\u3ci\u3eAMBYSTOMA\u3c/i\u3e: PERSPECTIVES ON ADAPTATION AND THE EVOLUTION OF VERTEBRATE GENOMES

Tiger salamanders, and especially the Mexican axolotl (Ambystoma mexicanum), are important model organisms in biological research. This dissertation describes new genomic resources and scientific results that greatly extend the utility of tiger salamanders. With respect to new resources, this dissertation describes the development of expressed sequence tags and assembled contigs, a comparative genome map, a web-portal that makes genomic information freely available to the scientific community, and a computer program that compares structure features of organism genomes. With respect to new scientific results, this dissertation describes a quantitative trait locus that is associated with ecologically and evolutionarily relevant variation in developmental timing, the evolutionary history of the tiger salamander genome in relation to other vertebrate genomes, the likely origin of amniote sex chromosomes, and the identification of the Mexican axolotl sex-determining locus. This dissertation is concluded with a brief outline of future research directions that can extend from the works that are presented here

Identification of genes related to self-incompatibility in ryegrass (Lolium perenne L.)

Self-incompatibility (SI) is a mechanism that prevents self-pollination and inbreeding in many flowering plant species. SI in Lolium perenne is controlled by two multi-allelic loci, S and Z. The working of this SImechanism has important consequences for L. perenne breeding as it prevents the efficient production of inbred lines and hybrids. The aim of this study was to identify genes involved in the SI-response in L. perenne. Therefore, different approaches were applied. We investigated first whether homologues of genes involved in the SI-response of other plant families and grass species (Solanaceae, Brassicaceae and Phalaris coerulescens) are also present in L. perenne. Based on the results, we could conclude that no S-RNase based system (active in members of the Solanaceae) is present in ryegrass. This result was not surprising as very little features with regards to the SI-response are common for grasses and species displaying a S-RNase based system. On the other hand, a L. perenne homologue of SRK, the key component at the female side in members of the Brassicaceae, was identified. However, this gene was not only expressed in pistils but also in leaves indicating that it is not the recognition factor of the SI-response in ryegrass. This conclusion was also supported by its map position, as it mapped on LG4 and not on LG1 or LG2, where S and Z are located. Finally, we amplified in pistils and leaves of L. perenne the thioredoxin homologue known to be involved in the SI-response of Phalaris coerulescens, also a member of the Poaceae. The L. perenne thioredoxin homologue mapped in the neighbourhood of S (at 3cM), indicating that it is not S. Whether this thioredoxin gene is involved in the SI-response in ryegrass needs to be investigated. However, the involvement of thioredoxins in SI has been demonstrated in Brassica. Another objective of this study was to identify genes involved in the signalling cascade triggered by a SI-response in L. perenne. Therefore we used two differential expression techniques: cDNA-AFLP and the cDNAmicroarray. The genome-wide expression analysis carried out by cDNAAFLP allowed us to identify 479 TDFs (Transcript Derived Fragments) as putatively related to SI in L. perenne. All the expression profiles were quantified using AFLP Quantar Pro and clustered into groups of co-regulated genes. A subset of these genes was selected to study further. The selected genes clustered into 11 functional categories. Several of them corresponded to genes involved in general cellular processes but homology was also been found to proteins known to be involved in fertilization and SI-processes in other plant families such as ubiquitin-related and calcium-related proteins. The GRASP microarray was used to confirm the expression profiles of the TDFs selected by the cDNA-AFLP and to identify other genes related to SI. This microarray contains the TDFs selected by cDNA-AFLP and cDNA clones of genes involved in different biological processes. Using this technique, we were able to identify 371 differentially expressed genes putatively involved in the SI-response. Surprisingly the results from both techniques were contradictory. Confirmation of a selection of genes by Realtime RT-PCR revealed consistency between the results of Real-time RTPCR and those of the cDNA-AFLP but not with those of the microarray. Further investigation is needed to clarify this point. As a complementary approach, the genome location of the genes identified as putatively involved in SI was determined. Two mapping populations were used for this purpose: the ILGI population and the CLODvP population. Some of the mapped genes clustered around S and Z on LG1 and LG2, respectively. Several other markers mapped across the different linkage groups with a higher concentration on LG3 and LG4. Also a strong association was identified between LG1 and LG3 indicating that some genes of LG3 interact with LG1 during SI. Finally, combining all these data, a selection of 21 genes putatively involved in SI in L. perenne was made. Confirmation of their involvement is the subject for further research

Genetic Networks Modulating Retinal Injury

Many advances have and are being made with the advent of modern sciences. One area that remains resistant to significant advances is the recovery of the mammalian central nervous system (CNS) after injury. Considerable efforts were placed on understanding the cellular and biochemical changes occurring after injury and during wound healing. Aside from cataloging the changes that occur, little progress is being made in understanding the molecular cascades associated with the control of the healing process. The present series of studies define the global changes that occur after injury. They also define CNS repair mechanisms by identifying genetic networks that underlie the temporal changes of retinal wound healing. Gene expression changes in the injured rat retina were analyzed with microarray technology, genetic linkage analysis of gene expression, and higher-level bioinformatic analyses. This work yielded three complementary insights. First, groups of functionally related genes underlie the early, delayed, and sustained responses of wound healing. For example, transcriptional factors such as Fos and Egr1 define the early response, whereas, glial reactive markers such as Gfap and Cd81 define the sustained response. Second, three specific genomic loci modulate coordinated changes in gene expression in mouse brains: regulatory loci on chromosomes 6, 12, and 14. Of the three only the regulatory locus on chromosome 12 specifically modulates the expression of a group of genes involved in early wound-healing events (mainly, the regulation of transcription, differentiation, apoptosis, and proliferation). Third, candidate genes Id2 and Lpin1 are current modulators for the network controlled by the chromosome 12 locus. Higher levels of Id2 and Lpin1 correlated with higher levels of the survival gene Crygd, and with lower levels of acute phase genes Fos and Stat3, reactive gliosis genes Gfap and Cd81, and apoptotic gene Casp3. In this body of work I have moved beyond cataloging changes in the transcriptome to identifying candidate genes modulating the retinal response to injury. During this process I developed an integrated approach of gene expression profiling and higher-level bioinformatic analyses to define genetic networks. This work not only advances our understanding of the molecular networks controlling the CNS response to injury, but may also form the basis for interventions that can rescue injured neurons and re-establish lost CNS connections

A transcriptome analysis of apple (Malus x domestica Borkh.) cv ‘golden delicious’ fruit during fruit growth and development

Philosophiae Doctor - PhDThe growth and development of apple (Malus x domestica Borkh.) fruit occurs over a period of about 150 days after anthesis to full ripeness. During this period morphological and physiological changes occur defining fruit quality. These changes are a result of spatial and temporal patterns of gene expression during fruit development as regulated by environmental, genetic and environmental-by-genetic factors. A number of previous studies partially characterised the transcriptomes of apple leaf, fruit pulp, whole fruit, and peel plus pulp tissues, using cDNA micro arrays and other PCR based technologies. These studies, however, remain limited in throughput and specificity for transcripts of low abundance. Hence, the aim of this project was to apply a high throughput technique to characterise the full mRNA transcriptome of the ‘Golden Delicious’ fruit peels and pulp tissues in order to understand the molecular mechanisms underlying the morphophysiological changes that occur during fruit development

Heterogeneity, High Performance Computing, Self-Organization and the Cloud

application; blueprints; self-management; self-organisation; resource management; supply chain; big data; PaaS; Saas; HPCaa

Putting the Pieces Together: Exons and piRNAs: A Dissertation

Analysis of gene expression has undergone a technological revolution. What was impossible 6 years ago is now routine. High-throughput DNA sequencing machines capable of generating hundreds of millions of reads allow, indeed force, a major revision toward the study of the genome’s functional output—the transcriptome. This thesis examines the history of DNA sequencing, measurement of gene expression by sequencing, isoform complexity driven by alternative splicing and mammalian piRNA precursor biogenesis. Examination of these topics is framed around development of a novel RNA-templated DNA-DNA ligation assay (SeqZip) that allows for efficient analysis of abundant, complex, and functional long RNAs. The discussion focuses on the future of transcriptome analysis, development and applications of SeqZip, and challenges presented to biomedical researchers by extremely large and rich datasets

Advances in Wheat Genetics: From Genome to Field: Proceedings of the 12th International Wheat Genetics Symposium

plant genetics; plant genomics; agricultur