, Volume 173, Issue 3–4, pp 123–132 | Cite as

Quantification of nuclear DNA and G-C content in marine macroalgae by flow cytometry of isolated nuclei

  • Y. Le Gall
  • S. Brown
  • Dominique Marie
  • Meftaha Mejjad
  • B. Kloareg


The amounts of nuclear DNA in ten species of seaweeds belonging to the Rhodophyceae, Phaeophyceae, and Chlorophyceae were determined by flow cytometric analysis of nuclei isolated from protoplasts. Genome size was determined from the fluorescence of the nuclei stained with ethidium bromide. The size of the nuclear genome ranged from 0.13 pg per cell in the 1 C population ofUlva rigida to 3.40 pg per cell in the 2 C population ofSphacelaria sp. GC% analysis was based on staining with either Hoechst 33342 or mithramycin A, two fluorochromes specific for the bases A-T and G-C, respectively. Two models were used for the estimation of the proportion of guanine plus cytosine in the nuclear genome. The first one was based on the linear relationships mithramycin A fluorescence/G-C content and ethidium bromide fluorescence/total DNA content. The second model, based on the curvilinear relationships Hoechst 33342 fluorescence/A-T content and mithramycin A fluorescence/G-C content, resulted in comparatively more homogenous and consistent data and appears more accurate. Comparison with previous reports from other methods for the physical investigation of nuclear genomes shows that flow cytometry of nuclei isolated from protoplasts is an accurate, convenient and robust technique to assay for genome sizes and base pair composition in marine macroalgae.


Flow cytometry Genome size G-C composition Protoplasts Seaweeds 



nucleic bases adenine and thymine


chicken red blood cell


forward-angle light scatter


nucleic bases guanine and cytosine


sorbitol enzymatic incubation medium


sea water incubation medium


thermal denaturation temperature of DNA


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  1. Bliding C (1963) A critical survey of European taxa in Ulvales. Part I. Capsosiphon, Percursaria, Blidingia, Enteromorpha. Opera Bot 8: 1–160Google Scholar
  2. — (1968) A critical survey of European taxa in Ulvales. II. Ulva, Ulvaria, Monostroma, Kornmannia. Bot Nov 121: 535–629Google Scholar
  3. Bot PVM, Holton RW, Stam WT, van den Hoek C (1989 a) Molecular divergence between North Atlantic and Indo-West PacificCladophora albida (Cladophorales, Chlorophyta) isolates as indicated by DNA-DNA hybridization. Mar Biol 102: 307–313Google Scholar
  4. —, Stam WT, Boele-Bos SA, van den Hoek C, van Delden W (1989 b) Biogeographic and phylogenetic studies in three North Atlantic species of Cladophora (Cladophorales, Chlorophyta) using DNA-DNA hybridization. Phycologia 28: 159–168Google Scholar
  5. —, Brussaard CPD, Stam WT, van den Hoek C (1991) Evolutionary relationships between four species of Cladophora (Cladophorales, Chlorophyta) based on DNA-DNA hybridization. J Phycol 27: 617–623Google Scholar
  6. Brown S (1984) Analysis and sorting of plant material by flow cytometry. Physiol Vég 22: 341–349Google Scholar
  7. —, Bergounioux C, Tallet S, Marie D (1991) Flow cytometry of nuclei for ploidy and cell cycle analysis. In: Negrutiu I, GhartiChhetri G (eds) Practical guide to plant cellular and molecular techniques. Birkhäuser, Basel, pp 326–345Google Scholar
  8. Butler DM, Østgaard K, Boyen C, Evans LV, Jensen A, Kloareg B (1989) Isolation conditions for high yields of protoplasts fromLaminaria saccharina andL. digitata. J Exp Bot 40: 1237–1246Google Scholar
  9. —, Evans LV, Kloareg B (1990) Isolation of protoplasts from marine macroalgae. In: Akatsuka I (ed) Introduction to applied phycology. SPB Academic Publishing, The Hague, pp 647–668Google Scholar
  10. Cardinal A (1964) Etude sur les Ectocarpacées de la Manche. Beih Nova Hedwigia 15Google Scholar
  11. Clowes AW, Reidy MA, Clowes MM (1983) Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in absence of endothelium. Lab Invest 49: 327–333PubMedGoogle Scholar
  12. Dalmon J, Loiseaux S (1981) The deoxyribonucleic acids of two brown algae:Pylaiella littoralis (L.) Kjellm. andSphacelaria sp. Plant Sci Lett 21: 241–251Google Scholar
  13. Druehl LD, Robertson BR, Button DK (1989) Characterizing and sexing laminarialean meiospores by flow cytometry. Mar Biol 101: 451–456Google Scholar
  14. Ducreux G, Kloareg B (1988) Plant regeneration from protoplasts ofSphacelaria (Phaeophyceae). Planta 174: 25–29Google Scholar
  15. Dutcher JA, Sizemore RK, Kapraun DF (1990 a) Variation in nuclear DNA base composition (mol% G+C) in three genera of Gigartinales (Rhodophyta). Cryptogam Bot 1: 390–395Google Scholar
  16. —, Kapraun DF, Sizemore RK (1990 b) Inter-and intraspecific variation in nuclear DNA reassociation kinetics in the Gracilariales (Rhodophyta). J Appl Phycol 2: 259–267Google Scholar
  17. Ersland DR, Aldrich J, Cattolico RA (1981) Kinetic complexity, homogeneity, and copy number of chloroplast DNA from the marine algaOlithodiscus luteus. Plant Physiol 68: 1468–1473Google Scholar
  18. Fain SR, Druehl LD, Baillie DL (1988) Repeat and single copy sequences are differentially conserved in the evolution of kelp chloroplast DNA. J Phycol 24: 292–302Google Scholar
  19. Freshwater DW, Dutcher JA, Kapraun DF, Sizemore RK (1990) Variation in DNA nuclear base composition (mol% G+C) in three orders of marine green algae. Hydrobiologia 204/205: 167–172Google Scholar
  20. Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, Firoozabady E (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220: 1049–1051Google Scholar
  21. Godelle B, Cartier D, Marie D, Brown SC, Siljak-Yakovlev S (1990) Relationship between flow cytometric determination of nuclear DNA content and the amount of heterochromatin within theCrepis praemorsa complex. Biol Cell 70: 28 aGoogle Scholar
  22. Goff LJ, Coleman A (1984) Elucidation of fertilization and development in a red alga by quantitative DNA microspectrofluorometry. Dev Biol 102: 173–194PubMedGoogle Scholar
  23. Hinson TK, Kapraun DF (1991) Karyology and nuclear DNA quantification of four species of Chaetomorpha (Cladophorales, Chlorophyta) from the western Atlantic. Helgol Meeresunters 45: 273–285Google Scholar
  24. Hoek C van den, Flinterman A (1968) The life history ofSphacelaria furcigera Kütz. Blumea 16: 193–242Google Scholar
  25. Holm-Hansen O (1969) Algae: amounts of DNA and organic carbon in single cells. Science 163: 87–88PubMedGoogle Scholar
  26. Kapraun DF (1989) Recovery and development of parasexual fusion products inEnteromorpha linza (L.) J. Ag. (Ulvales, Chlorophyta). J Appl Phycol 1: 239–246Google Scholar
  27. —, Bailey JC (1992) Karyology and cytophotometric estimation of nuclear DNA variation in seven species of Ulvales (Chlorophyta). Jpn J Phycol (Sôrui) 40: 15–26Google Scholar
  28. —, Dutcher JA (1991) Cytophotometric estimation of inter- and intraspecific nuclear DNA content variation in Gracilaria and Gracilariopsis (Gracilariales, Rhodophyta). Bot Mar 34: 139–144Google Scholar
  29. —, Shipley MJ (1990) Karyology and nuclear DNA quantification in Bryopsis (Chlorophyta) from North Carolina, USA. Phycologia 29: 443–453Google Scholar
  30. —, Gargiulo MG, Tripodi MG (1988) Nuclear DNA and karyotype variation in species of Codium (Codiales, Chlorophyta) from the North Atlantic. Phycologia 27: 273–282Google Scholar
  31. —, Hinson TK, Lemus AJ (1991) Karyology and cytophotometric estimation of inter- and intraspecific nuclear DNA variation in four species of Porphyra (Rhodophyta). Phycologia 30: 458–466Google Scholar
  32. Kruth HS (1982) Flow cytometry: rapid biochemical analysis of single cells. Anal Biochem 125: 225–242PubMedGoogle Scholar
  33. Le Gall Y, Braud J-P, Kloareg B (1990) Protoplast production inChondrus crispus gametophytes (Gigartinales, Rhodophyta). Plant Cell Rep 8: 582–585Google Scholar
  34. Mejjad M, Loiseaux-de-Goër S, Ducreux G (1992) Protoplast isolation, development, and regeneration in different strains ofPilayella littoralis (L.) Kjellm. Protoplasma 169: 42–48Google Scholar
  35. Müller DG, Stache B (1989) Life history studies onPylaiella littoralis (L.) Kjellman (Phaeophyceae, Ectocarpales) of different geographic origin. Bot Mar 32: 71–75Google Scholar
  36. Ohri D, Khoshoo TN (1986) Plant DNA: contents and systematic. In: Dutta SK (ed) DNA systematics, vol 2. CRC Press, Boca Raton, pp 1–19Google Scholar
  37. Olsen JL, Stam WT, Bot PVM, Hoek C van den (1987) scDNA-DNA hybridization studies in Pacific and Caribbean isolates ofDictyosphaeria cavernosa (Chlorophyta) indicate a long divergence. Helgol Meeresunters 41: 377–383Google Scholar
  38. Pakhomova MV, Zajceva GN, Belozerskij AN (1968) Presence of 5-methylcytosine and 6-methylaminopurine in the DNA of some algae. Dokl Akad Nauk SSSR 182: 712–714PubMedGoogle Scholar
  39. Portugal J, Waring M (1988) Assignment of DNA binding sites for DAPI and bisbenzimide (Hoechst 33258). Comparative foot-printing study. Biochim Biophys Acta 949: 158–168PubMedGoogle Scholar
  40. Stam WT, Bot PVM, Boele-Bos SA, van Rooij JM, Hoek C van den (1988) Single-copy DNA-DNA hybridizations among five species of Laminaria (Phaeophyceae): phylogenetic and biogeographic implications. Helgol Meeresunters 42: 251–267Google Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • Y. Le Gall
    • 1
  • S. Brown
    • 2
  • Dominique Marie
    • 2
  • Meftaha Mejjad
    • 3
  • B. Kloareg
    • 1
  1. 1.Centre d'Etudes Océanographiques et de Biologie MarineCNRS (UPR 4601) and Université Paris VI, Station BiologiqueRoscoff
  2. 2.Service de CytométrieISV, CNRSGif-sur-Yvette
  3. 3.Laboratoire de Morphogénèse Végétale ExpérimentaleCNRS, URA 115, Université Paris XIOrsay

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