Flow Cytometric Analysis and Sorting of Plant Chromosomes

A general outline of the procedure for flow cytometric analysis and sorting of plant chromosomes consists of the following steps: 

  1. accumulation of cells in metaphase 
  2. preparation of chromosome suspensions 
  3. flow analysis and sorting 
  4. processing of flow-sorted chromosomes 

Although plant chromosome suspensions have also been prepared from cultured cells and mesophyll protoplasts, the most frequently used procedure, developed by Dolezel et al. (1992), involves the use of root tips.

Protocols that describe inpidual steps of chromosome analysis and sorting are given here. Support protocols are also included describing the estimation of cell-cycle synchrony in root tips, instrument alignment, and estimation of purity of flow-sorted chromosome fractions.

Key Methodological Papers

  • Dolezel J, Cihalikova J, Lucretti S. A high-yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba. L. Planta 188: 93 - 98 (1992) 
    * First report on metaphase accumulation and chromosome isolation from fixed root tips.
  • Dolezel J, Cihalikova J, Weiserova J, Lucretti S. Cell cycle synchronization in plant root meristems. Methods in Cell Science 21: 95 - 107 (1999)
  • Dolezel J, Macas J, Lucretti S. Flow analysis and sorting of plant chromosomes. In: Robinson JP, Darzynkiewicz Z, Dean PN, Dressler LG, Orfao A, Rabinovitch PS, Stewart CC, Tanke HJ, Wheeless LL (eds.). Current Protocols in Cytometry. Pp 5.3.1. - 5.3.33. John Wiley & Sons, Inc., New York, 1999.
  • Kubalakova M, Macas J, Dolezel J. Mapping of repeated DNA sequences in plant chromosomes by PRINS and C-PRINS. Theoretical and Applied Genetics 94: 758 - 763 (1997)
  • Lucretti S, Dolezel J. Bivariate flow karyotyping in broad bean (Vicia faba). Cytometry 28: 236 - 242 (1997) 
    * First report on bivariate flow karyotiping in plants.
  • Neumann P, Lysak M, Dolezel J, Macas J. Isolation of chromosomes from Pisum sativum L. hairy root cultures and their analysis by flow cytometry. Plant Science 137: 205 - 215 (1998)
  • Simkova H, Cihalikova J, Vrana J, Lysak MA, Dolezel J. Preparation of HMW DNA from plant nuclei and chromosomes isolated from root tips. Biologia Plantarum 46: 369 - 373 (2003) 
    * This work reports on isolation of high-molecular weight DNA from flow sorted plant chromosomes.
  • Valarik M, Bartos J, Kovarova P, Kubalakova M, de Jong H, Dolezel J. High-resolution FISH on super-stretched flow-sorted plant chromosomes. Plant Journal 37: 940 - 950 (2004) Abstract PDF 
    * This work describes FISH with 100-fold increased resolution when compared with FISH on "standard" metaphase chromosomes.
  • Safar J, Bartos J, Janda J, Bellec A, Kubalakova M, Valarik M, Pateyron S, Weiserova J, Tuskova R, Cihalikova J, Vrana J, Simkova H, Faivre-Rampant P, Sourdille P, Caboche M, Bernard M, Dolezel J, Chalhoub B. Dissecting large and complex genomes: flow sorting and BAC cloning of inpidual chromosomes from bread wheat. Plant Journal 39: 960 - 968 (2004) Abstract PDF 
    * First successful construction of chromosome specific BAC library in plants. The library specific for wheat chromosome 3B was prepared from 2 000 000 flow sorted chromosomes.

Recent Review Papers

  • Dolezel J, Kubalakova M, Bartos J, Macas, J. Flow cytogenetics and plant genome mapping. Chromosome Research 12: 77 - 91 (2004) Abstract
  • Dolezel J, Kubalakova M, Bartos J, Macas J. Chromosome flow sorting and physical mapping. In: Meksem K, Kahl G (eds.). The Handbook of Plant Genome Mapping. Genetic and Physical Mapping. Pp. 151 - 171. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2005.

Papers Reporting Chromosome Analysis and Sorting in Cereals

  • BARLEY (Hordeum vulgare
    • Lysak, M.A., Cihalikova, J., Kubalakova, M., Simkova, H., Kunzel, G., Dolezel, J.: Flow karyotyping and sorting of mitotic chromosomes of barley (Hordeum vulgare L.). Chrom. Res. 7: 431-444, 1999. 
    • Kubalakova M, Kovarova P, Suchankova P, Cihalikova J, Bartos J, Lucretti S, Watanabe N, Kianian SF, Dolezel J. Chromosome sorting in tetraploid wheat and its potential for genome analysis. Genetics 170: 823 - 829 (2005) Abstract PDF 
  • RYE (Secale cereale
    • Kubalakova M, Valarik M, Bartos J, Vrana J, Cihalikova J, Molnar-Lang M, Dolezel J. Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry. Genome 46: 893 - 905 (2003) Abstract 
  • WHEAT (Triticum aestivum
    • Vrana J, Kubalakova M, Simkova H, Cihalikova J, Lysak MA, Dolezel J. Flow-sorting of mitotic chromosomes in common wheat (Triticum aestivum L.). Genetics 156: 2033 - 2041 (2000) Abstract PDF 
    • Kubalakova M, Vrana J, Cihalikova J, Simkova H, Dolezel J. Flow karyotyping and chromosome sorting in bread wheat (Triticum aestivum L.). Theoretical and Applied Genetics 104: 1362 - 1372 (2002) Abstract

Papers Reporting Chromosome Analysis and Sorting in Legumes

  • CHICKPEA (Cicer arietinum
    • Vlacilova K, Ohri D, Vrana J, Cihalikova J, Kubalakova M, Kahl G, Dolezel J. Development of flow cytogenetics and physical genome mapping in chickpea (Cicer arietinum L.). Chromosome Research 10: 695 - 706 (2002) Abstract 
  • FIELD BEAN (Vicia faba
    • Lucretti S, Dolezel J, Schubert I, Fuchs J. Flow karyotyping and sorting of Vicia faba chromosomes. Theoretical and Applied Genetetics 85: 665 - 672 (1993) 
  • GARDEN PEA (Pisum sativum
    • Neumann P, Pozarkova D, Vrana J, Dolezel J, Macas J. Chromosome sorting and PCR-based physical mapping in pea (Pisum sativum L.). Chromosome Research 10: 63 - 71 (2002) Abstract