Noninvasive determination of genome size and ploidy level in fishes by flow cytometry: Detection of triploid Poecilia formosa

Background: In order to understand the evolutionary significance of single triploids among the mostly diploid Poecilia formosa we have developed a sim ple, noninvasive technique for DNA content and ploidy determination. Methods: From dorsal fin clips of 14 different fish species single cell sus pensions were obtained by chopping the material in 2.1% citric acid/0.5% Tw een20, passing it through a 0.6-gauge needle and incubating it for 20 min a t room temperature (RT) with gentle agitation. After overnight fixation in 70% ethanol, the cells were treated with 1ml 0.5% pepsin/0.1 M HCl for 15 m in at RT before adding DAPI to a final volume of 2 mi. The cells were stain ed for 1-3 h and then analyzed by flow cytometry. Results: We obtained good measurements with CVs ranging from 1.23%, to 3.36 %. The poeciliid species measured contain from 1.6 to 2.0 pg/nucleus, Oryzi as latipes (Medaka) exhibits a nuclear DNA content of 2.2 pg, Danio rerio ( zebrafish) 4.6 pg, Tetraodon fluviatilis (freshwater fugu) 0.70 pg. All val ues except zebrafish are in good agreement with the literature. Conclusions: The identification of Living specimens of different ploidy for breeding experiments, behavioral studies and tissue transplantations is no w made possible. With slight modifications the method can be extended to a field technique, providing therefore a useful tool for a variety of researc hers. Cytometry 39:91-95, 2000. (C) 2000 Wiley-Liss, Inc.