093 0.2218 0.2415 0.1478 0.1941 0.1542 0.1428 0.2057 0.1591 0.1842 0.2666
0.2029 0.1966 0.2064 0.2100 0.1975 0.1857 0.1806 0.1999 0.1841 0.2093 0.1641
0.3137 0.3339 0.3567 0.2140 0.2939 0.2356 0.2196 0.3073 0.2462 0.2719 0.4122
0.2867 0.2792 0.2919 0.2985 0.2806 0.2706 0.2644 0.2882 0.2652 0.2982 0.2197
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Table 3 Nei’s genetic identity of the roseroot individuals of the 10 North-Norwegian habitats based on 8 ISSR markers. Habitat
1
2
3
4
5
6
7
8
9
10
1 2 3 4 5 6 7 8 9 10
X
0.90 X
0.90 0.87 X
0.87 0.85 0.94 X
0.83 0.80 0.90 0.90 X
0.85 0.86 0.89 0.93 0.94 X
0.85 0.88 0.90 0.92 0.91 0.95 X
0.84 0.84 0.90 0.90 0.93 0.93 0.92 X
0.86 0.84 0.92 0.92 0.93 0.95 0.92 0.97 X
0.88 0.85 0.89 0.87 0.91 0.90 0.88 0.88 0.89 X
Fig. 2. A: Dendrogram of the 10 roseroot habitats assayed in this study generated by UPGMA cluster analysis of the similarity matrix obtained using Nei’s genetic distance based on ISSR data (Nei, 1973) B: Dendrogram using Ward linkage hierarchical cluster analysis with squared Euclidean distance based on the salidroside and rosavin content of the 10 habitats.
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365
30
mg/g dried material
25
20 salidroside 15
rosavine tot.rosavin
10
5
0 1
2
3
4
5
6
7
8
9
10
Habitat
Fig. 3. Salidroside, rosavin and total rosavins content of the 10 studied North-Norwegian habitats.
AMOVA (Table 4) of the 10 habitats and the 4 glycoside groups showed that the most molecular variation, 85% was found within habitats, and only 16% among habitats. When the habitats were analysed in four groups based on their glycoside content, the molecular variance due to the groups was 0.00%. 4. Discussion The aim of the present study was to estimate the genetic diversity of Rhodiola rosea with ISSR markers in 10 habitats in Northern-Norway. ISSR markers were sensitive enough and revealed polymorphism. Genetic variability within the populations detected with the ISSR method was somewhat higher than in the study of Kozyrenko et al. (2011). Shannon index for the 10 North-Norwegian habitats ranged between 0.21 and 0.36 while in the study of Kozyrenko et al. (2011) it ranged between 0.14 and 0.26. However, since only half of the primers used are the same in the two studies comparison of the results is not fully appropriate. The genetic diversity among the habitats seems to be much
Fig. 4. Classification plot of the discriminant scores of the first two canonical discriminant functions from the discriminant function analysis of the 10 habitats (P1-P10). Different symbols denote groups with different amount of compounds such as high salidroside-low rosavins (HS_LR), higher rosavins-low salidroside (LS_HR); middle salidroside and rosavins (MS_MR) and outstandingly low compound (OLC).
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Table 4 Results of molecular variance (AMOVA) for 10 Rhodiola rosea habitats based on ISSR markers and 4 groups based on the glycoside contents. Source of variation
Degrees of freedom
Sum of squares
Estimated variance
% of total variance
Fixation index (V)
Significance (P)
Among groups Among populations Within population Total
3 6 48 57
41.43 82.37 314.93 438.72
0 1.25 6.56 7.81
0 16 84 100
0.00a 0.16b 0.16c
0.534 0.001 0.001
a, b, c Fixation indices are calculated as the correlations. a among groups. b among populations. c within population relative to the total.
higher in the present study. Shannon index was 0.41 while in the study of Kozyrenko et al. (2011) it was just 0.29. On the other hand this would be very strange, considering that in the present study the sampling area was far much smaller, than in the study of Kozyrenko et al. (2011). This phenomenon can be due to the subjective evaluation of the ISSR gels. Earlier two Northern-Finnish roseroot populations were compared with ISSR method (György et al., 2012) and Shannonindexes of 0.31 and 0.23 were calculated within the two populations, while 0.34 among the populations. These are comparable values to the present results. Generally all these Shannon index values are similar to those of Rhodiola crenulata from the Hengduan Mountains (0.17–0.33, Lei et al., 2006) and somewhat higher than of Rhodiola alsia (0.07–0.22, Xia et al., 2005) and Rhodiola chrysanthemifoila (0.08–0.24, Xia et al., 2007) in the Tibetian Plateau, indicating rather low genetic diversity in the studied populations. Results of chemical analysis of these 10 habitats showed also big differences. Salidroside content varied between 0.46 and 2.61% and the content of total rosavins varied between 0.67 and 2.7% comparing the 10 habitats. Both the lowest salidroside level and also the lowest level of rosavins were detected in Habitat 10 (Fjeldal et al., 2010). This habitat was also found to be separated based on its genetic background in the present study. This separation is approved by the geographics, as this habitat is located on an island. 4 groups were formed based on the glycoside content. AMOVA was performed to see if the molecular variance is in correlation with the glycoside content. The results showed that molecular variance has no effect on the glycoside content, it is only effected by environmental factors. In conclusion, using ISSR markers we were able to assess genetic diversity of roseroot of 10 habitats from NorthernNorway. 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