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Laboratory of flow cytometry Průhonice J. Suda 1,2 & A. Krahulcová 2 1 Department of Botany, Faculty of Science, Charles University 2 Institute of Botany,

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Presentation on theme: "Laboratory of flow cytometry Průhonice J. Suda 1,2 & A. Krahulcová 2 1 Department of Botany, Faculty of Science, Charles University 2 Institute of Botany,"— Presentation transcript:

1 Laboratory of flow cytometry Průhonice J. Suda 1,2 & A. Krahulcová 2 1 Department of Botany, Faculty of Science, Charles University 2 Institute of Botany, Academy of Sciences of the Czech Republic

2 E q u i p m e n t Partec PA II HBO lamp (UV, DAPI staining) air-cooled argon ion laser (488 nm, PI staining) 2 parameters - FL1, FL2

3 B a s i c d a t a Startup: September 2000 Total number of samples: 10 517 Botanical Institute: 5 509 (cca 52 %) Department of Botany: 5 008 (cca 48 %) Operating: ca 940 days Out of order: ca 200 days (!) (13.9. 02 - 23.1. 03) (6.6. 03 - 14.8. 03)

4 M a t e r i a l a n a l y z e d Other taxa Agropyron, Agrostis, Allium, Asplenium, Aster, Betula, Butomus, Carduus, Centaurea, Dryopteris, Echium, Lythrum, Orchis, Phragmites, Poa, Ranunculus, Tragopogon, Vaccinium, Valerianella Most frequent taxa Hieracium subg. Pilosella - 4 494 Taraxacum - 1 205 Macaronesian endemics - 1 082 Galeobdolon - 696 Oxycoccus - 467 Campanula - 418 Empetrum - 326 Reynoutria - 297 Anthoxanthum - 149

5 A p p l i c a t i o n s Nuclear DNA amount analysis Ploidy estimation Determination of closely related taxa Determination of hybrid plants Detection of rare cytotypes Sympatric growth of various cytotypes Breeding mode detection Absolute nuclear DNA content Correlation with environmental conditions and/or evolutionary history Discrimination of plants with the same chromosome number Genome composition in allopolyploid taxa

6 M e t h o d s Nuclei isolation (chopping in hypotonic buffer) Staining (DAPI or PI) Fluorescence analysis Two step procedure Otto I + II (Otto 1990) (with centrifugation) Simplified Otto I + II (without centrifugation) LB01 (Doležel et al. 1989) Matzk (seeds) (Matzk et al. 2000) Tris-MgCl 2 (Pfosser et al. 1995)

7 G r a n t p r o j e c t s 2002-6 (GAAV): The influence of environmental parameters on the genetic structure of young agamic complex: Hieracium subgen. Pilosella (project leader: F. Krahulec) 2002-4 (GAČR): Evolution within hybridogenous population of Tragopogon: does Czech population differ from those ones in US? (project leader: F. Krahulec) 2002 (FRVŠ): An employment of flow cytometry and isozyme analyses in the study of the genus Taraxacum (project leader: L. Záveský) 2003 (FRVŠ): Genome size in the genus Reynoutria – stable or variable? (project leader: J. Suda, researcher: Z. Hoták) 2002 (FRVŠ): A biosystematic study of the polyploid Campanula patula L. (project leader: J. Suda, researcher: H. Látalová) 2001 (FRVŠ): An employment of flow cytometry in the taxonomic study of the genus Galeodolon (project leader: J. Suda, researcher: R. Rosenbaumová) 2002-4 (GAUK): Genome size – a key to the knowledge of relationships among central European taxa from Hieracium subg. Pilosella (project leader: J. Suda, researchers: A. Krahulcová, T. Peckert, R. Rosenbaumová) 2001-3 (GAUK): A biosystematic study of the genus Empetrum (project leader: J. Suda)

8 M a s t e r T h e s e s 2000 - 2:R. Rosenbaumová: A taxonomic study of the genus Galeobdolon Hudson (supervisor: J. Suda) 2000 - 2:S. Papoušková: Reproduction mode variability in the facultatively apomictic hawkweed Hieracium rubrum (supervisor: A. Krahulcová) 2001 - ?:V. Košťálová: Hybridization between Hieracium bauhinii and H. pilosella in mixed populations (supervisor: F. Krahulec) 2003 - ?:T. Urfus: Sources of variation in Hieracium pilosella (supervisor: F. Krahulec) 2003 - ?:T. Mandáková: Biosystematic and ecological study of various cytotypes in Aster amellus (supervisor: Z. Münzbergová) 2003 - ?:K. Mozolová: Cytogeography and population structure in Pimpinella saxifraga (supervisor: J. Suda) 2003 - ?:L. Grattová: Morphological and phytocoenological differentiation of the taxa in the genus Empetrum (supervisor: J. Suda) 2003 - ?:R. Holubová: Morphological and karyological study of the Dryopetris dilatata alliance (supervisor: J. Suda) 2003 - ?:P. Vít: Biosystematic study of the Czech endemic apomicts from the genus Sorbus (supervisor: J. Suda)

9 P h. D. T h e s e s 2000 - 3/4:J. Suda: An employment of flow cytometry in the biosystematics of vascular plants (supervisor: J. Kirschner) 2001 - ?:T. Peckert: A taxonomic study of Hieracium sect. Echinina in Central Europe (supervisor: J. Chrtek jun.) 2001 - ?:L. Záveský: Apomixis vs. sexuality: different approaches to the investigation in the genus Taraxacum Wigg (supervisor: J. Štěpánek) 2001 - ?:V. Mahelka: An effect of potential hybridization between crops and weedy species: a risk of genetic material transfer (with Elytrigia spp. as a model organism) 2002 - ?:R. Rosenbaumová: Relationships between breeding mode and population structure in Hieracium subg. Pilosella (supervisor: F. Krahulec)

10 P r o s a n d C o n s  high accuracy of measurements (when properly adjusted)  sub-optimal design of the instrument non-professional Partec service absolute incompetence of the Biotech (Czech representative)

11 P e r s p e c t i v e s new instrument (Cy Flow with green laser) utilization of forward scatter image cytometry volumetric measurements (Coulter counter)

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