Biologia Plantarum

, Volume 51, Issue 1, pp 43–48 | Cite as

Chromosome analysis and sorting in Vicia sativa using flow cytometry

  • P. Kovářová
  • A. Navrátilová
  • J. Macas
  • J. DoleželEmail author
Original Papers


Procedures were developed for flow cytometric analysis and sorting of mitotic chromosomes (flow cytogenetics) of common vetch (Vicia sativa L., 2n=12). Suspensions of intact chromosomes were prepared from root tips after cell cycle synchronization, formaldehyde fixation, and mechanical homogenization. On average, 3 × 105 morphologically intact chromosomes could be isolated from 25 root tips. Flow cytometric analysis of DAPI-stained chromosomes resulted in histograms of relative fluorescence intensity (flow karyotypes) containing four peaks, representing particular chromosomes and/or pairs of chromosomes with similar relative DNA content. Peaks I and II were assigned to chromosomes 6 and 5, respectively. These chromosomes could be sorted with a purity exceeding 90 %. The two remaining peaks on the flow karyotype were composite, each of them representing a pair of chromosomes. Chromosomes 1 and 3 were assigned to composite peak III while chromosomes 2 and 4 were assigned to composite peak IV. The chromosomes could be sorted with a purity of 99 % from both composite peaks. Bivariate flow karyotyping after simultaneous staining of chromosomes with DAPI and mithramycin was not found helpful in discriminating additional chromosomes. This study extends the number of legume species for which flow cytogenetics is available and provides a new tool for targeted and effective analysis and mapping of common vetch genome.

Additional key words

cell cycle synchronization chromosome suspension common vetch metaphase accumulation physical genome mapping 





fluorescence in situ hybridization




primed in situ DNA labelling


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ahmed, S., Akem, C., Abd El Moneim, A.M.: Sources of resistance to downy mildew in narbon (Vicia narbonensis) and common (Vicia sativa) vetches.-Genet. Resour. Crop Ev. 47: 153–156, 2000.CrossRefGoogle Scholar
  2. Arumuganathan, K., Slaterry, J.P., Tanksley, S.D., Earle, E.D.: Preparation and flow cytometric analysis of metaphase chromosomes of tomato.-Theor. appl. Genet. 82: 101–111, 1991.CrossRefGoogle Scholar
  3. Chowdhury, D.M.S., Rathjen, J.M., Tate, M.E., McDonald, G.: Genetics of colour traits in common vetch (Vicia sativa L.).-Euphytica 136: 249–255, 2004.CrossRefGoogle Scholar
  4. Conia, J., Bergounioux, C., Perennes, C., Muller, P., Brown, S., Gadal, P.: Flow cytometric analysis and sorting of plant chromosomes from Petunia hybrida protoplasts.-Cytometry 8: 500–508, 1987.PubMedCrossRefGoogle Scholar
  5. Doležel, J.: KARYOSTAR: Microcomputer program for modelling of monoparametric flow karyotypes.-Biológia 46: 1059–1064, 1991.Google Scholar
  6. Doležel, J., Binarová, P., Lucretti, S.: Analysis of nuclear DNA content in plant cells by flow cytometry.-Biol. Plant. 31: 113–120, 1989.Google Scholar
  7. Doležel, J., Číhalíková, J., Lucretti, S.: A high-yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba L.-Planta 188: 93–98, 1992.CrossRefGoogle Scholar
  8. Doležel, J., Číhalíková, J., Weiserová, J., Lucretti, S.: Cell cycle synchronization in plant root meristems.-Methods Cell Sci. 21: 95–107, 1999.PubMedCrossRefGoogle Scholar
  9. Doležel, J., Kubaláková, M., Bartoš, J., Macas, J.: Flow cytogenetics and plant genome mapping.-Chromosome Res. 12: 77–91, 2004.PubMedCrossRefGoogle Scholar
  10. Doležel, J., Kubaláková, M., Bartoš, J., Macas, J.: Chromosome flow sorting and physical mapping.-In: Meksem, K., Kahl, G. (ed.): The Handbook of Plant Genome Mapping. Genetic and Physical Mapping. Pp. 151–171. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 2005.CrossRefGoogle Scholar
  11. Doležel J., Novák, F.J.: A standard method of measurement of nuclear Feulgen-DNA content in Allium sativum L. cells by absorption cytophometry.-Mikroskopie 41: 335–341, 1984.Google Scholar
  12. Elmoneim, A.: Selection for non-shattering common vetch (Vicia sativa L.).-Plant Breed. 100: 168–171, 1993.CrossRefGoogle Scholar
  13. Gamborg, O.L., Wetter, L.R.: Plant Tissue Culture Method.-Nat. Res. Council of Canada, Saskatoon 1975.Google Scholar
  14. Gray, J.W., Langlois, R.G., Carrano, A.V., Burkhart-Schulte, K., Van Dilla, M.A.: High resolution chromosome analysis: one and two parameter flow cytometry.-Chromosoma 73: 9–27, 1979.CrossRefGoogle Scholar
  15. Gualberti, G., Doležel, J., Macas, J., Lucretti, S.: Preparation of pea (Pisum sativum L.) chromosomes and nucleus suspensions from single root tips.-Theor. appl. Genet. 92: 744–751, 1996.CrossRefGoogle Scholar
  16. Ibrahim, S.F., Van den Engh, G.: High-speed chromosome sorting.-Chromosome Res. 12: 5–14, 2004.PubMedCrossRefGoogle Scholar
  17. Kubaláková, M., Valárik, M., Bartoš, J., Vrána, J., Číhalíková, J., Molnár-Lang, M., Doležel, J.: Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry.-Genome 46: 893–905, 2003.PubMedCrossRefGoogle Scholar
  18. Lucretti, S., Doležel, J.: Bivariate flow karyotyping in broad bean (Vicia faba).-Cytometry 28: 236–242, 1997.PubMedCrossRefGoogle Scholar
  19. Lucretti, S., Doležel, J., Schubert, I., Fuchs, J.: Flow karyotyping and sorting of Vicia faba chromosomes.-Theor. appl. Genet. 85: 665–672, 1993.CrossRefGoogle Scholar
  20. Macas, J., Navrátilová, A., Mészáros, T.: Sequence subfamilies of satellite repeats related to rDNA intergenic spacer are differentially amplified on Vicia sativa chromosomes.-Chromosoma 112: 152–158, 2003.PubMedCrossRefGoogle Scholar
  21. Macas, J., Požárková, D., Navrátilová, A., Nouzová, M., Neumann, P.: Two new families of tandem repeats isolated from genus Vicia using genomic self-priming PCR.-Mol. gen. Genet. 263: 741–751, 2000.PubMedCrossRefGoogle Scholar
  22. Navrátilová, A., Neumann, P., Macas, J.: Karyotype analysis of four Vicia species using In situ hybridization with repetitive sequences.-Ann. Bot. 91: 921–926, 2003.PubMedCrossRefGoogle Scholar
  23. Neumann, P., Lysák, M.A., Doležel, J., Macas, J.: Isolation of chromosomes from Pisum sativum L. hairy root cultures and their analysis by flow cytometry.-Plant Sci. 137: 205–215, 1998.CrossRefGoogle Scholar
  24. Neumann, P., Požárková, D., Vrána, J., Doležel, J., Macas, J.: Chromosome sorting and PCR based physical mapping in pea (Pisum sativum L.).-Chromosome Res. 10: 63–71, 2002.PubMedCrossRefGoogle Scholar
  25. Nouzová, M., Neumann, P., Navrátilová, A., Galbraith, D.W., Macas, J.: Microarray-based survey of repetitive genomic sequences in Vicia spp.-Plant mol. Biol. 45: 229–244, 2001.PubMedCrossRefGoogle Scholar
  26. Portugal, J., Waring, M.J.: Assignment of DNA binding sites for 4′,6-diaminidino-2-phenylindole and bisbenzimide (Hoechst 33258). A comparative footprinting study.-Biochim. biophys. Acta 949: 158–168, 1988.PubMedGoogle Scholar
  27. Schifino-Wittmann, M.T.: The cytogenetics and evolution of forage legumes from Rio Grande do Sul: a review.-Genet. mol. Biol. 23: 989–995, 2000.CrossRefGoogle Scholar
  28. Shiran, B., Raina, S.N.: Evidence of rapid evolution and incipient speciation in Vicia sativa species complex based on nuclear and organellar RFLPs and PCR analysis.-Genet. Resour. Crop Ev. 48: 519–532, 2001.CrossRefGoogle Scholar
  29. Tate, M.E., Enneking, D.: A mess of red pottage.-Nature 359: 357–358, 1992.PubMedCrossRefGoogle Scholar
  30. Van de Wouw, M., Maxted, N., Chabane, K., Ford-Lloyd, B.V.: Molecular taxonomy of Vicia ser. Vicia based on Amplified Fragment Lenght Polymorphisms.-Plant Syst. Evol. 229: 91–105, 2001.CrossRefGoogle Scholar
  31. Van de Wouw, M., Maxted, N., Ford-Lloyd, B.V.: Agromorphological characterisation of common vetch and its close relatives.-Euphytica 130: 281–292, 2003a.CrossRefGoogle Scholar
  32. Van de Wouw, M., Maxted, N., Ford-Lloyd, B.V.: A multivariate and cladistic study of Vicia L. ser. Vicia (Fabaceae) based on analysis of morphological characters.-Plant Syst. Evol. 237: 19–39, 2003b.CrossRefGoogle Scholar
  33. Vláčilová, K., Ohri, D., Vrána, J., Číhalíková, J., Kubaláková, M., Kahl, G., Doležel, J.: Development of flow cytogenetics and physical mapping in chickpea (Cicer arietinum L.).-Chromosome Res. 10: 695–706, 2002.PubMedCrossRefGoogle Scholar

Copyright information

© Institute of Experimental Botany, ASCR 2007

Authors and Affiliations

  • P. Kovářová
    • 1
  • A. Navrátilová
    • 2
  • J. Macas
    • 2
  • J. Doležel
    • 1
    • 3
    Email author
  1. 1.Laboratory of Molecular Cytogenetics and CytometryInstitute of Experimental BotanyOlomoucCzech Republic
  2. 2.Institute of Plant Molecular BiologyČeské BudějoviceCzech Republic
  3. 3.Department of Cell Biology and GeneticsPalacký UniversityOlomoucCzech Republic

Personalised recommendations