Abstract
We carried out the first cytogenetic characterisation of Mediterranean species of Juniperus (Cupressaceae); to date, nuclear DNA amount and chromosome numbers were known, but a fine karyotype analysis had been only done in three eastern Asian taxa. We performed chromomycin A3 banding, for the first time in the family, and fluorescent in situ hybridisation (FISH) in order to detect 18S-5.8S-26S (also termed 35S and 45S) rRNA genes in five species of the genus, four diploid (Juniperus communis, Juniperus oxycedrus, Juniperus phoenicea and Juniperus sabina) and the only Mediterranean one that is exclusively tetraploid (Juniperus thurifera). The results show a very homogeneous pattern, with one chromosome pair with chromomycin-positive bands at the secondary constrictions and colocalised 35S rDNA signals. In diploid species, this model agrees with that of the two Asian taxa previously investigated with FISH. In the tetraploid species, conversely, the results are different: in the Asian taxa, signal number and location are exactly double in tetraploid than in diploid taxa, whereas in our case, the tetraploid species bears the same number of signals (not twice) as diploid ones. This fact can be interpreted as accounting for the age of polyploidy in J. thurifera: after the polyploidisation event and the stabilisation of the doubled genome, there has been time left for genome rearrangements implying a loss of GC-rich chromatin and one of the rDNA loci; this argument is a support for the ancient and relict character attributed to this taxon.
References
Adams RP, Schwarzbach AE (2013) Phylogeny of Juniperus using nrDNA and four cpDNA regions. Phytologia 95:179–187
Ahuja MR (2005) Polyploidy in gymnosperms: revisited. Silvae Genet 54:59–69
Bennett MD (1998) Plant genome values: how much do we know? Proc Natl Acad Sci U S A 95:2011–2016
Besendorfer V, Krajačić-Sokol I., Jelenić S, Puizina J, Mlinarec J, Sviben T, Papeš D (2005) Two classes of 5S rDNA unit arrays of the silver fir, Abies alba Mill.: structure, localization and evolution. Theor Appl Genet 110:730–741
Bogunić F, Siljak-Yakovlev S, Muratović E, Ballian D (2011) Different karyotype patterns among allopatric Pinus nigra (Pinaceae) populations revealed by molecular cytogenetics. Plant Biol 13(1):194–200
Bou Dagher-Kharrat M, Grenier G, Bariteau M, Brown S, Siljak-Yakovlev S, Savouré A (2001) Karyotype analysis reveals interspecific differentiation in the genus Cedrus despite genome size and base composition constancy. Theor Appl Genet 103:846–854
Cai Q, Zhang D, Liu ZL, Wang XR (2006) Chromosomal localization of 5S and 18S rDNA in five species of subgenus Strobus and their implications for genome evolution of Pinus. Ann Bot 97:715–722
Cui L, Wall PK, Leebens-Mack JH, Lindsay BG, Soltis DE, Doyle JJ, Soltis PS, Carlson JE, Arumuganathan K, Barakat A, Albert VA, Ma H, dePamphilis CW (2006) Widespread genome duplications throughout the history of flowering plants. Genome Res 16:738–749
Cullis CA, Creisson GP, Gorman SW, Teasdale RD (1988) The 25S, 18S and 5S ribosomal RNA genes from Pinus radiata D.Don. Proceedings of the 2nd IUFRO WP on Molecular genetics of forest trees. Canadian Forest Service, Petawawa National Forest Institute, 33–40
Galián JA, Rosato M, Rosselló JA (2012) Early evolutionary colocalization of the nuclear ribosomal 5S and 45S gene families in seed plants: evidence from the living fossil gymnosperm Ginkgo biloba. Heredity 108:640–646
Garcia S, Kovařík A (2013) Dancing together and separate again: gymnosperms exhibit frequent changes of fundamental 5S and 35S rRNA gene (rDNA) organisation. Heredity 111:23–33
Gauquelin T (2006) Les Genévriers du monde et du bassin occidental de la Méditerranée : diversité, stabilité, expansion, régression. l; Actas del III coloquio internacional sobre sabinares y enebrales, Soria 24–26 de Mayo de 2006; Junta de Castilla y León; Ponencia inaugural, 25–32
Gauquelin T, Bertaudière V, Montes N, Badri W, Asmode J-F (1999) Endangered stands of thuriferous juniper in the western Mediterranean basin: ecological status, conservation and management. Biodivers Conserv 8:1479–1498
Goryachkina OV, Badaeva ED, Muratova EN, Zelenin AV (2013) Molecular cytogenetic analysis of Siberian Larix species by fluorescence in situ hybridization. Plant Syst Evol 299:471–479
Heslop-Harrison JS, Schwarzacher T, Anamthawat-Jonsson K, Leitch IJ (1991) In situ hybridization with automated chromosome denaturation techniques. J Methods Cell Mol Biol 3:109–116
Hizume M (1995) Physical mapping of 5S rRNA genes in Cycas revoluta. Cytologia 60:389–393
Hizume M (1996) Localization of 18S ribosomal RNA genes in the meiotic chromosomes of Cunninghamia lanceolata and Cryptomeria japonica, Taxodiaceae. Chromosome Inf Serv 60:22–24
Hizume M, Akiyama M (1992) Size variation of chromomycin A3-band in chromosomes of Douglas fir, Pseudotsuga menziesii. Jpn J Genet 67:425–435
Hizume M, Ishida F, Kondo K (1992) Differential staining and in situ hybridization of nuclear organizers and centromeres in Cycas revoluta chromosomes. Jpn J Genet 67:381–387
Hizume M, Kondo K, Yang Q, Hong D, Wang F, Tanaka R (1994) Fluorescent chromosome banding in six Chinese Pinaceae species: Abies faxoniana, Larix principis-rupprechtii, Picea asperata, Picea wilsonii, Pinus tabuleaformis and Pinus bungeana. In: Tanaka R (ed) Karyomorphological and cytogenetical studies in plants common to Japan and China. Hiroshima University, Hiroshima, pp 33–54
Hizume M, Kuzukawa Y, Kondo K, Yang Q, Hong D, Tanaka R (1995) Localization of rDNAs and fluorescent bandings in chromosomes of Larix potaninii var. macrocarpa collected in Sichuan, China. La Kromosomo II 78:2689–2694
Hizume M, Shibata F, Kondo T (1998) Fluorescence in situ hybridization of ribosomal RNA gene in Cryptomeria japonica, Taxodiaceae. Chrom Sci 2:99–102
Hizume M, Shibata F, Matsusaki Y, Kondo K (1999) Mapping of 45S rRNA and 5S rRNA loci in Cryptomeria japonica, Cunninghamia lanceolata and Ginkgo biloba. Chrom Sci 3:142
Hizume M, Shibata F, Matsusaki Y, Garajova Z (2002) Chromosome identification and comparative karyotypic analyses of four Pinus species. Theor Appl Genet 105:491–497
Horjales M, Redondo N, Blanco A, Rodríguez MA (2003) Cantidades de DNA nuclear en árboles y arbustos. Nova Acta Cient Compost (Biol) 13:23–33
Islam-Faridi MN, Nelson CD, Kubisiak TL (2007) Reference karyotype and cytomolecular map for loblolly pine (Pinus taeda L.). Genome 50:241–251
Jacobs MD, Gardner RC, Murray BG (2000) Cytological characterization of heterochromatin and rDNA in Pinus radiata and P. taeda. Plant Syst Evol 223:71–79
Jensen H, Levan A (1941) Colchicine-induced tetraploidy in Sequoia gigantea. Hereditas 27:220–224
Kokubugata G, Kondo K (1998) Comparative karyotype analysis of Ceratozamia mexicana and Microcycas calocoma (Zamiaceae) using fluorochrome banding (CMA/DAPI) and fluorescence in situ hybridization of ribosomal DNA. Plant Syst Evol 210:41–50
Kondo K, Kokubugata G, Honda Y (1996) Marking and identification of certain chromosomes in wild Chrysanthemus and Cycads by fluorescence in situ hybridization using pTa71 rDNA probe. Genet Pol 37:24–26
Levin DA (2002) The role of chromosomal changes in plant evolution. Oxford University Press, Oxford
Loureiro J, Capelo A, Brito G, Rodriguez E, Silva S, Pinto G, Santos C (2007) Micropropagation of Juniperus phoenicea from adult plant explants and analysis of ploidy stability using flow cytometry. Biol Plant 51:7–14
Lubaretz O, Fuchs J, Ahne R, Meister A, Schubert I (1996) Karyotyping of three Pinaceae species via fluorescent in situ hybridization and computer-aided chromosome analysis. Theor Appl Genet 92:411–416
Mao K, Hao G, Liu J, Adams RP, Milne RI (2010) Diversification and biogeography of Juniperus (Cupressaceae): variable diversification rates and multiple intercontinental dispersals. New Phytol 188:254–272
Montesinos D (2007) Juniperus thurifera: una especie dioica, vecera y relíctica. Ecosistemas 16:172–185
Muratović E, Bogunić F, Šolić EM, Soljan D, Siljak-Yakovlev S (2004) 2C DNA values in genus Juniperus: genome size revealed the rare case of polyploidy in Conifers. Poster. XI OPTIMA Meeting, Organization for Phyto Taxonomic Investigation of the Mediterranean Area. Belgrad, Serbia
Murray BG, Friesen N, Heslop-Harrison JS (2002) Molecular cytogenetic analysis of Podocarpus and comparison with other gymnosperm species. Ann Bot 89:483–489
Murray BG, Leitch IJ, Bennett MD (2012) Gymnosperm DNA C-values database (release 5.0, Dec. 2012) http://www.kew.org/cvalues (accessed 22 Aug 2013)
Nagano K, Umeda Т, Toda Y (2000) Karyomorphological study of Juniperus. In: Guttenberger H, Borzan Ž, Schlarbaum SE, Hartman TPV (eds) Cytogenetic studies of forest trees and shrubs review, present status, and outlook on the future. Arbora, Zvolen, pp 143–159
Nagano K, Matoba H, Yonamura K, Matsuda Y, Murata T, Hoshi Y (2007) Karyotype analysis of three Juniperus species using fluorescence in situ hybridization (FISH) with two ribosomal genes. Cytologia 72:37–42
Nakao Y, Taira T, Horiuchi S, Kawase K, Mukai Y (2005) Chromosomal difference between male and female trees of Ginkgo biloba examined by karyotype analysis and mapping of the rDNA on the chromosomes by fluorescence in situ hybridization. J Jpn Soc Hortic Sci 74:275–280
Puizina J, Sviben T, Krajacic-Sokol I, Zoldos-Pecnik V, Siljak-Yakovlev S, Papes D, Besendorfer V (2008) Cytogenetic and molecular characterization of the Abies alba genome and its relationship with other members of the Pinaceae. Plant Biol 10:256–267
Rai HS, Reeves PA, Peakall R, Olmstead RG, Graham SW (2008) Inference of higher-order conifer relationships from a multi-locus plastid data set. Botany 86:658–669
Romo A, Hidalgo O, Boratyński A, Sobierajska K, Jasińska AK, Vallès J, Garnatje T (2013) Genome size and ploidy levels in highly fragmented habitats: the case of western Mediterranean Juniperus (Cupressaceae) with special emphasis on J. thurifera L. Tree Genet Genom 9:587–599
Sax K, Sax HJ (1933) Chromosome number and morphology in the conifers. J Arnold Arboretum 14:356–375
Schweizer D (1976) Reverse fluorescent chromosome banding with chromomycin and DAPI. Chromosoma 58:307–324
Siljak-Yakovlev S, Peruzzi L (2012) Cytogenetic characterization of endemics: past and future. Plant Biosyst 146(3):694–702
Siljak-Yakovlev S, Cerbah M, Coulaud J, Stoian V, Brown SC, Jelenic S, Papes D (2002) Nuclear DNA content, base composition, heterochromatin and rDNA in Picea omorika and Picea abies. Theor Appl Genet 104:505–512
Siljak-Yakovlev S, Pustahija F, Šolić EM, Bogunić F, Muratović E, Bašić N, Catrice O, Brown SC (2010) Towards a genome size and chromosome number database of Balkan Flora: C-values in 343 taxa with novel values for 242. Adv Sci Lett 3:190–213
Tagashira N, Kondo K (2001) Chromosome phylogeny of Zamia and Ceratozamia by means of Robertsonian changes detected by fluorescence in situ hybridization (FISH) technique of rDNA. Plant Syst Evol 227:145–155
Terrab A, Schönswetter P, Talavera S, Vela E, Stuessy TF (2008) Range-wide phylogeography of Juniperus thurifera L., a presumptive keystone species of western Mediterranean vegetation during cold stages of the Pleistocene. Mol Phylogenet Evol 48:94–102
Villar L (2013) Le Genévrier thurifère, vieil arbre compagnon de l’homme en Méditerranée occidentale. Ecol Medit 39:7–16
Vischi M, Jurman I, Bianchi G, Morgante M (2003) Karyotype of Norway spruce by multicolor FISH. Theor Appl Genet 107:591–597
Zonneveld BJM (2012) Genome sizes of 172 species, covering 64 out of the 67 genera, range from 8 to 72 picogram. Nord J Bot 30:490–502
Acknowledgments
Miquel Veny (Institut Botànic de Barcelona) is thanked for taking cure of the living plants collection, and Samuel Pyke (Jardí Botànic de Barcelona) for the revision of the English text. The collaboration of the Proyecto Forestal Ibérico forest tree nursery is acknowledged. This work has been subsidised by Ministerio de Ciencia e Innovación, Spanish government, Spain (projects CGL2010-22234-C02-01 and 02/BOS, CGL2013-49097-C2-2-P), Generalitat de Catalunya-Catalan government, Catalonia (2009/SGR/00439, 2014/SGR/514) and funds from CNRS and Université de Paris-Sud (Orsay). JV received a grant (2012BE100508) from AGAUR (Catalan government’s agency for university and research grants) for a research stay in the Laboratoire Ecologie, Systématique, Evolution, Université Paris-Sud at Orsay, in the frame of a sabbatical semester attributed to him by the Universitat de Barcelona.
Data archiving statement
FISH data are available at Plant rDNA database (http://www.plantrdnadatabase.com).
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by Z. Kaya
Topical Collection on Evolution
Rights and permissions
About this article
Cite this article
Vallès, J., Garnatje, T., Robin, O. et al. Molecular cytogenetic studies in western Mediterranean Juniperus (Cupressaceae): a constant model of GC-rich chromosomal regions and rDNA loci with evidences for paleopolyploidy. Tree Genetics & Genomes 11, 43 (2015). https://doi.org/10.1007/s11295-015-0860-3
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11295-015-0860-3