Master of Science / Genetics Interdepartmental Program / Sanzhen Liu / The major component of complex genomes is repetitive elements, which remain recalcitrant to characterization. Using maize as a model system, we analyzed whole genome shotgun (WGS) sequences for the two maize inbred lines B73 and Mo17 using k-mer analysis to quantify the differences between the two genomes. Significant differences were identified in highly repetitive sequences, including centromere, 45S ribosomal DNA (rDNA), knob, and telomere repeats. Genotype specific 45S rDNA sequences were discovered. The B73 and Mo17 polymorphic k-mers were used to examine allele-specific expression of 45S rDNA in the hybrids. Although Mo17 contains higher copy number than B73, equivalent levels of overall 45S rDNA expression indicates that transcriptional or post-transcriptional regulation mechanisms operate for the 45S rDNA in the hybrids. Using WGS sequences of B73xMo17 doubled haploids, genomic locations showing differential repetitive contents were genetically mapped, revealing differences in organization of highly repetitive sequences between the two genomes. In an analysis of WGS sequences of HapMap2 lines, including maize wild progenitor, landraces, and improved lines, decreases and increases in abundance of additional sets of k-mers associated with centromere, 45S rDNA, knob, and retrotransposons were found among groups, revealing global evolutionary trends of genomic repeats during maize domestication and improvement.

Os peixes apresentam uma grande diversidade quanto a sua morfologia, seus habitats e também sua biologia. São encontrados em lagos, córregos, estuários e oceanos, constituindo assim mais de 50% do número total das espécies de vertebrados conhecidas atualmente. Essa fauna tem sido objeto de um número expressivo de estudos citogenéticos e moleculares, tendo-se já conhecimento não só das relações cromossômicas, mas também da sistemática de vários grupos. Essas pesquisas têm investigado não somente o número e fórmula cromossômica, mas também a presença de cromossomos sexuais diferenciados, presença de cromossomos supranumerários, padrões de distribuição da heterocromatina, localização das regiões organizadoras de nucléolo, padrões de bandamento de restrição e replicação, permitindo a localização de diferentes classes de DNAs repetitivos, bem como a identificação de homeologias cromossômicas que auxiliam a compreensão da evolução cariotípica dos grupos. Os estudos moleculares, por sua vez, têm se tornado cada vez mais importantes nesse grupo e têm fornecido dados fundamentais não só no que diz respeito à filogenia dos grupos, como também em relação a regiões repetitivas do DNA e sua importância no genoma. A união dessa área com a Citogenética tem permitido uma maior e melhor compreensão sobre os processos evolutivos associados às alterações de seqüências específicas do genoma visíveis tanto a níveis cromossômicos, quanto moleculares. O gênero Gymnotus (Teleostei: Gymnotiformes) inclui representantes com características biológicas peculiares, o que os torna objeto de estudo de diversas áreas da Biologia. Estudos sobre o gênero incluem sua caracterização cariotípica, estudo das regiões organizadoras de nucléolo (RONs) polimórficas, bem como estudos envolvendo marcadores moleculares, os quais conjuntamente com a Citogenética permitiram a análise de filogenética molecular, com inferência na evolução cromossômica, permitindo uma melhor compreensão das relações dentro do gênero. No presente trabalho foram levados a efeito estudos sobre as regiões heterocromáticas e os DNAs repetitivos desse grupo, para uma melhor compreensão da organização e localização dessas seqüências no genoma e a identificação de possíveis marcadores moleculares. Foram efetuados ainda, estudos envolvendo a evolução cariotípica das espécies G. carapo e G. sylvius, localização de genes ribossômicos e análise molecular do gene ribossômico 5S juntamente com seu espaçador não transcrito, propiciando uma melhor compreensão da evolução dessa família gênica em Gymnotus. / Fishes present a great diversity in relation to their morphology, habitat and biology. They are found in lakes, rivers, estuaries and oceans, comprising more than 50% of the total number of known vertebrates. Cytogenetic and molecular aspects of the fish fauna have been extensively studied, providing information about their chromosomal relationships and also about the systematic status of several groups. These researches have focused on the description of both chromosomal number and formula as well as the presence of differentiated sex chromosomes, occurrence of B-chromosomes, patterns of heterochromatin distribution, localization of nucleolar organizer regions, restriction or replication banding profiles allowing to locate distinct classes of repetitive DNAs and to identify chromosomal homeologies in order to understand the karyotypic evolution in distinct groups. On the other hand, molecular studies have become of utmost importance in this group, providing essential data about phylogeny of many groups and about repetitive DNA regions and their role in the genome. The union between this approach and cytogenetics has favored a better comprehension about the evolutionary processes associated with visible alterations in specific sequences within the genome at both chromosomal and molecular levels. The genus Gymnotus is composed of representatives with peculiar biological features, which turn them suitable for studies in a variety of biology approaches. Genetic studies in this genus comprise karyotype characterization, analysis of polymorphic NORs, besides studies of molecular markers that, coupled with cytogenetics, have fostered molecular phylogenetic analyzes with inferences on their chromosomal evolution, which have led to a better understanding about the interrelationships in the group. In the present work, we carried out studies about the heterochromatic regions and the repetitive DNAs in this group for a better comprehension about the organization and localization of these sequences in the genome and identification of potential molecular markers. Furthermore, studies related to the karyotype evolution in the species G. carapo and G. sylvius, location of ribosomal genes and molecular analysis of both 5S ribosomal gene and its non-transcribed spacer were performed to provide a better comprehension about the evolution of this gene family in Gymnotus.

Microsatellites are simple repetitive DNA sequences that are used as genetic markers throughout the biological sciences. The high levels of variation observed at microsatellite loci contribute to their utility in studies at the population and individual levels. This variation is a consequence of mutations that change the length of microsatellite repeat tracts. Current understanding suggests that most mutations are caused by polymerase slippage during DNA replication and lead to changes of a single repeat unit in length, but some changes involving multiple repeats can also occur. Despite this simplistic overview, there is evidence for considerable heterogeneity in mutation processes between species, loci and alleles. Such complex patterns suggest that other mechanisms, including those associated with DNA recombination, are also involved in the generation of microsatellite mutations. Understanding which mutational mechanisms are responsible for variation at microsatellite markers is essential to enable accurate data interpretation in genotyping projects, as many commonly used statistics assume specific mutation models. I developed microsatellite markers specific to the X and Y chromosomes and an autosome in the tammar wallaby, Macropus eugenii, and investigated their evolutionary properties using two approaches: indirectly, as inferred from population data, and directly, from observation of mutation events. First, I found that allelic richness increased with repeat length and that two popular mutation models, the stepwise mutation model and the infinite allele model, were poor at predicting the number of alleles per locus, particularly when gene diversity was high. These results suggest that neither model can account for all mutations at tammar wallaby microsatellites and hint at the involvement of more complex mechanisms than replication slippage. I also determined levels of variation at each locus in two tammar wallaby populations. I found that allelic richness was highest for chromosome 2, intermediate for the X chromosome and lowest for the Y chromosome in both populations. Thus, allelic richness varied between chromosomes in the manner predicted by their relative exposure to recombination, although these results may also be explained by the relative effective population sizes of the chromosomes studied. Second, I used small-pool PCR from sperm DNA to observe de novo mutation events at three of the most polymorphic autosomal markers. To determine the reliability of my observations I developed and applied strict criteria for scoring alleles and mutations at microsatellite loci. I observed mutations at all three markers, with rate variation between loci. Single step mutations could not be distinguished because of the limitations of the approach, but 24 multi-step mutations, involving changes of up to 35 repeat units, were recorded. Many of these mutations involved changes that could not be explained by the gain or loss of whole repeat units. These results imply that a large number of mutations at tammar wallaby microsatellites are caused by mechanisms other than replication slippage and are consistent with a role for recombination in the mutation process. Taken as a whole, my results provide evidence for complex mutation processes at tammar wallaby microsatellites. I conclude that careful characterisation of microsatellite mutation properties should be conducted on a case-by-case basis to determine the most appropriate mutation models and analysis tools for each locus. In addition, my work has provided a set of chromosome-specific markers for use in macropod genetic studies, which includes the first marsupial Y chromosome microsatellites. Sex chromosome microsatellites open a new range of possibilities for population studies, as they provide opportunities to investigate gene flow in a male context, to complement data from autosomal and maternally-inherited mitochondrial markers.

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Agradecemos a compreensão. on 2017-10-26T16:14:49Z (GMT) / Submitted by MARIANI CRISTINA ALVES PICCOLI null (marianicpiccoli@gmail.com) on 2017-10-26T18:42:01Z No. of bitstreams: 1 versaofinalfinal.pdf: 2319438 bytes, checksum: 6f431d0bb6380c6dd4298885b7f2a00b (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-10-26T18:53:20Z (GMT) No. of bitstreams: 1 piccoli_mca_me_rcla.pdf: 2319438 bytes, checksum: 6f431d0bb6380c6dd4298885b7f2a00b (MD5) / Made available in DSpace on 2017-10-26T18:53:20Z (GMT). No. of bitstreams: 1 piccoli_mca_me_rcla.pdf: 2319438 bytes, checksum: 6f431d0bb6380c6dd4298885b7f2a00b (MD5) Previous issue date: 2017-08-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os DNAs repetitivos representam uma porção significante do genoma e podem estar envolvidos com os processos de variação cromossômica e reorganização genômica. Estes DNAs podem ser organizados em tandem ou dispersos, apresentando ampla variabilidade de composição e localização, sendo em muitos casos os principais constituintes da heterocromatina. A utilização dessas sequências como marcadores para a análise da evolução cromossômica é ampla em várias ordens de insetos, enquanto que para as abelhas esse conhecimento ainda é bastante restrito. Na tribo Meliponini o gênero Melipona se encontra dividido em quatro subgrupos e é o mais caracterizado cromossomicamente, sendo as espécies do gênero divididas ainda em dois grupos que apresentam marcante diferença na distribuição da heterocromatina. Um grupo é representado por espécies com pouca heterocromatina (principalmente centromérica) enquanto no segundo grupo as espécies apresentam heterocromatina dispersa ao longo de todos os cromossomos. Sendo assim, este gênero apresenta-se como um potencial modelo para estudos de amplificação e diversificação de DNAs repetitivos e sequências heterocromáticas. Neste contexto, utilizamos a espécie Melipona scutellaris que possui grande quantidade de heterocromatina nos cromossomos como modelo, buscando entender possíveis causas e eventos evolutivos envolvidos na ampla diferenciação heterocromática. Foram mapeadas distintas sequências de DNAs repetitivos por FISH, tais como DNAr 18S, DNAsn U2 e microssatélites, todos com localização eucromática, sugerindo que as sequências testadas não estão envolvidas com a amplificação da heterocromatina. A fração C0t-DNA e DOP-PCR evidenciou que a heterocromatina é composta por sequências altamente repetitivas, enquanto que a hibridização em membrana dessa fração em outras Meliponas demonstrou que a heterocromatina dentro do gênero difere de acordo com cada subgrupo. / Repetitive DNAs represent a significant portion of the genome and may be involved in the processes of chromosome variation and genomic rearrangement. These DNAs can be organized in tandem or dispersed, presenting wide variety of composition and location, being in many cases the main constituents of heterochromatin. An application as sequences as markers for an analysis of chromosomal evolution is broad in several orders of insects, whereas for bees this knowledge is still quite restricted. In the Meliponini tribe, the genus Melipona is divided into four subgroups and is the most characteristic chromosomally, being as species of the genus divided into two groups that present a marked differential in the distribution of heterochromatin. One group is represented by species with little heterochromatin (mainly centromeric) while not second group as species presented in heterochromatin dispersed throughout all the chromosomes. Thus, this genus presents itself as a potential model for amplification and diversification studies of repetitive DNAs and heterochromatic sequences. In this context, it uses a species Melipona scutellaris that has large amount of heterochromatin in the chromosomes as model, seeking to understand possible evolutionary events involved in the wide heterochromatic differentiation. Different FISH repeating DNA sequences have been mapped, such as 18S rDNA, U2 DNAs and microsatellites, all with euchromatic location, suggesting that as tested sequences are not involved with heterochromatin amplification. The C0t-DNA and DOP-PCR fraction showed that heterochromatin is composed of highly repetitive sequences, whereas a membrane hybridization of this fraction in other Meliponas demonstrated that heterochromatin within the genus differs according to each subgroup.

Orientadores: Shirlei Maria Recco-Pimentel, Luciana Bolsoni Lourenço Morandini / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T17:27:31Z (GMT). No. of bitstreams: 1 Vittorazzi_StenioEder_M.pdf: 1698813 bytes, checksum: 820a78de37442254ed35e58e413de026 (MD5) Previous issue date: 2010 / Resumo: O gênero Physalaemus é atualmente constituído de 42 espécies, distribuídas em sete grupos. No grupo de Physalaemus cuvieri, além dessa, estão alocadas outras oito espécies. Estudos citogenéticos disponíveis até momento para algumas espécies do gênero nos mostram um número diplóide de 2n = 22, sendo a morfologia cromossômica similar entre elas. O objetivo desse trabalho foi desenvolver novos marcadores citogenéticos para o estudo do grupo de Physalaemus cuvieri. Foram estudadas três populações de P. cuvieri e duas outras espécies do grupo, P. albonotatus e P. centralis. Foram isoladas três sequências de DNA repetitivo, as quais foram denominadas PcP190EcoRI, PcP883EcoRI e PcP174PstI. A primeira sequência apresentou similaridade com os DNAr 5S disponíveis no GenBank e assim, adicionalmente, foi feito experimento para isolar o DNAr 5S de P. cuvieri. Foram obtidos dois fragmentos com cerca 201 e 690pb, localizados por double- FISH em cromossomos distintos. A sequência PcP190EcoRI mostrou similaridade de aproximadamente 70% com a região de transcrição de ambos os tipos de DNAr 5S, sugerindo a origem dessa sequência a partir do DNAr 5S. A hibridação in situ com sondas das três sequências de DNA repetitivo isoladas, nas três populações de P. cuvieri, em P. albonotatus e em P. centralis, mostrou que todas as regiões cromossômicas marcadas são coincidentes com regiões de banda C detectadas em estudo prévio. No entanto, as populações de P. cuvieri e as espécies estudadas diferem entre si pelo número e localização de marcações com sondas das três sequências. Essas sequências representam bons marcadores cromossômicos, já que permitiram demonstrar tanto variações interpopulacionais em P. cuvieri como também diferenças interespecíficas, corroborando a sugestão prévia de que as diferentes populações de P. cuvieri podem se tratar de um complexo de espécies crípticas. / Abstract: The genus Physalaemus is currently composed of 42 species, divided into seven groups. Within the Physalaemus cuvieri group are eight other species. Cytogenetic studies for some of the species within the genus show a diploid number of 2n = 22 and similar chromosome morphology. The aim of this research was to develop new cytogenetic markers to study the group of Physalaemus cuvieri. We studied three populations of P. cuvieri in addtion to two other species: P. albonotatus and P. centralis. We isolated three repetitive DNA sequences, which were named PcP190EcoRI, PcP883EcoRI and PcP174PstI. The former sequence showed similarities with the 5S rDNA available in GenBank and, therefore, an experiment was done to isolate the 5S rDNA of P. cuvieri. We obtained two fragments of about 201 bp and 690 bp, which were localized by double-FISH to distinct chromosomes. The PcP190EcoRI sequence showed approximately 70% similarity with the transcription regions of both types of 5S rDNA, suggesting the PcP190EcoRI sequence originated from the 5S rDNA. The three repetitive DNA sequences were detected by in situ hybridization to chromosomes from P. albonotatus, P. centralis and the three populations of P. cuvieri. These chromosome regions are coincident with C-bands detected in a previous study. Populations of P. cuvieri and others two species, however, differ by the number and location of the three sequences. These sequences seem to be good chromosomes markers since they were used to show interpopulational variation in P. cuvieri, as well interspecific differences. This supports the previous suggestion that different populations of P. cuvieri may be a complex of cryptic species. / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

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No. of bitstreams: 1 Dissertação - Sabrina Emanuela de Melo Araújo.pdf: 1064742 bytes, checksum: bea4234c7d69e414fb2af0d8af97b7cf (MD5) Previous issue date: 2016-02-17 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / Among the Chiropteran, the Phyllostomidae family is the most diverse clade of the Neotropics and in Amazon are found about 80 species. From a chromosomal point of view, Phyllostomidae stands out for presenting many karyotype variation, with diploid numbers ranging from 2n = 14 in Vampyressa melissa (Stenodermatinae) to 2n = 46 in Macrotus waterhousii (Macrotinae). There are several genetic mechanisms or processes that can result in numeric/structural chromosomal abnormalities and often repetitive DNA sequences are involved in this process. In order to understand the variety and karyotype evolution of this family, classical and molecular cytogenetic analyzes were performed on mitotic chromosomes of four species belonging to four subfamilies of distinct phylogenetic clades: Artibeus obscurus, Carollia perspicillata, Desmodus rotundus and Phylostomus elongatus. Artibeus obscurus presented NF = 56 and 2 N = 30/31, with 22m + 6st + XX / XY1Y2; C. perspicillata NF = 36 and 2N = 20/21, with 14m-sm + 4st + XX / XY1Y2; D. rotundus NF = 52 and 2 N = 28, and 26m-sm + XX / XY; P. elongatus NF = 60 and 2 N = 32, and 28m-sm + 2a + XX / XY. The distribution pattern of constitutive heterochromatin revealed a signal in the pericentromeric region in all chromosomes of the four analyzed species and intraspecific variations were observed when compared the results of this work with the one in existing literature. Regarding the signal of ribosomal DNA sites 18S and nucleolus organizer regions active in A. obscurus were shown in the terminal region of the pairs 5, 6 and 7; C. perspicillata in pericentromeric region of the X chromosome; D. rotundus in the centromeric region of pair 8 and in P. elongatus in the centromeric/terminal region of the pair 15. Conspicuous telomeric signals were observed in D. rotundus and P. elongatus, while in A. obscurus and C. perspicillata the terminals signals are blurred. Interstitial telomeric sites were absent in P. elongatus and present in other species, which may indicate mergers. The LINE-1 retroelement presented scattered signals, however in some chromosomes they are identical to the patterns of dark bands evident in the band G and is also accumulated on chromosome X. If compared the phylogenetic position of the studied species, it is noted that the most derived taxons accumulate high karyotype variation as repetitive elements. / Dentre os Chiropteros, a família Phyllostomidae constitui o clado mais diversificado do neotrópico e na Amazônia são encontradas cerca de 80 espécies. Do ponto de vista cromossômico, Phyllostomidae destaca-se por apresentar grande variação cariotípica, com números diplóides que vão de 2n=14 em Vampyressa melissa (Stenodermatinae) a 2n=46 em Macrotus waterhousii (Macrotinae). São vários os processos ou mecanismos genéticos que podem resultar em alterações cromossômicas numéricas/estruturais e muitas vezes sequências repetitivas de DNA estão envolvidas neste processo. Visando compreender a variedade e a evolução cariotípica desta família, foram realizadas análises citogenéticas clássicas e moleculares em cromossomos mitóticos de quatro espécies, pertencentes a quatro subfamílias de clados filogenéticos distintos: Artibeus obscurus, Carollia perspicillata, Desmodus rotundus e Phylostomus elongatus. Artibeus obscurus apresentou NF=56 e 2N=30/31, sendo 22m+6st+XX/XY1Y2; C. perspicillata NF=36 e 2N=20/21, sendo 14m-sm+4st+XX/XY1Y2; D. rotundus NF=52 e 2N=28, sendo 26m-sm+XX/XY; P. elongatus NF=60 e 2N=32, sendo 28m-sm+2a+XX/XY. O padrão de distribuição da heterocromatina constitutiva revelou marcação na região pericentromérica em todos os cromossomos das quatro espécies analisadas e variações intraespecíficas foram observadas quando comparados os resultados deste trabalho com o existente na literatura. Em relação à marcação de sítios de DNA ribossomal 18S e regiões organizadoras de nucléolo ativas, em A. obscurus foram evidenciados na região terminal dos pares 5, 6 e 7; em C. perspicillata na região pericentromérica do cromossomo X; em D. rotundus na região centromérica do par 8 e em P. elongatus na região centromérica/terminal do par 15. Marcações teloméricas conspícuas foram visualizadas em D. rotundus e P. elongatus, enquanto que em A. obscurus e C. perspicillata as marcações terminais são tênues. Sítios teloméricos intersticiais foram ausentes em P. elongatus e presente nas demais espécies, podendo ser indicativo de fusões. O retroelemento LINE-1 revelou marcação dispersas, porém em alguns cromossomos são coincidentes com os padrões de bandas escuras evidenciados na banda G, sendo também acumulados no cromossomo X. Se comparada a posição filogenética as espécies estudadas, nota-se que os táxons mais derivados, acumulam maior variação cariotípica, assim como os elementos repetitivos.

Os peixes apresentam uma grande diversidade quanto a sua morfologia, seus habitats e também sua biologia. São encontrados em lagos, córregos, estuários e oceanos, constituindo assim mais de 50% do número total das espécies de vertebrados conhecidas atualmente. Essa fauna tem sido objeto de um número expressivo de estudos citogenéticos e moleculares, tendo-se já conhecimento não só das relações cromossômicas, mas também da sistemática de vários grupos. Essas pesquisas têm investigado não somente o número e fórmula cromossômica, mas também a presença de cromossomos sexuais diferenciados, presença de cromossomos supranumerários, padrões de distribuição da heterocromatina, localização das regiões organizadoras de nucléolo, padrões de bandamento de restrição e replicação, permitindo a localização de diferentes classes de DNAs repetitivos, bem como a identificação de homeologias cromossômicas que auxiliam a compreensão da evolução cariotípica dos grupos. Os estudos moleculares, por sua vez, têm se tornado cada vez mais importantes nesse grupo e têm fornecido dados fundamentais não só no que diz respeito à filogenia dos grupos, como também em relação a regiões repetitivas do DNA e sua importância no genoma. A união dessa área com a Citogenética tem permitido uma maior e melhor compreensão sobre os processos evolutivos associados às alterações de seqüências específicas do genoma visíveis tanto a níveis cromossômicos, quanto moleculares. O gênero Gymnotus (Teleostei: Gymnotiformes) inclui representantes com características biológicas peculiares, o que os torna objeto de estudo de diversas áreas da Biologia. Estudos sobre o gênero incluem sua caracterização cariotípica, estudo das regiões organizadoras de nucléolo (RONs) polimórficas, bem como estudos envolvendo marcadores moleculares, os quais conjuntamente com a Citogenética permitiram a análise de filogenética molecular, com inferência na evolução cromossômica, permitindo uma melhor compreensão das relações dentro do gênero. No presente trabalho foram levados a efeito estudos sobre as regiões heterocromáticas e os DNAs repetitivos desse grupo, para uma melhor compreensão da organização e localização dessas seqüências no genoma e a identificação de possíveis marcadores moleculares. Foram efetuados ainda, estudos envolvendo a evolução cariotípica das espécies G. carapo e G. sylvius, localização de genes ribossômicos e análise molecular do gene ribossômico 5S juntamente com seu espaçador não transcrito, propiciando uma melhor compreensão da evolução dessa família gênica em Gymnotus. / Fishes present a great diversity in relation to their morphology, habitat and biology. They are found in lakes, rivers, estuaries and oceans, comprising more than 50% of the total number of known vertebrates. Cytogenetic and molecular aspects of the fish fauna have been extensively studied, providing information about their chromosomal relationships and also about the systematic status of several groups. These researches have focused on the description of both chromosomal number and formula as well as the presence of differentiated sex chromosomes, occurrence of B-chromosomes, patterns of heterochromatin distribution, localization of nucleolar organizer regions, restriction or replication banding profiles allowing to locate distinct classes of repetitive DNAs and to identify chromosomal homeologies in order to understand the karyotypic evolution in distinct groups. On the other hand, molecular studies have become of utmost importance in this group, providing essential data about phylogeny of many groups and about repetitive DNA regions and their role in the genome. The union between this approach and cytogenetics has favored a better comprehension about the evolutionary processes associated with visible alterations in specific sequences within the genome at both chromosomal and molecular levels. The genus Gymnotus is composed of representatives with peculiar biological features, which turn them suitable for studies in a variety of biology approaches. Genetic studies in this genus comprise karyotype characterization, analysis of polymorphic NORs, besides studies of molecular markers that, coupled with cytogenetics, have fostered molecular phylogenetic analyzes with inferences on their chromosomal evolution, which have led to a better understanding about the interrelationships in the group. In the present work, we carried out studies about the heterochromatic regions and the repetitive DNAs in this group for a better comprehension about the organization and localization of these sequences in the genome and identification of potential molecular markers. Furthermore, studies related to the karyotype evolution in the species G. carapo and G. sylvius, location of ribosomal genes and molecular analysis of both 5S ribosomal gene and its non-transcribed spacer were performed to provide a better comprehension about the evolution of this gene family in Gymnotus.

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Malaria is a life-threatening disease caused by Plasmodium parasites that are transmitted through the bites of infected females of a few Anopheles mosquito species. Understanding the chromosome evolution and mechanisms of speciation can shed light on developing novel ecological-friendly vector control techniques. Sibling species of the An. gambiae complex provide an excellent model system for these topics. To understand the mechanisms of speciation, we investigated the cellular basis and phenotypes of hybrid male sterility in species crosses of the An. gambiae complex. By performing inter-species crosses of An. coluzzii/An. gambiae and An. merus lab strains, we found an asymmetric pattern of hybrid male sterility existed in sons from reciprocal interspecies crosses. Compared with pure species, hybrid males from crosses of ♀An. merus  ♂An. gambiae/An. coluzzii were normal in the morphology of male reproductive tracts; however, the testes of which that process the reductional meiotic division failed to produce primary spermatocytes and were accompanied with unpaired and insufficiently condensed chromosomes. As a result, primary spermatocytes undergo a mitosis-like anaphase division, producing nonmotile and malfunctional diploid sperm with two tails. However, individuals can mate with females normally and form the mating plug to induce the female monogamy. In contrast, hybrid males from the opposite crosses manifest severely underdeveloped reproductive tracts and a premeiotic arrest of germline stem cells in the testis, accompanied by a strong suppression of premeiotic and meiotic genes. In addition, hybrid males from this cross suffered from a shorter copulation time and failed to form mating plugs to induce female monogamous behaviors, albeit the expression of male accessory gland specific genes were similar between hybrids and pure species. To figure out chromosome evolution in the An. gambiae complex, we studied the molecular organization of heterochromatin and investigated the spatial organizations of autosomal regions of polytene chromosomes in soma and germline cells. We found that molecular composition of pericentrometric autosome and sex chromosome repetitive DNA differs among sibling species of An. gambiae complex with highly similarity between An. coluzzii and An. arabiensis. In addition, heterochromatin blocks of chromosomes have distinct compositions of satellite DNA sequences. Next, in order to address the relationship between inter-chromosomal (Chr-Chr) contacts and chromosome-nuclear envelope (Chr-NE) attachments during the development of the organism, we conducted microscopic analyses of the 3D organization of polytene chromosome in An. gambiae, An. coluzzii, and An. merus. Our quantitative study on chromosome territories in larval salivary gland cells and adult ovarian nurse cells showed that, compared with autosomal arms, the X chromosome has a significantly smaller volume and occupies more compact territories. The number of Chr-Chr contacts and the percentage of Chr-NE attachment were conserved among the species within the same cell type. Our data also demonstrated that there is a significantly and consistently inverse relationship between the frequencies of Chr–NE and Chr–Chr attachments on autosomes of two cell types in all tested species. / Doctor of Philosophy / Malaria is a life-threatening disease caused by Plasmodium parasites that are transmitted through the bites of infected females of a few Anopheles mosquito species. Despite being treatable and preventable, malaria is estimated to cause large numbers of deaths every year. Since 2015, the malaria elimination program has stalled largely due to increased insecticide resistance. Novel transgenic techniques have a huge potential in reducing malaria transmission more effectively. However, there are large concerns about the potential negative effects of releasing genetically modified mosquitoes, such as a possibility of accidental spread to non-target species with incomplete reproductive barriers and unpredicted ecological damage. Understanding the mechanisms of speciation about how reproductive isolation occurred and developed as well as chromosome evolution can not only empower the development of ecologically friendly vector control techniques but also improve our basic knowledge. To study mechanisms of speciation, we mated males and females from different closely related species in the Anopheles gambiae complex to investigate the fecundity of hybrid generations. Our study identified two different types of reproductive abnormalities leading to hybrid male sterility. Hybrid males from female An. merus and male An. gambiae or An. coluzzii have normal appearing testes and male accessary glands but the testes produce abnormal sperms, which cannot move and have two tails. Hybrid males from female An. gambiae or An. coluzzii and An. merus have severely underdeveloped testes and male accessary glands. The sperm producing process stops unusually very early in their tiny underdeveloped testes. We also investigated chromosome evolution in species of An. gambiae complex. We found that chromosomal parts containing repetitive DNA, the sequence in the genome not producing proteins, evolve rapidly in An. coluzzii, An. arabiensis, An. quadriannulatus, and An. merus. In contrast, chromosome territories of gene rich regions in giant polytene chromosomes from larval salivary gland cells and adult ovarian nurse cells of An. gambiae, An. coluzzii, and An. merus, were relatively conserved within the same cell type among different species. However, the chromosomal 3D distribution pattern is different among various cell types in these species.

Short interspersed nuclear elements (SINEs) are small non-autonomous and heterogeneous retrotransposons, widespread in animals and plants and usually differentially propagated in related species resulting in genome-specific copy numbers. Within the monocots, the Poaceae (sweet grasses) is the largest and economically most important plant family. The distribution of 24 Poaceae SINE (PoaS) families, five of which showing a subfamily structure, was analyzed in five important cereals (Oryza sativa, Triticum aestivum, Hordeum vulgare, Sorghum bicolor, Zea mays), the energy crop Panicum virgatum and the model grass Brachypodium distachyon. The comparative investigation of SINE abundance and sequence diversity within Poaceae species provides insights into their species‐specific diversification and amplification. The PoaS families and subfamilies fall into two length and structural categories: simple SINEs of up to 180 bp and dimeric SINEs larger than 240 bp. Of 24 PoaS families, 20 are structurally related across species, in particular either in their 5′ or 3′ regions. Hence, reshuffling between SINEs, likely caused by nested insertions of full-lengh and truncated copies, is an important evolutionary mechanism of SINE formation. Most striking, the recently evolved homodimeric SINE family PoaS‐XIV occurs exclusively in wheat (T. aestivum) and consists of two tandemly arranged PoaS‐X.1 copies. Exemplary for deciduous tree species, the evolutionary history of SINE populations was examined in six Salicaceae genomes (Populus deltoides, Populus euphratica, Populus tremula, Populus tremuloides, Populus trichocarpa, Salix purpurea). Four of eleven Salicaceae SINE (SaliS) families exhibit a subfamily organization. The SaliS families consist of two groups, differing in their phylogenetic distribution pattern, sequence similarity and 3’ end structure. These groups probably emerged at different evolutionary periods of time: during the ‘salicoid duplication’ (~ 65 million years ago) in the Salix-Populus progenitor, and during the separation of the genus Salix (~ 45 - 65 million years ago), respectively. Similar to the PoaS families, the majority of the 20 SaliS families and subfamilies share regions of sequence similarity, providing evidence for SINE emergence by reshuffling. Furthermore, they also contain an evolutionarily young dimeric SINE family (SaliS-V), amplified only in two poplar genomes. The special feature of the Salicaceae SINEs is the contrast of the conservation of 5’ start motifs across species and SINE families compared to the high variability of 3’ ends within the SINE families, differing in sequence and length, presumably resulting from mutations in the poly(A) tail as a possible route for SINE elongation. Periods of increased transpositional activity promote the dissemination of novel 3’ ends. Thereby, evolutionarily older motifs are displaced leading to various 3’ end subpopulations within the SaliS families. Opposed to the PoaS families with a largely equal ratio of poly(A) to poly(T) tail SINEs, the SaliS families are exclusively terminated by adenine stretches. Among retrotransposon-based markers, SINEs are highly suitable for the development of molecular markers due to their unidirectional insertion and random distribution mainly in euchromatic genome regions, together with an easy and fast detection of the heterogeneous SINE families. As a prerequisite for the development of SINE-derived inter-SINE amplified polymorphism (ISAP) markers, 13 novel Theaceae SINE families (TheaS-I - TheaS-VII, TheaS-VIII.1 and TheaS-VIII.2, TheaS-IX - TheaS-XIII) were identified in the angiosperm tree species Camellia japonica. Moreover, six Pinaceae SINE families (PinS-I.1 and PinS-I.2, PinS-II – PinS-VI) were detected in the gymnosperm species Larix decidua. Compared to the SaliS and PoaS families, structural relationships are less frequent within the TheaS families and absent in the PinS families. The ISAP analysis revealed the genetic identity of Europe’s oldest historical camellia (C. japonica) trees indicating their vegetative propagation from the same ancestor specimen, which was probably the first living camellia on European ground introduced to England within the 18th century. Historical sources locate the native origin of this ancestral camellia specimen either in the Chinese province Yunnan or at the Japanese Gotō Islands. Comparative ISAPs showed no accordance to the Gotō camellia sample pool and appropriate Chinese reference samples were not available. However, the initial experiments demonstrated the potential of ISAP to resolve variations among natural populations. The ISAP application on angiosperm trees also concerned fast growing Populus clones grown in short rotation coppice plantations for energy production. The species-specific P. tremula ISAP primers might also be applied for the discrimination of hybrid poplar clones involving P. tremuloides genome portions, since SINEs of these two species are highly related. However, due to lineage-specific SINE evolution during speciation, cross-species applications are generally only successful to limited extent. The analysis of poplar hybrids composed of P. maximowiczii with either P. trichocarpa or P. nigra based on P. tremula ISAP primers showed a strongly reduced resolution. In forestry, hybrid larch (e.g. Larix × eurolepis) genotypes have to be selected from the offspring of Japanese (Larix kaempferi) and European larch (Larix decidua) crosses, as they exhibit superior growth rates compared to the parental species. Initial ISAP-based examinations of European larch genotypes provided less polymorphic banding patterns, probably resulting from general high levels of synteny and collinearities reported for gymnosperm species. Hence, the ISAP was combined with the AFLP technique to the novel marker system inter-SINE-restriction site amplified polymorphism (ISRAP). The amplicons originating from genomic regions between SINEs and EcoRI cleavage sites were visualized with the sensitive capillary gel electrophoresis. The ISRAP assays, based on EcoRI adapter primers combined with two different SINE-derived primers, resulted in a sufficient number of polymorphic peaks to distinguish the L. decidua genotypes investigated. Compared to ISAPs, the ISRAP approach provides the required resolution to differentiate highly similar larch genotypes.