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Molecular Plant Taxonomy pp 279–307Cite as

The Application of Flow Cytometry for Estimating Genome Size and Ploidy Level in Plants

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1115))

Abstract

Over the years, the amount of DNA in a nucleus (genome size) has been estimated using a variety of methods, but increasingly, flow cytometry (FCM) has become the method of choice. The popularity of this technique lies in the ease of sample preparation and in the large number of particles (i.e., nuclei) that can be analyzed in a very short period of time. This chapter presents a step-by-step guide to estimating the nuclear DNA content of plant nuclei using FCM. Attempting to serve as a tool for daily laboratory practice, we list, in detail, the equipment required, specific reagents, and buffers needed, as well as the most frequently used protocols to carry out nuclei isolation. In addition, solutions to the most common problems that users may encounter when working with plant material and troubleshooting advice are provided. Finally, information about the correct terminology to use and the importance of obtaining chromosome counts to avoid cytological misinterpretations of the FCM data are discussed.

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References

  1. Doležel J, Bartoš J, Voglmayr H et al (2003) Nuclear DNA content and genome size of trout and human. Cytometry A 51A:127–128

    Article  Google Scholar 

  2. Ogur M, Erickson RO, Rosen GU et al (1951) Nucleic acids in relation to cell division in Lilium longiflorum. Exp Cell Res 2:73–89

    Article  CAS  Google Scholar 

  3. Bennett MD, Leitch IJ (2012) Plant DNA C-values database (release 6.0, Dec. 2012). http://data.kew.org/cvalues/

  4. Kron P, Suda J, Husband BC (2007) Applications of flow cytometry to evolutionary and population biology. Annu Rev Ecol Evol Syst 38:847–876

    Article  Google Scholar 

  5. Loureiro J, Travnicek P, Rauchova J et al (2010) The use of flow cytometry in the biosystematics, ecology and population biology of homoploid plants. Preslia 82:3–21

    Google Scholar 

  6. Suda J, Kron P, Husband BC et al (2007) Flow cytometry and ploidy: applications in plant systematics, ecology and evolutionary biology. In: Doležel J, Greilhuber J, Suda J (eds) Flow cytometry with plants cells. Wiley-VCH, Weinheim, pp 103–130

    Chapter  Google Scholar 

  7. Leus L, Van Laere K, Dewitte A et al (2009) Flow cytometry for plant breeding. Acta Hortic 836:221–226

    Google Scholar 

  8. Doležel J, Greilhuber J, Suda J (2007) Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc 2:2233–2244

    Article  PubMed  Google Scholar 

  9. Suda J, Krahulcova A, Travnicek P et al (2006) Ploidy level versus DNA ploidy level: an appeal for consistent terminology. Taxon 55:447–450

    Article  Google Scholar 

  10. Noirot M, Barre P, Louarn J et al (2002) Consequences of stoichiometric error on nuclear DNA content evaluation in Coffea liberica var. dewevrei using DAPI and propidium iodide. Ann Bot 89:385–389

    Article  CAS  PubMed  Google Scholar 

  11. Noirot M, Barre P, Louarn J et al (2000) Nucleus-cytosol interactions: a source of stoichiometric error in flow cytometric estimation of nuclear DNA content in plants. Ann Bot 86:309–316

    Article  CAS  Google Scholar 

  12. Noirot M, Barre P, Duperray C et al (2003) Effects of caffeine and chlorogenic acid on propidium iodide accessibility to DNA: consequences on genome size evaluation in coffee tree. Ann Bot 92:259–264

    Article  CAS  PubMed  Google Scholar 

  13. Noirot M, Barre P, Duperray C et al (2005) Investigation on the causes of stoichiometric error in genome size estimation using heat experiments. Consequences on data interpretation. Ann Bot 95:111–118

    Article  CAS  PubMed  Google Scholar 

  14. Price HJ, Hodnett G, Johnston JS (2000) Sunflower (Helianthus annuus) leaves contain compounds that reduce nuclear propidium iodide fluorescence. Ann Bot 86:929–934

    Article  CAS  Google Scholar 

  15. Loureiro J, Rodriguez E, Doležel J et al (2006) Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content. Ann Bot 98:515–527

    Article  CAS  PubMed  Google Scholar 

  16. Bennett MD, Price HJ, Johnston JS (2008) Anthocyanin inhibits propidium iodide DNA fluorescence in Euphorbia pulcherrima: implications for genome size variation and flow cytometry. Ann Bot 101:777–790

    Article  PubMed  Google Scholar 

  17. Loureiro J, Suda J, Doležel J (2007) FLOWer: a plant DNA flow cytometry database. In: Dolezel J, Greilhuber J, Suda J (eds) Flow cytometry with plant cells. Wiley-VCH, Weinheim, pp 423–438

    Chapter  Google Scholar 

  18. Loureiro J, Rodriguez E, Santos C et al (2008) FLOWer: a plant DNA flow cytometry database (release 1.0, May 2008). http://flower.web.ua.pt/

  19. Garnatje T, Canela MÁ, Garcia S et al (2011) GSAD: a genome size in the Asteraceae database. Cytometry A 79A:401–404

    Article  CAS  Google Scholar 

  20. Greilhuber J, Doležel J, Lysak MA et al (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

    Article  CAS  PubMed  Google Scholar 

  21. Leitch IJ, Kahandawala I, Suda J et al (2009) Genome size diversity in orchids: consequences and evolution. Ann Bot 104:469–481

    Article  CAS  PubMed  Google Scholar 

  22. Torrell M, Valles J (2001) Genome size in 21 Artemisia L. species (Asteraceae, Anthemideae): systematic, evolutionary, and ecological implications. Genome 44: 231–238

    Article  CAS  PubMed  Google Scholar 

  23. Garcia S, Sanz M, Garnatje T et al (2004) Variation of DNA amount in 47 populations of the subtribe Artemisiinae and related taxa (Asteraceae, Anthemideae): karyological, ecological, and systematic implications. Genome 47:1004–1014

    Article  CAS  PubMed  Google Scholar 

  24. Leitch IJ, Bennett MD (2004) Genome downsizing in polyploid plants. Biol J Linn Soc Lond 82:651–663

    Article  Google Scholar 

  25. Lysák MA, Lexer C (2006) Towards the era of comparative evolutionary genomics in Brassicaceae. Plant Syst Evol 259:175–198

    Article  Google Scholar 

  26. Poggio L, Burghardt AD, Hunziker JH (1989) Nuclear DNA variation in diploid and polyploid taxa of Larrea (Zygophyllaceae). Heredity 63:321–328

    Article  Google Scholar 

  27. Sliwinska E, Pisarczyk I, Pawlik A et al (2009) Measuring genome size of desert plants using dry seeds. Botany 87:127–135

    Article  CAS  Google Scholar 

  28. Sliwinska E, Zielinska E, Jedrzejczyk I (2005) Are seeds suitable for flow cytometric estimation of plant genome size? Cytometry A 64A:72–79

    Article  Google Scholar 

  29. Kron P, Husband BC (2012) Using flow cytometry to estimate pollen DNA content: improved methodology and applications. Ann Bot 110:1067–1078. doi:10.1093/aob/mcs1167

    Article  CAS  PubMed  Google Scholar 

  30. Suda J, Travnicek P (2006) Reliable DNA ploidy determination in dehydrated tissues of vascular plants by DAPI flow cytometry: new prospects for plant research. Cytometry A 69A:273–280

    Article  Google Scholar 

  31. Bainard JD, Husband BC, Baldwin SJ et al (2011) The effects of rapid desiccation on estimates of plant genome size. Chromosome Res 19:825–842

    Article  CAS  PubMed  Google Scholar 

  32. Kolář F, Lučanová M, Těšitel J et al (2012) Glycerol-treated nuclear suspensions: an efficient preservation method for flow cytometric analysis of plant samples. Chromosome Res 20:303–315

    Article  PubMed  Google Scholar 

  33. Doležel J, Binarova P, Lucretti S (1989) Analysis of nuclear DNA content in plant cells by flow cytometry. Biol Plant 31:113–120

    Article  Google Scholar 

  34. Pfosser M, Amon A, Lelley T et al (1995) Evaluation of sensitivity of flow-cytometry in detecting aneuploidy in wheat using disomic and ditelosomic wheat-rye addition lines. Cytometry 21:387–393

    Article  CAS  PubMed  Google Scholar 

  35. Galbraith DW, Harkins KR, Maddox JM et al (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220:1049–1051

    Article  CAS  PubMed  Google Scholar 

  36. Loureiro J, Rodriguez E, Doležel J et al (2007) Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species. Ann Bot 100:875–888

    Article  CAS  PubMed  Google Scholar 

  37. Arumuganathan K, Earle ED (1991) Nuclear DNA content of some important plant species. Plant Mol Biol Rep 9:208–218

    Article  CAS  Google Scholar 

  38. Bino RJ, Lanteri S, Verhoeven HA et al (1993) Flow cytometric determination of nuclear replication stages in seed tissues. Ann Bot 72: 181–187

    Article  Google Scholar 

  39. De Laat AMM, Blaas J (1984) Flow cytometric characterization and sorting of plant chromosomes. Theor Appl Genet 67:463–467

    Article  PubMed  Google Scholar 

  40. Ebihara A, Ishikawa H, Matsumoto S et al (2005) Nuclear DNA, chloroplast DNA, and ploidy analysis clarified biological complexity of the Vandenboschia radicans complex (Hymenophyllaceae) in Japan and adjacent areas. Am J Bot 92:1535–1547

    Article  CAS  PubMed  Google Scholar 

  41. Matzk F, Meister A, Brutovská R et al (2001) Reconstruction of reproductive diversity in Hypericum perforatum L. opens novel strategies to manage apomixis. Plant J 26:275–282

    Article  CAS  PubMed  Google Scholar 

  42. Otto F (1992) Preparation and staining of cells for high-resolution DNA analysis. In: Radbruch A (ed) Flow cytometry and cell sorting. Springer, Berlin, pp 101–104

    Google Scholar 

  43. Baranyi M, Greilhuber J (1995) Flow cytometric analysis of genome size variation in cultivated and wild Pisum sativum (Fabaceae). Plant Syst Evol 194:231–239

    Article  Google Scholar 

  44. Mishiba KI, Ando T, Mii M et al (2000) Nuclear DNA content as an index character discriminating taxa in the genus Petunia sensu Jussieu (Solanaceae). Ann Bot 85:665–673

    Article  CAS  Google Scholar 

  45. Loureiro J, Rodriguez E, Doležel J et al (2006) Comparison of four nuclear isolation buffers for plant DNA flow cytometry. Ann Bot 98:679–689

    Article  CAS  PubMed  Google Scholar 

  46. Greilhuber J, Temsch EM, Loureiro J (2007) Nuclear DNA content measurement. In: Doležel J, Greilhuber J, Suda J (eds) Flow cytometry with plant cells. Wiley-VCH, Weinheim, pp 67–102

    Chapter  Google Scholar 

  47. Hörandl E, Dobes C, Suda J et al (2011) Apomixis is not prevalent in subnival to nival plants of the European Alps. Ann Bot 108:381–390

    Article  PubMed  Google Scholar 

  48. Greilhuber J, Leitch IJ (2013) Genome size and the phenotype. In: Leitch IJ, Doležel J, Wendel JF, Greilhuber J (eds) Plant genome diversity, vol 2, physical structure, behaviour and evolution of plant genomes. Springer, Wein, pp 323–344

    Google Scholar 

  49. Bennett MD, Smith JB (1991) Nuclear DNA amounts in angiosperms. Philos Trans R Soc Lond Ser B 334:309–345

    Article  CAS  Google Scholar 

  50. Doležel J, Sgorbati S, Lucretti S (1992) Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiol Plant 85:625–631

    Article  Google Scholar 

  51. Doležel J, Dolezelova M, Novak FJ (1994) Flow cytometric estimation of nuclear DNA amount in diploid bananas (Musa acuminata and M. balbisiana). Biol Plant 36:351–357

    Article  Google Scholar 

  52. Marie D, Brown SC (1993) A cytometric exercise in plant DNA histograms, with 2C values for 70 species. Biol Cell 78:41–51

    Article  CAS  PubMed  Google Scholar 

  53. Obermayer R, Leitch IJ, Hanson L et al (2002) Nuclear DNA C-values in 30 species double the familial representation in pteridophytes. Ann Bot 90:209–217

    Article  CAS  PubMed  Google Scholar 

  54. Lysák MA, Doležel J (1998) Estimation of nuclear DNA content in Sesleria (Poaceae). Caryologia 52:123–132

    Article  Google Scholar 

  55. Doležel J, Greilhuber J, Lucretti S et al (1998) Plant genome size estimation by flow cytometry: inter-laboratory comparison. Ann Bot 82(Suppl A):17–26

    Article  Google Scholar 

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Author information

Authors and Affiliations

  1. Jodrell Laboratory, Royal Botanic Gardens, Surrey, UK

    Jaume Pellicer & Ilia J. Leitch

Authors
  1. Jaume Pellicer
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  2. Ilia J. Leitch
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Editors and Affiliations

  1. UMR C53 PVBMT Université de la Réunion-Cirad, Université de la Réunion, Ile de la Réunion, France

    Pascale Besse

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Pellicer, J., Leitch, I.J. (2014). The Application of Flow Cytometry for Estimating Genome Size and Ploidy Level in Plants. In: Besse, P. (eds) Molecular Plant Taxonomy. Methods in Molecular Biology, vol 1115. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-767-9_14

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  • DOI: https://doi.org/10.1007/978-1-62703-767-9_14

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-766-2

  • Online ISBN: 978-1-62703-767-9

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