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Plant DNA Standards

It is generally agreed that flow cytometric estimation of nuclear DNA amount in absolute units should be performed using internal standard (the nuclei of a standard are isolated, stained and analysed simultaneously with the nuclei of a sample).

DNA content of the sample is then calculated:


C DNA Amount (pg) = sample G1 peak mean × standard 2C DNA amount (pg) standard G1 peak mean 

To estimate nuclear DNA content in plants, most laboratories prefer to use plant DNA standards. When choosing a standard, it is advisable to select a taxon whose DNA amount is not very different from that of a sample (this will decrease a risk of errors due to nonlinearity of the instrument). Several species are thus needed to cover the large range of genome size known for plants. Unfortunately, there is no general agreement on DNA standards for plant flow cytometry. This laboratory has been using the following plant cultivars:

List of DNA standards suitable for plant DNA flow cytometry

Species Cultivar 2C DNA Content (pg)* 1C Genome Size (Mbp)** Reference
Allium cepa Alice 34.89 17 061 Dolezel et al. (1998)
Vicia faba ssp. faba var.equina Inovec 26.90 13 154 Dolezel et al. (1992)
Secale cereale Dankovske 16.19 7 917 Dolezel et al. (1998)
Pisum sativum Ctirad 9.09 4 445 Dolezel et al. (1998)
Zea mays CE-777 5.43 2 655 Lysak and Dolezel (1998)
Glycine max Polanka 2.50 1 223 Dolezel et al. (1994)
Lycopersicon esculentum Stupicke polni tyckove rane 1.96 958 Dolezel et al. (1992)
Raphanus sativus Saxa 1.11 543 Dolezel et al. (1998)

*) Nuclear DNA content was established using human male leukocytes (2C = 7.0 pg DNA; Tiersch et al. 1989) as a primary reference standard. 
**) 1 pg DNA = 978 Mbp (Dolezel et al. 2003)

All cultivars fulfil the criteria for plant DNA standards as they are: a) suitable for flow cytometric analysis of DNA content; b) seed propagated; c) available as elite lines from breeders. Upon request, this laboratory can provide reasonable amounts of seeds free of charge. Please, send your requests to: dolezelatueb [dot] cas [dot] cz

References

  • Dolezel J, Bartos J, Voglmayr H, Greilhuber J. Nuclear DNA content and genome size of trout and human. Cytometry 51: 127 - 128 (2003) Abstract PDF
  • Dolezel J, Dolezelova M, Novak FJ. Flow cytometric estimation of nuclear DNA amount in diploid bananas (Musa acuminata and M. balbisiana). Biologia Plantarum 36: 351 - 357 (1994)
  • Dolezel J, Greilhuber J, Lucretti S, Meister A, Lysak M A, Nardi L, Obermayer R. Plant genome size estimation by flow cytometry: Inter-laboratory comparison. Annals Botany 82 (Suppl. A): 17 - 26 (1998)
  • Dolezel J, Sgorbati S, Lucretti S. Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiologia Plantarum 85: 625 - 631 (1992)
  • Lysak MA, Dolezel J. Estimation of nuclear DNA content in Sesleria (Poaceae). Caryologia 52: 123 - 132 (1998)
  • Tiersch TR, Chandler RW, Wachtel SSM, Ellias S. Reference standards for flow cytometry and application in comparative studies of nuclear DNA content. Cytometry 10: 706 - 710 (1989)