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 simple, noninvasive technique for DNA content and ploidy determination.


METHODS
From dorsal fin clips of 14 different fish species single cell suspensions were obtained by chopping the material in 2.1% citric acid/0.5% Tween20, passing it through a 0. 6-gauge needle and incubating it for 20 min at 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 min at RT before adding DAPI to a final volume of 2 ml. The cells were stained 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, Oryzias 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 values 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 now made possible. With slight modifications the method can be extended to a field technique, providing therefore a useful tool for a variety of researchers.
Lamatsch
D K
DK
Lehrstuhl für Physiologische Chemie I, Theodor-Boveri-Institut, Würzburg, Germany. lamatsch@biozentrum.uni-wuerzburg.de
Steinlein
C
C
Schmid
M
M
Schartl
M
M
D000818:Animals; D004247:DNA / Q000032:analysis; D005260:Female; D005399:Fishes / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods*; D016678:Genome; D007211:Indoles; D007621:Karyotyping; D009990:Oryzias / Q000235:genetics; D011003:Ploidies; D015027:Zebrafish
D007211:Indoles; C007293:DAPI; D004247:DNA