- ABSTRACT - Among vertebrates, reptiles represent the ideal group for the study of sex determination. Reptiles include lineages with environmental sex determination (ESD) as seen in crocodiles and tuatara, lineages with genotypic sex determination (GSD), like e.g. iguanas, chameleons, skinks, lacertid lizards and birds, and few groups which possess variability in sex determination mechanisms, i.e. geckos, dragon lizards and turtles. This thesis is focused on the evolution of sex chromosomes and sex determination in turtles. The majority of turtle species exhibit ESD, which is considered the ancestral sex determination system of this group, while GSD either as male or female heterogamety evolved independently at least five times. We investigated the presence of sex chromosomes in representative species of turtles by cytogenetic analyses. The analyses included the reconstruction of karyotypes, distribution of constitutive heterochromatin (C-banding, methylation analysis) and repetitive elements (fluorescence in situ hybridization) and comparative genome hybridization (CGH), which often characterize the degenerated Y or W and can be helpful in the identification of "cryptic" sex chromosomes. We described XX/XY sex chromosomes in seven previously unstudied Australasian chelids (Pleurodira) from the genera...

Orb-weavers (Araneidae) are a diversified spider family comprising more than 3,100 species in more than 170 genera. Together with 13 other families, they con- stitute to superfamily Araneoidea. The presented thesis focuses on karyotype evo- lution of Araneidae, including its comparison with a related family Tetragnathidae. The results obtained from 19 araneid and four tetragnathid species confirm previ- ously postulated hypothesis that the ancestral karyotype of Araneoidea (including Araneidae) consists of 24 acrocentric chromosomes in males, including two acro- centric X chromosomes of system X1X20. However, there is a tendency of 2n decrease in some araneids due to centric fusions. In these cases, centric fusions affected most autosomes (and sometimes gonosomes as well); number of chromosome pairs de- creased from 11 to six. Three independent reduction events were detected in this thesis. Furthermore, pattern of nucleolar organizer regions (NORs) was studied in this thesis using fluorescent in situ hybridization, since data on evolution of this marker are scarce in spiders. Striking variability in NORs number was discovered, ranging from one to 13 loci. Remarkably, multiple centric fusions were always ac- companied by considerable increase of NORs number. In araneids and tetragnathids possessing...

The thesis focuses mostly on discovering unrevealed causes of pathologic phenotype symptoms of patients with whom no pathologic changes in genetic material were detected by common cytogenetic methods. All samples were examined by a chip method array-CGH (comparative genomic hybridization) which detects aberrant spots without any knowledge of where they are located in the genome. In some cases the method was used to verify or specify the finding that was diagnosed during previous genetic testing. The patients were examined by this method in an accredited genetics laboratory GENvia, s.r.o. in 2013 and partly also in 2014. The theoretical part of the thesis focuses on the role of the common cytogenetic method and its diagnostic use. I also describe the basic principle of the array-CGH method and its use in prenatal and postnatal diagnostics. The practical part of the thesis describes results of all examined patients. But mostly it focuses on pediatric patients where the diagnosis correlates with their clinical symptoms. All the results were verified by another method used for the particular diagnosis. Some results were verified by FISH (fluorescence in situ hybridization) method, other ways of the results verification are described as well. In total 8 pathologic findings were discovered in patients up to 12...

Cytogenetics of spiders of the infraorder Mygalomorphae is largely unknown. My thesis is focused on the karyotype evolution of spiders of families Hexathelidae Dipluridae, which are basal groups of the superfamily Avicularioidea. In this thesis the evolution of diploid chromosome number, chromosome morfology, and also sex chromosomes are studied. The karyotypes of the mygalomorphs are quite interesting for their diversity. Many members of the family Hexathelidae exhibit extreme systems of a chromosomal sex determination which are composed by many gonosomes X. The meiotic division of males is analyzed. Some species exhibit modifications of this division such as achiazmatic meiosis. Selected markers have been detected by fluorescence in situ hybridization. Keywords: evolution, fluorescent in situ hybridization, karyotype, nucleolus organizer region, sex chromosomes, spider

Die Verbindung von genetischen, physikalischen und zytologischen Daten ist entscheidend für die Genom- und Chromosomenanalyse. Obwohl Beta vulgaris (2n = 18) als wichtige Kulturpflanze und Untersuchungsobjekt der Grundlagenforschung eine intensiv analysierte Art darstellt, existiert bisher keine Verknüpfung zwischen Kopplungsgruppen (LG) und Chromosomen. B.-vulgaris-Chromosomen können zudem aufgrund fehlender morphologischer Unterscheidungsmerkmale bisher nicht einzeln identifiziert und klassifiziert werden. Somit sind zytogenetisch gewonnene Ergebnisse nicht ohne weiteres auf genetische Kopplungsgruppen und physikalische Karten übertragbar. Zytogenetische Methoden können zur Analyse struktureller Chromosomenveränderungen, zur Identifizierung und Lokalisierung von repetitiver DNA sowie zur Kartierung schwierig zu positionierender Marker verwendet werden. Ziel dieser Arbeit war es daher, ein FISH (Fluoreszenz-in-situ-Hybridisierung)-Verfahren zu etablieren, das die Kopplungsgruppen und Chromosomen der Zuckerrübe korreliert und die mikroskopische Identifizierung aller Chromosomenarme ermöglicht. Im Rahmen dieser Arbeit wurde ein FISH-Referenzkaryotyp der Zuckerrübe entwickelt. Durch ein Sondenset aus 18 BACs (bacterial artificial chromosome) sind alle Chromosomenarme der Zuckerrübe identifizierbar und werden mit den nördlichen und südlichen Enden der genetischen Kopplungsgruppen verknüpft. Somit ist eine einheitliche Nummerierung von Kopplungsgruppen und Chromosomen möglich. Durch die gleichzeitige Hybridisierung von chromosomenspezifischen BACs und den Satelliten-DNA-Sonden pAv34 und pBV VI beziehungsweise pEV und pBV wurden die Verteilungsmuster der Sequenzfamilien auf den Chromosomen ermittelt. Die gleichzeitige Hybridisierung aller vier repetitiven Sonden ergab ein chromosomenspezifisches Muster aus subtelomerischen, interkalaren und zentromerischen Signalen. Damit ist die Identifizierung aller B.-vulgaris-Chromosomen in einem einzelnen FISH-Experiment möglich. Zudem wurden dadurch die Chromosomen mit hohem Anteil an tandemartig angeordneten repetitiven Sequenzen identifiziert und die Chromosomenregionen lokalisiert, welche die Sequenzassemblierung behindern können. Sowohl das entwickelte BAC-Set als auch der Sondenpool aus repetitiver DNA unterscheiden die somatischen Metaphasechromosomen erstmals unabhängig von trisomen Linien. Da mit Hilfe der Satelliten-DNA-Sonden alle Chromosomen gleichzeitig markiert werden können, waren die spezifischen physikalischen Längen ermittelbar. Sie wurden mit den genetischen Längen der Kopplungsgruppen in Verbindung gebracht und deckten eine kopplungs-gruppenspezifische Rekombinationshäufigkeit zwischen 0,73 und 1,14 Mb/cM auf. Durch Hybridisierung der BACs und subtelomerischer beziehungsweise telomerischer Sonden auf Pachytänchromosomen wurde der Abstand der BACs sowie der in ihnen enthaltenen genetischen Marker zum physikalischen Chromosomenende abgeschätzt. An fünf Chromo-somenenden wurde ein deutlicher Abstand zwischen den Signalen des BACs und der terminalen Sonden festgestellt. Die zugehörigen Kopplungsgruppen sind demnach erweiterbar. Zudem wurden drei BACs mit nicht detektierbarem Abstand zum Chromosomenende durch FISH an gestreckten Chromatinfasern näher untersucht. Einer der drei BACs wurde eindeutig in unmittelbarer Nähe des Telomers nachgewiesen. Für dieses Ende (Chr 2N) ist die Wahrscheinlichkeit gering, dass die Kopplungsgruppe durch zusätzliche Marker erweitert werden kann; sie wird darum als abgeschlossen angesehen. Für die Enden Chr 4S und Chr 9S war der Abstand zwischen BAC und terminaler Sonde zu groß, um ihn durch Fiber-FISH zu ermitteln. Für sie sind weitere distal zu positionierende Marker wahrscheinlich. Weiterhin wurden bioinformatische Analysen an der verfügbaren B.-vulgaris-Genomsequenz RefBeet 1.0 durchgeführt. Scaffolds, welche die genetischen terminalen Marker enthalten, wurden bioinformatisch identifiziert und auf ihren Gehalt subtelomerischer und telomerischer Sequenzen untersucht. Vorhandene terminale Sequenzen sind ein Nachweis für eine terminale Lokalisierung der in-silico-Chromosomenabschnitte. Für drei Scaffolds mit zuvor ungeklärter Lage wurde dadurch das in-silico-Chromosom ermittelt beziehungsweise die nördliche oder südliche Position auf dem Chromosom dargestellt. Durch die Lokalisierung dieser Bereiche innerhalb der Sequenz in Bezug zum genetischen Marker und unter Berücksichtigung der Ergebnisse der Pachytän-FISH wurde die Strangorientierung von 16 Scaffolds ermittelt. Auf 14 Scaffolds wurden die Abstände der Marker zu den terminalen Sequenzen bestimmt. Der Median betrug etwa 196 kb. Für alle Kopplungsgruppenenden außer dem Norden von LG 2 und LG 4 ist das Vorhandensein weiterer distaler genetischer Marker wahrscheinlich. Satelliten-DNA ist innerhalb einer Art meist homogen, kann jedoch chromosomenspezifische Varianten ausbilden. Auf dem BAC-Marker für Chr 2N wurde durch Southern-Hybridisierung die subtelomerische Sequenzfamilie pAv34 detektiert. Von dem betreffenden BAC wurde eine Subklonbank erstellt. Durch Southern-Hybridisierung wurde der pAv34-Gehalt der Subklone analysiert. Positive Klone wurden sequenziert. Dabei wurden vier verschiedene vollständige pAv34-2N-Monomere detektiert. Im Vergleich mit pAv34-Volllängenmotiven aus der RefBeet 1.0 und dem Datensatz der nicht assemblierten Sequenzen der RefBeet 0.2 bilden die pAv34-2N-Einheiten mit pAv34-Kopien, die verschiedenen in-silico-Chromosomen und Contigs zugeordnet sind, eine Subfamilie. Aus den Sequenzen der Subklone wurden zwei Subklon-Contigs gebildet, die im in-silico-Chromosomenabschnitt von Chr 2N (Bvchr2.un.sca001) positioniert wurden. Dadurch wurden Regionen bisher unbekannter Sequenz entschlüsselt. Abweichungen zwischen den assemblierten Daten und den Subklonsequenzen deuten auf Assemblierungsfehler der Genomsequenz in repetitiven Bereichen hin. Die in dieser Arbeit erzielten Ergebnisse ermöglichen erstmalig die eindeutige Identifizierung aller B.-vulgaris-Chromosomen unabhängig vom Zellzyklusstadium und im Einklang mit genetischen Informationen. Zytogenetische sind jetzt mit molekularen Daten integrierbar und können verwendet werden, um den chromosomenspezifischen Satelliten-DNA-Gehalt aufzudecken und mögliche chromosomenspezifische Subfamilien zu identifizieren. Sie erlauben, physikalische Abstände zwischen Markern zu ermitteln und die Abdeckung von Kopplungsgruppen im terminalen Bereich zu untersuchen. Die Ergebnisse tragen dazu bei, Marker und nicht zugeordnete Contigs und Scaffolds zu kartieren, Ursachen für Lücken aufzudecken und damit die Sequenzdaten des Zuckerrübengenoms zu einer fortlaufenden, hochqualitativen Sequenz zu assemblieren. Die zytogenetischen Daten bilden zudem die Basis für zukünftige Untersuchungen struktureller Umbauten von Chromosomen, die während der Genomevolution stattfanden. / The correlation of genetic, physical and cytological data is crucial for interdisciplinary genome and chromosome analyses. Beta vulgaris (2n = 18) is an important crop and an object of basic research. Although it is an intensely analysed species, its genetic linkage groups (LG) have not been assigned to chromosomes. Additionally, sugar beet chromosomes lack distinct morphological features and could therefore not be identified and classified individually. Consequently, results generated by cytogenetic methods can not be readily applied to genetic and physical maps. Cytogenetic approaches enable analysing structural chromosomal changes, identifying and localizing repetitive DNA, and mapping of markers which are difficult to place within linkage maps. Therefore, the main objective of this work has been the development of a FISH (fluorescence in situ hybridization) procedure that correlates LGs with chromosomes of sugar beet and that allows the microscopic identification of individual chromosome arms. In this work a FISH reference karyotype for sugar beet has been established. A set of 18 BACs (bacterial artificial chromosome) allows the unequivocal identification of each sugar beet chromosome and assigns them to the southern and northern ends of LGs. Hence, the chromosomes are numbered in accordance with the genetic map. The arm-specific BACs and the satellite DNA families pBV and pBV VI or pEV and pAv34 have been hybridized simultaneously to assign the distribution patterns of the highly abundant sequence families to chromosomes. Simultaneous hybridization of the four repetitive probes revealed a chromosome-specific pattern of subtelomeric, intercalary and centromeric signals. Thus, each of the sugar beet chromosomes can be identified in a single FISH experiment. Furthermore, chromosomes with a high content of repetitive DNA have been identified and chromosomal regions that may hinder the correct sequence assembly have been localized. The BAC set as well as the pooled satellite DNA probes discriminate the somatic chromosomes for the first time independently from trisomic lines. Since the chromosomes are differentially labelled with the satellite DNA probes their physical distances could be determined and correlated with genetic distances of the corresponding LGs. A LG-specific recombination frequency from 0.73 to 1.14 Mb/cM has been disclosed. BACs and subtelomeric or telomeric sequences have been hybridized simultaneously on pachytene chromosomes to estimate distances between BACs plus the markers they contain and the physical chromosome ends. Five BACs showed substantial distances to the physical chromosome ends; the corresponding LGs could thus be extended by additional markers. Furthermore, three BACs showing only minor distances to chromosome ends have been investigated in detail by fiber-FISH. One of these BACs was localized closely adjacent to the telomere. For this chromosome end (Chr 2N) it is unlikely that the LG could be extended distally by additional markers and is therefore considered to be closed. The BACs for the chromosome ends Chr 4S and Chr 9S have been too distant from the terminal probe to be bridged by fiber-FISH. For them it is likely that further markers can be placed distally. Furthermore, the B. vulgaris genomic sequence RefBeet 1.0 has been investigated. Scaffolds containing terminal genetic markers have been identified bioinformatically and analysed for the content of subtelomeric and telomeric sequences. The occurrence of terminal sequences confirms the terminal localization of in silico chromosome segments. Three scaffolds with an initially unknown position could thus be allocated to in silico chromosomes and to the northern or southern position on the chromosome. The strand orientation of 16 scaffolds has been determined based on the localization of terminal sequences in relation to the genetic marker considering the results of FISH on pachytene chromosomes. The distance between markers and terminal sequences has been determined for 14 scaffolds. The median is 196 kb. It is likely that further markers can be placed distally from all LG ends except for the north of LG 2 and LG 4. Satellite DNA is usually homogenous within one species; however, it can form chromosome-specific variants. Southern hybridization revealed that the BAC marker for Chr 2N contains the subtelomeric sequence family pAv34. The BAC has been subcloned and the pAv34 content of the subclones has been analysed by Southern hybridization. Positive clones have been sequenced. Thereby, four pAv34-2N monomeres have been detected. Compared to full-length pAv34 motives derived from the RefBeet 1.0 and from unassembled sequence data of the RefBeet 0.2 the pAv34-2N units form a subfamily together with pAv34 copies assigned to different in silico chromosomes and contigs. The subclone sequences have been assembled to two subclone contigs, which have been positioned within the in silico chromosome segment of Chr 2N (Bvchr2.un.sca001). Thereby, regions of unknown sequence have been decoded and probable misassemblies in repetitive regions within the RefBeet 1.0 have been disclosed. The results obtained in this work enable the identification of all sugar beet chromosomes independently from their stage of cell division and in accordance with genetic information. Cytogenetic data are integrated with molecular data and can be used for identifying the chromosome-specific distribution of repeats and chromosome-specific repeat variants. They enable determining physical distances between markers and investigating the terminal coverage of LGs. The results support the correct mapping of markers and unassigned contigs, uncover reasons for gaps within maps and sequence assemblies, and thus contribute to assembling data into a continuous high quality genome sequence of sugar beet. Moreover, the cytogenetic data represent the basis for future investigations of structural chromosomal changes that took place during evolution.

Structure of the karyotype of three species in ermine moths (Yponomeuta: Yponomeutidae, Lepidoptera) was investigated by means of several cytogenetic methods (DAPI staining, orcein staining, CGH and GISH with telomeric probe) focusing on their sex chromosomes. Additionally, the origin of the Z2 chromosome in the WZ1Z2/Z1Z1Z2Z2 sex chromosome system (specific for this family of butterflies) was tested using southern hybridization.

This master’s thesis is focused on digital image analysis of mitotic chromosomes. It deals with the design of the processing of digital images - from image preprocessing to clasification of each chromosomes, including testing on a set of images. This work introduces used cytogenetic methods, that are used to visualize chromosomes. In its practical part describes morphology operations and clasification procedure. Classification of the chomosomes was divided into 5 groups (A-G). All algorithms were created in the MATLAB program.

Changes in chromosome number and structure may cause serious diseases. Cytogenetic tests leadin to set of karyotype are done for detecting these abnormalities. Chromosomes are visualised with proper methods and karyotype is made up most often. Manual karyotyping is time-consuming and expensive task. Because of this, researchers have been developing automated karyotyping systems. Karyotyping systems classify chromosomes into classes based on their characteristic features. Overlapping and bent chromosomes are limitations for automatic classification since they ocur at almost every mitosis. Accuracy and reliability of karyotyping systems still depend on the human intervention. Overcoming of these problems and development of fully automated system is the aim of modern approaches.

The development in modern medicine has allowed to study human genome and detect predispositions to several diseases. One of very promising techniques is the analysis of human karyotype, i.e., the number and appearance of chromosomes in the cell nucleus. The most important step in the karyotype analysis is the chromosome detection and categorization. In this work, a new algorithm for detection of chromosomes from an image of microscopic DNA sample and their categorization into seven groups was developed. The algorithm was implemented in Matlab. The accuracy of segmentation and classification was tested on a set of images from two databases with 117 and 38 images, respectively. The sensitivity of the developed segmentation reached 88% while the value of positive predictivity of segmentation reached 92%. The success rate of chromosome pairing achieves 77%.

Squamate reptiles in general possess an unusual variability in karyotype and sex determining mechanisms. However, in these two aspects, iguanas (Pleurodonts) are considered as a relatively conservative group of lizards. So far only genotypic sex determination with male heterogamety has been detected in this lineage. However, the sex chromosomes have not been revealed in many species by classical cytogenetics, probably due to their homomorphy. Significant variability in karyotype was observed only in the species-rich genera Anolis, Sceloporus and Liolaemus. The aim of this thesis was to investigate the genome of available species from the main iguanid lineages using classical and molecular cytogenetic methods. As well as finding the karyotype characteristics, which may represent synapomorphies of main iguanid lineages, the other aim was to try to identify sex chromosomes. This study confirmed previously published karyotypes of 13 species and established new descriptions of karyotype for eight species. The chromosomes of all studied specimens were examined by methods of classical and molecular cytogenetics, 21 species covering eight iguanid families were analysed in this thesis. The majority of studied species shared the apparently ancestral karyotype of the group, with diploid chromosome number...