Knowledge of phylogenetic relationships among taxa is essential for comparative and evolutionary genomic research. Here, we report reconstruction of the phylogenetic tree of the genus Gossypium containing cultivated cottons of importance in agriculture by using variation of nuclear repetitive DNA sequences. Genomic DNA was isolated from 87 available accessions of 35 species representing all eight basic genome groups of the genus Gossypium and analyzed to infer phylogeny of the genus and genome origin of its polyploid species. Twenty-two interspersed repeated sequence clones derived from G. hirsutum, each representing a repeated sequence family, were hybridized to the genomic DNA of the 35 species, respectively. Southern hybridization showed that 15 of the repetitive DNA sequences could be detected in all of the eight diploid genome groups, five were A genome-specific, and two were detected in some of the non D-genome groups. A total of 642 major restriction bands of repeated sequences were used for phylogenetic analysis of the species. A phylogenetic tree of the species was constructed, based on the parsimony method and evaluated by the bootstrap approach. The tree was consistent with those previously constructed with different methods in major clades in which the genealogical lineages of species are largely congruent with genome designations and geographical distribution; but significantly different branching among some of the species was observed. These results not only further confirm the previously phylogenetic analysis of the species and the utility of repetitive DNA sequences for phylogenetic analysis of the genus Gossypium, but also provide new insights into the phylogeny of the genus.

Abstract The genetic structure at different spatial scales and growing habitats was studied on Silene tatarica, using AFLP and microsatellite markers. S. tatarica is a rare perennial plant occurring along riverbanks and shores of two annually flooding rivers in Finland. Regional scale analysis based on AFLP fragment analysis showed that at Oulanka River population structure represented mostly classical metapopulation model. In general, colonization-extinction processes had an important role, dispersal between subpopulations was limited and genetic differentiation was independent of geographic location. The same subpopulations were partly used to study spatial genetic structuring within subpopulations. Spatial autocorrelation revealed clear spatial genetic structure in each subpopulation. Paternity analysis in an isolated subpopulation showed small amounts of inbreeding, restricted seed dispersal and pollen flow through the subpopulation. Factors affecting the creation and maintenance of spatial genetic structure within subpopulation were most likely colonization events and restricted seed dispersal. The impact of river regulation on the genetic structure of populations was studied by comparing results from Oulanka River to the results obtained from second main growing area, Kitinen River. Oulanka River is a natural river system, whereas Kitinen is a regulated river. The overall regional scale studies did not indicate major differences between river systems. There were some clear population genetic differences between rivers but there were no clear evidence that those would have been caused by river regulation. More likely differences were related to the marginal location of Kitinen population at the edge of the distribution range. Studies indicated that regardless of the species rarity in Finland, active management measures are not currently needed in either S. tatarica growing area. Species specific microsatellite loci were isolated to complement AFLP studies. During the microsatellite isolation, an interesting amplification pattern was detected and studied further. It was suggested that there were repetitive areas within genome containing microsatellites resulting in unusual amplification. The most likely explanation for this phenomenon would be transposable elements containing proto-microsatellite areas. The microsatellites isolated could have evolved mostly from those proto-microsatellites.

O DNA repetitivo constitui uma fração considerável do genoma de muitos organismos eucarióticos. Composto tanto por sequências funcionais, como os genes ribossômicos, quanto não codificantes, como é o caso dos elementos transponíveis, mini/microssatélites e o DNA satélite, essa porção do genoma tem sido amplamente utilizada como objeto de estudo, uma vez que sequências repetitivas podem estar associadas, por exemplo, a processos de diferenciação sexual. Esses estudos têm auxiliado tanto na melhor compreensão da dinâmica dessas regiões cromossômicas, como salientado a importância, a conservação e a evolução da porção repetitiva no genoma. O gênero Eigenmannia (Gymnotiformes, Sternopygidae) compreende espécies crípticas do ponto de vista morfológico que exibem variação no número cromossômico e podem apresentar sistemas sexuais XY ou ZW nos quais os elementos do par sexual diferem pela presença de blocos heterocromáticos maiores do que os encontrados em cromossomos autossomos, ou sistemas múltiplos envolvendo translocação Y-autossomo. O presente trabalho tem por objetivos o estudo sobre do gene Citocromo Oxidase I (COI), de forma a verificar a capacidade discriminatória desse gene mitocondrial e sugerir possíveis espécies dos então cariomorfos do gênero Eigenmannia no estado de São Paulo, continuidade do estudo do DNA repetitivo no gênero Eigenmannia, tanto de regiões funcionais do genoma, no caso o gene ribossômico 5S, bem como de elementos transponíveis, permitindo assim uma melhor compreensão sobre a distribuição, conservação nos cariomorfos e verificar sua eventual participação no processo de diferenciação não só de cromossomos sexuais, mas também na evolução cariotípica do grupo. Os resultados obtidos com o gene COI, assim como aqueles obtidos pelo gene ribossômico 5S evidenciam distâncias genéticas consistentes com a hipótese de que os cinco cariomorfos possam ser considerados como espécies distintas. Além disso, a hibridação in situ do gene ribossômico 5S forneceu uma nova evidência para a fusão cromossômica que deu origem ao cromossomo sexual Y, já descrita na literatura, enquanto que a hibridação de sequências teloméricas não forneceu evidências de processos de fusão recentes envolvendo os cariomorfos. Com relação aos elementos transponíveis foi possível verificar padrões distintos nos elementos TC1 e Rex1 no que diz respeito às sequências, uma vez que o elemento TC1 delimitou dois grandes grupos o que pode indicar uma invasão simultânea nos grupos e no retrotransposon Rex1 a invasão tenha ocorrido em um ancestral comum a todos os cariomorfos / The repetitive DNA constitutes a considerable fraction of the genome of many eukaryotic organisms. Compound by both functional sequences, such as ribosomal genes, and non-coding, such as transposable elements, mini / microsatellite DNA and the satellite, this portion of the genome has been widely used as a study object, since the repetitive sequences may be associated with, for example, the processes of sexual differentiation. These studies helped to understand the dynamics of these chromosomal regions, pointing the importance, conservation and evolution of the repetitive portion of the genome. The genus Eigenmannia (Gymnotiformes, Sternopygidae) comprises a morphological cryptic species that exhibit variation in chromosome number and may have sexual XY or ZW systems in which the elements of sexual pair differ by the presence of heterochromatic blocks larger than those found in chromosomes autosomes, or systems involving multiple Y-autosome translocation. The present work aims to study the gene Cytochrome Oxidase I (COI) to verify the discriminatory capacity of this mitochondrial gene and suggest possible species of the so called karyomorphs of the genus Eigenmannia in the state of São Paulo. The study of repetitive DNA in Eigenmannia genus, includes 5S ribosomal gene and transposable elements, thus allowing a better understanding of the distribution, conservation in karyomorphs and verify their possible participation in the process of differentiation not only of sex chromosomes, karyotypic evolution but also in the group. The results obtained with the COI gene, as well as those obtained by the 5S ribosomal gene demonstrate genetic distances consistent with the hypothesis that the five karyomorphs can be regarded as separate species. In addition, in situ hybridization of ribosomal 5S gene provided new evidence for chromosomal fusion which led to the Y sex chromosome, as described in the literature, whereas hybridization of telomeric sequences did not provide evidence of recent fusion events involving the karyomorphs. Regarding transposable elements, it could be verified distinct sequence patterns between TC1 and Rex1 elements, since the TC1 element delimited two groups which may indicate a simultaneously invasion in those groups and retrotransposon Rex1 invasion has occurred in a common ancestor to all karyomorphs

O DNA repetitivo constitui uma fração considerável do genoma de muitos organismos eucarióticos. Composto tanto por sequências funcionais, como os genes ribossômicos, quanto não codificantes, como é o caso dos elementos transponíveis, mini/microssatélites e o DNA satélite, essa porção do genoma tem sido amplamente utilizada como objeto de estudo, uma vez que sequências repetitivas podem estar associadas, por exemplo, a processos de diferenciação sexual. Esses estudos têm auxiliado tanto na melhor compreensão da dinâmica dessas regiões cromossômicas, como salientado a importância, a conservação e a evolução da porção repetitiva no genoma. O gênero Eigenmannia (Gymnotiformes, Sternopygidae) compreende espécies crípticas do ponto de vista morfológico que exibem variação no número cromossômico e podem apresentar sistemas sexuais XY ou ZW nos quais os elementos do par sexual diferem pela presença de blocos heterocromáticos maiores do que os encontrados em cromossomos autossomos, ou sistemas múltiplos envolvendo translocação Y-autossomo. O presente trabalho tem por objetivos o estudo sobre do gene Citocromo Oxidase I (COI), de forma a verificar a capacidade discriminatória desse gene mitocondrial e sugerir possíveis espécies dos então cariomorfos do gênero Eigenmannia no estado de São Paulo, continuidade do estudo do DNA repetitivo no gênero Eigenmannia, tanto de regiões funcionais do genoma, no caso o gene ribossômico 5S, bem como de elementos transponíveis, permitindo assim uma melhor compreensão sobre a distribuição, conservação nos cariomorfos e verificar sua eventual participação no processo de diferenciação não só de cromossomos sexuais, mas também na evolução cariotípica do grupo. Os resultados obtidos com o gene COI, assim como aqueles obtidos pelo gene ribossômico 5S evidenciam distâncias genéticas consistentes com a hipótese de que os cinco cariomorfos possam ser considerados como espécies distintas. Além disso, a hibridação in situ do gene ribossômico 5S forneceu uma nova evidência para a fusão cromossômica que deu origem ao cromossomo sexual Y, já descrita na literatura, enquanto que a hibridação de sequências teloméricas não forneceu evidências de processos de fusão recentes envolvendo os cariomorfos. Com relação aos elementos transponíveis foi possível verificar padrões distintos nos elementos TC1 e Rex1 no que diz respeito às sequências, uma vez que o elemento TC1 delimitou dois grandes grupos o que pode indicar uma invasão simultânea nos grupos e no retrotransposon Rex1 a invasão tenha ocorrido em um ancestral comum a todos os cariomorfos / The repetitive DNA constitutes a considerable fraction of the genome of many eukaryotic organisms. Compound by both functional sequences, such as ribosomal genes, and non-coding, such as transposable elements, mini / microsatellite DNA and the satellite, this portion of the genome has been widely used as a study object, since the repetitive sequences may be associated with, for example, the processes of sexual differentiation. These studies helped to understand the dynamics of these chromosomal regions, pointing the importance, conservation and evolution of the repetitive portion of the genome. The genus Eigenmannia (Gymnotiformes, Sternopygidae) comprises a morphological cryptic species that exhibit variation in chromosome number and may have sexual XY or ZW systems in which the elements of sexual pair differ by the presence of heterochromatic blocks larger than those found in chromosomes autosomes, or systems involving multiple Y-autosome translocation. The present work aims to study the gene Cytochrome Oxidase I (COI) to verify the discriminatory capacity of this mitochondrial gene and suggest possible species of the so called karyomorphs of the genus Eigenmannia in the state of São Paulo. The study of repetitive DNA in Eigenmannia genus, includes 5S ribosomal gene and transposable elements, thus allowing a better understanding of the distribution, conservation in karyomorphs and verify their possible participation in the process of differentiation not only of sex chromosomes, karyotypic evolution but also in the group. The results obtained with the COI gene, as well as those obtained by the 5S ribosomal gene demonstrate genetic distances consistent with the hypothesis that the five karyomorphs can be regarded as separate species. In addition, in situ hybridization of ribosomal 5S gene provided new evidence for chromosomal fusion which led to the Y sex chromosome, as described in the literature, whereas hybridization of telomeric sequences did not provide evidence of recent fusion events involving the karyomorphs. Regarding transposable elements, it could be verified distinct sequence patterns between TC1 and Rex1 elements, since the TC1 element delimited two groups which may indicate a simultaneously invasion in those groups and retrotransposon Rex1 invasion has occurred in a common ancestor to all karyomorphs

Despite their homology and analogous function, the Hox gene clusters of vertebrates and invertebrates are subject to different constraints on their structural organization. This is demonstrated by a drastically different distribution of repetitive DNA elements in the Hox cluster regions. While gnathostomes have a strong tendency to exclude repetitive DNA elements from the inside of their Hox clusters, no such trend can be detected in the Hox gene clusters of protostomes. Repeats “invade” the gnathostome Hox clusters from the 5′ and 3′ ends while the core of the clusters remains virtually free of repetitive DNA. This invasion appears to be correlated with relaxed constraints associated with gene loss after cluster duplications.

Retrotransposon mobilization is a major source of genome evolution. However, the functional consequences of these events, and particularly their influence upon transcriptional activity, are poorly defined. The extent of retrotransposon transcription, as well as that of other repetitive elements, has eluded systematic study due to difficulties in discriminating elements copied in multiple genomic loci. Moreover, the potential regulatory effects of retrotransposon transcription upon the expression of neighbouring protein-coding genes are also largely unknown. This thesis develops methods to survey repetitive element expression and assess the functions of retrotransposons in the mouse and human genomes. Chapter 1 summarises the complex transcriptional output of the mammalian genome, the functional annotation of this expression and the genomic and bioinformatic tools available for its detection. Chapter 2 explores the capacity of short sequence tags to discern transcription from individual repetitive elements, as well as from protein-coding genes. It is based upon a publication that critiqued the bioinformatics associated with Cap Analysis Gene Expression (CAGE) and developed novel methodologies to resolve repetitive element transcription. Chapter 3 describes the development of an updated CAGE mapping pipeline for the fourth stage of the international Functional Annotation of Mouse (FANTOM) project, which lead to the generation of a research article and a book chapter. These works demonstrated the enhanced utility of CAGE when coupled with next-generation sequencing, highlighted the benefits of CAGE when applied to systems biology and profiled the temporal expression of human repetitive elements. Chapter 4 presents an in-depth analysis of repetitive element transcription in the mouse and human genomes. Using CAGE, approximately 250,000 retrotransposon associated transcription start sites were defined, many of which were tissue-specific. Retrotransposons were found to frequently function as alternative promoters for protein-coding genes and/or express non-coding RNAs. Furthermore, when retrotransposons were found within the 3’UTR of protein-coding genes, there was strong evidence for the reduced expression of the corresponding transcripts. A genome-wide screen for strong expression correlation between repetitive elements and neighbouring protein-coding genes identified approximately 23,000 candidate regulatory regions derived from retrotransposons, including several hundred putative boundary elements. These were in addition to more than two thousand examples of bidirectional transcription found in retrotransposons, which are known to be a source of double stranded RNAs involved in RNA interference. Chapter 5 explores the proportion of the mouse embryonic stem cell transcriptome comprised of repeat-derived transcripts, using next-generation RNA sequencing. This study defined the dynamic expression of repetitive elements at the greatest resolution achieved to date and demonstrated that repetitive elements are an intrinsic part of the mammalian transcriptional landscape.

Repetitive DNA comprises substantial part of the eukaryotic genome. ?Junk DNA?, as it was originally understood at the beginning of its discovery has attracted a lot of attention lately due to many studies proving its functional perspectives. Analysis of its dynamics, characteristics and distribution has been widely studied in organisms with monocentric chromosomes. Holokinetic system, however, was left behind in these efforts and whole image of repetitive DNA distribution and dynamics in this system remains to be elucidated. In this thesis various approaches were used to isolate and characterise repetitive DNA in the genome of the codling moth, Cydia pomonella. Satellite DNA CPSAT-1 was successfully isolated, characterised with Dot blot and Southern blot and mapped with FISH in the genome of C. pomonella. 17 microsatellite probes were used to localize microsatellite arrays in the genome of C. pomonella. Method of microsatellite FISH revealed distribution of all tested microsatellites in C. pomonella complement.

Orientador: Patrícia Parise Pasquali-Maltempi / Resumo: Os anfíbios anuros correspondem ao segundo grupo de vertebrados com maior número de espécies, perdendo apenas para os peixes. Só no Brasil são mais de 1000 espécies descritas, com novas descrições e revalidações acontecendo todo ano. Esse grupo está envolvido em diversas problemáticas taxonômicas, em parte por ser altamente polimórfico e ao mesmo tempo conter muitas espécies crípticas, em parte por ainda haver muitas lacunas no conhecimento acerca desse grupo, dado o tamanho da sua diversidade. Resolver problemáticas envolvendo esse grupo de vertebrados é fundamental para melhor estimar o tamanho real dessa diversidade, entender os caminhos evolutivos e as relações filogenéticas no grupo e poder auxiliar na conservação das espécies, traçando planos de manejo adequados, já que o declínio de anfíbios devido à perda e fragmentação de habitat, contaminação de ambientes naturais e disseminação de doenças tem aumentado vertiginosamente. Para isso, a taxonomia moderna conta com a soma de diferentes informações a respeito dos grupos, além dos dados morfológicos já tradicionalmente utilizados. Uma das áreas que tem se mostrado promissora como uma ferramenta de auxílio na taxonomia é a citogenética, especialmente depois do advento da citogenômica, além da sua importância para o entendimento da evolução cromossômica dos grupos. No entanto, trabalhos citogenéticos, especialmente aqueles aliados à biologia molecular, são escassos em anuros. Seguindo essa linha, esse trabalho buscou obter ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Anuran amphibians corresponds to one of the most diverse vertebrate groups, second only to fishes. In Brazil alone there are more than 1000 described species, with new descriptions and revalidations happening every year. This group is involved in several taxonomic issues, partially because it is highly polymorphic and at the same time contains many cryptic species, partially because there are still many gaps in knowledge about this group, given the size of its diversity. To solve these problems is a fundamental step to better estimate its true size, to understand the evolutionary pathways and phylogenetic relationships in the group and also to be able to contribute to the conservation of the species by helping to draw appropriate management plans, as the decline of amphibians due to habitat loss and fragmentation, contamination of natural environments and the spread of disease has increased dramatically. For this, modern taxonomy relies on the sum of different data about the groups, besides the morphological data traditionally used. One of the areas that has shown promise as a taxonomy tool is cytogenetics, especially after the advent of cytogenomics, in addition to its importance for understanding the chromosomal evolution of groups. However, cytogenetic studies, especially those allied to molecular biology, are scarce in anurans. Following this line, this work sought, through the cytogenetic study, using conventional molecular markers, and cytogenomic, with the help of bioi... (Complete abstract click electronic access below) / Doutor

Retrons are genetic elements encoding reverse transcriptase (RT) usually located on the chromosome of a wide variety of mostly Gram-negative bacteria. Here we describe a new retron, designated Ne144, found in the chromosome of the myxobacterium Nannocystis exedens. This element codes for a 515-amino-acid RT that is most closely related to those found in other myxobacterial retrons. The RT is responsible for the production of a small satellite DNA called msDNA. This msDNA is composed of a 144 base, single-stranded DNA that is linked to a 72 base single-stranded RNA. The RNA strand is joined to the 5′ end of the DNA chain via a 2′-5′ linkage that occurs from the 2′ position of an internal guanosine residue in the RNA. In addition to the retron element, the chromosome of N. exedens also contains several partial copies of the msDNA sequence as revealed by DNA hybridization experiments using msDNA as a probe. One of these partial copies was characterized from a chromosome restriction fragment and found to contain a sequence that matches the last 82 bases of the DNA strand and five bases of the RNA strand in msDNA-Ne144. This partial copy of msDNA is very likely a retrotransposed sequence that was generated by reverse transcription using an RNA (the primer-template RNA for msDNA) as a template and the 3′ end of a nick in the chromosome as a primer, followed by incorporation into an open reading frame. The presence of this truncated copy of msDNA is strong evidence of retrotransposition in N. exedens causing an alteration in the bacterial genome.

Die Gattung Beta gehört zur Familie der Chenopodiaceae und wird in die vier Sektionen Beta, Corollinae, Nanae und Procumbentes unterteilt, wobei die Zuckerrübe der Sektion Beta zugeordnet wird. Aus dem Genom der Zuckerrübe und verwandter Wildarten konnten bereits eine Vielzahl von repetitiven DNA-Familien kloniert und untersucht werden. Mit der monosomen Fragmentadditionslinie PRO1 stand eine Chromosomenmutante zur Verfügung, die neben den 18 B. vulgaris-Chromosomen ein Chromosomenfragment der Wildrübe Beta procumbens enthält. Da dieses als Minichromosom bezeichnete Fragment mitotische Stabilität aufweist, muss es ein funktionelles Centromer besitzen, das auch im genetischen Hintergrund von Beta vulgaris aktiv ist. Mit der Erstellung einer BAC (bacterial artifical chromosome)-Bank von PRO1 wurde die molekulare Charakterisierung von Ty3-gypsy-Retrotransposons eines einzelnen Wildrüben-Centromers möglich. Die für die Wildrübe Beta procumbens spezifischen Satellitenrepeats pTS5 und pTS4.1 dienten der Selektion von BACs aus der Centromer-Region des PRO1-Minichromosoms. Die Identifizierung eines unikalen genomischen Locus, mit einer Verschachtelung von zwei nicht homologen LTR-Retrotransposons, ermöglichte die gerichtete Isolation der LTR-Retrotransposons Beetle1 und Beetle2. Das Retrotransposon Beetle1 hat eine Gesamtlänge von 6736 bp und wird von LTR-Sequenzen begrenzt, die eine Länge von 1091 bp (5’-LTR) bzw. 1089 bp (3’-LTR) aufweisen. Das LTR-Retrotransposon Beetle2 weist mit 6690 bp eine ähnliche Gesamtlänge wie Beetle1 auf. Es wird von deutlich kürzeren LTR-Sequenzen mit einer Länge von 774 bp begrenzt. Aufgrund der Reihenfolge der Polyproteingene lassen sich Beetle1 und Beetle2 in die Gruppe der Ty3-gypsy-Retrotransposons (Metaviridae) einordnen. Beide Retrotransposon-Familien besitzen ein einziges offenes Leseraster (open reading frame; ORF) mit fusionierten gag- und pol-Genen. Datenbankrecherchen zeigten hohe Homologien von Beetle1 und Beetle2 mit den centromerischen Ty3-gypsy-Retrotransposons CRM aus Zea mays, CRR aus Oryza sativa und cereba aus Hordeum vulgare. Diese centromerischen Retrotransposons (CRs) sind in den Poaceae stark konserviert und stellen neben Satellitenrepeats eine hochabundante Sequenzklasse der Centromere der Süßgräser dar. Da sie im 3’-Bereich des gag-pol-Polyproteins eine Chromodomäne aufweisen, werden sie der eigenständigen Gruppe der Chromoviren zugeordnet. Chromodomänen sind zur Bindung von Proteinen und DNA befähigt und spielen eine wichtige Rolle in der Chromatin-Modifikation und der Bildung von Heterochromatin-Regionen. Beetle1 und Beetle2 besitzen Motive einer Chromodomäne, die vermutlich für eine gerichtete Transposition in die Centromer-Region verantwortlich ist. Neben der geringen Divergenz von Beetle1- und Beetle2-Sequenzen sowohl im Genom von Beta procumbens als auch in den anderen Arten der Sektion Procumbentes spricht auch das junge Alter von 100 000 bis 350 000 Jahren und die Transkriptionsaktivität für eine Einordnung dieser Ty3-gypsy-Retrotransposons in die Gruppe der Chromoviren. Sowohl die Southern-Hybridisierung als auch die Fluoreszenz-in situ-Hybridisierung zeigten, dass Beetle1 und Beetle2 nur für die Sektion Procumbentes spezifisch sind und dort in hoher Kopienzahl vorkommen. Untersuchungen mit methylierungssensitiven Restriktionsendonukleasen veranschaulichten den hohen Grad an Cytosin-Methylierung von Beetle1 und Beetle2.