BACKGROUND: Black rot is a destructive bacterial disease causing large yield and quality losses in Brassica oleracea. To detect quantitative trait loci (QTL) for black rot resistance, we performed whole-genome resequencing of two cabbage parental lines and genome-wide SNP identification using the recently published B. oleracea genome sequences as reference. RESULTS: Approximately 11.5 Gb of sequencing data was produced from each parental line. Reference genome-guided mapping and SNP calling revealed 674,521 SNPs between the two cabbage lines, with an average of one SNP per 662.5 bp. Among 167 dCAPS markers derived from candidate SNPs, 117 (70.1%) were validated as bona fide SNPs showing polymorphism between the parental lines. We then improved the resolution of a previous genetic map by adding 103 markers including 87 SNP-based dCAPS markers. The new map composed of 368 markers and covers 1467.3 cM with an average interval of 3.88 cM between adjacent markers. We evaluated black rot resistance in the mapping population in three independent inoculation tests using F₂:₃ progenies and identified one major QTL and three minor QTLs. CONCLUSION: We report successful utilization of whole-genome resequencing for large-scale SNP identification and development of molecular markers for genetic map construction. In addition, we identified novel QTLs for black rot resistance. The high-density genetic map will promote QTL analysis for other important agricultural traits and marker-assisted breeding of B. oleracea.

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The theory of "genomic islands of speciation" has been extensively debated during the last decade. This thesis not only supports this theory, but provides evidence that challenges previous beliefs on the level of admixture between species. The recently published Heliconius melpomene genome project reported apparent genomic paraphyly of H. pardalinus with regard to H. elevatus (Heliconius Genome Consortium 2012). Here, we investigate this pair of butterfly species more fully, firstly by using whole-genome resequence data, and secondly by analyzing additional geographic populations of both species, as well as outgroup taxa. Using a nuclear whole-genome phylogenetic analysis we also confirm that H. elevatus is paraphyletic. The genome-wide phylogenetic signal in H. pardalinus and H. elevatus does not indicate expected mutual monophyly of each species as it seems strongly distorted by a high level of admixture. However, several regions of the genome remain differentiated and do show the presumably original phylogenetic signal with mutual monophyly of H. pardalinus and H. elevatus. The genomic background is so homogenized that its level of differentiation (FST ~ 0.03) virtually implies panmixia. The pattern of a high level of homogenization across the genome with several regions of differentiation was consistent with a number of other statistics such as absolute divergence Dxy, nucleotide polymorphism π, number of fixed differences and with a sliding window phylogeny. The identified genomic islands of divergence comprise genes responsible for wing-patterning and chemosensation in Heliconius and some of these genes are found to be under positive selection, suggesting possible candidates of speciation.

Human autoimmune diseases are hallmarked by inappropriate loss-of-tolerance and self-attacking response of the immune system. Studies included in this thesis are focusing on the implication and functional impact of genetic factors in three autoimmune diseases rheumatoid arthritis (RA), asthma, and systemic lupus erythematosus (SLE). Using genetic association studies, we found in study I and II that sequence variants of the interferon regulatory factor 5 (IRF5) gene were associated with RA and asthma, and the associations were more pronounced in certain disease subtypes. Distinct association patterns or risk alleles of the IRF5 gene variants were revealed in different diseases, indicating that IRF5 contributes to disease manifestations in a dose-dependent manner. In study III, we found that seven out of eight genetic risk loci for SLE, which were originally identified in East Asian populations, also conferred disease risk with the same risk alleles and comparable magnitudes of effect sizes in Caucasians. Remarkable differences in risk allele frequencies were observed for all associated loci across ethnicities, which seems to be the major source of genetic heterogeneity for SLE. In study IV we explored an exhaustive spectrum of sequence variants in the genes inhibitor of kappa light polypeptide gene enhancer in B-cells kinase epsilon (IKBKE) and interferon induced with helicase C domain 1 (IFIH1) by gene resequencing, and identified nine variants in IKBKE and three variants in IFIH1 as genetic risk factors for SLE. One of the associated variants may influence splicing of IKBKE mRNA. In study V we provided genome-wide transcriptional regulatory profiles for IRF5 and signal transducer and activator of transcription 4 (STAT4) using chromatin immunoprecipitation-sequencing (ChIP-seq). The target genes of IRF5 and STAT4 were found to play active roles in pathways related with inflammatory response, and their expression patterns were characteristic for SLE patients. We also identified potential cooperative transcription factors for IRF5 and STAT4, and disease-associated sequence variants which may affect the regulatory function of IRF5 and STAT4. In conclusion, this thesis illuminates the contribution of several genetic risk factors to susceptibility of human autoimmune diseases, which facilitates our understanding of the genetic basis of their pathogenesis.

Rice is one of the most important cereal crops for the world population. Flowering time and seed development of rice are directly related to plant regional and ecological adaptions, and productive yield. In this dissertation, to gain knowledge of seed development in rice, the status of post-translational modifications (PTMs) in developing rice seeds was investigated. Numerous modified lysine sites in developing rice seeds were identified utilizing antibody-based affinity enrichment approaches and nano-HPLC/MS/MS analyses of acetylated, succinylated, crotonylated and 2-hydroxyisobutyrylated peptides. Functional annotation analyses indicated that a wide variety of vital biological processes were targeted by lysine PTMs. A number of modified histone and non-histone proteins were found to harbor multiple PTMs, and our findings showed that many modified histone sites were conserved across plant, human, and animal systems. Comprehensive analyses of lysine modification sites illustrated that the sites were highly sequence-specific for distinct motifs. Overall, this study provides a systematic analysis of lysine PTM proteome in plants, which will serve as the basis for future investigations of the regulatory mechanisms and functions of lysine PTMs. The mechanisms of flowering time variances in response to different photoperiods were further studied in the rice mutant, HSS. QTL-seq analysis identified a major effect on chromosome 6 responsible for the phenotypic divergence between Nipponbare (wild-type) and HSS rice. Sequence and mRNA expression analyses confirmed that allelic variants of Hd1 make HSS plants less sensitive to photoperiod by altering expression level of Hd3a. Diurnal expression pattern analyses revealed that DTH8 transcripts were largely affected by Hd1 expression level in both LD and SD. This result suggested that Hd1 may able to regulate DTH8 and DTH8-Hd1 complex abundance in response to day length in rice flowering time regulation. In addition, we discussed the functions of PTMs in flowering time regulation in rice.

Brachydactyly type A1 (BDA1) is a rare autosomal dominant trait characterized by the shortening of the middle phalanges of digits 2-5 and of the proximal phalange of digit 1 in both hands and feet. Many of the brachymesophalangies including BDA1 have been associated with genetic perturbations along the BMP-SMAD signaling pathway. The goal of this thesis is to identify the molecular pathways that are associated with the BDA1 phenotype through the genetic assessment of BDA1-affected families. We identified four missense mutations that are clustered with other reported BDA1 mutations in the central region of the N-terminal signaling peptide of IHH. We also identified a missense mutation in GDF5 cosegregating with a semi-dominant form of BDA1. In two families we reported two novel BDA1-associated sequence variants in BMPR1B, the gene which codes for the receptor of GDF5. In 2002, we reported a BDA1 trait linked to chromosome 5p13.3 in a Canadian kindred (BDA1B; MIM %607004) but we did not discover a BDA1-causal variant in any of the protein coding genes within the 2.8 Mb critical region. To provide a higher sensitivity of detection, we performed a targeted enrichment of the BDA1B locus followed by high-throughput sequencing. We report the identification of a novel 9.5 Kb intergenic tandem duplication in two unrelated BDA1-affected families. In-vitro and in-vivo reporter assays demonstrated the enhancer activity of noncoding conserved sequence elements found within the microduplication. We also show an upregulation of the neighboring genes, NPR3 and PDZD2, in the patients' fibroblasts that suggests a gain-of-function through the duplication of cis-regulatory elements on dose sensitive genes. By expanding the repertoire of BDA1-causing mutations in IHH, GDF5, BMPR1B and at the BDA1B locus, we have begun to elucidate a common genetic pathway underlying phalangeal formation and elongation.

Next generation sequencing technologies are changing the way scientific experiments and diseases diagnostics are performed and thus will allow what is called personalized medicine. The sense of presented thesis is to make survey of new approaches to DNA sequencing and demonstrate usage and constraints of bioinformatic analytical tools available to day. Discussed techniques are then applied to the case study of finding molecular basis for rare hereditary disease. Introductory part deals with overview of commercially available sequencing techniques (454 Life Science, Applied Biosystems, Illumina, Helicos). Fundamentals of each method are described and possible further development is outlined. Post sequencing data analysis is than discussed in details. In practical section we demonstrate genome analysis techniques successfully used to reveal causal mutation in the gene responsible for adult form of autozomal neuronal ceroid lipofuscinosis (ANCL). Combination of linkage analysis (Merlin), copy number variant analysis (Genome-Wide Human SNP Array 6.0), analysis of expression profiles (HumanRef-8 v2 Expression BeadChips) and exome sequencing (SOLiD™ 4 System) has been applied to members of one ANCL family. We also paid attention to comparison, evaluation and selection of available mapping algorithms used in...

This thesis describes the development and use of a new class of molecular tools called Selector probes, and its potential for investigations of genetic variation. The Selector technology provides multiplex amplification of targeted DNA sequences with a high specificity, and an enrichment factor in the same order of magnitude as PCR. A common feature in this thesis work is to focus the analysis on DNA regions of interest. For example, this technique can be implemented in analysing candidate regions found by whole genome studies that need validation (global to local analysis), and applications requiring detection of rare alleles (common to rare allele), important in for example cancer samples. An assay is presented that allows for fast and simple quantification of relative copy-number variations. The method was proven to be able to detect aneuploidy in chromosome 13, 18, 21 and X, with a resolution enough to distinguish between 4 and 5 copies. The method was successfully applied to solve a biological question regarding a copy-number variation, that explains the Ridge phenotype typical for the dog bread Rhodesian Ridgebacks. The Selector strategy was able to detect and map a tandem duplication with a size of 133 kb, which was characterized with base-pair resolution. A readout platform that facilitates simultaneous digital quantitative analysis of a large numbers of biomolecules is further introduced. The work involves arraying amplified product from successful selection and decoding each molecule by hybridization of fluorophore labeled oligonucleotides. Finally, a genome partitioning method which is applied upstream of next generation sequencing platforms is presented. It is shown that the method provides successful enrichment with 98 % coverage and 94 % specificity and high enrichment uniformity. The technique was applied for mutation analysis of 26 cancer-related genes in tumor cell-lines and tissue.

[pt] Este trabalho apresenta técnicas de reordenação para minimização de banda, perfil e frente de malhas de elementos finitos. Um conceito unificado relacionando as malhas de elementos finitos, os grafos associados e as matrizes correspondentes é proposto. As informações geométricas, disponíveis nos programans de elemnetos finitos, são utilizadas para aumentar a eficiência dos algoritmos heurísticos. Com base nestas idéias, os algoritmos são classificados em topológicos, geométricos, híbridos e espectrais. Um Grafo de Elementos Finitos - Finite Element Graph (FEG)- é definido coo um grafo nodal(G), um garfo dual(G) ou um grafo de comunicação(G.), associado a uma dada malha de elementos finitos. Os algoritmos topológicos mais utilizados na literatura técnica, tais como, Reverse- CuthiiMcKee (RCM), Collins, Gibbs-Poole-Stockmeyer(GPS), Gibbs-King (GK), Snay e Sloan, são inventigados detalhadamente. Em particular, o algoritmo de Collins é estendido para consideração de componentes não conexos nos grafos associados e a numeração é invertida para uma posterior redução do perfil das matrizer correspondentes. Essa nova versão é denominada Modified Reverse Collins (MRCollins). Um algoritmo puramente geométrico, denominado Coordinate Based Bandwidth and Profile Reduction (CBBPR), é apresentado. Um novo algoritmo híbrido (HybWP) para redução de frente e perfil é proposto. A matriz Laplaciana [L(G), L(G) ou L (G.)], utilizada no estudo de propriedades espectrais de grafos, é construída a partir das relações usuais de adjacências entre vértices e arestas. Um algoritmo automático, baseado em propriedades espectrais de FEGs, é proposto para reordenação de nós e/ou elementos das malhas associadas. Este algoritmo, denominado Spectral FEG Resequencing (SFR), utiliza informações globais do grafo; não depende da escolha de um vértice pseudo- periférico; e não utiliza o conceito de estrutura de níveis. Um novo algoritmo espectral para determinação de vértices pseudo-periféricos em grafos também é proposto. Os algoritmos apresentados neste trabalho são implementados computacionalmente e testados utilizando- se diversos exemplos numéricos. Finalmente, conclusões são apresentadas e algumas sugestões para trabalhos futuros são propostas. / [en] This work presents resequencing techniques for minimizing bandwidth, profile and wavefront of finite element meshes. A unified approach relating a finite element mesh, its associated graphs, and the corresponding matrices is proposed. The geometrical information available from conventional finite element program is also used in order to improve heuristic algorithms. Following these ideas, the algorithms are classified here as a nodal graph (G), a dual graph (G) or a communication graph (G.) associated with a generic finie element mesh. The most widely used topological algorithms, such as Reverse-Cuthill-McKee (RCM), Collins, Gibbs-Poole-Stockmeyer (GPS), Gibbs-King (GK), Snay, and Sloan, are investigated in detail. In particular, the Collins algorithm is extended to consider nonconnected components in associated graph and the ordering provide by this algorithm is reverted for improved profile. This new version is called Modified Reverse Collins (MRCollins). A purely geometrical algorithm, called Coordinate Based Bandwidth and Profile Reduction (CBBPR), is presented. A new hybrid reordering algorithm (HybWP) for wavefront and profile reduction is proposed. The Laplacian matrix [L(G), L(G) or L(G.)], used for the study of spectral properties of an FEG, is constructed from usual vertex and edge conectivities of a graph. An automatic algorithm, based on spectral properties of an FEG, is proposed to reorder the nodes and/or elements of the associated finite element meshes. The new algorithm, called Spectral FEG Resequencing (SFR), uses global information in the graph; it does not depende on a pseudoperipheral vertex in the resequencing process; and it does not use any kind of level structure of the graph. A new spectral algorithm for finding pseudoperipheral vertices in graphs is also proposed. The algorithmpresented herein are computationally implemented and tested against several numerical examples. Finally, conclusions are drawn and directions for futue work are given.