Chapter 1 (General Introduction), Chapter 2 (Polyspermy produces viable haploid/diploid mosaics in sturgeon), Chapter 3 (First evidence of viable progeny from three interspecific parents in sturgeon), Chapter 4 (General discussion, English Summary, Czech Summary, Acknowledgements, List of Publications, Training and Supervision Plan during Study, Curriculum Vitae). The thesis is written in English, contains 71 pages. The results were published in two scientific journals. The actuality of work: The majority of sturgeons are critically endangered and at the same time they are producers of the most expensive food product - black caviar. Presently, sturgeon restoring and conservation are depended on artificial reproduction in hatcheries. However, the way of fertilization is not clear: monospermy, physiological or pathological polyspermy? Moreover, during last decades it was believed that under artificial fertilizations, can appear atypically divided embryos like 3 or 6 cells, which die prior hatching stage or sometimes develop into malformed shape fry with soon death due to deformations. Atypically divided embryos, their origin and the reason of appearance was not studied well. Investigation of fertilization aspects in economically significant and endangered fishes is extremely important. Requires attention and studies a hybridization abilities in sturgeons, as hybridization plays an important role in sturgeon evolution. The purposes of the study were: to find a way of fertilization in sturgeons (monospermy/polyspermy); to understand the reasons of atypical division of cells during artificial breeding; to investigate if atypically divided embryos are able to develop beyond the feeding stage, to analyze their ploidy; to classify the types of atypically divided embryos and their frequency; to investigate sturgeon hybridization plasticity, to produce first interspecific hybrid from three parents. Novelty and scientific originality: In this research two ways of fertilization were found in sturgeons: physiological polyspermy (fertilization typical for caudate amphibians) and karyogamy with an additional plasmogamy, which results in production of viable multiple-sperm mosaics with atypical division on the 2-4 cell stage and mosaic ploidy). Conducted research is important for avoiding of negative effect on sturgeon propagation programs, due to uncontrolled fertilization and releasing of multiple-sperm mosaics into the wild, which can cause appearance of sturgeons with irregular ploidy and induce a detrimental genetic effects on sturgeon populations. However, multiple-sperm mosaics, which were discovered in this study and their easy way of production can be used as a beneficial tool for a rapid manufacture of isogenic strains in sturgeons. In this study was shown that applying of just one part of body (blood, tail, etc.) for ploidy determination is not giving a full view of a real ploidy of the studied individual. A huge ability for hybridization and plasticity were described in sturgeon. It was possible to generate a first viable hybrids from three interspecific parents.

Oxymonads are a group of flagellate protists living in low oxygen environments - mainly the guts of insects and vertebrates. In this study, we focus on the analysis of ploidy and karyotype of various species of oxymonads using Fluorescence In Situ Hybridization (FISH) with probes against single copy genes and telomeric repeats as well as estimating the DNA content in the nuclei of these oxymonads using flow cytometry. Using specific FISH probes against SufDSU gene, which is present in a single copy in the haploid genome, we showed that all studied strains are probably haploid. From the genome of Monocercomonoides exilis strain PA203 we know that oxymonads have the ancestral type of telomeric repeat (TTAGGG). Using a probe against these repeats we tried to label chromosome ends and estimate the number of chromosomes for seven strains (five species) of Monocercomonoides. With a single exception, the average number of signals per nucleus was below 20 indicating number of chromosomes below 10. In the strains of M. mercovicensis, we observed much higher number of signals suggesting that the cells have much higher number of chromosomes. Finally, we established the DNA content for several strains using flow cytometry. We used as a standard M. exilis strain PA203 knowing that the haploid genome size is...

This thesis is focused on the chromosome-number studies in alveolate alga C. velia. This Apicomplexa-related photosynthetic organism proved to be a unique instrument for the study of Apicomplexa and their unique organelle apicoplast. To better understand the similarities and differences within these two phyla, a further chromosome analysis was needed. The first part of this thesis sums up the present knowledge about ploidy, life cycle and genome organization within C. velia and the close-relative phyla of Apicomplexa and Dinoflagellata. The second part describes our attempt to examine the ploidy and total number of chromosomes in C. velia using fluorescence in situ hybridization. The ploidy of C. velia was successfully determined and we also made a significant progress in the determination of the total chromosome number. (max. 4000 znaků)

The Master's thesis studied the Odontites vernus group in the Czech Republic. The group was presented by two different taxa based on the seasonal types and the ploidy levels: an early flowering tetraploid Odontites vernus subsp. vernus (2n = 4x = 40) and a late-flowering diploid Odontites vernus subsp. serotinus (2n = 2x = 18). Plants from 33 populations were sampled for measuring the ploidy level and 27 morphological characters for morphological analysis. Two ploidy levels were confirmed in the Czech Republic and a new late flowering tetraploid taxon (2n = 4x = 40) was found. The three taxa were separated from each other based on the seasonal variation, ploidy level, morphology and ecology.

The ploidy level of diploid and induced triploid tench, Tinca tinca, was verified using flow cytometry to determine relative DNA content of 4',6-diamidino-2-phenylindole (DAPI)-stained erythrocyte nuclei. The C-value (haploid nuclear DNA content; pgDNA.nucleus-1) of these same individuals was determined by means of Feulgen image analysis densitometry, in comparison to the chicken standard (Gallus gallus domesticus; 1.25 pgDNA.nucleus-1, P < 0.05), using three different approaches. Highly similar mean C-values were obtained, thus confirming the possibility of using tench blood as standard in European pond aquaculture for ploidy and DNA content determination in fishes. Feulgen image analysis densitometry (FIAD), flow cytometry (FC) and confocal laser scanning microscopy (CLSM) were used to study the relationship between the DNA content (pgDNA.nucleus-1), nuclear area (?m2), nuclear volume (?m3) and 3-D structure of erythrocyte nuclei in a series of fish ploidy level models: diploid tench (Tinca tinca) (2n), Cuban gar (Atractosteus tristoechus) (2n), triploid tench (3n), evolutionary tetraploid sterlet (Acipenser ruthenus) and stellate sturgeons (A. stellatus) (4n), evolutionary octaploid Siberian sturgeon (A. baerii) and Russian sturgeon (A. gueldenstaedtii) (8n), spontaneous triploid Siberian and Russian sturgeons exhibiting dodecaploidy (12n), evolutionary 12n shortnose sturgeon (A. brevirostrum), and experimentally obtained sturgeon hybrids that were tetraploid, hexaploid (6n), heptaploid (7n), octaploid (8n), decaploid (10n), dodecaploid (12n) and/or tetradecaploid (14n). Standards used for FIA were blood smears of chicken (2.5 pgDNA.nucleus-1), diploid and induced triploid tench (2.04 and 3.1 pgDNA.nucleus-1, respectively). All ploidy levels were first verified by means of FC. Increase in ploidy was accompanied by growth of the nucleus and an increase in the number of flattened ellipsoid nuclei with increased transverse diameter. The volume (Vvoxel) of erythrocyte nuclei, as the sum of voxels calculated from live cells, seems more accurate than volume (Vaxis) calculated from measuring the major and minor axis, especially at higher and odd ploidy levels. Data of absolute and relative DNA content were in agreement with previously published reports. Species of the same ploidy level, however differing in their DNA content, exhibited a similar mean erythrocyte nuclear area, as could be demonstrated on A. ruthenusand and A. stellatus (19.27 and 19.79 ?m2 with a respective mean DNA content of 3.72 and 4.68 pgDNA.nucleus-1) and volume as could be demonstrated on a A. ruthenus and hybrid of A. ruthenus and H. huso(48.3 and 48.9 ?m3 with a respective mean DNA content of 3.74 and 3.10 pg DNA.nucleus-1). Similar relationship was found for the ploidy 6n, 8n, 10n, 12n. The 0.46-1.58 pgDNA increments in DNA content of erythrocytes thus had no effect on their nuclear area/volume. With increasing ploidy level, the DNA concent ration (pgDNA per 1 ?m3 of erythrocyte nuclear volume) as well as surface-to-volume ratio was found not to increase linearly. Nuclear DNA content appeared to be more condensed with an increase of the ploidy level. Observed results deduce properties of whole cell and particularly of the nuclei in series of ploidy levels fishes, adding conformations of nucleotypic hypothesis in context of cell/nuclear size and genome size relationships, as well as taxonomic position of sturgeons.

Die vorliegende Arbeit befasst sich mit züchtungsmethodischen Untersuchungen zur Schaffung blattvariegater Pelargonium x zonale-Hybriden auf tetraploidem Leistungsstand. Basierend auf dem Wirkstoff Trifluralin konnte eine effektive Behandlungsvariante zur somatischen Polyploidisierung periklinalchimärischer Pelargonium x zonale-Klone etabliert werden. Unter Ausnutzung biparentaler Erbgänge wurden fünf ausgewählte Plasmotypen an das Leistungsniveau moderner Sortimente herangeführt. Daneben erbrachten Kreuzungen innerhalb der Sektion Ciconium hybridvariegate F1-Pflanzen. Die Möglichkeit der Ausnutzung von Kern-Plasma-Wechselwirkungen in der Pelargonienzüchtung wird diskutiert. Im Hinblick auf den Aufbau eines Protoplastenregenerationssystems konnten Zellsuspensionskulturen etabliert werden. Im Anschluss an enzymatische Verdauungen wurde die Regeneration von Kallus beobachtet. Variegate Pflanzen aus Mutationsversuchen mit NMH (Nitroso-Methyl-Harnstoff), einer weiteren experimentellen Variante, erwiesen sich als steril, so dass eine weiterführende Züchtungsarbeit auf diesem Weg bisher noch nicht möglich war / The study analyzes breeding schemes concerning the development of variegated tetraploid Pelargonium x zonale-hybrids (Pelargonium x hortorum). With a focus on practical relevance breeding methods for periclinal chimeric leaf patterns are discussed. Trifluralin-induced tetraploid Pelargonium x zonale-hybrids were successfully crossed with modern cultivars. Via biparental mode of inheritance five defined plasmotypes were transfered to the karyological background of current high-performance Pelargonium series. In a crossing-program within the section Ciconium hybrid-variegation was detected. The possibility of using nucleo-plasmatic interactions in developing new Pelargonium cultivars is discussed. First steps concerning a biotechnological approach to create variegated plants included the establishment of cell-suspension-cultures as the base for a protoplast regeneration system. Following the enzymatic digestion of Pelargonium-liquid cultures up to now, callus regeneration was achieved. Variegated plants resulting from mutagenic treatments with NMU (Nitroso-methylurea) proved to be sterile.

La conservation au niveau population est extrêmement nécessaire pour limiter la perte de biodiversité au sein d'une espèce ou d'un complexe d'espèces. Ainsi, l'évaluation de la variabilité inter-populationnelle dans le complexe est reconnue comme première étape importante pour bien définir les plans de conservation des espèces menacées. Arenaria grandiflora form un complexe d'espèces herbacées pérennes à courte durée de vie (4 ans en moyenne) menacé dans certains sites de ses zones de distribution en Europe. A ce jour, sa taxonomie n'est pas bien résolue, ce qui entraîne des problèmes potentiels pour mettre en oeuvre une conservation efficace de ce taxon. Une variation inter-populationnelle du complexe d'espèces A. grandiflora est présentée dans cette étude aux niveaux génétiques, cytogénétiques et morphométriques. Quatre méthodes ont été utilisées : des marqueurs microsatellites nucléaires, une approche cytogénétique, la cytométrie en flux, et enfin la morphométrie sur les feuilles. De plus, les études phénotypiques de variation de taux de germination entre stocks de graines ont été développées. Une différenciation significative entre les profils de variations moléculaires, cytogénétiques et phénotypiques a été détectée dans le complexe d'espèces. Deux cytotypes (diploïdes 2n=2x=22 et tétraploïdes 2n = 4x = 44) ont été mis en évidence en utilisant à la fois des méthodes classiques et des méthodes plus récentes (marqueurs microsatellites, nombres chromosomiques et cytométrie de flux). Le complexe d'espèces d'A; grandiflora présente une forte variation de la valeur de l'ADN 2C, la taille du génome varie de 2.11 ± 0.74 pg à 2.70 ± 0.11 pg pour les populations diploïdes et de 4.30 ± 1.51 pg à 5.27 ± 0.14 pg pour les populations de tétraploïdes. En outre, les grains de tétraploïdes germent significativement mieux que les graines des diploïdes. Les feuilles diffèrent considérablement entre les diploïdes (aciculaires et linéaires) et les tétraploïdes (lancéolées). Cette étude peut être considérée comme préliminaire pour une révision taxonomique de ce complexe d'espèces. D'autre part, grâce à l'ensemble des résultats obtenus, il est également possible de revisiter le concept d'unités évolutives significatives (ESUs) dans le complexe d'espèces A. grandiflora et donc de définir les groupes de populations devant faire l'objet de mesures distinctes. Ainsi, il est possible d'évaluer la pertinence de plans déjà entrepris et de proposer de nouveaux plans de restauration efficaces pour l'avenir de ce complexe d'espèces. / Population-level conservation is being extremely required to restrain the biodiversity loss within a species. So, the assessment of the variability within the species complex is being renowned as an important first step to well implement the future conservation settings for threatened species. The species complex of Arenaria grandiflora is a short-lived perennial herbaceous and a threatened taxon in certain of sites of its distribution areas in Europe, with unresolved gentics and taxonomy, which lead to potential problems in the conservation and utilization of the resource. A differenciation among populations of the species complex of A. grandiflora is presented in this study based on the genetic, cytogenetic and phenotypic patterns. Intraspecific ploidy level varaition is an important aspect of numerous species, so, the present study explores this phenomenon within the A. grandiflora species complex in some type of populations (27 natural populations). To infer the intraspecific genetic and cytogenetic patterns of variability among the studied natural populations of the investigated species complex (A. grandiflora), three methods were used : nuclear microsatellite markers, cytogenetic and flow cytometry approaches. Moreover, the phenotypic patterns of variation among both the stock of seeds and the herbarium materials of A. grandiflora were defined. These patterns were detected using three methods of seed germination (in vitro culture, filter papers and potting soil) and morphometric approaches. A significant differentiation among populations' patterns of molecular, cytogenetic and phenotypic variation was detected within the A. grandiflora species complex. Presence of two closely related cytotypes (diploids 2n=2x=22 and tetraploids 2n=4x=44) was detected using both classical and more recent methods (chromosome number count and flow cytometry respectively). The species complex of A. grandiflora exhibits high variation in 2C-DNA value, the genome size ranges from 2.11 ± 0.74 pg to 2.70 ± 0.11 pg for the diploid populations and from 4.30 ± 1.51 pg to 5.27 ± 0.14 pg for the tetraploid populations. Moreover, the seeds of tetraploids germinate well and in high proportion than the seeds of the diploid ones. In addition, both acicular and linear leaves from the diploid populations differ significantly within the diploids and with the lanceolate leaves of the tetraploid ones. New protocol of seed germination for the tetraploids by in vitro culture after scarifying was described for th first time. The affected factors on seed germination percentages were determinated by an explanatory model of six predictors (altitude, longitude, latitude, ploidy levls, both period and condition of seed storage). Consequently, all these findings are fundamental for the determination of the evolutionarily significant units (ESUs) within A. grandiflora species complex and thus the definition of efficient restoration plans in the future. This study would consider as the preliminary signal for necessary revision for the intraspecific taxonomic keys problematic for this species complex.

Presence and range of phenotypic plasticity in the Agrostis stolonifera polyploid complex (Poaceae) was studied in the territory of the Czech Republic. Plants were cultivated under different experimental conditions. Stomatal size of different Agrostis stolonifera cytotypes was measured. Flow cytometry was applied for genome size estimation. Ploidy levels were determined for more than 150 samples of Agrostis stolonifera as well as several specimens of Agrostis canina, A. capillaris, A. gigantea, A. rupestris and A. vinealis. Absolute DNA content was estimated in all studied species. Isozyme analysis was used to test the possibility of the hybrid origin of pentaploid individuals of Agrostis stolonifera species.

The mode of reproduction can greatly influence the demography and the evolutionary success of the taxon. In the case of autonomous asexual formation seeds are apomictic taxa fully independent of pollinators and compatible partners. For sexual taxa with strict autoincompatibility it is the opposite, i.e. sexual taxa need pollinators and compatible partners for birth of offspring. Therefore, in marginal population and for more extreme situation with lower pollinating activity will have apomictic taxa a higher level of reproductive assurance than sexual taxa vascular plants. This hypothesis was tested in natural populations of apomictic and sexual taxa. In the diploma thesis we therefore investigate the method mode of reproduction and reproductive assurance of 52 taxa of the genus Hieracium s. str. (family Asteraceae) in Europe. Of these, 12 were diploid sexually diploid taxa and 42 polyploid apomictic reproductive taxa. From these taxa we harvested seeds from fully developed capitulum and we determined the potential (total number of seeds in the capitulum) and the realized (the percentage of well-developed seeds at the capitulum). The ploidy of the offspring (the embryos and the seedling) and method origins of seeds we examined using flow cytometry. The results show that the plants of diploid species...

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