el, J.
1 De Montfort University Norman Borlaug Centre for Plant Science, Institute of Experimental Botany, Olomouc, Czech Republic
Institute of Experimental Botany, Laboratory of Molecular Cytogenetics and Cytometry, Sokolovská 6, CZ-77200 Olomouc, Czech Republic. dolezel{at}risc.upol.cz
Flow cytometry is a convenient and rapid method that has been used extensively for estimation of nuclear genome size in plants. In contrast to general expectations, results obtained in different laboratories showed some striking discrepancies. The aim of this joint experiment was to test the reliability and reproducibility of methods. Care was taken to avoid a bias due to the quantity of DNA in the nucleus, the procedure for nuclei isolation or the type of instrument. Nuclear DNA content was estimated in nine plant species representing a typical range of genome size (2C = approx. 0·3–30 pg DNA). Each of the four laboratories involved in this study used a different buffer and/or procedure for nuclei isolation. Two laboratories used arc lamp-based instruments while the other two used laser-based instruments. The results obtained after nuclei staining with propidium iodide (a DNA intercalator) agreed well with those obtained using Feulgen densitometry. On the other hand, results obtained after staining with DAPI (binding preferentially to AT-rich regions) did not agree with those obtained using Feulgen densitometry. Small, but statistically significant, differences were found between data obtained with individual instruments. Differences between the same type of instruments were negligible, while larger differences were observed between lamp- and laser-based instruments. Ratios of fluorescence intensity obtained by laser instruments were higher than those obtained by lamp-based cytometers or by Feulgen densitometry. The results obtained in this study demonstrate that flow cytometry with DNA intercalators is a reliable method for estimation of nuclear genome size in plants. However, the study confirmed an urgent need for an agreement on standards. Given the small but systematic differences between different types of flow cytometers, analysis of very small differences in genome size should be made in the same laboratory and using the same instrument.
DAPI, Feulgen densitometry, flow cytometry, plant genome size, nuclear DNA content, propidium iodide, standardization
Submitted on November 4, 1997
© 1998 Annals of Botany Company
Plant Genome Size Estimation by Flow Cytometry: Inter-laboratory Comparison
el 1
2 Institute of Botany, University of Vienna, Vienna, Austria
3 ENEA, Casaccia Research Centre, INN Department, Plant Biotechnology Division, Rome, Italy
4 Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany
5 De Montfort University Norman Borlaug Centre for Plant Science, Institute of Experimental Botany, Olomouc, Czech Republic and Department of Botany, Palack
University, Olomouc, Czech Republic
Revised on April 9, 1998
Accepted on May 8, 1998
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  K. F.X. Mayer, S. Taudien, M. Martis, H. Simkova, P. Suchankova, H. Gundlach, T. Wicker, A. Petzold, M. Felder, B. Steuernagel, et al. Gene Content and Virtual Gene Order of Barley Chromosome 1H Plant Physiology, October 1, 2009; 151(2): 496 - 505. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Kolar, M. Stech, P. Travnicek, J. Rauchova, T. Urfus, P. Vit, M. Kubesova, and J. Suda Towards resolving the Knautia arvensis agg. (Dipsacaceae) puzzle: primary and secondary contact zones and ploidy segregation at landscape and microgeographic scales Ann. Bot., April 1, 2009; 103(6): 963 - 974. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Greilhuber Cytochemistry and C-values: The Less-well-known World of Nuclear DNA Amounts Ann. Bot., April 1, 2008; 101(6): 791 - 804. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. J. Leitch, L. Hanson, K. Y. Lim, A. Kovarik, M. W. Chase, J. J. Clarkson, and A. R. Leitch The Ups and Downs of Genome Size Evolution in Polyploid Species of Nicotiana (Solanaceae) Ann. Bot., April 1, 2008; 101(6): 805 - 814. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Smarda, P. Bures, L. Horova, B. Foggi, and G. Rossi Genome Size and GC Content Evolution of Festuca: Ancestral Expansion and Subsequent Reduction Ann. Bot., February 1, 2008; 101(3): 421 - 433. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Loureiro, E. Rodriguez, J. Dolezel, and C. Santos Two New Nuclear Isolation Buffers for Plant DNA Flow Cytometry: A Test with 37 Species Ann. Bot., October 1, 2007; 100(4): 875 - 888. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Leong-Skornickova, O. Sida, V. Jarolimova, M. Sabu, T. Fer, P. Travnicek, and J. Suda Chromosome Numbers and Genome Size Variation in Indian Species of Curcuma (Zingiberaceae) Ann. Bot., September 1, 2007; 100(3): 505 - 526. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Castro, J. Loureiro, C. Santos, M. Ater, G. Ayensa, and L. Navarro Distribution of Flower Morphs, Ploidy Level and Sexual Reproduction of the Invasive Weed Oxalis pes-caprae in the Western Area of the Mediterranean Region Ann. Bot., March 1, 2007; 99(3): 507 - 517. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. LOUREIRO, E. RODRIGUEZ, J. DOLEZEL, and C. SANTOS Comparison of Four Nuclear Isolation Buffers for Plant DNA Flow Cytometry Ann. Bot., September 1, 2006; 98(3): 679 - 689. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. SMARDA and P. BURES Intraspecific DNA Content Variability in Festuca pallens on Different Geographical Scales and Ploidy Levels Ann. Bot., September 1, 2006; 98(3): 665 - 678. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. PECINKA, P. SUCHANKOVA, M. A. LYSAK, B. TRAVNICEK, and J. DOLEZEL Nuclear DNA Content Variation among Central European Koeleria Taxa Ann. Bot., July 1, 2006; 98(1): 117 - 122. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. BENNETT and I. J. LEITCH Plant Genome Size Research: A Field In Focus Ann. Bot., January 1, 2005; 95(1): 1 - 6. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. BENNETT and I. J. LEITCH Nuclear DNA Amounts in Angiosperms: Progress, Problems and Prospects Ann. Bot., January 1, 2005; 95(1): 45 - 90. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. GREILHUBER Intraspecific Variation in Genome Size in Angiosperms: Identifying its Existence Ann. Bot., January 1, 2005; 95(1): 91 - 98. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. DOLEZEL and J. BARTOS Plant DNA Flow Cytometry and Estimation of Nuclear Genome Size Ann. Bot., January 1, 2005; 95(1): 99 - 110. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. C. ALBACH and J. GREILHUBER Genome Size Variation and Evolution in Veronica Ann. Bot., December 1, 2004; 94(6): 897 - 911. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. D. Eaton, J. Curley, R. C. Williamson, and G. Jung Determination of the Level of Variation in Polyploidy among Kentucky Bluegrass Cultivars by Means of Flow Cytometry Crop Sci., November 1, 2004; 44(6): 2168 - 2174. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. S. Jakob, A. Meister, and F. R. Blattner The Considerable Genome Size Variation of Hordeum Species (Poaceae) Is Linked to Phylogeny, Life Form, Ecology, and Speciation Rates Mol. Biol. Evol., May 1, 2004; 21(5): 860 - 869. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. SCHMUTHS, A. MEISTER, R. HORRES, and K. BACHMANN Genome Size Variation among Accessions of Arabidopsis thaliana Ann. Bot., March 1, 2004; 93(3): 317 - 321. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. BENNETT, I. J. LEITCH, H. J. PRICE, and J. S. JOHNSTON Comparisons with Caenorhabditis (~100 Mb) and Drosophila (~175 Mb) Using Flow Cytometry Show Genome Size in Arabidopsis to be ~157 Mb and thus ~25 % Larger than the Arabidopsis Genome Initiative Estimate of ~125 Mb Ann. Bot., April 1, 2003; 91(5): 547 - 557. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Pedrosa, N. Sandal, J. Stougaard, D. Schweizer, and A. Bachmair Chromosomal Map of the Model Legume Lotus japonicus Genetics, August 1, 2002; 161(4): 1661 - 1672. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. NOIROT, P. BARRE, J. LOUARN, C. DUPERRAY, and S. HAMON Consequences of Stoichiometric Error on Nuclear DNA Content Evaluation in Coffea liberica var. dewevrei using DAPI and Propidium Iodide Ann. Bot., April 1, 2002; 89(4): 385 - 389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Draper, L. A.J. Mur, G. Jenkins, G. C. Ghosh-Biswas, P. Bablak, R. Hasterok, and A. P.M. Routledge Brachypodium distachyon. A New Model System for Functional Genomics in Grasses Plant Physiology, December 1, 2001; 127(4): 1539 - 1555. [Abstract] [Full Text] [PDF]
|
|