title: Application of flow cytometry for genome size determination in Geosmithia fungi: a comparison of methods.
abstract: Genome size has played an important role in the evolution of plants and animals because changes in genome size seem to accompany if not facilitate evolutionary adaptation to environmental conditions. Flow cytometry (FCM) is a widespread method for determining genome size thanks to its high accuracy and speed of measurements. Nevertheless, only a few comparative studies of FCM methods exist in the field of mycology, and reviews are absent. In this study, we compared the suitability of several concentrations and RNAse A incubation times, fixatives and buffers for estimating genome size in fungi. We chose the genus Geosmithia as a model filamentous fungus. We also introduced a new standard, Aspergillus fumigatus CEA10, to determine absolute genome size. We found FCM to be an appropriate method for measuring genome size in fungi, but optimization steps showed that incorrect propidium iodide staining of nuclei can overestimate genome size due to cytoplasmic staining. We identified fixation with methanol:glacial acetic acid (3:1 v/v), 10% DMSO, 0.1% Triton-X 100, and 5 mM EDTA in combination with Tris-MgCl2 buffer as the best treatment.
authors: Department of Botany, Faculty of Science, Charles University, Czech Republic; Institute of Microbiology of the ASCR, 142 20 Praha 4, Czech Republic.
authors: Tereza
authors: T
authors: Veselská
authors: Jan
authors: J
authors: Svoboda
authors: Zaneta
authors: Z
authors: Růžičková
authors: Miroslav
authors: M
authors: Kolařík
mesh_terms: D002467:Cell Nucleus / Q000235:genetics; D032641:Eurotiales / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods*; D059646:Genome Size / Q000235:genetics*
keywords: RNAse; buffer; confocal microscopy; fixation; flow cytometry; fungi; genome size; standard