The Araneoidea superfamily is a diverse clade of spiders with a great species diversity. The whole superfamily displays considerable conservativeness of observed karyotypes. Most likely ancestral karyotype in males is 24 acrocentric chromosomes with X1X2 sex determination system. The goal of this study is to explore the karyotype diversity of two araneoid families - Araneidae and Mimetidae. The majority of studied species exhibit the ancestral karyotype. In some species of the aformentioned families was observed sudden increase in chromosome numbers, up to 2n♂ = 52 in Araneidae and up to 2n♂ = 57 in Mimetidae. The latter number is the highest chromosome count observed in Entelegynae so far. Increase in 2n goes hand in hand with increase in sex chromosome numbers, leading up to X1X2X3X40 system in Araneidae and up to X1X2X3X4X5X6X70 in Mimetidae. I suggest polyploidy as a possible mechanism of the increase. To test this hypothesis, I measured the size of the genome using flow cytometry and used fluorescence in situ hybridization for the detection of 18S rRNA and 5S rRNA genes. For one species, probe for U2 snRNA gene was also optimized as part of this thesis. In many species studied, these techniques were used for the first time ever. In the case of the family Mimetidae, the largest genomes in...

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.

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Due to their phylogenetic position, Psychidae play an important role in the investigation of the W chromosome origin in Lepidoptera. Several species of Psychidae were tested for the presence of sex-chromatin and investigated via comparative genomic hybridization. Furthermore, odd chromosome numbers and a Z univalent were observed in females. Overall, this study brings tangible evidence for the absence of the W chromosome in Psychidae, thus contributes to complex knowledge of the W chromosome evolution. In addition, karyotypes of the given species were analyzed using 18S rDNA and histone H3 probes. The results indicate relative stability of their karyotypes.

Cytogenetics of mesothelid spiders is largely unkown. The presented diploma thesis is focused on the karyotype evolution of these spiders. As it is the most basal group of spiders, the analysis of its cytogenetics can bring important data about ancestral spider karyotype. In the framework of my thesis, I analysed diploid chromosome numbers, chromosome morphology, meiotic division, sex chromosomes and the pattern of selected molecular markers that were detected by fluorescence in situ hybridization. According to my results, mesothelid spiders have a high number of chromosomes and the prevalence of monoarmed chromosomes. Unlike other spiders, mesothelids have little differentiated sex chromosomes. Key words: evolution, spider, chromosome, karyotype, fluorescence in situ hybridization, nucleolar organiser region, sex chromosomes

1 Abstract: The diploma thesis focuses on study of sex chromosomes evolution and repetitive organized genes of chosen mygalomorph and araneomorph spiders. Spiders are characterized by complexicity of sex chromosome systems, their karyotypes contain multiple sex chromosomes X. Besides multiple X chromosomes they also contain a pair or two pairs of nondiferentiated sex chromosomes X and Y. The used methods include methods of classical cytogenetics (preparation of chromosome slides, C-banding) and methods of molecular cytogenetics (fluorescent in situ hybridization and comparative genome hybridization). Complex sex systems were discovered in the studied Theraphosidae spiders. In Theraphosidae spiders Atropothele socotrana and Poecilotheria vittata neo-sex chromosomes were found. Analysis of molecular differentiation of sex chromosomes suggests low differentiation of Y chromosome in neo-sex chromosomes and pair of nondifferentiated sex chromosomes XY. In haplogyne spider Kukulcania aff. hibernalis (X1X2Y), the Y chromosome was significantly differentiated, male specific signal covered the whole chromosome. Detection of 18S rDNA showed that karyotypes of majority of analysed Theraphosidae spiders and haplogyne spiders contain low number (1 or 2) of nucleolar organizing regions localized terminally, which...

Karyotypes of mygalomorph spiders are not satisfactorily known. This thesis is focused on the basic cytogenetic analysis of selected species of African clades of theraphosid mygalomorphs. It includes four subfamilies: Eumenophorinae, Harpactirinae, Ischnocolinae and Stromatopelminae. Diploid numbers, chromosome morphology, sex chromosome systems and chromosome behaviour in male germline in the selected species of African theraphosid subfamilies were studied. The findings support published results, that refer of high karyotype diversity in Theraphosidae. Diploid chromosome number reduction is probably a basic trend of theraphosid karyotype evolution. The majority of analysed species exhibited one, two or three sex chromosomes. In some species neo-sex chromosome systems were found. In some species one or two sex chromosome pairs (SCP), composed of chromosomes which lack morphological differentiation were detected. Nucleolus organizer regions were detected by fluorescent in situ hybridization in several species. Constitutive heterochromatin detection was performed by C-banding in two species. Keywords: constitutive heterochromatin, diploid number, karyotype, fluorescence in situ hybridization, Mygalomorphae, nucleolus organizer region, SCP, sex chromosome, spider, Theraphosidae

Karyotype analysis of selected species from arachnid orders Amblypygi and Uropygi Whip spiders (Amblypygi) and whip scorpions (Uropygi) represent relict arachnid orders which has been found already at Upper Carboniferous strata. Although cytogenetic data from amblypygids and uropygids might be important to reconstruct karyotype evolution of arachnids, cytogenetics of these orders is almost unknown. Presented study is aimed in analysis of karyotype and meiosis in 16 species of Amblypygi and 4 species of Uropygi. Both groups are characterized by considerable range of diploid chromosome numbers (2n = 24 - 86 in Amblypygi and 36 - 66 in Uropygi). Analysed species does not exhibit morfologically differentiated sex chromosomes. Differentiation of sex chromosomes on molecular level was revealed in amblypygid Paraphrynus mexicanus by comparative genome hybridization. Obtained data indicate XY/XX sex chromosome system in this species. Comparison of karyotype data indicates reduction of chromosome numbers during evolution of both orders. In Amblypygi, this reduction was accompanied by increase of number of biarmed chromosomes. This trend is not apparent in Uropygi. Karyotypes of most analysed amblypygids and uropygids are also characterized by low amount of heterochromatin. Most studied species exhibit two pairs...

Cytogenetics of mesothelid spiders is largely unkown. The presented diploma thesis is focused on the karyotype evolution of these spiders. As it is the most basal group of spiders, the analysis of its cytogenetics can bring important data about ancestral spider karyotype. In the framework of my thesis, I analysed diploid chromosome numbers, chromosome morphology, meiotic division, sex chromosomes and the pattern of selected molecular markers that were detected by fluorescence in situ hybridization. According to my results, mesothelid spiders have a high number of chromosomes and the prevalence of monoarmed chromosomes. Unlike other spiders, mesothelids have little differentiated sex chromosomes. Key words: evolution, spider, chromosome, karyotype, fluorescence in situ hybridization, nucleolar organiser region, sex chromosomes