No description available.

No description available.

Cette thèse présente le développement, en vue de l'analyse statistique des séquences d'ADN, de nouveaux modèles permettant de prendre en compte l'hétérogénéité de ces séquences : les chaînes de Markov régulées (DMM pour drifting Markov model). Afin d'éviter l'homogénéité supposé par les modèles de Markov et de Markov cachés, nous permettons à la matrice de transition de varier du début à la fin de la séquence. A chaque position, nous avons une matrice de transition différente. Ces modèles peuvent être vus comme une alternative mais aussi comme un outil complémentaire aux modèles de Markov cachés. Nous avons considéré des dérives polynomiales ainsi que des dérives par splines polynomiales. Nous avons estimé nos modèles de multiples manières puis évalué la qualité de ces estimateurs avant de les utiliser en vue d'applications telle la recherche de mots exceptionnels. Nous avons mis en oeuvre le software DRIMM, dédié à l'estimation de nos modèles. / This document propose the conception, in the way of statistical analysis of DNA sequences, of new models which permit to take into account the heterogeneity of these sequences : the drifting Markov models (DMM). In order to avoid homogeneity of Markov models or hidden Markov models, we allow the transition matrix to vary from the beginning to the end of the sequence. At each position, we obtain a different transition matrix. DMM can be seen as a competitive model to the HMM one but it over all can be understood as a complementary tool: the hidden models of an HMM, usually fixed Markov chains can be replaced by DMM. Along this work, we consider polynomial drift or drift by polynomial splines. We estimate our models by different ways, evaluate their qualities and used them in biological applications such as the search of rare words. We develop the software DRIMM, dedicated to estimation of DMM.

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.

No description available.

No description available.

Phylogeography of temperate plant species with the focus on Central Europe Ph.D. Thesis Hana Daneck Charles University Prague Faculty of Science Department of Botany Supervisor: Prof. RNDr. Karol Marhold, CSc. Consultant: Mgr. Tomáš Fér, Ph.D. Praha 2012 2 Summary This thesis presents contribution to clarification of postglacial history of temperate plant taxa in Europe with the focus on especially interesting region of Central Europe, for which diverse roles in postglacial plant histories were suggested. The first part of the thesis summarises general phylogeographical views and methodological approaches with the respect to species history after the last ice age in Europe. Further, the most important aspects of phylogeography of European temperate plant taxa are discussed. The second part contains a set of papers dealing with selected European temperate plant species, for which phylogeographical patterns throughout their present distribution area were inferred, including assumptions on the origin of their contemporary Central European populations and comparisons with another previously studied species. Paper 1: Phylogeographic pattern of the European forest grass species Hordelymus europaeus: cpDNA evidence. This paper presents phylogeographical pattern based on chloroplast haplotype variation covering the...

The objective of this dissertation is the isolation of DNA sequences that are differentially represented in males and females of the Oriental fruit fly Bactrocera dorsalis, specifically by initiating a molecular characterization of Y chromosome sequences in this species. Cytological observations have established the presence of a diminutive Y chromosome in B. dorsalis males. To isolate DNA sequences from the Y chromosome, a special method of genomic DNA isolation known as Representational Difference Analysis (RDA) was utilized to obtain DNA sequences unique to the B. dorsalis male genome. Genomic DNA from B. dorsalis males served as the "tester" DNA and female genomic DNA as the "driver" DNA. Six distinct RDA products were obtained following two complete rounds of DNA hybridization and difference enrichment via the Polymerase Chain Reaction (PCR). One ofthese products (RDA product 1) was used to isolate a genomic DNA clone (3.1a) from a B. dorsalis male genomic DNA minilibrary. This sequence shows similarity to the reverse transcriptase of R1 retrotransposable elements. The presence of R1 elements in the Tephritid insects has heretofore been undescribed, although these elements have been previously described in the genomes of other Dipteran species. Oligonucleotide primers for PCR were designed for the 3.1a clone. These primers consistently produce different amplification patterns in PCRs ofgenomic DNA from B. dorsalis males vs. females. Amplification using male genomic DNA produces 325 bp and 2.6 kb products while only a 2.6 kb product is obtained from female DNA. The amplification products obtained with these primers are also produced in PCRs of genomic DNA from B. dorsalis embryos and third instar larvae, suggesting the ability of this method to infer sex at pre-adult stages ofthe B. dorsalis life cycle. Similar amplification products have also been obtained in other Bactrocera species. Both the 325 bp male PCR product and the 2.6 kb products have regions of sequence similarity to R1 elements. The 2.6 kb product contains a putative 1.7 kb open reading frame (ORF) encoding 583 amino acids. Three amino acid motifs found in Drosophila R1 element reverse transcriptases are present in comparable locations within the hypothetical ORF product. Both of these sequences are also repetitively represented in the B. dorsalis male and female genomes. However, the 325 bp male product produces some bands that are male specific when used as a probe for Southern blots of B. dorsalis male and female genomic DNA. The amplification pattern produced by the 3.1a primers is consistent with what would be expected if the 2.6 kb and 325 bp PCR products originated from the B. dorsalis X and Y chromosomes, respectively. Thus, the cloned male-specific sequence recovered here is potentially useful both as a gateway into the relatively uncharacterized B. dorsalis Y chromosome and as a tool for the characterization of other aspects of the B. dorsalis genome.

A Ordem Diptera é constituída de milhares de espécies, cujo tempo de divergência pode chegar a 250 milhões de anos entre representantes das Sub-Ordens Brachycera e Nematocera. Esta janela temporal, no entanto, não é suficiente para explicar o aparecimento de estruturas cromossômicas terminais alternativas aos telômeros canônicos, conservados desde eucariontes unicelulares até mamíferos. Alguns autores admitem que estruturas não canônicas tenham sido geradas e selecionadas a partir de eventos mutacionais que resultaram na perda da telomerase em espécies ancestrais. Especulações deste tipo, embora pertinentes, não levam em conta que o número de espécies de dípteros estudados até aqui quanto a telômeros e sub-telômeros está longe de representar a diversidade na Ordem. Como evidência negativa não constitui prova, a possível existência de dípteros dotados de telômeros canônicos não pode ser descartada. Também neste sentido, a possível ocorrência de retrotransposons especificamente terminais, como vistos em Drosophila, em espécies ainda não estudadas pode ser vista como possibilidade em aberto apesar da evidência negativa documentada no presente trabalho em R. Americana e em T. pubescens. Outra idéia que tem ganhado corpo com dados procedentes de telômeros não canônicos refere-se à possibilidade de que um organismo apresente mais de uma sequência de DNA terminal. Pouco comentada, esta noção teve início em meados da década de 1980 quando foram publicados os primeiros trabalhos sobre o DNA telomérico de espécies da família Chironomidae. O aparecimento destes dados na literatura praticamente coincidiu com a publicação da descoberta da telomerase. Mais tarde, estudos em Drosophila têm mostrado que três retrotransposons com sequências diferentes podem compor o DNA telomérico nesta espécie. Além disto, repetições aparentemente terminais em Anopheles hibridam em um único telômero, sugerindo que sequências desconhecidas estejam presentes em outros telômeros. Dados obtidos neste trabalho mostram em R. Americana uma nova repetição em tandem que apresenta características de sequência telomérica, a exemplo de duas outras caracterizadas com antecedência nesta espécie. Assim, R. Americana poderia ser vista como exemplo adicional de organismo dotado de mais de uma sequência de DNA terminal. No final da presente exploração, não foi possível a identificação de sequências comuns às extremidades cromossômicas de T. pubescens. Os resultados obtidos sugerem que esta espécie apresenta uma estrutura cromossômica terminal distinta se comparada àquelas de dípteros estudados até então. Uma das hipóteses levantadas a partir dos resultados observados é a de que sequências teloméricas estão presentes em todas as extremidades cromossômicas de T. pubescens; o problema estaria na impossibilidade de visualizá-las através de hibridação in situ em função do comprimento do DNA telomérico que estaria abaixo do limite da técnica de detecção. Isto implicaria deixar de lado os métodos usualmente empregados pelo laboratório na busca de DNA repetitivo terminal. Caso esta espécie apresentasse repetições terminais curtas como aquelas caracterizadas em R. Americana, uma das alternativas seria a de sequênciar massivamente clones obtidos por microdissecção e DOP-PCR até encontrarmos sequências com estas características. Em seguida, o ensaio com Bal-31 seria decisivo na determinação da posição cromossômica das mesmas. Finalmente, os dados sintetizados nos três capítulos deste trabalho reforçam a afirmação de que a Ordem Diptera é uma fornte privilegiada de diversidade quanto a estruturas cromossômicas terminais. Apesar das dificuldades inerentes à exploração de organismos não modelares, a continuidade dos estudos sobre telômeros e sub-telômeros nestas espécies certamente ampliará o conhecimento sobre alternativas de manutenção da integridade cromossômica a partir de suas extremidades / The vast majority of eukaryotic organisms have short tandem repeats and telomerase as components of telomeric structures. However, in dipteran species, this structural conservation is not found. While in Drosophila telomeres are composed of specific retrotransposons, complex tandem repeats are found in the genus Anopheles and in species of Chironomus. In Rhynchosciara americana (family Sciaridae), short repetitions (16 and 22 base pairs) arranged in tandem are observed at chromosome terminal regions. Moreover, in situ hybridization using RNA probes suggests that a third repetition enriched with homopolymeric (dA)/(dT) could occupy significant portions of this chromosomal region in R. americana. In addition, a retroelement named \"RaTART\" was described at chromosomal ends of R. americana; this element enables telomeric maintenance by retrotransposon action in basal dipterans. In this thesis, we present results that rule out the possibility that the \"RaTART\" element occupies the chromosome terminal structure in R. americana. Additional analyses were performed with the sequence RaTART from GenBank and primers designed for the amplification of significant portions of the regions 5′UTR, 3′UTR, and the coding region for reverse transcriptase (RT) of this retroelement. The sequencing and in situ hybridization of these three fragments obtained after PCR (5′UTR, 3′UTR, and RT) indicate that the retroelement RaTART corresponds to a chimeric genomic clone, consisting of distinct repetitive elements, of which only one might be present at the apparent enrichedment terminal. In the second phase of this thesis, we describe a method for the isolation and characterization of the homopolymeric (dA)/(dT) sequence described in the chromosome terminal regions of R. americana. Named T-14, this sequence shares some similarity with canonical telomeric repeats, suggesting that R. americana represents an additional example of an organism where more than one DNA sequence may extend toward the chromosome terminal regions. Finally, we present the results of heterologous hybridization to elucidate structural aspects of conservation and/or divergence in Sciaridae terminal heterochromatin. Three species of the family Sciaridae were assessed by in situ hybridization of polytene chromosomes: R. americana, Rhynchosciara milleri, and Trichosia pubescens. The DNA probe used was obtained by microdissection of non-centromeric chromosome ends from R. americana and T. pubescens, followed by amplification and labeling by degenerate oligonucleotide primed (DOP-PCR). When each probe was hybridized to own chromosome complement, two patterns were observed: (i) hybridization at all non-centromeric ends (R. americana), and most surprisingly, (ii) hybridization only at the end that gave the product of microdissection (T. pubescens). Probes obtained from R. americana produced no hybridization signals on chromosomes of T. pubescens and R. milleri. Unexpected results were obtained when the probe obtained from T. pubescens was hybridized with chromosomes of R. americana and R. milleri. Surprisingly, this probe, which scored only one end in its own chromosome complement, generated hybridization signals in all non-centromeric chromosomal ends of R. americana, as well as in its pericentromeric-centromeric heterochromatin. In R. milleri, the probe from T. pubescens clearly hybridized with centromere-associated heterochromatin of its chromosome C. The data obtained in this study support a process of chromosomal divergence between these three species of Sciaridae occurred by differential amplification of sequences of heterochromatin components, and point to an unusual structure in T. pubescens compared to other dipterans studied for terminal chromosome structure

Camarea St.-Hil. (Malpighiaceae) é um gênero endêmico da América do Sul, constituído por nove espécies. O objetivo do trabalho foi a reconstrução da filogenia do gênero, por meio de evidências químicas e moleculares. Foram avaliados nove terminais, sete dos quais são espécies correntemente reconhecidas, um é uma espécie que entrou em sinonímia (C. triphylla = C. axillaris) e outro é um suposto híbrido. Como grupos externos, foram utilizadas as espécies Peixotoa reticulata e Janusia guaranitica. Foram analisados os n-alcanos das ceras epicuticulares e os flavonóides de todas as espécies. Os n-alcanos principais foram C29, C31 e C33, todos da série normal. Como flavonóides característicos de Camarea, foram identificados glicosídeos de apigenina, luteolina, crisoeriol, campferol e quercetina. A análise de agrupamentos baseada na distribuição de alcanos, usando UPGMA e distâncias euclideanas, resultou em dois grupos principais, um com C29 como homólogo principal, constituído por C. hirsuta, C. affinis x C. hirsuta, C. affinis e C. ericoides. O outro grupo caracteriza-se por homólogos principais C31 ou C33, e é formado por C. elongata, C. humifusa, C. sericea, C. axillaris e C. triphylla (= C. axillaris). Esses dois principais agrupamentos contêm grupos internos menores. Uma análise de UPGMA usando coeficiente de DICE e baseada na distribuição de agliconas de flavonóides forneceu um dendrograma com alguns agrupamentos coerentes com as afinidades reveladas pela distribuição de alcanos, como as associações: 1) C. hirsuta, C. affinis e C. affinis x hirsuta; 2) C. elongata e C. axillaris; 3) C. sericea e C. humifusa. Uma inferência filogenética molecular foi obtida com seqüências de duas regiões do cloroplasto (trnL-F e rps16) e uma nuclear (ITS). Dentre as análises com um só marcador, resultados mais consistentes foram conseguidos com ITS, que forneceu 49 caracteres filogeneticamente informativos, enquanto trnL-F e rps16 forneceram 10 e 18 caracteres informativos, respectivamente. A análise de consenso estrito de quatro árvores mais parcimoniosas de uma análise combinando-se as três seqüências resultou em um cladograma em que Camarea é um grupo monofilético, com \"bootstrap\" (BS) 100, várias politomias e clados com baixa consistência. Foi realizada análise por AFLP utilizando quatro combinações de iniciadores seletivos, obtendo-se 217 fragmentos polimórficos. Uma análise combinando as evidências moleculares resultantes de seqüências de DNA e marcadores AFLP forneceu uma única árvore mais parcimoniosa com uma politomia agrupando C. affinis, C. ericoides, C. hirsuta e C. affinis x C. hirsuta, mas boa resolução e elevada sustentação em outros clados. A combinação de todas as evidências moleculares e químicas (estas compreendendo três caracteres derivados da análise de alcanos e cinco de flavonóides) resultou numa única árvore mais parcimoniosa completamente resolvida. Os resultados apóiam a fusão de C. triphylla em sinonímia com C. axillaris e indicam forte associação entre: 1) C. humifusa e C. sericea (BS 94); 2) C. affinis, C. affinis x hirsuta, C. hirsuta e C. ericoides (BS 83); 3) C. axillaris e C. elongata (BS 100). C. affinis, C. hirsuta e C. affinis x C. hirsuta compartilham a presença crisoeriol. C. affinis e C. affinis x C. hirsuta, compartilham também luteolina e formam um clado com BS 70. O presente trabalho demonstra a utilidade de caracteres químicos para melhorar a resolução de filogenias e elevar a sustentação de clados. / Camarea St.-Hil. (Malpighiaceae) is a genus with eight species endemic in South America. The purpose of the present work was the attainment of a phylogenetic inference of the genus by means of chemical and molecular evidences. Nine accessions were analyzed: seven correspond to currently recognized species; one is a species sunk into synonymy (C. triphylla = C. axillaris); and a last one is a hypothesized hybrid. Peixotoa reticulata and Janusia guaranitica were used as out-groups. n-Alkanes from epicuticular waxes and flavonoids were analyzed from all species. The main alkanes of all distributions were either C29 or C31 or C33, all from the normal series. The characteristic flavonoids of Camarea were shown to be apigenin, luteolin, chrysoeriol, kaempferol and quercetin. A cluster analysis based on the alkane distribution using UPGMA and Euclidean distances provided two main clusters. One cluster is characterized by C29 as main homologue and is formed by C. hirsuta, C. affinis, C. affinis x hirsuta and C. ericoides. The other cluster has species with either C31 or C33 as main homologue and is formed by C. elongata, C. humifusa, C. sericea, C. axillaris and C. triphylla (= C. axillaris). These two main clusters contain smaller inner clusters. An analysis using UPGMA and DICE coefficients and based on the distribution of flavonoid aglycones provided a dendrogram with clusters congruent with affinities revealed by the alkane evidence, such as the groupings: 1) C. hirsuta, C. affinis and C. affinis x hirsuta; 2) C. elongata and C. axillaris; 3) C. sericea and C. humifusa. A phylogenetic molecular inference was obtained with sequences from two chloroplast (trnL-F and rps16) and one nuclear (ITS) DNA regions. Among the analyses based on a single marker, more consistent results were obtained with ITS, which provided 49 informative phylogenetic characters, while trnL-F and rps16 provided 10 and 18 informative characters, respectively. A strict consensus analysis based on four more parsimonious trees from an analysis combining sequences of the three DNA regions gave a cladogram showing Camarea as a monophyletic group with bootstrap support (BS) 100. The cladogram contains several polytomies and clades with low support. An AFLP analysis, using four combinations of selective primers, provided 217 polymorphic fragments. Data from sequencing and AFLP were combined in a phylogenetic analysis. A sole more parsimonious tree was obtained, with most clades completely resolved with and high support. A polytomy remained, grouping C. affinis, C. ericoides, C. hirsuta and C. affinis x hirsuta. The combination of all molecular and chemical evidences (the latter comprising three alkane and five flavonoid characters) was used for a phylogenetic analysis. A sole more parsimonious tree was obtained, completely resolved and with clades highly supported. The results support sinking C. triphylla into synonymy of C. axillaris and indicate strong kinship: 1) between C. humifusa and C. sericea (BS 94); 2) among C. affinis, C. affinis x C. hirsuta, C. hirsuta and C. ericoides (BS 83); 3) between C. axillaris and C. elongata (BS 100). C. affinis, C. hirsuta and C. affinis x C. hirsuta share the possession of chrysoeriol. C. affinis and C. affinis x C. hirsuta share the possession also of luteolin and form a clade with BS 70. The present work reveals the utility of chemical characters to improve resolution of phylogenies and increment clade support.