The assembly of a robust structural genomics system requires the development and integration of multiple types of genome maps. This research focused on the development of a relatively new means of plant genome mapping, radiation hybrid mapping, for use in cotton genomics. Simple sequence repeat markers were genotyped onto an existing wide-cross whole-genome radiation hybrid panel for genome mapping of the Gossypium barbadense line ‘3-79’. A new mapping panel was created for genome mapping of the G. hirsutum line ‘TM-1’. Carthagene software was compared to RHMAP and found to be superior in most regards. A total of 92 simple sequence repeat markers were genotyped onto the mapping panel for G. barbadense. Data from 64 of the 92 markers were deemed robust and combined with pre-existing data to develop an expanded framework map, which provides partial coverage of 7 chromosomes and three unidentified linkage groups. A new mapping population was created to allow mapping of the G. hirsutum genome. The population was developed by treatment of TM-1 pollen with 8 krad of radiation, which was used to make more than 1000 controlled cross-pollinations. From these, 979 bolls were harvested and seeds were planted until a population of 115 viable plants was obtained. Of these, 92 were selected at random for inclusion in the mapping panel. Carthagene genome mapping software was evaluated and compared to the previously utilized RHMAP. Carthagene compared favorably in ease of use, calculation speed, and reliability of results. As such, it is recommended for use for the RH mapping project.

High-resolution physically ordered gene maps for the horse (Equus caballus, ECA) are essential to the identification of genes associated with hereditary diseases and traits of interest like fertility, coat color, and disease resistance or susceptibility. Such maps also serve as foundations for genome comparisons across species and form the basis to study chromosome evolution. In this study seven equine chromosomes (ECA6, 7, 10, 15, 18, 21 and X) corresponding to human chromosomes (HSA) 2, 19 and X were selected for high-resolution mapping on the basis of their potential involvement in diseases and conditions of importance to horses. To accomplish this, gene- and sequence-specific markers were generated and genotyped on the TAMU 5000rad horse x hamster RH panel. Additionally, screening of a BAC library by overgoes and subsequent STS content mapping and fingerprinting approaches were used to assemble and verify a BAC contig along a ~5 Mb span on ECA21. Dense gene maps were generated for each of the seven equine chromosomes by adding 408 new markers (285 type I and 123 type II) to the current maps of these chromosomes, thereby greatly improving overall map resolution to one mapped marker every 960kb on average (range: 700 kb – 1.3 Mb). Moreover, the contig on ECA21 contained 47 markers (42 genes and 5 microsatellites) as well as 106 STS markers distributed along 207 BAC clones. Comparisons of these maps with other species revealed a remarkably high level of horse-human X chromosome conservation, as well as two evolutionary breakpoints unique to Perissodactyls or Equids for the equine homologues of HSA19 and HSA2, one of which has been more precisely localized by the ECA21 contig. Thus, high resolution maps developed for these chromosomes i) provide a basis to map traits of interest rapidly to specific chromosomal regions, ii) facilitate searches for candidate genes for these traits by fine comparisons of the equine regions with corresponding segments in other species, and iii) enable understanding the evolution of the chromosomes. Expansion of this work to the entire equine genome will be important for developing novel strategies for diagnosis, prevention, and treatment of equine diseases.

La construction de cartes de génomes consiste à baliser les chromosomes par des repères : les marqueurs. Plus une carte est dense en marqueurs régulièrement espacés, plus elle est informative et donc plus les applications ultérieures sont nombreuses. Parmi les différentes stratégies de cartographie, celle exploitant les hybrides d'irradiation dite carte RH, présente de nombreux avantages. Ainsi, des marqueurs polymorphes tels que les microsatellites, utiles pour les analyses de liaisons génétiques et des marqueurs de gènes, polymorphes ou non polymorphes permettant d'établir des cartes comparées avec d'autres génomes et définir des zones de conservation ou de rupture de synténie, peuvent être localisés sur une carte RH. Ces cartes comparées sont utiles non seulement pour l'identification de gènes d'intérêts mais également pour l'étude de l'évolution des génomes. Parmi les nombreux vertébrés d’intérêt, nous nous sommes particulièrement attachés à la construction de cartes RH de poissons et de cichlidés en particulier. Ceux-ci constituent en effet un modèle génétique très intéressant du point de vue économique et évolutif. La réalisation de cartes des génomes de plusieurs poissons devrait aider à l'identification de gènes impliqués dans des traits phénotypiques ou pathologiques voire même des marqueurs liés aux stress et à la reproduction. Mon travail de thèse a consisté à construire une carte du génome du bar, un panel RH de tilapia et la carte RH attenante qui a été utilisée dans la phase finale de l’assemblage des données de séquence du génome de Tilapia. Par ailleurs nous avons construit un panel RH d’esturgeon et un autre d’huitre. La cartographie du génome de tilapia, réalisée dans le cadre d’un consortium international, nous a donné un accès privilégié aux données de séquences des génomes de cinq cichlidés (O. niloticus, P. nyererei, H. burtoni, N. brichardi et M. zebra) et nous a permis de participer à l’annotation de ces séquences génomiques en nous intéressant plus particulièrement à l’identification des répertoires des gènes codant pour les récepteurs olfactifs (OR) et les récepteurs connus sous le vocable de TAAR pour ‘ Trace Amine-Associated Receptors’. C’est ainsi que nous avons identifié et caractérisé 158, 88, 90, 69, 102 gènes OR et 45, 19, 23, 12, 20 gènes TAAR dans les génomes de ces cinq poissons (O. niloticus, P. nyererei, H. burtoni, N. brichardi et M. zebra) / The construction of genome maps consists in placing tags, called markers, on the chromosomes. The denser in evenly spaced markers is a map, the more it is informative, leading to the development of more future applications. Among the different mapping strategies, those using radiation hybrids (RH) have numerous advantages. Indeed, polymorphic markers such as microsatellites, useful for linkage analyses, as well as gene markers, polymorphic or not and allowing comparative mapping with other genomes and definition of synteny breaks and conservation, can be localized on a RH map. Not only these maps are useful to identify genes of interest but they are also essential tools to study genome evolution. Among numerous vertebrates of interest, we constructed RH maps of fishes and cichlids in particular. Indeed these fishes constitute interesting genetic models from economical and evolution points of view. Having genome maps of several of these fishes would help to identify genes implicated in phenotypical or pathological traits, or even markers linked to stress and reproduction. My thesis work consisted in the construction of a genome map of the sea bass, a tilapia RH panel and its RH map, a sturgeon RH panel as well as an oyster RH panel. The tilapia map was used for the assembly of the sequencing data of the tilapia genome. Thanks to this last work realized with an international consortium we had a privileged access to the sequencing data of five cichlid genomes (O. niloticus, P. nyererei, H. burtoni, N. brichardi and M. zebra). We then participated to the annotation of these genomic sequences. In particular we have identified and characterized the olfactory receptor gene (OR) repertoires and of the trace amine-associated receptor (TAAR) gene repertoires of these five cichlids. Which are then made of 158, 88, 90, 69, 102 OR genes and 45, 19, 23, 12, 20 TAAR genes respectively.

The first part of this research focused on wide-cross whole-genome radiation hybrid (WWRH) mapping of the cotton (Gossypium) genome. Radiation hybrid mapping has been used extensively to map the genomes of human and certain animal species, but not plant species. In lieu of in vitro hybrid cell line technologies for plants, we developed a novel approach for radiation hybrid mapping based on wide-cross in vivo hybridization. Flowers from one species of cotton, either G. hirsutum or G. barbadense, were -irradiated and then used to pollinate the other species. The resulting hybrid plants were assessed as a mapping tool. Two WWRH mapping panels were constructed from 5- and 8-krad -irradiation treatments. Both panels demonstrated that the WWRH mapping method can be used to map the cotton genome, and that this method complements traditional linkage mapping approaches. The second part of this research focused on the identification of cold-responsive genes using spotted oligo-gene microarray analysis. Increased cold-tolerance in cotton would promote early and uniform seedling establishment, expand the growing season, decrease susceptibility to fungal infections and certain diseases, and increase fiber yield and quality. BLAST searches of the cotton database using amino acid sequences of 93 drought/cold-related genes from Arabidopsis and several other plant species led to 806 cotton orthologous cDNAs and expressed sequence tags (ESTs). Eight hundred and six cotton 70-mer oligos were designed and included in an oligo-gene microarray containing 1,536 70-mer oligos, each representing a cDNA or EST from cotton, or one of 121 chloroplast genes or 66 mitochondrial genes from Arabidopsis. Thirty-eight cotton cDNAs and ESTs were identified as cold-responsive genes based on experimental treatment and oligo-gene microarray analysis. Expression was up-regulated for 36 genes and down-regulated for two genes by cold treatment. Results from microarray analysis were tested and confirmed by northern blot analysis for 16 genes. Our data suggest that Arabidopsis orthologous genes can be used to identify homologous cotton genes. The oligo-gene microarray is a valid approach to study transcriptional changes in cotton.

The first part of this research focused on wide-cross whole-genome radiation hybrid (WWRH) mapping of the cotton (Gossypium) genome. Radiation hybrid mapping has been used extensively to map the genomes of human and certain animal species, but not plant species. In lieu of in vitro hybrid cell line technologies for plants, we developed a novel approach for radiation hybrid mapping based on wide-cross in vivo hybridization. Flowers from one species of cotton, either G. hirsutum or G. barbadense, were -irradiated and then used to pollinate the other species. The resulting hybrid plants were assessed as a mapping tool. Two WWRH mapping panels were constructed from 5- and 8-krad -irradiation treatments. Both panels demonstrated that the WWRH mapping method can be used to map the cotton genome, and that this method complements traditional linkage mapping approaches. The second part of this research focused on the identification of cold-responsive genes using spotted oligo-gene microarray analysis. Increased cold-tolerance in cotton would promote early and uniform seedling establishment, expand the growing season, decrease susceptibility to fungal infections and certain diseases, and increase fiber yield and quality. BLAST searches of the cotton database using amino acid sequences of 93 drought/cold-related genes from Arabidopsis and several other plant species led to 806 cotton orthologous cDNAs and expressed sequence tags (ESTs). Eight hundred and six cotton 70-mer oligos were designed and included in an oligo-gene microarray containing 1,536 70-mer oligos, each representing a cDNA or EST from cotton, or one of 121 chloroplast genes or 66 mitochondrial genes from Arabidopsis. Thirty-eight cotton cDNAs and ESTs were identified as cold-responsive genes based on experimental treatment and oligo-gene microarray analysis. Expression was up-regulated for 36 genes and down-regulated for two genes by cold treatment. Results from microarray analysis were tested and confirmed by northern blot analysis for 16 genes. Our data suggest that Arabidopsis orthologous genes can be used to identify homologous cotton genes. The oligo-gene microarray is a valid approach to study transcriptional changes in cotton.

No description available.

The objective of this thesis was to construct radiation hybrid (RH) maps and estimate linkage disequilibrium (LD) using high density SNP markers on chromosomes 19 (BTA19) and 29 (BTA29) and use these as a tool to detect QTL in dairy and beef cattle. We have constructed RH maps of BTA19 and BTA29 consisting of 555 and 253 SNP markers respectively using a 12,000 rad whole genome RH panel. When aligned with the third draft of bovine genome sequence assembly, there was a significant internal rearrangement of the markers involving displacement, inversion and flips within the scaffolds with some scaffolds being misplaced in the genome assembly. Many of these mapped markers (370 and 186 SNP markers on BTA19 and 29 respectively) were further utilized to quantify the extent of LD using the square of the correlation coefficient (r2) and to study the pattern of selection signatures in beef (Angus) and dairy (Holstein) breeds of Bos taurus. Along the chromosomes, patterns of LD were variable in both breeds and a minimum of 30,000 informative and evenly spaced markers would be required for whole genome association studies in cattle. In addition, chromosomal regions showing evidence of selection for economically important traits in Angus and Holstein were identified. Furthermore, the dense SNP markers were used to perform chromosome-wide scan to detect QTL for different economically important traits in beef and dairy cattle. Two approaches, single marker LD regression and Bayesian Monte Carlo Markov Chain, were used to map QTL. QTL for 10 and 5 traits in dairy cattle and for 2 and 1 trait in beef cattle on BTA19 and 29 respectively were detected using both approaches of QTL mapping. The QTL detected in this study are a step towards the identification of positional candidate genes controlling these traits. In addition, we have detected several SNPs influencing economically important traits in both beef and dairy cattle. Some SNPs have been validated in an independent cattle population and has the potential of being utilized in the marker assisted selection of cattle. / Animal Science

River buffalo are economically important to many countries and only recently has their genome been explored for the purpose of mapping genetic variation in traits of economic and biologic interest. The purpose of this research is to characterize the genetic and evolutionary profile of Toll-like receptor 5 (TLR5), which mediates the mammalian innate immune response to bacterial flagellin. This study is comprised of three parts: 1) generating a radiation hybrid (RH) map of river buffalo chromosome 5 (BBU5) where the TLR5 gene is located and building a comparative map with homologous cattle chromosomes; 2) conducting a single-nucleotide polymorphism (SNP) survey of the TLR5 gene to reveal variation within river buffalo and other species; and 3) performing an evolutionary study by inferring phylogenetic trees of TLR5 across multiple taxa and determining the possible evolutionary constraints within the TLR5 coding region. River buffalo chromosome 5 is a bi-armed chromosome with arms corresponding to cattle chromosomes 16 and 29. A BBU5 RH map was developed using the previously published river buffalo RH mapping panel and cattle-derived markers. The RH map developed in this study became an integral part of the first river buffalo whole genome RH map. Genetic variation of the TLR5 gene was evaluated in a small domestic herd of river buffalo. Sequencing of the TLR5 coding region and partial associated 5'- and 3'-untranslated regions yielded 16 novel SNPs. Six SNPs were identified as non-synonymous with one predicted to potentially code for a functionally altered product. For the evolutionary study of the TLR5 coding region, phylogenetic trees were inferred based on TLR5 variation across multiple orders and another for artiodactyla. Species that are closely related to river buffalo appear to have undergone negative selection in TLR5 while those that diverged from river buffalo earlier may be retaining alleles that river buffalo are removing from the population. In conclusion, putative chromosomal rearrangements were identified between river buffalo and cattle, the variation that was uncovered in the TLR5 coding region could potentially lead to differential immunity across species, and there appears be some evolutionary flexibility in the DNA sequence of the TLR5 coding region.

Made available in DSpace on 2014-06-11T19:29:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-25Bitstream added on 2014-06-13T20:19:15Z : No. of bitstreams: 1 santana_am_me_jabo.pdf: 1036969 bytes, checksum: 219ad2d9cc666bb8a7c481a54c0a4cf9 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O búfalo de rio (Bubalus bubalis) é uma espécie de interesse econômico pertencente à família Bovidae, utilizado para a produção de carne e leite, além de força de trabalho no campo, constituindo-se em um animal de tripla aptidão. Recentemente, a construção de um painel de células somáticas híbridas irradiadas (linhagens celulares) contendo fragmentos do genoma do búfalo de rio, incorporado ao genoma de hamster (BBURH5000), está permitindo, pela primeira vez, o mapeamento de todos os cromossomos bubalinos. A utilização desse painel de células associado a análises estatísticas, está gerando mapas genômicos de alta resolução contendo diferentes tipos de marcadores moleculares. O presente trabalho teve por objetivo utilizar este painel de células para gerar grupos de ligação com os marcadores mapeados no cromossomo 9 bubalino e assim comparar a organização dos grupos de ligação obtidos com aqueles já descritos para o cromossomo 7 bovino. Para tal, foram testados com DNA bubalino, seqüências de “primers” para PCR de 26 marcadores moleculares previamente mapeados no cromossomo 7 bovino, o qual foi descrito na literatura como o homólogo ao cromossomo 9 de búfalo. Do total de marcadores testados, 18 geraram produtos de PCR adequados ao mapeamento utilizando o painel BBURH5000, sendo que destes 18, foi possível a genotipagem final de 10 marcadores. A partir das análises de ligação com os 10 marcadores genotipados, foi possível distribuir os mesmos em três grupos de ligação no BBU9. A análise comparativa entre os grupos de ligação do BBU9 e do BTA7 revelou a presença dos mesmos grupos de ligação nos cromossomos de ambas espécies, evidenciando conservação de sintenia. / The river buffalo (Bubalus bubalis), a member of the Bovidae family, is an economically important livestock specie useful to produce milk and meat, as well as source of labor, comprising an animal of triple aptness. The recent construction of a whole-genome 5000-rad radiation hybrid somatic cell panel for the river buffalo genome (BBURH5000), is allowing for the first time the mapping of all chromosomes from the specie. The use of BBURH5000 panel associated with statistical analyses is generating high-resolution RH maps integrating different types of molecular markers. The goal of this study was to genarate linkage groups with the markers mapped on the river buffalo chromosome 9 (BBU9), using the BBURH5000 panel, and then compare these groups with those already described in BTA7. Previous studies have identified bovine chromosome 7 as homologous to BBU9. Cattle derived PCR primers from 26 markers, previously mapped in cattle, were tested with buffalo DNA. A total of 18 of the 26 markers amplified PCR products suitable for RH mapping. From these 18 markers, it was possible the genotyping of 10. The analyses of linkage with the 10 markers genotyped turned possible to distribute them in three linkage groups on the BBU9. The comparative analyses between the linkage groups of BBU9 and BTA7 revealed the presence of the same linkage groups on the chromosome of both species, revealing conservation of synteny between them.

Resumo: O búfalo de rio (Bubalus bubalis) é uma espécie de interesse econômico pertencente à família Bovidae, utilizado para a produção de carne e leite, além de força de trabalho no campo, constituindo-se em um animal de tripla aptidão. Recentemente, a construção de um painel de células somáticas híbridas irradiadas (linhagens celulares) contendo fragmentos do genoma do búfalo de rio, incorporado ao genoma de hamster (BBURH5000), está permitindo, pela primeira vez, o mapeamento de todos os cromossomos bubalinos. A utilização desse painel de células associado a análises estatísticas, está gerando mapas genômicos de alta resolução contendo diferentes tipos de marcadores moleculares. O presente trabalho teve por objetivo utilizar este painel de células para gerar grupos de ligação com os marcadores mapeados no cromossomo 9 bubalino e assim comparar a organização dos grupos de ligação obtidos com aqueles já descritos para o cromossomo 7 bovino. Para tal, foram testados com DNA bubalino, seqüências de "primers" para PCR de 26 marcadores moleculares previamente mapeados no cromossomo 7 bovino, o qual foi descrito na literatura como o homólogo ao cromossomo 9 de búfalo. Do total de marcadores testados, 18 geraram produtos de PCR adequados ao mapeamento utilizando o painel BBURH5000, sendo que destes 18, foi possível a genotipagem final de 10 marcadores. A partir das análises de ligação com os 10 marcadores genotipados, foi possível distribuir os mesmos em três grupos de ligação no BBU9. A análise comparativa entre os grupos de ligação do BBU9 e do BTA7 revelou a presença dos mesmos grupos de ligação nos cromossomos de ambas espécies, evidenciando conservação de sintenia. / Abstract: The river buffalo (Bubalus bubalis), a member of the Bovidae family, is an economically important livestock specie useful to produce milk and meat, as well as source of labor, comprising an animal of triple aptness. The recent construction of a whole-genome 5000-rad radiation hybrid somatic cell panel for the river buffalo genome (BBURH5000), is allowing for the first time the mapping of all chromosomes from the specie. The use of BBURH5000 panel associated with statistical analyses is generating high-resolution RH maps integrating different types of molecular markers. The goal of this study was to genarate linkage groups with the markers mapped on the river buffalo chromosome 9 (BBU9), using the BBURH5000 panel, and then compare these groups with those already described in BTA7. Previous studies have identified bovine chromosome 7 as homologous to BBU9. Cattle derived PCR primers from 26 markers, previously mapped in cattle, were tested with buffalo DNA. A total of 18 of the 26 markers amplified PCR products suitable for RH mapping. From these 18 markers, it was possible the genotyping of 10. The analyses of linkage with the 10 markers genotyped turned possible to distribute them in three linkage groups on the BBU9. The comparative analyses between the linkage groups of BBU9 and BTA7 revealed the presence of the same linkage groups on the chromosome of both species, revealing conservation of synteny between them. / Orientadora: Maria Elisabete Jorge Amaral / Coorientadora: Vera Fernanda Martins H. de Lima / Banca: Humberto Tonhati / Banca: Simone Cristina Méo Niciura / Mestre