Biologia Plantarum

, Volume 56, Issue 2, pp 283–291 | Cite as

Genetic diversity in white clover and its progenitors as revealed by DNA fingerprinting

Original Papers


The genetic diversity and ancestral relationships of a number of Trifolium species was revealed by using the amplified fragment length polymorphism (AFLP) and the random amplified polymorphic DNA (RAPD) markers. Both markers produced few species-specific markers. Using distance and parsimony methods, in NTSYS-pc and PAUP software programs, we clearly differentiated the accessions of white clover from other closely related progenitors. The phylogenetic trees, produced by PAUP, also reinforced the close affinity of T. nigrescens and the allopolyploid white clover in support of former views that this diploid species could have been the donor of one of two genomes of the allotetraploid T. repens. In addition, the dendrograms, produced by NTSYS-pc, also indicated close affinity of T. nigrescens and T. occidentale to the accessions of T. repens. These data is congruent with karyological and phylogenetic affinities between the white clover and T. occidentale. The relationships between the examined accessions, in the T. repens gene pool, may be regarded to indicate the presence of shared alleles between T. repens, T. occidentale and T. uniflorum. Further, T. occidentale showed close phylogenetic relations to T. pallescens.

Additional key words

AFLP ancestry progenitors RAPD Trifolium repens 



amplified fragment length polymorphism


random amplified polymorphic DNA


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.



The authors express their thanks and gratitude to Professor Linda Watson, Chairman of Botany Department, Oklahoma State University for providing facilities for AFLP analysis and for linguistic advice on the manuscript. We also thank the Fulbright Foundation for funding AB visit to conduct the AFLP analysis at Miami University, Oxford, Ohio, USA.


  1. Abberton, M.T.: Interspecific hybridization in the genus Trifolium. — Plant Breed. 126: 337–342, 2007.CrossRefGoogle Scholar
  2. Abberton, M.T., Marshal, A.H.: Progress in breeding perennial clovers for temperate agriculture. — Centenary Rev. J. agr. Sci. 143: 117–135., 2005.CrossRefGoogle Scholar
  3. Ansari, H.A., Ellison, N.W., Griffiths, A.G., Williams, W.M.: A lineage-specific centromeric satellite sequence in the genus Trifolium. — Chromosome Res. 12: 357–367, 2004.PubMedCrossRefGoogle Scholar
  4. Ansari, H.A., Ellison, N.W., Reader, S.M., Badeva, E.D., Friebe, B., Miller, T.E., Williams, W.M.: Molecular cytogenetic organization of 5S and 18S-26S rDNA loci in white clover (Trifolium repens L) and related species. — Ann. Bot. 83:199-206, 1999.Google Scholar
  5. Badr, A., Sayed-Ahmed, H., El-Shanshouri, A., Watson, L.E.: Ancestors of white clover (Trifolium repens L.), as revealed by isozyme polymorphisms. — Theor. appl. Genet. 106: 143–148, 2002.PubMedGoogle Scholar
  6. Badr, A., El-Shazly, H.H., Watson, L.E.: Origin and ancestry of Egyptian clover (Trifolium alexandrinum L.) as revealed by AFLP markers. — Genet. Res. Crop Evol. 55: 21–31, 2008.CrossRefGoogle Scholar
  7. Badr, A., Müller, K., Schäfer-Pregl, R., El-Rabey, H., Effgen, S., Ibrahim, H.H., Pozzi, C., Rohde, W., Salamini. F.: On the origin and domestication history of barley (Hordeum vulgare L.). — Mol. Biol. Evol. 17: 499–510, 2000.PubMedGoogle Scholar
  8. Bortolini, F., Miguel, D., Schifino-Wittmann, M.: Molecular characterization of the USDA white clover (Trifolium repens L.) core collection by RAPD markers. — Genet. Res. Crop Evol. 53: 1081–1087, 2006.CrossRefGoogle Scholar
  9. Brewbaker, J.L., Keim, W.F.: A fertile interspecific hybrid in Trifolium. — Amer. Natur. 87: 323–326, 1953.CrossRefGoogle Scholar
  10. Chen, C.C., Gibson, P.B.: Chromosome pairing in two interspecific hybrids of Trifolium. — Can. J. Genet. Cytol. 12: 190–794, 1970.Google Scholar
  11. Chen, C.C., Gibson, P.B.: Barriers to hybridization of Trifolium repens with related species. — Can. J. Genet. Cytol. 14: 591–595, 1972.Google Scholar
  12. Darlington, C.D., La Cour, F.: Handling of Chromosomes. 6th Ed. — George Allen and Unwin, London.Google Scholar
  13. Ellison, N.W., Liston, A., Steiner, J.J., Williams, W.M., Taylor, N.L.: Molecular phylogenetics of the clover genus (Trifolium-Leguminosae). — Mol. Phylogen. Evol. 39: 688–705, 2006.CrossRefGoogle Scholar
  14. Felsenstein, J.: Confidence limits on phylogenies: an approach using the bootstrap. — Evolution 39: 783–791, 1985.CrossRefGoogle Scholar
  15. Gibson, P.G., Beinhart, G.: Hybridization of Trifolium occidentale with two other species of clover. — J. Hered. 60: 93–96, 1969.Google Scholar
  16. Gillett, J.M., Taylor, N.L.: The World of Clovers. — Iowa State University Press, Ames 2001.Google Scholar
  17. Hand, M.L., Ponting, R.C., Drayton, M.C., Lawless, K.A., Cogan, N.O.I., Brummer, C.E., Sawbridge, T.I., Spangenberg, G.C., Smith, K.F. Forster, J.W.: Identification of homologous, homoeologous and paralogous sequence variants in an outbreeding allopolyploid species based on comparison with progenitor taxa. — Mol. Genet. Genom. 280: 293–304, 2008.CrossRefGoogle Scholar
  18. Hargreaves, S., Maxted, N., Hirano, R., Abberton, M., Skøt, L., Ford-Lloyd, B.V.: Islands as refugia of Trifolium repens genetic diversity. — Conserv. Genet. 11: 1317–1326, 2009.CrossRefGoogle Scholar
  19. Heun, M., Schäfer-Pregl, R., Klawan, D., Castagna, R., Accerbi, M., Borghi, B., Salamini, F.: Site of einkorn wheat domestication identified by DNA finger-printing. — Science 278: 1312–1314, 1997.CrossRefGoogle Scholar
  20. Hussain, S.W., Williams, W.M.: Evidence of functional unreduced gametes in Trifolium repens L. — Euphytica 97: 21–24, 1997.CrossRefGoogle Scholar
  21. Hussain, S.W., Williams, W.M., Mercer, C.F., White. D.W.R.: Transfer of clover cyst nematode resistance from Trifolium nigrescens Viv. to T. repens by interspecific hybridisation. — Theor. appl. Genet. 95: 1274–1281, 1997.CrossRefGoogle Scholar
  22. Jahufer, M.Z.Z., Cooper, M., Ayres, J.F., Bray, R.A.: Identification of research to improve the efficiency of breeding strategies for white clover in Australia: a review. — Aust. J. agr. Res. 53: 239–257, 2002.CrossRefGoogle Scholar
  23. Kakes, P., Chardonnens, A.N.: Cyanotypic frequencies in adjacent and mixed populations of Trifolium occidentale Coombe and Trifolium repens L. are regulated by different mechanisms. — Biochem. Syst. Ecol. 28: 633–649, 2000.PubMedCrossRefGoogle Scholar
  24. Kakes, P., Hakvoort, H.W.J.: On the origin of the cyanogenic polymorphism in Trifolium repens L. — J. Evol. Biol. 7: 201–215, 1994.CrossRefGoogle Scholar
  25. Kazimierski, T., Kazimierska, E.M.: Investigation of hybrids in the genus Trifolium L. V. Fertility and cytogenetics of the hybrid Trifolium nigrescens and T. isthmocarpum. Brot. — Acta. Soc. Bot. Pol. 42: 567–589, 1973.Google Scholar
  26. Marshall, A.H., Abberton, M.T., Michaelson-Yeates, T.P.T., Bowen, C.: The application of molecular markers to monitor introgression of seed yield traits from Trifolium nigrescens into T. repens (white clover). — In: D. Loch (ed.): Proc. 5th International Herbage Seed Conference. Pp 79–83. IHSG, Gatton 2003.Google Scholar
  27. Marshall, A.H., Holdbrook-Smith, K., Michaelson-Yeates, T.P.T., Abberton, M.T., Rhodes, I.: Growth and reproductive characteristics in backcross hybrids derived from Trifolium repens L. × T. nigrescens Viv. interspecific crosses. — Euphytica 104: 61–66, 1998.CrossRefGoogle Scholar
  28. Marshall, A.H., Michaelson-Yeates, T.P.T., Abberton, M.T.: Introgression of reproductive traits from Trifolium nigrescens increases the seed yield of white clover (T. repens). — Plant Breed. 127: 597–601, 2008.CrossRefGoogle Scholar
  29. Marshall, A.H., Michaelson-Yeates, T.P.T., Aluka, P., Meredith, M.: Reproductive characters of interspecific hybrids between Trifolium repens L. and T. nigrescens Viv. — Heredity 74: 136–145, 1995.CrossRefGoogle Scholar
  30. Marshall, A.H., Williams, T.A., Olyott, P., Abberton, M.T., Michaelson-Yeates, T.P.T.: Forage yield and persistency of Trifolium repens × Trifolium nigrescens hybrids under rotational sheep grazing. — Grass Forage Sci. 60: 68–73, 2005.CrossRefGoogle Scholar
  31. Michaelson-Yeates, T.P.T., Marshall, A.H., Abberton, M.T.: Self-incompatibility and heterosis in white clover (Trifolium repens L.). — Euphytica 94: 341–348, 1997.CrossRefGoogle Scholar
  32. Mo, X.Y., Long, T., Liu, Z., Lin, H., Liu, X.Z., Yang, Y.M., Zhang, H.Y. — AFLP analysis of somaclonal variations in Eucalyptus globules. Biol. Plant. 53: 741–744, 2009.CrossRefGoogle Scholar
  33. Olsen, K.M., Sutherland, B.L., Small, L.L.: Molecular evolution of the Li/li chemical defense polymorphism in white clover (Trifolium repens L.). — Mol. Ecol. 16: 4180–4193, 2007.PubMedCrossRefGoogle Scholar
  34. Piluzza, G., Pecetti, L., Bullitta, S., Piano, E.: Discrimination among subterranean clover (Trifolium subterraneum L. complex) accessions using RAPD markers. — Genet. Res. Crop. Evol. 52: 193–199, 2005.CrossRefGoogle Scholar
  35. Rohlf, F.J.: NTSYS-pc. Numerical Taxonomy and Multivariate Analysis System. Version 2.1. — Applied Biostatistics, New York 2002.Google Scholar
  36. Saghai-Maroof, N.A., Soliman, K.M., Jorgensen, R.A., Allard, R.: Ribosomal RNA spacer-length polymorphism in barley: Mendelian inheritance, chromosomal location and population dynamics. Proc. nat. Acad. Sci. USA 81: 8014–8018, 1984.PubMedCrossRefGoogle Scholar
  37. Saitou, N., Nei, M.: The neighbor-joining method: a new method for reconstructing phylogenetic trees. — Mol. Biol. Evol. 4: 406–425, 1987.PubMedGoogle Scholar
  38. Sciacca, F., Fichera, C., Di Silvestro, S., Conte, E., Palumbo, M.: Genetic diversity of durum wheat as determined by AFLP in fluorescence. — Biol. Plant. 54: 435–442, 2010.CrossRefGoogle Scholar
  39. Sokal, R.R., Michener, C.D.: A statistical method for evaluating systematic relationships. — Univ. Kansas Sci. Bull. 28: 1409–1438, 1958.Google Scholar
  40. Sprent, J.I.: Nodulation in Legumes. — Royal Botanic Gardens, Kew 2001.Google Scholar
  41. Swofford, D.L.: PAUP* Phylogenetic Analysis Using Parsimony* and Other Methods, Version 4. — Sinauer Associates, Sunderland 2002.Google Scholar
  42. Vos, P., Hogers, R., Bleckers, M., Beijans, M., Van de Lee, T., Hornes, M., Frijiters, A., Pot. J., Oeleman, J., Zabeau, M.: AFLP: New technique for DNA fingerprinting. — Nucl. Acids Res. 23: 4407–4414, 1995.PubMedCrossRefGoogle Scholar
  43. Williams, J.G.K., Kubelk, R., Livark, K.J., Rafalski, J.A. Tingey, S.V.: DNA polymorphism amplified by arbitrary primers are useful as genetic markers. — Nucl. Acids Res. 18: 6231–6235, 1990.CrossRefGoogle Scholar
  44. Williams, W.M.: Genetics and breeding. — In: Baker, M.J., Williams, W.M. (ed.): White Clover. Pp. 343–419. CAB International, Wallingford — Oxon 1987.Google Scholar
  45. Williams, W.M., Ansari, H.A., Ellison, N.W., Hussain, S.W.: Evidence of three subspecies in Trifolium nigrescens Viv. — Ann. Bot. 87: 683–691, 2001.CrossRefGoogle Scholar
  46. Williams, W.M., Ansari, H.A., Hussain, S.W., Ellison, N.W., Williamson, M.L., Verry, I.M.: Hybridization and introgression between two diploid wild relatives of white clover, Trifolium nigrescens Viv. and T. occidentale Coombe. — Crop Sci. 48: 139–148, 2008.CrossRefGoogle Scholar
  47. Zabeau, M., Vos, P.: Selective restriction fragment amplification: a general method for DNA fingerprinting. — European Patent Application No. 92402629-7, 1993.Google Scholar
  48. Zohary, M., Heller, D.: The Genus Trifolium. — The Israel Academy of Science and Humanities, Jerusalem 1984.Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  1. 1.Botany Department, Faculty of ScienceTanta UniversityTantaEgypt
  2. 2.Faculty of EducationAin Shams University, Roxy, HeliopolisCairoEgypt
  3. 3.Botany Department, Faculty of ScienceSuiz Canal UniversityIsmailiaEgypt

Personalised recommendations