, Volume 117, Issue 2, pp 159–167 | Cite as

Molecular and cytogenetic evidence for an allotetraploid origin of Trifolium dubium (Leguminosae)

  • Helal A. Ansari
  • Nicholas W. Ellison
  • Warren M. Williams
Research Article


Suckling clover, Trifolium dubium Sibth., is a European grassland legume that has spread to many parts of the world. The present work shows that it is an allotetraploid (2n = 4x = 30) combining the genomes of T. campestre Schreb. (2n = 2x = 14) and T. micranthum Viv. (2n = 2x = 16), two diploid species of similar geographic distribution. T. dubium has two nuclear ITS sequences that closely match those of T. campestre and T. micranthum. Genomic in situ hybridisation using genomic DNA of T. campestre and T. micranthum as probes has differentiated the ancestral sets of chromosomes in T. dubium cells. Comparative fluorescence in situ hybridisation analyses of 5S and 18S-26S rDNA loci were also consistent with an allotetraploid structure of the T. dubium genome. A marked preponderance of ITS repeats from T. campestre over those from T. micranthum indicated that concerted evolution has resulted in partial homogenisation of these sequences by depletion of the T. micranthum-derived 18S-26S rDNA repeats. In parallel with this, the epigenetic phenomenon of nucleolar dominance has been observed in T. dubium such that the chromatin associated with the 18S-26S rDNA loci derived from T. campestre is decondensed (transcriptionally active), whilst that from T. micranthum remains highly condensed throughout the cell cycle. T. dubium, therefore, appears to have arisen by way of hybridisation between forms of the diploid species T. campestre and T. micranthum accompanied by chromosome doubling. The observed genomic changes in rDNA resulting from interspecific hybridisation provide evidence for the process of genome diploidisation in T. dubium.


Internal Transcribe Spacer Secondary Constriction Nucleolar Dominance Active NORs rDNA Chromatin 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We thank Prof B. S. Gill, Prof. B. Friebe and Dr R. G. Kynast, Kansas State University, Manhatten, KS, USA, for providing guidance in the GISH technique in their laboratory and Prof. I. Schubert, Leibniz-Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany for helpful comments on an earlier version of the manuscript. This work was supported in part by the Foundation for Research, Science and Technology, New Zealand.


  1. Ansari HA, Ellison NW, Reader SM, Badaeva ED, Friebe B, Miller TE, Williams WM (1999) Molecular cytogenetic organization of 5S and 18S-26S rDNA loci in white clover (Trifolium repens L.) and related species. Ann Bot 83:199–206CrossRefGoogle Scholar
  2. Baldwin BG, Sanderson MJ, Porter JM, Wojciechowski MF, Campbell CS, Donoghue MJ (1995) The ITS region of nuclear ribosomal DNA: a valuable source of evidence on angiosperm phylogeny. Ann Mo Bot Gard 82:247–277CrossRefGoogle Scholar
  3. Baum BR, Bailey G (2000) The 5S rDNA units in Kengylia (Poaceae: Triticeae): diversity of the nontranscribed spacer and genomic relationships. Can J Bot 78:1571–1579CrossRefGoogle Scholar
  4. Baumel A, Ainouche ML, Levasseur JE (2001) Molecular investigations in populations of Spartina C.E. Hubbard (Poaceae) invading coastal Brittany (France). Mol Ecol 10:1689–1701PubMedCrossRefGoogle Scholar
  5. Benabdelmouna A, Shi Y, Abirached-Darmency M, Darmency H (2001) Genomic in situ hybridization (GISH) discriminates between the A and the B genomes in diploid and tetraploid Setaria species. Genome 44:685–690PubMedCrossRefGoogle Scholar
  6. Clarkson JJ, Lim KY, Kovarik A, Chase MW, Knapp S, Leitch AR (2005) Long-term genome diploidization in allopolyploid Nicotiana section Repandae (Solonaceae). New Phyto 168:241–252CrossRefGoogle Scholar
  7. Cleveland RW (1985) Reproductive cycle and cytogenetics. In: Taylor NL (ed) Clover Science and Technology. American Society of Agronomy, Inc., Crop Science Society of America, Inc., Soil Science Society of America, Inc., Madison, Wisconsin, pp 71–110Google Scholar
  8. Cronn RC, Zhao X, Patterson AH, Wendel JF (1996) Polymorphism and concerted evolution in a tandemly repeated gene family: 5S ribosomal DNA in diploid and allopolyploid cottons. J Mol Evol 42:685–705PubMedCrossRefGoogle Scholar
  9. Earley K, Lawrence RJ, Pontes O, Reuther R, Enciso AJ, Silva M, Neves N, Gross M, Viegas W, Pikaard CS (2006) Erasure of histone deacetylation by Arabidopsis HDA6 mediates large-scale gene silencing in nucleolar dominance. Genes Dev 20:1283–1293PubMedCrossRefGoogle Scholar
  10. Elder JF, Turner BJ (1995) Concerted evolution of repetitive DNA sequences in eukaryotes. Q Rev Biol 70:279–320CrossRefGoogle Scholar
  11. Ellison NW, Liston A, Steiner JJ, Williams WM, Taylor NL (2006) Molecular phylogenetics of the clover genus (Trifolium-Leguminosae). Mol Phylogenet Evol 39:688–705PubMedCrossRefGoogle Scholar
  12. Franzke A, Mummenhoff K (1999) Recent hybrid speciation in Cardamine (Brassicaceae)—conversion of nuclear ribosomal ITS sequences in statu nascendi. Theor Appl Genet 98:831–834CrossRefGoogle Scholar
  13. Fulnecek J, Lim KY, Leitch AR, Kovarik A, Matyasek R (2002) Evolution and structure of 5S rDNA loci in allotetraploid Nicotiana tabacum and its putative parental species. Heredity 88:19–25PubMedCrossRefGoogle Scholar
  14. Grummt I, Pikaard CS (2003) Epigenetic silencing of RNA polymerase I transcription. Nat Rev Mol Cell Biol 4:641–649PubMedCrossRefGoogle Scholar
  15. Guggisberg A, Bretagnolle, F, Mansion G (2006) Allopolyploid origin of the Mediterranean endemic, Centaurium bianoris (Gentianaceae), inferred by molecular markers. Syst Bot 31:368–379CrossRefGoogle Scholar
  16. Hasterok R, Draper J, Jenkins G (2004) Laying the cytotaxonomic foundations of a new model grass, Brachypodium distachyon (L.) Beauv. Chromosom Res 12:397–403CrossRefGoogle Scholar
  17. Hasterok R, Wolny E, Hosiawa M, Kowalczyk M, Kulak-Ksiazczyk S, Ksiazczyk T, Heneen W, Maluszynska J (2006) Comparative analysis of rDNA distribution in chromosomes of various species of Brassicaceae. Ann Bot 97:205–216PubMedCrossRefGoogle Scholar
  18. Kovarik A, Pires JC, Leitch AR, Lim KY, Sherwood AM, Matyasek R, Rocca J, Soltis DE, Soltis PS (2005) Rapid concerted evolution of nuclear ribosomal DNA in two Tragopogon allopolyploids of recent and recurrent origin. Genetics 169:931–944PubMedCrossRefGoogle Scholar
  19. Lawrence RJ, Earley K, Pontes O, Silva M, Chen ZJ, Neves N, Viegas W, Pikaard CS (2004) A concerted DNA methylation/histone methylation switch regulates rRNA gene dosage control and nucleolar dominance. Mol Cell 13:599–609PubMedCrossRefGoogle Scholar
  20. Lefort F, Douglas GC (1999) An efficient micro-method of DNA isolation from mature leaves of four hardwood tree species Acer, Fraxinus, Prunus and Quercus. Ann For Sci 56:259–263CrossRefGoogle Scholar
  21. Li CB, Zhang S, Lu BR, Hong D (2001) Identification of genome constitution of Oryza malampuzhaensis, O. minuta, and O. punctata by multicolour in situ hybridization. Theor Appl Genet 103:204–211CrossRefGoogle Scholar
  22. Lim KY, Kovarik A, Matyášek R, Bezděk M, Lichtenstein CP, Leitch AR (2000) Gene conversion of ribosomal DNA in Nicotiana tabacum is associated with undermethylated, decondensed and probably active gene units. Chromosoma 109:161–172PubMedCrossRefGoogle Scholar
  23. Lim KY, Skalicka KS, Sarasan V, Clarckson JJ, Chase MW, Kovarik A, Leitch AR (2006) A genetic appraisal of a new synthetic Nicotiana tabacum (Solonaceae) and the Kostoff synthetic tobacco. Am J Bot 93:875–883CrossRefGoogle Scholar
  24. Liu B, Vega JM, Segal G, Abbo S, Rodova M, Feldman M (1998) Rapid genomic changes in newly synthesized amphiploids of Triticum and Aegilops. I. Changes in low-copy non-coding DNA sequences. Genome 41:272–277CrossRefGoogle Scholar
  25. Lysak MA, Lexer C (2006) Towards the era of comparative evolutionary genomics in Brassicaceae. Plant Syst Evol 259:175–198CrossRefGoogle Scholar
  26. Ma X-F, Gustafson JP (2005) Genome evolution of allopolyploids: a process of cytological and genetic diploidization. Cytogenet Genome Res 109:236–259PubMedCrossRefGoogle Scholar
  27. Maluszynska J, Hasterok R (2005) Identification of individual chromosomes and parental genomes in Brassica juncea using GISH and FISH. Cytogenet Genome Res 109:310–314PubMedCrossRefGoogle Scholar
  28. Matyasek R, Lim KY, Kovarik A, Leitch AR (2003) Ribosomal DNA evolution and gene conversion in Nicotiana rustica. Heredity 91:268–275PubMedCrossRefGoogle Scholar
  29. Pikaard CS (1999) Nucleolar dominance and silencing of transcription. Trends Plant Sci 4:478–483PubMedCrossRefGoogle Scholar
  30. Pontes O, Lawrence RJ, Neves N, Silva M, Lee J-H, Chen J, Viegas W, Pikaard CS (2003) Natural variation in nucleolar dominance reveals the relationship between nucleolus organizer chromatin topology and rRNA gene transcription in Arabidopsis. Proc Natl Acad Sci USA 100:11418–11423PubMedCrossRefGoogle Scholar
  31. Pontes O, Neves N, Silva M, Lewis MS, Madlung A, Comai L, Viegas W, Pikaard CS (2004) Chromosomal locus rearrangements are a rapid response to formation of the allotetraploid Arabidopsis suecica genome. Proc Natl Acad Sci USA 101:18240–18245PubMedCrossRefGoogle Scholar
  32. Raap RA, Wendel JF (2005) Epigenetics and plant evolution. New Phytol 168:81–91CrossRefGoogle Scholar
  33. Santoro R (2005) The silence of the ribosomal RNA genes. Cell Mol Life Sci 62:2067–2079PubMedCrossRefGoogle Scholar
  34. Seijo JG, Lavia GI, Fernández A, Krapovickas A, Ducasse D, Moscone EA (2004) Physical mapping of the 5S and 18S-26S rRNA genes by FISH as evidence that Arachis duranensis and A. ipaensis are the wild diploid progenitors of A. hypogaea (Leguminosae). Am J Bot 91:1294–1303CrossRefGoogle Scholar
  35. Snowdon RJ, Kohler W, Friedt W, Kohler A (1997) Genomic in situ hybridization in Brassica amphidploids and interspecific hybrids. Theor Appl Genet 95:1320–1324CrossRefGoogle Scholar
  36. Taylor NL, Gillett JM, Giri N (1983) Morphological observations and chromosome numbers in Trifolium L. section Chronosemium Ser. Cytologia 48:671–677Google Scholar
  37. Vizintin L, Javornik B, Bohanec B (2006) Genetic characterization of Trifolium species as revealed by nuclear DNA content and ITS rDNA analysis. Plant Sci 170:859–866CrossRefGoogle Scholar
  38. Volkov RA, Borisjuk NV, Panchuk II, Schweizer D, Hemleben V (1999) Elimination and rearrangement of parental rDNA in the allotetraploid Nicotiana tabacum. Mol Biol Evol 16:311–320PubMedGoogle Scholar
  39. Volkov RA, Komarova NY, Hemleben V (2007) Ribosomal DNA in plant hybrids: inheritance, rearrangement, expression. Systematics & Biodiversity 5:261–276CrossRefGoogle Scholar
  40. Weiss-Schneeweiss H, Schneeweiss GM, Stuessy TF, Mabuchi T, Park J, Jang C, Sun B (2007) Chromosomal stasis in diploid contrasts with genome sctructure in auto- and allopolyploid taxa of Hepatica (Ranunculaceae). New Phytol 174:669–682PubMedCrossRefGoogle Scholar
  41. Wendel JF, Schnabel A, Seelanan T (1995) Bidirectional interlocus concerted evolution following allopolyloid speciation in cotton (Gossypium). Proc Natl Acad Sci USA 92:280–284PubMedCrossRefGoogle Scholar
  42. Widmer A, Baltisberger M (1999) Molecular evidence for allopolyploid speciation and a single origin of the narrow endemic Draba ladina (Brassicaceae). Am J Bot 86:1282–1289PubMedCrossRefGoogle Scholar
  43. Williams WM, Ansari HA, Ellison NW, Hussain SW (2001) Evidence of three subspecies in Trifolium nigrescens Viv. Ann Bot 87:683–691CrossRefGoogle Scholar
  44. Zohary M, Heller D (1984) The genus Trifolium. Israel Academy of Sciences and Humanities, JerusalemGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Helal A. Ansari
    • 1
  • Nicholas W. Ellison
    • 1
  • Warren M. Williams
    • 1
  1. 1.Grasslands Research CentreAgResearch Ltd.Palmerston NorthNew Zealand

Personalised recommendations