Abstract
Three European representatives of the genus Fraxinus were studied for the first time for their rDNA and heterochromatin patterns. The physical mapping of two rRNA gene families 5S and 18S–5.8S–26S (45S) and the distributional pattern of GC-rich regions in the chromosomes have been established by means of fluorescence in situ hybridization (FISH) and fluorochrome banding with chromomycin A3. The genome size was assessed by flow cytometry. Heterochromatin and rDNA organization was conserved and almost identical for two species from Fraxinus section (F. angustifolia and F. excelsior). The number and position of rDNA loci in F. ornus (section Ornus) were similar; however, the organization of genes was quite different. In this species the 5S and 45S rRNA genes were colocalized at the level of satellites of two chromosome pairs bearing nucleolar organizing regions (NORs). One 5S locus was also observed under the 45S one of one chromosome pair. In F. angustifolia and F. excelsior, only 45S loci were situated at the level of satellites and secondary constrictions, while 5S was located just under 45S in the distal part of the short arm of one out of two marked pairs. The number and position of GC-rich DNA correspond to those of 45S loci. The genome size (2C value) was of 1.54 and 1.68 pg for F. angustifolia and F. excelsior, respectively. Fraxinus ornus possessed the highest 2C DNA value (1.98 pg). In the light of these cytogenetic features the clear differentiation between two sections (Fraxinus and Ornus) was observed both at the rDNA and genome size levels.
Similar content being viewed by others
References
Al-Bermani AKKA, Al-Shammary KIA, Gornall RJ, Bailey JP (1993) Contribution to a cytological catalogue of the British and Irish flora. Watsonia 19:169–171
Arca M, Hinsinger DD, Cruaud C, Tillier A, Bousquet J, Frascaria-Lacoste N (2012) Deciduous trees and the application of universal DNA barcodes: a case study on the circumpolar Fraxinus. PLoS ONE 7:e34089
Bareka P, Siljak-Yakovlev S, Kamari G (2012) Molecular cytogenetics of Bellevalia (Hyacinthaceae) species occurring in Greece. Plant Syst Evol 298(2):421–430
Benmiloud-Mahieddine R, Abirached-Darmency M, Brown SC, Kaid-Harche M, Siljak-Yakovlev S (2011) Genome size and cytogenetic characterization of the Algerian Retama species. Tree Genet Genomes 7:987–998
Bennett MD, Leitch IJ (2005) Nuclear DNA amounts in angiosperms – progress, problems and prospects. Ann Bot 95:45–90
Bennett MD, Leitch IJ (2011) Nuclear DNA amounts in angiosperms: targets, trends and tomorrow. Ann Bot 107:467–590
Besnard G, Garcia-Verdugo C, Rubio De Casas R, Treier UA, Galland N, Vargas P (2008) Polyploidy in the olive complex (Olea europaea): evidence from flow cytometry and nuclear microsatellite analyses. Ann Bot 101:25–30
Bitonti MB, Cozza R, Chiappetta A, Contento A, Minelli S, Ceccarelli M et al (1999) Amount and organization of the heterochromatin in Olea europaea and related species. Heredity 83:188–195
Bogunić F, Muratović E, Brown S, Siljak-Yakovlev S (2003) Genome size and base composition of five Pinus species from Balkan region. Plant Cell Rep 22:59–63
Bogunić F, Muratović E, Ballian D, Siljak-Yakovlev S, Brown S (2007) Genome size stability among five subspecies of Pinus nigra Arnold s.l. Environ Exp Bot 59:354–360
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, Savoure A (2001) Karyotype analysis revealed interspecific differentiation in Cedrus genus despite genome size and base composition constancy. Theor Appl Genet 103:846–854
Brito G, Loureiro J, Lopes T, Rodriguez E, Santos C (2008) Genetic characterisation of olive trees from Madeira Archipelago using flow cytometry and microsatellite markers. Genet Resour Crop Ev 55:657–664
Cai Q, Zhang D, Liu Z-L, Wang X-R (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
Cerbah M, Coulaud J, Brown SC, Siljak-Yakovlev S (1999) Evolutionary DNA variation in the genus Hypochaeris. Heredity 82:261–266
Cerbah M, Montreau E, Brown S, Siljak-Yakovlev S, Bertrand H, Lambert C (2001) Genome size variation and species relationships in the genus Hydrangea. Theor Appl Genet 103:45–51
Chiche J, Brown SC, Leclerc J-C, Siljak-Yakovlev S (2003) Genome size, heterochromatin organisation and ribosomal gene mapping in four species of Ribes. Can J Bot 81:1049–1057
Dobrowolska D, Hein S, Oosterbaan A, Wagner S, Clark J, Skovsgaard JP (2011) A review of European ash (Fraxinus excelsior L.): implications for silviculture. Forestry 84:133–148
Doležel J, Bartoš J (2005) Plant DNA flow cytometry and estimation of nuclear genome size. Ann Bot 95:99–110
Doležel J, Bartoš J, Voglmayr H, Greilhuber J (2003) Nuclear DNA content and genome size of trout and human. Cytometry 51:127–128
Doležel J, Greilhuber J, Suda J (2007) Flow cytometry with plant cells. Analysis of genes, chromosomes and genomes. Wiley, Weinheim
Fernandez-Manjarres JF, Gerard PR, Dufour J, Raquin C, Frascaria-Lacoste N (2006) Differential patterns of morphological and molecular hybridization between Fraxinus excelsior L. and Fraxinus angustifolia Vahl (Oleaceae) in eastern and western France. Mol Ecol 15:3245–3257
Fraxigen (2005) Ash Species in Europe: Biological Characteristics and Practical Guidelines for Sustainable Use. Oxford Forestry Institute, University of Oxford, Oxford, UK, p 128
Fukarek P (1960) Narrow-leaved ash and its morphological variability. Fraxinus angustifolia Vahl (= Fr. oxycarpa Willd.). Glas Šum Pokuse 14:133–258
Galbraith D, Harkins K, Maddox J, Ayres N, Sharma D, Firoozabady E (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220:1049–1051
Garcia S, Garnatje T, Hidalgo O, Mcarthur ED, Siljak-Yakovlev S, Vallès J (2007) Extensive ribosomal DNA (18S–5.8S–26S and 5S) colocalization in the North American endemic sagebrushes (subgenus Tridentatae, Artemisia, Asteraceae) revealed by FISH. Plant Syst Evol 267:79–92
Garcia S, Lim KY, Chester M, Garnatje T, Pellicer J, Vallès J, Leitch AR, Kovarik A (2009) Linkage of 35 S and 5 S rRNA genes in Artemisia (family Asteraceae): first evidence from angiosperms. Chromosoma 118:85–97
Garnatje T, Hidalgo O, Vitales D, Pellicer J, Vallès J, Robin O, Garcia S, Siljak-Yakovlev S (2012) Swarm of terminal 45S in Cheirolophus (Asteraceae, Centaureinae). Genome 55(7):529–535
Geber G, Schweizer D (1987) Cytochemical hetero-1 chromatin differentiation in Sinapis alba (Cruciferae) using a simple air-drying technique for producing chromosome spreads. Plant Syst Evol 158:97–106
Gerard PR, Fernandez-Manjarres JF, Frascaria-Lacoste N (2006a) Temporal cline in a hybrid zone population between Fraxinus excelsior L. and Fraxinus angustifolia Vahl. Mol Ecol 15:3655–3667
Gerard PR, Fernandez-Manjarres JF, Bertolino P, Dufour J, Raquin C, Frascaria-Lacoste N (2006b) New insights in the recognition of the European ash species Fraxinus excelsior L. and Fraxinus angustifolia Vahl as useful tools for forest management. Ann For Sci 63(7):733–738
Gerard PR, Temunović M, Sannier J et al (2013) Chilled but not frosty: understanding the role of climate in the hybridization between the Mediterranean Fraxinus angustifolia Vahl and the temperate Fraxinus excelsior L. (Oleaceae) ash trees. J Biogeogr. doi:10.1111/jbi.12021
Gerlach WL, Dyer TA (1980) Sequence organization of the repeated units in the nucleus of wheat, which contains 5S rDNA genes. Nucl Acids Res 8:4851–4865
Gregory TR (2005) The C-value enigma in plants and animals: a review of parallels and an appeal for partnership. Ann Bot 95:133–146
Greilhuber J (2005) Intraspecific variation in genome size in angiosperms: identifying its existence. Ann Bot 95:91–98
Greilhuber J, Doležel J, Lysak MA, Bennett MD (2005) The origin, evolution and proposed stabilization of the terms “genome size” and “C-value” to describe nuclear DNA contents. Ann Bot 95:255–260
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
Heuertz M, Carnevale S, Fineschi S et al (2006) Chloroplast DNA phylogeography of European ashes, Fraxinus sp. (Oleaceae): roles of hybridization and life history traits. Mol Ecol 15:2131–2140
Hidalgo O, Garcia-Jacas N, Garnatje T, Romashchenko K, Susanna A, Siljak-Yakovlev S (2008) Extreme environmental conditions and phylogenetic inheritance: systematics of Myopordon and Oligochaeta (Asteraceae, Cardueae-Centaureinae). Taxon 57:769–778
Hinsinger DD (2010) Diversité et évolution du genre Fraxinus. University of Paris-Sud France and University of Laval, Québec, Dissertation
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 (Bioloxía) 13:23–33
Ivanova D, Stanimirova P, Vladimirov V (2005) Reports (1445–1456). In: Kamari G, Blanche C, Garbari F (eds), Mediterranean chromosome number reports 15. Flora Mediter 15:719–728
Ivanova D, Dimitrova D, Vladimirov V (2006) Chromosome numbers of selected woody species from the Bulgarian flora. Phytol Balcan 12(1):79–84
Jarni K, Westergren M, Kraigher H, Brus R (2011) Morphological variability of Fraxinus angustifolia Vahl in the north-western Balkans. Acta Soc Bot Pol 80(3):245–252
Jiang J, Gill B (1994) New 18S–26S ribosomal gene loci: chromosomal landmarks for the evolution of polyploid wheats. Chromosoma 103:179–185
Johnson LAS (1954) A review of the family Oleaceae. Contrib New South Wales Natl Herb 2:395–418
Katsiotis A, Hagidimitriou M, Douka A, Hatzopoulos P (1998) Genomic organization, sequence interrelationships, and physical localization using in situ hybridization of two tandemly repeated DNA sequences in the genus Olea. Genome 41:527–534
Knight CA, Ackerly DD (2002) Variation in nuclear DNA content across environmental gradients: a quantile regression analysis. Ecol Lett 5:66–76
Marie D, Brown SC (1993) A cytometric exercise in plant DNA histograms with 2C values for 70 species. Biol Cell 78:41–51
Minelli S, Maggini F, Gelati MT, Angiolillo A, Cionini PG (2000) The chromosome complement of Olea europaea L.: characterization by differential staining of the chromatin and in-situ hybridization of highly repeated DNA sequences. Chromosom Res 8:615–619
Morand-Prieur ME, Vedel F, Raquin C, Brachet S, Sihachakr D, Frascaria-Lacoste N (2002) Maternal inheritance of a chloroplast microsatellite marker in controlled hybrids between Fraxinus excelsior and Fraxinus angustifolia. Mol Ecol 11:613–617
Mortreau E, Siljak-Yakovlev S, Cerbah M, Brown S, Bertrand H, Lambert C (2010) Cytogenetic characterization of Hydrangea involucrata and H. aspera complex: genetic, evolutional and taxonomic implication. Tree Genet Genomes 6:137–148
Moscone EA, Klein F, Lambru M, Fuchs J, Schweizer D (1999) Quantitative karyotyping and dual-color FISH mapping of 5S and 18S–25S rDNA probes in the cultivated Phaseolus species (Leguminosae). Genome 42:1224–1233
Muratović E (2003) Molekularno–citogenetička i anatomska diferencijacija taksona Lilium bosniacum (Beck) Beck ex Fritsch i Lilium carniolicum Bernh. ex W. Koch. M.Sc. thesis, Prirodnomatematički fakultet Univerziteta u Sarajevu, Sarajevo
Muratović E, Robin O, Bogunić F, Šoljan D, Siljak-Yakovlev S (2010) Speciation of European lilies from Liriotypus section based on karyotype evolution. Taxon 59:165–175
Olszewska MJ, Osiecka R (1984) Relationship between 2C DNA content, systematic position and level of DNA endoreplication during differentiation of root parenchyma in dicot shrubs and trees—comparison with herbaceous sp. Biochem Physiol Pflanzen 179:641–657
Pellicer J, Garnatje T, Molero J, Pustahija F, Siljak-Yakovlev S, Vallès J (2010) Origin and evolution of the South American endemic Artemisia species (Asteraceae): evidence from molecular phylogeny, ribosomal DNA and genome size data. Aust J Bot 58:605–616
Petit RJ, Csaikl UM, Bordàcs S, Burg K, Coart E, Cottrell J et al (2002) Chloroplast DNA variation in European white oaks: phylogeography and patterns of diversity based on data from over 2600 populations. Forest Ecol Manage 156:5–26
Petit RJ, Bodenes C, Ducousso A, Roussel G, Kremer A (2004) Hybridization as a mechanism of invasion in oaks. New Phytol 161:151–164
Price HJ, Johnston JS (1996) Analysis of plant DNA content by Feulgen microspectrophotometry and flow cytometry. In: Jauhar PP (ed) Method of genome analysis in plants. CRC Press, Boca Raton, FL, pp 115–132
Probatova NS, Sokolovskaja AP, Rudyka EG (1991) Chromosome numbers in some species of vascular plants from the Soviet Far East and other regions of the USSR. Bot Žhurn (Moscow & Leningrad) 76:1174–1178
Raquin C, Jung-Muller B, Dufour J, Frascaria-Lacoste N (2002) Rapid seedling obtaining from European ash species Fraxinus excelsior (L.) and Fraxinus angustifolia (Vahl). Ann For Sci 59:219–224
Ribeiro T, Barão A, Viegas W, Morais-Cecílio L (2008) Molecular cytogenetics of forest trees. Cytogenet Genome Res 120:220–227
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 Genomes 9:587–599
Sánchez A, Gallego MF, Navarro FA (1987) Datos cariológicos de algunas plantas salmantinas. Stud Bot Univ Salamanca 6:169–171
Schweizer D (1976) Reverse fluorescent chromosome banding with chromomycin and DAPI. Chromosoma 58:307–324
Seijo GJ, Lavia GI, Fernandez A, Krapovickas A, Ducasse D, Moscone EA (2004) Physical mapping of the 5S and 18S–25S rRNA genes by FISH as evidence that Arachis duranensis and A. ipaensis are the wild diploid progenitors of A. hypogaea (Leguminosae). Am J Bot 91(9):1294–1303
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, Robin O, Muratović E, Bogunić F, Zoldoš V (2004) Interindividual variability of chromosomal organization In Lilium bosniacum growing on the peridotit substratum. XI OPTIMA Meeting, Belgrade September 2004
Siljak-Yakovlev S, Šolić ME, Catrice O, Brown SC, Papeš D (2005) Genome size and chromosome number in some Centaurea (Asteraceae: Cardueae) from Dalmatia region. Plant Biol 7:397–404
Siljak-Yakovlev S, Stevanović V, Tomasević M, Brown S, Stevanović B (2008) Genome size variation and polyploidy in the resurrection plant genus Ramonda: cytogeography of living fossils. Environ Exper Bot 62:101–112
Siljak-Yakovlev S, Pustahija F, Šolić ME, Bogunić F, Muratović E, Bašić N, Catrice O, Brown CS (2010) Towards a database of genome size and chromosome number of Balkan flora: C-values in 343 taxa with novel values for 252. Adv Sci Lett (USA) 3:190–213
Suda J, Kyncl T, Jarolimova V (2005) Genome size variation in Macaronesian angiosperms: forty percent of the Canarian endemic flora completed. Plant Syst Evol 252:215–238
Taylor H (1945) Cyto-taxonomy and phylogeny of the Oleaceae. Brittonia 5:337–367
Temunović M, Franjić J, Satovic Z, Grgurev M, Frascaria-Lacoste N, Fernández-Manjarrés JF (2012) Environmental heterogeneity explains the genetic structure of Continental and Mediterranean populations of Fraxinus angustifolia Vahl. PLoS ONE 7:e42764
Torrell M, Cerbah M, Siljak-Yakovlev S, Vallès J (2001) Etude cytogénétique de trois taxons du complexe d'Artemisia campestris L. (Asteraceae): localisation de l'hétérochromatine et de l'ADN ribosomique. Bocconea 13:623–628
Torrell M, Cerbah M, Siljak-Yakovlev S, Vallès J (2003) Molecular cytogenetics of the genus Artemisia (Asteraceae, Anthemideae): fluorochrome banding and fluorescence in situ hybridization: I. Subgenus Seriphidium and related taxa. Plant Syst Evol 239:141–153
Vidic T, Greilhuber J, Vilhar B, Dermastia M (2009) Selective significance of genome size in a plant community with heavy metal pollution. Ecol Appl 19(6):1515–1521
Wallander E (2001) Evolution of wind-pollination in Fraxinus (Oleaceae) — an ecophylogenetic approach. PhD thesis, Botanical Institute, Goteborg University, Sweden
Wallander E (2008) Systematics of Fraxinus genus (Oleaceae) and evolution of dioecy. Plant Syst Evol 273:25–49
Wallander E, Albert VA (2000) Phylogeny and classification of Oleaceae based on rps16 and trnL-F sequence data. Am J Bot 87(12):1827–1841
Wright JW (1944) Genotypic variation in white ash. J For 42:489–495
Wright (1957) New chromosome counts in Acer and Fraxinus. Morris Arb Bull 8:33–34
Zhang L, Zhang BB, Wang HG (2007) The Karyotype Analysis of Fraxinus velutina. J Wuhan Bot Res 25:513–514
Zoldoš V, Papeš D, Brown S, Panaud O, Siljak-Yakovlev S (1998) Genome size and base composition of seven Quercus species: inter- and intra-population variation. Genome 41:162–168
Zoldos V, Muratović E, Bogunić F, Birus I, Robin O, Horvat T, Siljak-Yakovlev S (2007) Anatomic, cytogenetic and molecular variations between non-serpentine and serpentine populations of endemic Lilium bosniacum. Chromosome Res 15:38
Acknowledgments
We thank Mickael Bourge and Fatima Pustahija for technical assistance in cytometry at the Imagif and IFR87/IBiSA. The authors also thank Samuel Pyke for the English revision of the manuscript, and two anonymous reviewers for valuable comments that improved the quality of the paper.
Data archiving statement
New data concerning 2C DNA values (genome size) will be incorporated in the Kew plant DNA C-values database (http://data.kew.org/cvalues) and number of rDNA gene loci in rDNA database (http://www.plantrdnadatabase.com).
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by D. Neale
Rights and permissions
About this article
Cite this article
Siljak-Yakovlev, S., Temunović, M., Robin, O. et al. Molecular–cytogenetic studies of ribosomal RNA genes and heterochromatin in three European Fraxinus species. Tree Genetics & Genomes 10, 231–239 (2014). https://doi.org/10.1007/s11295-013-0654-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11295-013-0654-4