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

The amounts of nuclear DNA in ten species of seaweeds belonging to the Rhodophyceae, Phaeophyeeae, 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 fi'om 0.13 pg per cell in the 1 C population of Ulva rigida to 3.40 pg per cell in the 2 C population of Sphacetaria 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, respectiveIy. 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 ethidinm 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, Abbreviations: A-T nucleic bases adenine and thymine; CRBC chicken red blood cell; FA LS forward-angle light scatter; G-C nucleic bases guanine and cytosine; SEIM sorbitoI enzymatic incubation medium; SWIM sea water incubation medium; Tm thermal denaturation temperature of DNA.

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