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Využití cytometrických metod k rozeznání mezidruhových hybridů jeseterů od jeseterů čistého druhu
MIKUŠKOVÁ, Nikola
The purpose of this master thesis is comparison of DNA content measured by flow cytometry and image cytometry to distinguish pure sturgeon species (Acipenser ruthenus, A. baerii, A. gueldenstaedtii) from artificial interspecific F1 hybrids (A. ruthenus x A. baerii, A. baerii x A. ruthenus, A. baerii x A. gueldenstaedtii, A. gueldenstaedtii x A. baerii) originating from Genetic Fisheries Centre, RIFCH in Vodnany (FFPW USB). The relative DNA content was measured by flow cytometry (FCM) with using fluorescent dye DAPI (4,6-diamidin-2-phenylin-dole). Fin tissue was used as a sample for this method. Consequently, results of measuring relative DNA content were converted to absolute DNA content. The representative method of image cytometry was a Feulgen image analysis (FIA). Blood samples were already prepared, stored but still unprocessed. Absolute DNA content was gained by measuring of IOD (integrated optical density) of erythrocyte nuclei. In this experiment, 20 individuals were consequently analysed from each group/ population. These individuals were not identical and not tested by both methods at the same time. Therefore, it is possible that more ploidy levels were detected by FCM method than by FIA method and vice versa. Based on the obtained results, it can be said that the use of these cytometric methods to distinguish interspecific hybrids from clear sturgeons species only based on measurement of DNA content is complicated. Due to statistical analysis, a significant difference (p < 0,05) was found only between DNA contents corresponding to different ploidy levels. It means, that groups with interspecific hybrids or group with pure species with the same or very similar DNA content corresponding the same ploidy level were not statistically different (p > 0,05) from each other, and it does not matter which cytometric method was used. The crucial factor will be probably spontaneous polyploidy or interspecific hybridization. Between methods, not any statistically significant difference (p > 0,05) was found. Therefore, one the one hand, the hypothesis of underestimating the results measured by the FCM method with the fluorescent dye DAPI and the subsequent recalculation can be to some extent rejected. On the other hand, if the FCM - DAPI method can be preferred to the more time-consuming FIA method for measuring DNA content in practice, further investigation is required. It is recommended to provide similar experiment again. Also flow cytometry with fluorescent dye propidium iodide (PI) will be applied because with this method is possible to measure directly absolute DNA content, and finally compare these three methods by statistical analysis.
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