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Genetic variation in Danish and Norwegian germplasm collections of hops
Biochemical Systematics and Ecology 52 (2014) 53–59
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Genetic variation in Danish and Norwegian germplasm collections of hops Svein Øivind Solberg a, *, Agnese Kolodinska Brantestam a, Madeleine Kylin a, Gitte Kjeldsen Bjørn b, Mette Goul Thomsen J c a
Nordic Genetic Resource Centre, Smedjevägen 3, 22227 Alnarp, Sweden AgroTech, Denmark c Norwegian Institute for Agricultural and Environmental Research, Norway b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 October 2013 Accepted 23 December 2013 Available online 9 January 2014
The germplasm collections of hops (Humulus lupulus L.) in Denmark and Norway are maintained in clonal archives funded by the national authorities. The plants have been collected over the last decades as part of a strategy to conserve plant genetic resources for future generations. The major part of the various collections consist of plants collected in villages and gardens. About 20% are plants used for breeding, mainly kept in a collection at Carlsberg, Denmark. In order to identify any duplicates and with a view to learning more about the various collections, a DNA fingerprinting study was initiated, analysing 62 Danish and 34 Norwegian clones with a set of five amplified fragment length polymorphism (AFLP) markers. The AFLP analyses resulted in 41 polymorphic bands and were able to separate the majority of the Danish and the Norwegian accessions. UPGMA dendrograms showed 21 accession groups, and potential duplicates were found within 13 of these groups. Principal coordinates analysis revealed that plants were differentiated according to country of origin. In addition, regional separation of the plants within each country was also detected, and similar levels of diversity were found in the Danish and the Norwegian collections. Compared to the rest of the plants, there was less diversity within the Carlsberg material. For the Norwegian as well as a part of the Danish collection, morphological characterisation and chemical analysis was carried out, allowing a comparison of these to the AFLP data. A correlation with AFLP bands and both morphological and chemical characteristics was detected. The most promising results for further breeding was an association of AFLP bands with the content of colupulon in the cones, measured by relative values compared to the total alpha acid content. Further studies are needed to verify such an association with the potential to develop a PCR-based marker for hop breeding carried out in the clones now analysed with AFLP markers, making it possible to search for any association between AFLP data and phenotypic data. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: AFLP Alpha acid Chemical content Colupulon Humulus lupulus Morphology
1. Introduction Hops (Humulus lupulus L.) originate in China (Neve, 1991; Murakami et al., 2006), but have been cultivated in the Nordic region since mediaeval times (Zachrisson 2000). Under the Nidaros archbishopric, farmers in the fifteenth century were obliged
* Corresponding author. Tel.: þ46 702798991. E-mail address: [email protected] (S.Ø. Solberg). 0305-1978/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2013.12.014
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S.Ø. Solberg et al. / Biochemical Systematics and Ecology 52 (2014) 53–59
to clear land for ten new hop plants every year (Borgen, 1999). This requirement was later reduced to six plants, but the legalisation remained under the Danish and Norwegian Union until the mid-eighteenth century. The climate permitted hop cultivation all the way to the Lofoten islands (68 N, 14 E), Northern Norway. Danish and Norwegian hop production declined markedly in the nineteenth century as a consequence of the centralisation of breweries and the importation of cheaper hops from Germany. This can be illustrated by figures from Denmark. Hop production fell from 1103 ha in 1881 to 200 ha only 20 years later, before almost disappearing in the twentith century (ibid). Hop production in Scandinavia is now very much related to historical practices, but new trends may lead to changes in this regards. Culinary concepts such as New Nordic Food and Local food have focused on unique and local ingredients, with beer being one of the products that form part of this trend. In beer production only the female hop plants are used; the inflorescences (cones) are used as flavour and as a preservative. The actual compounds belong to the alpha-acids (humulone, adhumulone, colupulon and cohumulone) and the essential oils (humulene, myrcene, caryophyllene and farnesene). Among the alpha acids, cohumulone has been regarded as unpleasant bitterness, while the other alpha acids are regarded as more favourus. Furthermore, on the basis of the content of alpha acid, cultivars are divided into two groups; aroma hops and high alpha acid hops. The content in what is called aroma hops is assumed to be similar to the wild hop, while the latter group has been developed to meet specific needs for the brewing industry. Hop plants can be found throughout both Denmark and Norway, often as relics from earlier cultivations, and resembling the finds of hops in Sweden described by Karlsson Strese et al. (2012). The plants may be old cultivars (whose identity is lost) or local, naturalised clones, or even populations. Since the identity and properties of the plants are mostly unknown, plants have been collected and maintained in clonal archives for further characterisation and evaluation. Our study is part of this work. The objectives were to assess any potential duplicates in these collections, but also to find out more about the genetic variation within and among the collections. We also wanted to search for any correlation between genetic characteristics and geographical, morphological or chemical data. Genetic studies of germplasm hop collections have been carried out before (see for example Jakse et al., 2001; Patzak et al., 2010; Karlsson Strese et al., 2012), but our study is the first on the Danish and Norwegian collections conserved under the national programmes for conservation and use of plant genetic resources. 2. Material and methods 2.1. Plant material A total of 100 single plant samples (clones) were included in the study, 62 from Denmark (D), 34 from Norway (N) (Fig. 1), and 1 from Sweden (S), Finland (F), Germany (G) and England (E) each. The Danish collection includes 13 clones from the
Fig. 1. Geographical origin of the plants Local plants from Denmark (number and D) and Norway (number and N) and the breeding material from Carlsberg and Winge in Denmark (number and D). In addition there was one sample from Sweden (8S), one from Finland (41F), one from Germany (58G) and one from England (59E).
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Fig. 2. PCOORD based on ALFP data. Norway: Southern Norway local plants (B), Eastern Norway local plants (6),Western Norway local plants (7), Northern Norway local plants (,), Mid Norway local plants (>). Denmark: Carlsberg breeding material (),Winge breeding material (þ)Funen local plants (C), Zeeland local plants (:), Jutland local plants (;),Sweden ( ), Finland ( ), Germany (,), England ( ).
Carlsberg breeding program, collected from the garden at “Gammel Carlsberg vej”. The places of origin of the Carlsberg clones are unknown. However, nine of the same clones are believed also to be in the Winge collection at Fuglebjergaard (Denmark), and these clones were included in the study. The rest of the Danish clones are locally collected plants from the regions of Zeeland (Sjælland), Funen (Fyn) and Jutland (Jylland). The Norwegian plants are all plants collected locally from the country’s
Fig. 3. UPGMA dendrogram based on AFLP data Danish (D); Norwegian (N), Swedish (S), Finish (F); German (G) and English (E) hops; breeding material is indicated in the italic shrift. * – groups containing accessions of breeding material, Group 1: N1; Group 2: 4N, 27D; Group 3: 58G; Group 4: 8N; Group 5: 11N; Group 6: 2N, 8S, 17D,37N, 18D; 1D, 5D,66D, 13D, 67D, 2D, 8D, 64D, 10D, 12D, 68D, 6D, 7D, 63D, 65D, 60D, 61D, 11D, 54D, 150D, 4D, 62D, 50D, 5N, 74D, 75D, 16D,70D, 71D, 77D; Group 7: 56D, 86D, 91D, 59E, 81D, 85D; Group 8: 6N, 40N, 36N, 28N, 80D, 9D; Group 9: 35D, 15D, 38N; Group 10:100D;Group 11:26N, 57D, 98D, 95D; Group 12: 9N, 15N, 20N, 12N, 14N, 16N, 32N, 13N, 17N, 19N, 29N, 30N, 31N; Group 13: 10N, 18N, 25N, 33N;Group 14: 48D, 96D, 78D; Group 15: 73D; Group 16: 7N, 21N, 39N; Group 17: 26D, 41D, 37D, 84D, 79D; Group 18: 41(F); Group 19: 82D,83D, 97D, 92D, 99D, 94D; Group 20: 34D, 38D; Group 21: 3N.
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Table 1 Results from AFLP analysis expressed by Shannon–weaver diversity index and number of polymorphic bands of hop plants from different regions or subcollections in Denmark and Norway. Origin of plants
Number of clones
Shannon–weaver diversity index (I)
Number of polymorphic bands
Denmark Funen, local plants Jutland, local plants Zeeland, local plants Carlsberg, breeding material Winge, breeding material Denmark, total
19 11 11 14 9 64
0.377 0.319 0.387 0.138 0.109 0.369
29 24 29 14 10 33
Norway Mid- and Northern Norway, local plants Southern Norway, local plants Western Norway, local plants Eastern Norway, local plants Norway, total
9 2 9 14 34
0.290 0.152 0.338 0.355 0.373
25 9 27 33 37
major geographic regions Southern Norway (Sørlandet), Eastern Norway (Østlandet), Western Norway (Vestlandet), (Mid Norway (Midt-Norge) and Northern Norway (Nord Norge). 2.2. AFLP analysis Using the Doyle and Doyle protocol (1990), DNA was extracted from young leaves. Amplified fragment length polymorphism (AFLP) analysis was carried out according to Vos et al. (1995). EcoRI and MseI restriction enzymes and adapters were used. The primers were selected based on Patzak (2001) and own studies (unpublished). The following primers were selected; E38/M47, E36/M49, E37/M62, E37/M47 and E37/M50. The E37/M50 showed no polymorphic bands and was excluded in further analysis. The products were separated by electrophoresis using polyacrylamide gel and visualised with silver staining according to Bassam et al. (1991). 2.3. Phenotypic characters All the Norwegian and 17 of the Danish accessions are described morphologically (Dragland et al., 2003), including main shoot colouration (clear green, greenish, reddish, clear red), main shoot rib colouration (clear green, greenish, reddish, clear red), leaf shape, leaf size (length and width, measured 2 m from the ground), stipule shape (upright or downward), cone bract and bracteole shape (six categories each), cone size (measured) and cone shape (length/width) (Table 3). The Norwegian
Fig. 4. UPGMA dendrogram, close-up of group 6.
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Table 2 Pearson coefficients of the significant correlations detected between AFLP bands and morphological, chemical and disease resistance properties of Norwegian accessions. AFLP band
Morphology Rib colour
E37M47-110bp E37m47-219bp E38M47-205bp E38M47-223bp E38M47-460bp E36M49-300bp E37M62-217bp E38M47-590bp E38M47-150bp E37M62-230bp E37M62-440bp
Stipule shape
Leaf length
Cone length
Cone shape
0.442*
0.515*
Resist. S. humeli
Cohumlon (%)
Adlupulon (%)
Cohumlon (Rel)
Colupulon (Rel)
0.439* 0.429*
0.475* 0.487*
0.470*
0.493* 0.499*
L0.584** 0.479*
0.422* 0.422* 0.477* L0.619**
0.467*
0.467* 0.437* 0.529**
0.405* 0.561**
*p < 0.01; **p < 0.001 marked also with bold text.
Table 3 Morphological and chemical characters and disease resistance to Sphaerotheca humeli (1 ¼ low infection and 9 ¼ high infection rate, average over 2 years) of the Norwegian hop clones. Only the characters relevant for the correlation analysis with the AFLP markers are shown. Clone
Rib colour
Stipule shape
Leaf length mm
Cone length mm
Cone shape l/w
Resistance S. humeli scale 1–9
Cohumulon (%)
Colupulon (%)
Adlupulon (%)
Total alpha acid (%)
Cohumlon (rel. value)
Colupulon (rel. value)
1N 2N 3N 4N 5N 6N 7N 8N 9N 10N 11N 12N 13N 14N 15N 16N 17N 18N 19N 20N 21N 25N 26N 27N 28N 29N 30N 31N 32N 33N 34N 35N 36N 37N 38N 39N 40N 41N
R R R R R R R R R Rw R R R R R R R R R R R R R R R R R R R R R R R R R R R R
U U U D U U U D U D D U U U U U U U U U U D U U U U U U/D U D U D D U U U D D
95 100 88 82 95 107 97 76 65 105 93 99 74 107 82 105 104 112 93 82 81 87 136 97 101 93 110 106 103 101 103 118 126 83 79 113 118 119
28 37 29 31 28 35 31 25 26 19 29 37 37 28 34 32 26 30 30 43 33 31 28 33 34 42 27 28 30 36 33 36 40 33 28 30 40 36
1,3 1,5 1,3 1,5 1,5 1,6 1,4 1,4 1,4 1,1 1,6 1,6 1,5 1,5 1,6 1,6 1,4 1,3 1,3 2,1 1,7 1,6 1,6 1,9 2,1 1,6 1,5 1,4 1,5 1,8 1,4 1,9 1,9 1,6 1,2 1,6 2.0 1,8
2,5 3 3 3 2,5 3,5 2,5 3,5 2 4,5 4 3 3,5 4 4 3,5 4 3 3 3,5 2,5 2,5 2,5 3 3 4,5 3,5 3,5 4,5 3,5 3 3,5 2 4 4 2,5 2 2
1,5 1,5 1 1,3 0,8 1,3 2,1 0,8 0,6
2,9 1,6 2,5 1,6 1,6 1,5 3,4 2 1,5
4,2 1,6 4,3 1,7 2,4 2,3 3,8 3 2,1
6,7 5,4 4,9 4,4 3,9 5,7 7,2 3,4 2,5
22,4 27,8 20,4 29,5 20,5 22,8 29,2 23,5 24
40,8 50 36,8 48,5 40 39,5 47,2 40 41,7
0,6 0,7 0,4 0,8 0,6 0,6 1,5 1,2 0,4 1,3 0,6 1,7 1,2 1,7 0,9 0,6 0,4 0,6 0,9 1,3 0,6 1 1 1,3 0,6 1,2 1,2 0,5
2,2 1,6 1,7 1,9 1,7 2,2 2,8 2 1,5 1,7 1,4 1,7 2,4 2,1 1,7 2,1 1,4 2,1 2,1 3,1 1,9 3,4 1,3 2,1 2,3 2,4 2,5 1,5
3,2 1,7 3,3 2,5 2,4 3,3 3,1 2,8 2,9 2,1 1,9 2,1 3,4 2,8 2,6 4 2,6 4,1 2,8 3,1 3,4 4,7 1,6 2,7 4,3 2,3 2,8 2,2
2,8 2,5 2,3 3,6 2,5 2,9 5,8 5,3 2,3 5 2,5 5,9 4,5 6,4 3,6 3,1 1,9 3,1 4,5 4,4 2,9 4,3 4,2 5,6 3,3 4,7 4,5 2,5
21,4 28 14,7 22,2 24 20,7 25,9 22,6 17,4 26 24 28,8 26,7 26,6 25 19,4 21,1 19,4 20 29,5 20,7 23,3 23,8 23,2 18,2 25,5 26,7 20
40,7 48,5 34 43,2 41,5 40 47,5 41,7 34,1 44,7 42,4 44,7 41,4 42,9 39,5 34,4 35 33,9 35,6 50 35,8 42 44,8 43,8 34,8 51,1 47,2 40,5
Rib colour of main shoot; red (R)), reddish (Rw) or green (G). Stipule: shape; upright (U) or downward (D), Cone shape; length/width. The chemical data relates to content in weight-% and the relative value of cohumulon and colupulon.
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plants were also scored for pests and disease resistance and analysed for cone chemical content. The chemical analyses were carried out at NateCO2GmbH & Co, Wolnzach, Germany. The following measurements were carried out: cone contents of cohumulon, colupulon, adhumulon, total alpha acids, total beta acids and etheric oils. 2.4. Data analysis Electrophoretic pattern of PCR products were scored as either presence (1) or absence (0) of bands. Using Jaccard’s similarity coefficient, a similarity matrix was calculated. The NTSYS (version 2.21c) (Rohlf, 2009) was used for cluster analysis and unweighted pair group method with arithmetic mean (UPGMA) dendrogram construction (Sneath and Sokal, 1973). Principal Coordinate Analysis (PCOORD) was also carried out, using NTSYS (Rohlf, 2009). The genetic variation was expressed by Shannon–Weaver diversity Indexes and Number of polymorphic bands (Hutchenson, 1970; King and Schaal, 1989). The results were associated with geographical origin (country and region), and the results from the Norwegian material was also associated to morphological and chemical characters. Using Minitab 16 software, a Pearson correlation analysis was carried out on AFLP data, morphological data and chemical data. 3. Results and discussion 3.1. Genetic diversity The AFLP analyses resulted in 41 polymorphic bands and were able to separate the majority of the Danish and the Norwegian accessions (Fig. 2). Most of the Norwegian plants were found in quadrant II of PCOORD, excepting only seven accessions, which were found in quadrant I. The Danish plants were principally located in quadrant I, III and IV, whereas nine plants in quadrant II overlapped with the Norwegian plants. Both national and regional grouping within the Norwegian and the Danish plants were revealed (Fig. 2), suggesting that there is a tendency for hop plants in one region to differ from plants from another region and that the majority of Danish hops are different from the Norwegian clones (Figs. 2 and 3). Data revealed that the German and English hop collects are related to Danish clones. Further studies including more plants are required in order to verify historical origin. The breeding material from the two sub-collections at Carlsberg and Winge Fuglebjergaard differed from the majority of other Danish plants (Figs. 2 and 3), and demonstrated the lowest diversity values (Table 1). In the UPGMA dendrogram 21 groups are presented (Fig.3). Among these, 13 groups included potential duplicates. For example, detailed cluster analysis of Group 6 (Fig. 4) reveals the potential duplicates within this group. This group includes many collects, and almost all of the breeding material is gathered here (except for one clone found in group 8). The following collects of breeding materials from Denmark are potential duplicates; 13D and 67D; 6D and 7D; 63D and 65D; as well as the collect 1D, 5D and 66D. Potential duplicates within the Norwegian collection were: 29N and 30N (both from Northern Norway), and 6N and 40N (both from Western Norway). However, based on the morphology data, the above-mentioned collects from Northern Norway vary with regard to cone character, and the collects from Western Norway vary in terms of leaf character. As far as this study shows, the decision to exclude plants from the collections (apart from potential duplicates with identical morphology) continues to be open for debate. Our study has shown that most of the clones are genetically unique. Furthermore, we should highlight the results from inventories done on monastery ruins and mediaeval sites. These showed that hop plants can be found from possible relict cultivations (Løjtnant 2007). The historical aspect should definitely be included in the debate, and a broad approach, including morphology and genetics, but also cultural history and cultural value of the plants, should be considered (Solberg et al., 2013). (Table 2). 3.2. Association analysis For the Norwegian and part of the Danish plants, phenotypic characterisation and chemical analysis have been carried out in the accessions analysed with AFLP markers. The collections include clones with both green and the red shoot colouration. The Danish collection had a few clones with green shoot ribs, while all the clones in the Norwegian collection had red shoot ribs. The clones in both collections showed variations in leaf and leaf size, and cone size and form. Only the Norwegian plants have been analysed for content of alpha acids and other aromatic compounds. Association between the AFLP data and stipule shape was found (p < 0.01, see Table 3). However, the association between the AFLP data and some of the chemical measurement may be more relevant for breeders. For example, a correlation of two AFLP band presences and the relative content of colupulon (as a percentage of total Alpha acids) was detected (p < 0.01) as well as association between one band and the relative content of cohumulone (also p < 0.01). When analysis was done based only on the four bands in the PCOORD analyses associated with Colupulon values, groups of accessions were revealed, separating hops with higher and lower values (figure not included). For verification of these associations, further studies should be carried out, using plants with different genetic backgrounds. Identification of quantitative trait loci (QTLs) for alpha acid content and yield in hop has been reported by Cerenak et al. (2006, 2009) and our findings support the potential for a PCR-based marker for diagnostic use in hop breeding.
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Acknowledgements The work was funded by the Nordic Council of Ministers. Furthermore, we would also like to thank Steinar Dragland for his work on the Norwegian hop collection, and for all the work he did for the scientifically-based cultivation and use of herbs and medicinal plants. References Bassam, B.J., Caetano-Anollks, G., Gresshoff, P., 1991. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal. Bioanal. Chem. 196, 80–83. http:// dx.doi.org/10.1016/0003-2697(91)90120-1. Borgen, L., 1999. Glimt frå humlens og humlehagenes historie i Norge. Våre nyttevekster 2, 45–56 (Norwegian text). Cerenak, A., Satovic, Z., Javornik, B., 2006. Identification of QTLs for alpha acid content and yield in hops(Humulus lupulus L.). Euphytica 170, 141–154. http:// dx.doi.org/10.1007/s10681-099-9920-9. Cerenak, A., Satovic, Z., Javornik, B., 2009. Genetic mapping of hop (Humulus lupulus L.) applied to detection of QTLs for alpha-acid content. Genome 49, 485–494. http://dx.doi.org/10.1139/G06-007. Doyle, J.J., Doyle, J.L., 1990. Isolation of plant DNA from fresh tissue. Focus 12, 13–15. Dragland, S., Bjørn, G.K., Suojala, T., 2003. Sluttrapport fra Nordisk Humleprosjekt (Humulus lupulus L.). Planteforsk, Kise, Norway (in Norwegian). Hutchenson, K., 1970. A test for comparing diversity based on Shannon formula. J. Theor. Biol. 29, 151–154. Jakse, J., Kindlhofer, K., Javornik, B., 2001. Assessment of genetic variation and differentiation of hop genotypes by microsatelite and AFLP markers. Genome 44, 773–782. http://dx.doi.org/10.1139/gen-44-5-733. Karlsson Strese, E.M., Karsvall, O., Tollin, C., 2012. Inventory methods for finding historically cultivated hop (Humulus lupulus L.) in Sweden. Genet. Resour. Crop Evol. 57, 219–227. King, D., Schaal, B.A., 1989. Ribosomal DNA variation and distribution in Rudbeckia mossouriensis. Evolution 43, 1117–1119. http://dx.doi.org/10.2307/ 2409592. Løjtnant, B., 2007. Levende levn. Tidsskr. Skalk, No. 4, 11–15 (Danish text). Murakami, A., Darby, P., Javornik, B., Pais, M.S.S., Seigner, E., Lutz, A., Svoboda, P., 2006. Microsatellite DNA analysis of wild hops, Humulus lupulus L. Genet. Resour. Crop Evol. 53, 1553–1562 http://dx.doi.org/10.1007/s10722-005-7765-1. Neve, R.A., 1991. Varieties and breeding. In: Neve, R.A. (Ed.), Hops. Chapman and Hall, London, pp. 196–223. Patzak, J., 2001. Comparison of RAPD, STS, ISSR and AFLP molecular methods used for assessment of genetic diversity in hop (Humulus lupulus L.). Euphytica 121, 9–18. http://dx.doi.org/10.1023/A:1012099123877. Patzak, J., Nesvadba, V., Henychova, A., Krofta, K., 2010. Assessment of the genetic diversity of wild hops (Humulus lupulus L.) in Europe using chemical and molecular analyses. Biochem. Syst. Ecol. 38, 136–145. http://dx.doi.org/10.1016/j:bse.2009.12.023. Rohlf, F.J., 2009. NTSYSpc: Numerical Taxonomy System, Ver. 2.21c. Exeter Software, Setauket, New York. Sneath, P., Sokal, P., 1973. Numerical Taxonomy. The Principles and Practice of Numerical Classifications. Freeman, San Francisco. Solberg, S.Ø., Ansebo, L., Persson, E., 2013. Cultural relict plants – a living heritage. J. Nord. Mus. (1), 24–35. Nordisk Museologi. Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23, 4407–4414. http://dx.doi.org/10.1093/nar/23.21.4407. Zachrisson, S., 2000. Humle – laglig beordrad gröda. Stud. Ethnobiol. 13, 55–61 (Swedish text).
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Genetic variation in Danish and Norwegian germplasm collections of hops Svein Øivind Solberg a, *, Agnese Kolodinska Brantestam a, Madeleine Kylin a, Gitte Kjeldsen Bjørn b, Mette Goul Thomsen J c a
Nordic Genetic Resource Centre, Smedjevägen 3, 22227 Alnarp, Sweden AgroTech, Denmark c Norwegian Institute for Agricultural and Environmental Research, Norway b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 October 2013 Accepted 23 December 2013 Available online 9 January 2014
The germplasm collections of hops (Humulus lupulus L.) in Denmark and Norway are maintained in clonal archives funded by the national authorities. The plants have been collected over the last decades as part of a strategy to conserve plant genetic resources for future generations. The major part of the various collections consist of plants collected in villages and gardens. About 20% are plants used for breeding, mainly kept in a collection at Carlsberg, Denmark. In order to identify any duplicates and with a view to learning more about the various collections, a DNA fingerprinting study was initiated, analysing 62 Danish and 34 Norwegian clones with a set of five amplified fragment length polymorphism (AFLP) markers. The AFLP analyses resulted in 41 polymorphic bands and were able to separate the majority of the Danish and the Norwegian accessions. UPGMA dendrograms showed 21 accession groups, and potential duplicates were found within 13 of these groups. Principal coordinates analysis revealed that plants were differentiated according to country of origin. In addition, regional separation of the plants within each country was also detected, and similar levels of diversity were found in the Danish and the Norwegian collections. Compared to the rest of the plants, there was less diversity within the Carlsberg material. For the Norwegian as well as a part of the Danish collection, morphological characterisation and chemical analysis was carried out, allowing a comparison of these to the AFLP data. A correlation with AFLP bands and both morphological and chemical characteristics was detected. The most promising results for further breeding was an association of AFLP bands with the content of colupulon in the cones, measured by relative values compared to the total alpha acid content. Further studies are needed to verify such an association with the potential to develop a PCR-based marker for hop breeding carried out in the clones now analysed with AFLP markers, making it possible to search for any association between AFLP data and phenotypic data. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: AFLP Alpha acid Chemical content Colupulon Humulus lupulus Morphology
1. Introduction Hops (Humulus lupulus L.) originate in China (Neve, 1991; Murakami et al., 2006), but have been cultivated in the Nordic region since mediaeval times (Zachrisson 2000). Under the Nidaros archbishopric, farmers in the fifteenth century were obliged
* Corresponding author. Tel.: þ46 702798991. E-mail address: [email protected] (S.Ø. Solberg). 0305-1978/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2013.12.014
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to clear land for ten new hop plants every year (Borgen, 1999). This requirement was later reduced to six plants, but the legalisation remained under the Danish and Norwegian Union until the mid-eighteenth century. The climate permitted hop cultivation all the way to the Lofoten islands (68 N, 14 E), Northern Norway. Danish and Norwegian hop production declined markedly in the nineteenth century as a consequence of the centralisation of breweries and the importation of cheaper hops from Germany. This can be illustrated by figures from Denmark. Hop production fell from 1103 ha in 1881 to 200 ha only 20 years later, before almost disappearing in the twentith century (ibid). Hop production in Scandinavia is now very much related to historical practices, but new trends may lead to changes in this regards. Culinary concepts such as New Nordic Food and Local food have focused on unique and local ingredients, with beer being one of the products that form part of this trend. In beer production only the female hop plants are used; the inflorescences (cones) are used as flavour and as a preservative. The actual compounds belong to the alpha-acids (humulone, adhumulone, colupulon and cohumulone) and the essential oils (humulene, myrcene, caryophyllene and farnesene). Among the alpha acids, cohumulone has been regarded as unpleasant bitterness, while the other alpha acids are regarded as more favourus. Furthermore, on the basis of the content of alpha acid, cultivars are divided into two groups; aroma hops and high alpha acid hops. The content in what is called aroma hops is assumed to be similar to the wild hop, while the latter group has been developed to meet specific needs for the brewing industry. Hop plants can be found throughout both Denmark and Norway, often as relics from earlier cultivations, and resembling the finds of hops in Sweden described by Karlsson Strese et al. (2012). The plants may be old cultivars (whose identity is lost) or local, naturalised clones, or even populations. Since the identity and properties of the plants are mostly unknown, plants have been collected and maintained in clonal archives for further characterisation and evaluation. Our study is part of this work. The objectives were to assess any potential duplicates in these collections, but also to find out more about the genetic variation within and among the collections. We also wanted to search for any correlation between genetic characteristics and geographical, morphological or chemical data. Genetic studies of germplasm hop collections have been carried out before (see for example Jakse et al., 2001; Patzak et al., 2010; Karlsson Strese et al., 2012), but our study is the first on the Danish and Norwegian collections conserved under the national programmes for conservation and use of plant genetic resources. 2. Material and methods 2.1. Plant material A total of 100 single plant samples (clones) were included in the study, 62 from Denmark (D), 34 from Norway (N) (Fig. 1), and 1 from Sweden (S), Finland (F), Germany (G) and England (E) each. The Danish collection includes 13 clones from the
Fig. 1. Geographical origin of the plants Local plants from Denmark (number and D) and Norway (number and N) and the breeding material from Carlsberg and Winge in Denmark (number and D). In addition there was one sample from Sweden (8S), one from Finland (41F), one from Germany (58G) and one from England (59E).
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Fig. 2. PCOORD based on ALFP data. Norway: Southern Norway local plants (B), Eastern Norway local plants (6),Western Norway local plants (7), Northern Norway local plants (,), Mid Norway local plants (>). Denmark: Carlsberg breeding material (),Winge breeding material (þ)Funen local plants (C), Zeeland local plants (:), Jutland local plants (;),Sweden ( ), Finland ( ), Germany (,), England ( ).
Carlsberg breeding program, collected from the garden at “Gammel Carlsberg vej”. The places of origin of the Carlsberg clones are unknown. However, nine of the same clones are believed also to be in the Winge collection at Fuglebjergaard (Denmark), and these clones were included in the study. The rest of the Danish clones are locally collected plants from the regions of Zeeland (Sjælland), Funen (Fyn) and Jutland (Jylland). The Norwegian plants are all plants collected locally from the country’s
Fig. 3. UPGMA dendrogram based on AFLP data Danish (D); Norwegian (N), Swedish (S), Finish (F); German (G) and English (E) hops; breeding material is indicated in the italic shrift. * – groups containing accessions of breeding material, Group 1: N1; Group 2: 4N, 27D; Group 3: 58G; Group 4: 8N; Group 5: 11N; Group 6: 2N, 8S, 17D,37N, 18D; 1D, 5D,66D, 13D, 67D, 2D, 8D, 64D, 10D, 12D, 68D, 6D, 7D, 63D, 65D, 60D, 61D, 11D, 54D, 150D, 4D, 62D, 50D, 5N, 74D, 75D, 16D,70D, 71D, 77D; Group 7: 56D, 86D, 91D, 59E, 81D, 85D; Group 8: 6N, 40N, 36N, 28N, 80D, 9D; Group 9: 35D, 15D, 38N; Group 10:100D;Group 11:26N, 57D, 98D, 95D; Group 12: 9N, 15N, 20N, 12N, 14N, 16N, 32N, 13N, 17N, 19N, 29N, 30N, 31N; Group 13: 10N, 18N, 25N, 33N;Group 14: 48D, 96D, 78D; Group 15: 73D; Group 16: 7N, 21N, 39N; Group 17: 26D, 41D, 37D, 84D, 79D; Group 18: 41(F); Group 19: 82D,83D, 97D, 92D, 99D, 94D; Group 20: 34D, 38D; Group 21: 3N.
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Table 1 Results from AFLP analysis expressed by Shannon–weaver diversity index and number of polymorphic bands of hop plants from different regions or subcollections in Denmark and Norway. Origin of plants
Number of clones
Shannon–weaver diversity index (I)
Number of polymorphic bands
Denmark Funen, local plants Jutland, local plants Zeeland, local plants Carlsberg, breeding material Winge, breeding material Denmark, total
19 11 11 14 9 64
0.377 0.319 0.387 0.138 0.109 0.369
29 24 29 14 10 33
Norway Mid- and Northern Norway, local plants Southern Norway, local plants Western Norway, local plants Eastern Norway, local plants Norway, total
9 2 9 14 34
0.290 0.152 0.338 0.355 0.373
25 9 27 33 37
major geographic regions Southern Norway (Sørlandet), Eastern Norway (Østlandet), Western Norway (Vestlandet), (Mid Norway (Midt-Norge) and Northern Norway (Nord Norge). 2.2. AFLP analysis Using the Doyle and Doyle protocol (1990), DNA was extracted from young leaves. Amplified fragment length polymorphism (AFLP) analysis was carried out according to Vos et al. (1995). EcoRI and MseI restriction enzymes and adapters were used. The primers were selected based on Patzak (2001) and own studies (unpublished). The following primers were selected; E38/M47, E36/M49, E37/M62, E37/M47 and E37/M50. The E37/M50 showed no polymorphic bands and was excluded in further analysis. The products were separated by electrophoresis using polyacrylamide gel and visualised with silver staining according to Bassam et al. (1991). 2.3. Phenotypic characters All the Norwegian and 17 of the Danish accessions are described morphologically (Dragland et al., 2003), including main shoot colouration (clear green, greenish, reddish, clear red), main shoot rib colouration (clear green, greenish, reddish, clear red), leaf shape, leaf size (length and width, measured 2 m from the ground), stipule shape (upright or downward), cone bract and bracteole shape (six categories each), cone size (measured) and cone shape (length/width) (Table 3). The Norwegian
Fig. 4. UPGMA dendrogram, close-up of group 6.
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Table 2 Pearson coefficients of the significant correlations detected between AFLP bands and morphological, chemical and disease resistance properties of Norwegian accessions. AFLP band
Morphology Rib colour
E37M47-110bp E37m47-219bp E38M47-205bp E38M47-223bp E38M47-460bp E36M49-300bp E37M62-217bp E38M47-590bp E38M47-150bp E37M62-230bp E37M62-440bp
Stipule shape
Leaf length
Cone length
Cone shape
0.442*
0.515*
Resist. S. humeli
Cohumlon (%)
Adlupulon (%)
Cohumlon (Rel)
Colupulon (Rel)
0.439* 0.429*
0.475* 0.487*
0.470*
0.493* 0.499*
L0.584** 0.479*
0.422* 0.422* 0.477* L0.619**
0.467*
0.467* 0.437* 0.529**
0.405* 0.561**
*p < 0.01; **p < 0.001 marked also with bold text.
Table 3 Morphological and chemical characters and disease resistance to Sphaerotheca humeli (1 ¼ low infection and 9 ¼ high infection rate, average over 2 years) of the Norwegian hop clones. Only the characters relevant for the correlation analysis with the AFLP markers are shown. Clone
Rib colour
Stipule shape
Leaf length mm
Cone length mm
Cone shape l/w
Resistance S. humeli scale 1–9
Cohumulon (%)
Colupulon (%)
Adlupulon (%)
Total alpha acid (%)
Cohumlon (rel. value)
Colupulon (rel. value)
1N 2N 3N 4N 5N 6N 7N 8N 9N 10N 11N 12N 13N 14N 15N 16N 17N 18N 19N 20N 21N 25N 26N 27N 28N 29N 30N 31N 32N 33N 34N 35N 36N 37N 38N 39N 40N 41N
R R R R R R R R R Rw R R R R R R R R R R R R R R R R R R R R R R R R R R R R
U U U D U U U D U D D U U U U U U U U U U D U U U U U U/D U D U D D U U U D D
95 100 88 82 95 107 97 76 65 105 93 99 74 107 82 105 104 112 93 82 81 87 136 97 101 93 110 106 103 101 103 118 126 83 79 113 118 119
28 37 29 31 28 35 31 25 26 19 29 37 37 28 34 32 26 30 30 43 33 31 28 33 34 42 27 28 30 36 33 36 40 33 28 30 40 36
1,3 1,5 1,3 1,5 1,5 1,6 1,4 1,4 1,4 1,1 1,6 1,6 1,5 1,5 1,6 1,6 1,4 1,3 1,3 2,1 1,7 1,6 1,6 1,9 2,1 1,6 1,5 1,4 1,5 1,8 1,4 1,9 1,9 1,6 1,2 1,6 2.0 1,8
2,5 3 3 3 2,5 3,5 2,5 3,5 2 4,5 4 3 3,5 4 4 3,5 4 3 3 3,5 2,5 2,5 2,5 3 3 4,5 3,5 3,5 4,5 3,5 3 3,5 2 4 4 2,5 2 2
1,5 1,5 1 1,3 0,8 1,3 2,1 0,8 0,6
2,9 1,6 2,5 1,6 1,6 1,5 3,4 2 1,5
4,2 1,6 4,3 1,7 2,4 2,3 3,8 3 2,1
6,7 5,4 4,9 4,4 3,9 5,7 7,2 3,4 2,5
22,4 27,8 20,4 29,5 20,5 22,8 29,2 23,5 24
40,8 50 36,8 48,5 40 39,5 47,2 40 41,7
0,6 0,7 0,4 0,8 0,6 0,6 1,5 1,2 0,4 1,3 0,6 1,7 1,2 1,7 0,9 0,6 0,4 0,6 0,9 1,3 0,6 1 1 1,3 0,6 1,2 1,2 0,5
2,2 1,6 1,7 1,9 1,7 2,2 2,8 2 1,5 1,7 1,4 1,7 2,4 2,1 1,7 2,1 1,4 2,1 2,1 3,1 1,9 3,4 1,3 2,1 2,3 2,4 2,5 1,5
3,2 1,7 3,3 2,5 2,4 3,3 3,1 2,8 2,9 2,1 1,9 2,1 3,4 2,8 2,6 4 2,6 4,1 2,8 3,1 3,4 4,7 1,6 2,7 4,3 2,3 2,8 2,2
2,8 2,5 2,3 3,6 2,5 2,9 5,8 5,3 2,3 5 2,5 5,9 4,5 6,4 3,6 3,1 1,9 3,1 4,5 4,4 2,9 4,3 4,2 5,6 3,3 4,7 4,5 2,5
21,4 28 14,7 22,2 24 20,7 25,9 22,6 17,4 26 24 28,8 26,7 26,6 25 19,4 21,1 19,4 20 29,5 20,7 23,3 23,8 23,2 18,2 25,5 26,7 20
40,7 48,5 34 43,2 41,5 40 47,5 41,7 34,1 44,7 42,4 44,7 41,4 42,9 39,5 34,4 35 33,9 35,6 50 35,8 42 44,8 43,8 34,8 51,1 47,2 40,5
Rib colour of main shoot; red (R)), reddish (Rw) or green (G). Stipule: shape; upright (U) or downward (D), Cone shape; length/width. The chemical data relates to content in weight-% and the relative value of cohumulon and colupulon.
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plants were also scored for pests and disease resistance and analysed for cone chemical content. The chemical analyses were carried out at NateCO2GmbH & Co, Wolnzach, Germany. The following measurements were carried out: cone contents of cohumulon, colupulon, adhumulon, total alpha acids, total beta acids and etheric oils. 2.4. Data analysis Electrophoretic pattern of PCR products were scored as either presence (1) or absence (0) of bands. Using Jaccard’s similarity coefficient, a similarity matrix was calculated. The NTSYS (version 2.21c) (Rohlf, 2009) was used for cluster analysis and unweighted pair group method with arithmetic mean (UPGMA) dendrogram construction (Sneath and Sokal, 1973). Principal Coordinate Analysis (PCOORD) was also carried out, using NTSYS (Rohlf, 2009). The genetic variation was expressed by Shannon–Weaver diversity Indexes and Number of polymorphic bands (Hutchenson, 1970; King and Schaal, 1989). The results were associated with geographical origin (country and region), and the results from the Norwegian material was also associated to morphological and chemical characters. Using Minitab 16 software, a Pearson correlation analysis was carried out on AFLP data, morphological data and chemical data. 3. Results and discussion 3.1. Genetic diversity The AFLP analyses resulted in 41 polymorphic bands and were able to separate the majority of the Danish and the Norwegian accessions (Fig. 2). Most of the Norwegian plants were found in quadrant II of PCOORD, excepting only seven accessions, which were found in quadrant I. The Danish plants were principally located in quadrant I, III and IV, whereas nine plants in quadrant II overlapped with the Norwegian plants. Both national and regional grouping within the Norwegian and the Danish plants were revealed (Fig. 2), suggesting that there is a tendency for hop plants in one region to differ from plants from another region and that the majority of Danish hops are different from the Norwegian clones (Figs. 2 and 3). Data revealed that the German and English hop collects are related to Danish clones. Further studies including more plants are required in order to verify historical origin. The breeding material from the two sub-collections at Carlsberg and Winge Fuglebjergaard differed from the majority of other Danish plants (Figs. 2 and 3), and demonstrated the lowest diversity values (Table 1). In the UPGMA dendrogram 21 groups are presented (Fig.3). Among these, 13 groups included potential duplicates. For example, detailed cluster analysis of Group 6 (Fig. 4) reveals the potential duplicates within this group. This group includes many collects, and almost all of the breeding material is gathered here (except for one clone found in group 8). The following collects of breeding materials from Denmark are potential duplicates; 13D and 67D; 6D and 7D; 63D and 65D; as well as the collect 1D, 5D and 66D. Potential duplicates within the Norwegian collection were: 29N and 30N (both from Northern Norway), and 6N and 40N (both from Western Norway). However, based on the morphology data, the above-mentioned collects from Northern Norway vary with regard to cone character, and the collects from Western Norway vary in terms of leaf character. As far as this study shows, the decision to exclude plants from the collections (apart from potential duplicates with identical morphology) continues to be open for debate. Our study has shown that most of the clones are genetically unique. Furthermore, we should highlight the results from inventories done on monastery ruins and mediaeval sites. These showed that hop plants can be found from possible relict cultivations (Løjtnant 2007). The historical aspect should definitely be included in the debate, and a broad approach, including morphology and genetics, but also cultural history and cultural value of the plants, should be considered (Solberg et al., 2013). (Table 2). 3.2. Association analysis For the Norwegian and part of the Danish plants, phenotypic characterisation and chemical analysis have been carried out in the accessions analysed with AFLP markers. The collections include clones with both green and the red shoot colouration. The Danish collection had a few clones with green shoot ribs, while all the clones in the Norwegian collection had red shoot ribs. The clones in both collections showed variations in leaf and leaf size, and cone size and form. Only the Norwegian plants have been analysed for content of alpha acids and other aromatic compounds. Association between the AFLP data and stipule shape was found (p < 0.01, see Table 3). However, the association between the AFLP data and some of the chemical measurement may be more relevant for breeders. For example, a correlation of two AFLP band presences and the relative content of colupulon (as a percentage of total Alpha acids) was detected (p < 0.01) as well as association between one band and the relative content of cohumulone (also p < 0.01). When analysis was done based only on the four bands in the PCOORD analyses associated with Colupulon values, groups of accessions were revealed, separating hops with higher and lower values (figure not included). For verification of these associations, further studies should be carried out, using plants with different genetic backgrounds. Identification of quantitative trait loci (QTLs) for alpha acid content and yield in hop has been reported by Cerenak et al. (2006, 2009) and our findings support the potential for a PCR-based marker for diagnostic use in hop breeding.
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Acknowledgements The work was funded by the Nordic Council of Ministers. Furthermore, we would also like to thank Steinar Dragland for his work on the Norwegian hop collection, and for all the work he did for the scientifically-based cultivation and use of herbs and medicinal plants. References Bassam, B.J., Caetano-Anollks, G., Gresshoff, P., 1991. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal. Bioanal. Chem. 196, 80–83. http:// dx.doi.org/10.1016/0003-2697(91)90120-1. Borgen, L., 1999. Glimt frå humlens og humlehagenes historie i Norge. Våre nyttevekster 2, 45–56 (Norwegian text). Cerenak, A., Satovic, Z., Javornik, B., 2006. Identification of QTLs for alpha acid content and yield in hops(Humulus lupulus L.). Euphytica 170, 141–154. http:// dx.doi.org/10.1007/s10681-099-9920-9. Cerenak, A., Satovic, Z., Javornik, B., 2009. Genetic mapping of hop (Humulus lupulus L.) applied to detection of QTLs for alpha-acid content. Genome 49, 485–494. http://dx.doi.org/10.1139/G06-007. Doyle, J.J., Doyle, J.L., 1990. Isolation of plant DNA from fresh tissue. Focus 12, 13–15. Dragland, S., Bjørn, G.K., Suojala, T., 2003. Sluttrapport fra Nordisk Humleprosjekt (Humulus lupulus L.). Planteforsk, Kise, Norway (in Norwegian). Hutchenson, K., 1970. A test for comparing diversity based on Shannon formula. J. Theor. Biol. 29, 151–154. Jakse, J., Kindlhofer, K., Javornik, B., 2001. Assessment of genetic variation and differentiation of hop genotypes by microsatelite and AFLP markers. Genome 44, 773–782. http://dx.doi.org/10.1139/gen-44-5-733. Karlsson Strese, E.M., Karsvall, O., Tollin, C., 2012. Inventory methods for finding historically cultivated hop (Humulus lupulus L.) in Sweden. Genet. Resour. Crop Evol. 57, 219–227. King, D., Schaal, B.A., 1989. Ribosomal DNA variation and distribution in Rudbeckia mossouriensis. Evolution 43, 1117–1119. http://dx.doi.org/10.2307/ 2409592. Løjtnant, B., 2007. Levende levn. Tidsskr. Skalk, No. 4, 11–15 (Danish text). Murakami, A., Darby, P., Javornik, B., Pais, M.S.S., Seigner, E., Lutz, A., Svoboda, P., 2006. Microsatellite DNA analysis of wild hops, Humulus lupulus L. Genet. Resour. Crop Evol. 53, 1553–1562 http://dx.doi.org/10.1007/s10722-005-7765-1. Neve, R.A., 1991. Varieties and breeding. In: Neve, R.A. (Ed.), Hops. Chapman and Hall, London, pp. 196–223. Patzak, J., 2001. Comparison of RAPD, STS, ISSR and AFLP molecular methods used for assessment of genetic diversity in hop (Humulus lupulus L.). Euphytica 121, 9–18. http://dx.doi.org/10.1023/A:1012099123877. Patzak, J., Nesvadba, V., Henychova, A., Krofta, K., 2010. Assessment of the genetic diversity of wild hops (Humulus lupulus L.) in Europe using chemical and molecular analyses. Biochem. Syst. Ecol. 38, 136–145. http://dx.doi.org/10.1016/j:bse.2009.12.023. Rohlf, F.J., 2009. NTSYSpc: Numerical Taxonomy System, Ver. 2.21c. Exeter Software, Setauket, New York. Sneath, P., Sokal, P., 1973. Numerical Taxonomy. The Principles and Practice of Numerical Classifications. Freeman, San Francisco. Solberg, S.Ø., Ansebo, L., Persson, E., 2013. Cultural relict plants – a living heritage. J. Nord. Mus. (1), 24–35. Nordisk Museologi. Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23, 4407–4414. http://dx.doi.org/10.1093/nar/23.21.4407. Zachrisson, S., 2000. Humle – laglig beordrad gröda. Stud. Ethnobiol. 13, 55–61 (Swedish text).