Nuclei isolation to evaluate genome size of the red alga Gracilaria caudata using flow cytometry

Fernanda Bouvie¹, Zenilda L. Bouzon², Carmen Simioni², Fabiana Marchi³, Estela M. Plastino³, Leila Hayashi¹

¹Seaweed Laboratory of the Marine Shrimp Laboratory, Department of Aquaculture, Federal University of Santa Catarina, 88034-001, Florianópolis, Santa Catarina, Brazil

²Plant Cell Biology Laboratory, Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina, 88049-900, CP 476, Florianópolis, Santa Catarina, Brazil

³Department of Botany, Institute of Biosciences, University of São Paulo, Rua do Matão 277, São Paulo 05508-090, Brazil

Flow cytometry (FCM) is widely used to estimate genome size in terrestrial plants. However, there are few studies in seaweeds due to difficulties such as nucleus release due to the cell wall polysaccharides and selection of a standard reference species genomically similar to the evaluated sample. In this work, an FCM analysis of Gracilaria caudata gametophyte was made by adapting a methodology used for land plants for nuclei isolation to confirm an estimate of the genome size. Twenty milligrams of species were used for each replicated (n = 3). The nuclear isolation protocol followed Galbraith et al. (1983) in a sample immersed in buffer described by Otto (1992) with modifications. In addition, propidium Iodide diluted in the Otto II buffer was used along with RNase to stain the nuclei. Before FCM, samples were analyzed under fluorescence microscopy to ensure nuclei isolation. Kappaphycus alvarezii tetrasporophyte was used as a standard internal reference to calculate the C value further. Fluorescence microscopy showed disaggregated and viable nuclei. The average value of the G. caudata genome size was estimated at 1C = 0.23 pg, approaching the value found by Lopez-Bautista and Kapraun (1995) of 0.24 pg, using the microspectrophotometry technique. In conclusion, establishing one fast and viable FCM protocol for G. caudata opens possibilities for future studies related to strain selection, manipulation of ploidy, and seaweed species’ genetic understanding.