The value of idiosyncratic markers and changes to conserved tRNA sequences from the mitochondrial genome of hard ticks (Acari: Ixodida: Ixodidae) for phylogenetic inference

Idiosyncratic markers are features of genes and genomes that are so unusual that it is unlikely that they evolved more than once in a lineage of organisms. Here we explore further the potential of idiosyncratic markers and changes to typically conserved tRNA sequences for phylogenetic inference. Hard ticks were chosen as the model group because their phylogeny has been studied extensively. Fifty-eight candidate markers from hard ticks ( family Ixodidae) and 22 markers from the subfamily Rhipicephalinae sensu lato were mapped onto phylogenies of these groups. Two of the most interesting markers, features of the secondary structure of two different tRNAs, gave strong support to the hypothesis that species of the Prostriata ( Ixodes spp.) are monophyletic. Previous analyses of genes and morphology did not strongly support this relationship, instead suggesting that the Prostriata is paraphyletic with respect to the Metastriata ( the rest of the hard ticks). Parallel or convergent evolution was not found in the arrangements of mitochondrial genes in ticks nor were there any reversals to the ancestral arthropod character state. Many of the markers identified were phylogenetically informative, whereas others should be informative with study of additional taxa. Idiosyncratic markers and changes to typically conserved nucleotides in tRNAs that are phylogenetically informative were common in this data set, and thus these types of markers might be found in other organisms.

Prospects for whole genome linkage disequilibrium mapping in domestic dog breeds

Linkage disequilibrium (LD) mapping is commonly used as a fine mapping tool in human genome mapping and has been used with some success for initial disease gene isolation in certain isolated inbred human populations. An understanding of the population history of domestic dog breeds suggests that LID mapping could be routinely utilized in this species for initial genome-wide scans. Such an approach offers significant advantages over traditional linkage analysis. Here, we demonstrate, using canine copper toxicosis in the Bedlington terrier as the model, that LID mapping could be reasonably expected to be a useful strategy in low-resolution, genome-wide scans in pure-bred dogs. Significant LID was demonstrated over distances up to 33.3 cM. It is very unlikely, for a number of reasons discussed, that this result could be extrapolated to the rest of the genome. It is, however, consistent with the expectation given the population structure of canine breeds and, in this breed at least, with the hypothesis that it may be possible to utilize LID in a genome-wide scan. In this study, LD mapping confirmed the location of the copper toxicosis in Bedlington terrier gene (CT-BT) and was able to do so in a population that was refractory to traditional linkage analysis.

Identification of mating type genes in the bipolar basidiomycetous yeast rhodosporidium toruloides: first insight into the MAT Locus structure of the sporidiobolales

Eukaryotic Cell, Vol.7, Nº6

Impact of chromossomal structure on the evolution of Schizosaccharomyces pombe undergoing Mutation Accumulation

Large chromosomal rearrangements are common in natural populations and thought to be involved in speciation events. In this project, we used experimental evolution to determine how the speed of evolution and the type of accumulated mutations depend on the ancestral chromosomal structure and genotype. We utilized two Wild Type strains and a set of genetically engineered Schizosaccharomyces pombe strains, different solely in the presence of a certain type of chromosomal variant (inversions or translocations), along with respective controls. Previous research has shown that these chromosomal variants have different fitness levels in several environments, probably due to changes in the gene expression along the genome. These strains were propagated in the laboratory at very low population sizes, in which we expect natural selection to be less efficient at purging deleterious mutations. We then measured these strains’ changes in fitness throughout this accumulation of deleterious mutations, comparing the evolutionary trajectories in the different rearrangements to understand if the chromosomal structure affected the speed of evolution. We also tested these mutations for possible epistatic effects and estimated their parameters: the number of arising deleterious mutations per generation (Ud) and each one’s mean effect (sd).

Structured RNAs in the ENCODE selected regions of the human genome.

Functional RNA structures play an important role both in the context of noncoding RNA transcripts as well as regulatory elements in mRNAs. Here we present a computational study to detect functional RNA structures within the ENCODE regions of the human genome. Since structural RNAs in general lack characteristic signals in primary sequence, comparative approaches evaluating evolutionary conservation of structures are most promising. We have used three recently introduced programs based on either phylogenetic-stochastic context-free grammar (EvoFold) or energy directed folding (RNAz and AlifoldZ), yielding several thousand candidate structures (corresponding to approximately 2.7% of the ENCODE regions). EvoFold has its highest sensitivity in highly conserved and relatively AU-rich regions, while RNAz favors slightly GC-rich regions, resulting in a relatively small overlap between methods. Comparison with the GENCODE annotation points to functional RNAs in all genomic contexts, with a slightly increased density in 3'-UTRs. While we estimate a significant false discovery rate of approximately 50%-70% many of the predictions can be further substantiated by additional criteria: 248 loci are predicted by both RNAz and EvoFold, and an additional 239 RNAz or EvoFold predictions are supported by the (more stringent) AlifoldZ algorithm. Five hundred seventy RNAz structure predictions fall into regions that show signs of selection pressure also on the sequence level (i.e., conserved elements). More than 700 predictions overlap with noncoding transcripts detected by oligonucleotide tiling arrays. One hundred seventy-five selected candidates were tested by RT-PCR in six tissues, and expression could be verified in 43 cases (24.6%).

Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project.

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

The amphioxus genome and the evolution of the chordate karyotype.

Lancelets ('amphioxus') are the modern survivors of an ancient chordate lineage, with a fossil record dating back to the Cambrian period. Here we describe the structure and gene content of the highly polymorphic approximately 520-megabase genome of the Florida lancelet Branchiostoma floridae, and analyse it in the context of chordate evolution. Whole-genome comparisons illuminate the murky relationships among the three chordate groups (tunicates, lancelets and vertebrates), and allow not only reconstruction of the gene complement of the last common chordate ancestor but also partial reconstruction of its genomic organization, as well as a description of two genome-wide duplications and subsequent reorganizations in the vertebrate lineage. These genome-scale events shaped the vertebrate genome and provided additional genetic variation for exploitation during vertebrate evolution.

Copy number variation and chromatin structure

The functional consequences of structural variation in the human genome range from adaptation, to phenotypic variation, to predisposition to diseases. Copy number variation (CNV) was shown to influence the phenotype by modifying, in a somewhat dose-dependent manner, the expression of genes that map within them, as well as that of genes located on their flanks. To assess the possible mechanism(s) behind this neighboring effect, we compared histone modification status of cell lines from patients affected by Williams-Beuren, Williams-Beuren region duplication, Smith-Magenis or DiGeorge Syndrome and control individuals using a high-throughput version of chromatin immuno-precipitation method (ChIP), called ChlP-seq. We monitored monomethylation of lysine K20 on histone H4 and trimethylation of lysine K27 on histone H3, as proxies for open and condensed chromatin, respectively. Consistent with the changes in expression levels observed for multiple genes mapping on the entire length of chromosomes affected by structural variants, we also detected regions with modified histone status between samples, up- and downstream from the critical regions, up to the end of the rearranged chromosome. We also gauged the intrachromosomal interactions of these cell lines utilizing chromosome conformation capture (4C-seq) technique. We observed that a set of genes flanking the Williams-Beuren Syndrome critical region (WBSCR) were often looping together, possibly forming an interacting cluster with each other and the WBSCR. Deletion of the WBSCR disrupts the expression of this group of flanking genes, as well as long-range interactions between them and the rearranged interval. We conclude, that large genomic rearrangements can lead to changes in the state of the chromatin spreading far away from the critical region, thus possibly affecting expression globally and as a result modifying the phenotype of the patients. - Les conséquences fonctionnelles des variations structurelles dans le génome humain sont vastes, allant de l'adaptation, en passant par les variations phénotypiques, aux prédispositions à certaines maladies. Il a été démontré que les variations du nombre de copies (CNV) influencent le phénotype en modifiant, d'une manière plus ou moins dose-dépendante, l'expression des gènes se situant à l'intérieur de ces régions, mais également celle des gènes se trouvant dans les régions flanquantes. Afin d'étudier les mécanismes possibles sous-jacents à cet effet de voisinage, nous avons comparé les états de modification des histones dans des lignées cellulaires dérivées de patients atteints du syndrome de Williams-Beuren, de la duplication de la région Williams-Beuren, du syndrome de Smith-Magenis ou du syndrome de Di- George et d'individus contrôles en utilisant une version haut-débit de la méthode d'immunoprécipitation de la chromatine (ChIP), appelée ChIP-seq. Nous avons suivi la mono-méthylation de la lysine K20 sur l'histone H4 et la tri-méthylation de la lysine K27 sur l'histone H3, marqueurs respectifs de la chromatine ouverte et fermée. En accord avec les changements de niveaux d'expression observés pour de multiples gènes tout le long des chromosomes affectés par les CNVs, nous avons aussi détecté des régions présentant des modifications d'histones entre les échantillons, situées de part et d'autre des régions critiques, jusqu'aux extrémités du chromosome réarrangé. Nous avons aussi évalué les interactions intra-chromosomiques ayant lieu dans ces cellules par l'utilisation de la technique de capture de conformation des chromosomes (4C-seq). Nous avons observé qu'un groupe de gènes flanquants la région critique du syndrome de Williams-Beuren (WBSCR) forment souvent une boucle, constituant un groupe d'interactions privilégiées entre ces gènes et la WBSCR. La délétion de la WBSCR perturbe l'expression de ce groupe de gènes flanquants, mais également les interactions à grande échelle entre eux et la région réarrangée. Nous en concluons que les larges réarrangements génomiques peuvent aboutir à des changements de l'état de la chromatine pouvant s'étendre bien plus loin que la région critique, affectant donc potentiellement l'expression de manière globale et ainsi modifiant le phénotype des patients.

Trypanosoma cruzi: ancestral genomes and population structure

Although the genome of Trypanosoma cruzi has been completely sequenced, little is known about its population structure and evolution. Since 1999, two major evolutionary lineages presenting distinct epidemiological characteristics have been recognised: T. cruzi I and T. cruzi II. We describe new and important aspects of the population structure of the parasite, and unequivocally characterise a third ancestral lineage that we propose to name T. cruzi III. Through a careful analysis of haplotypes (blocks of genes that are stably transmitted from generation to generation of the parasite), we inferred at least two hybridisation events between the parental lineages T. cruzi II and T. cruzi III. The strain CL Brener, whose genome was sequenced, is one such hybrid. Based on these results, we propose a simple evolutionary model based on three ancestral genomes, T. cruzi I, T. cruzi II and T. cruzi III. At least two hybridisation events produced evolutionarily viable progeny, and T. cruzi III was the cytoplasmic donor for the resulting offspring (as identified by the mitochondrial clade of the hybrid strains) in both events. This model should be useful to inform evolutionary and pathogenetic hypotheses regarding T. cruzi.

Molecular genetics of rare hereditary diseases via genome-wide and integrated approaches

ABSTRACTThe Online Mendelian Inheritance in Man database (OMIM) reports about 3000 Mendelian diseases of known causal gene and about 2000 that remain to be mapped. These cases are often difficult to solve because of the rareness of the disease, the structure of the family (too big or too small) or the heterogeneity of the phenotype. The goal of this thesis is to explore the current genetic tools, before the advent of ultra high throughput sequencing, and integrate them in the attempt to map the genes behind the four studied cases. In this framework we have studied a small family with a recessive disease, a modifier gene for the penetrance of a dominant mutation, a large extended family with a cardiac phenotype and clinical and/or allelic heterogeneity and we have molecularly analyzed a balanced chromosomal translocation.RESUMELa base de données des maladies à transmission mendélienne, Online Mendelian Inheritance in Man (OMIM), contient environ 3000 affections à caractère mendélien pour lesquelles le gène responsable est connu et environ 2000 qui restent à élucider.Les cas restant à résoudre sont souvent difficiles soit par le caractère intrinsèquement rare de ces maladies soit à cause de difficultés structurelles (famille trop petite ou trop étendue) ou hétérogénéité du phénotype ou génétique. Cette thèse s'inscrit avant l'arrivée des nouveaux outils de séquençage à haut débit. Son but est d'explorer les outils génétiques actuels, et de les intégrer pour trouver les gènes impliqués dans quatre cas représentant chacun une situation génétique différente : nous avons étudié une famille de quatre individus avec une transmission récessive, recherché un gène modificateur de la pénétrance de mutations dominantes, étudié une famille étendue présentant un phénotype cardiaque cliniquement et/ou allèliquement hétérogène et nous avons fait l'analyse moléculaire d'une translocation chromosomique balancée.

Genome-wide association study identifies two loci strongly affecting transferrin glycosylation.

Polysaccharide sidechains attached to proteins play important roles in cell-cell and receptor-ligand interactions. Variation in the carbohydrate component has been extensively studied for the iron transport protein transferrin, because serum levels of the transferrin isoforms asialotransferrin + disialotransferrin (carbohydrate-deficient transferrin, CDT) are used as biomarkers of excessive alcohol intake. We conducted a genome-wide association study to assess whether genetic factors affect CDT concentration in serum. CDT was measured in three population-based studies: one in Switzerland (CoLaus study, n = 5181) and two in Australia (n = 1509, n = 775). The first cohort was used as the discovery panel and the latter ones served as replication. Genome-wide single-nucleotide polymorphism (SNP) typing data were used to identify loci with significant associations with CDT as a percentage of total transferrin (CDT%). The top three SNPs in the discovery panel (rs2749097 near PGM1 on chromosome 1, and missense polymorphisms rs1049296, rs1799899 in TF on chromosome 3) were successfully replicated , yielding genome-wide significant combined association with CDT% (P = 1.9 × 10(-9), 4 × 10(-39), 5.5 × 10(-43), respectively) and explain 5.8% of the variation in CDT%. These allelic effects are postulated to be caused by variation in availability of glucose-1-phosphate as a precursor of the glycan (PGM1), and variation in transferrin (TF) structure.

Genetics for clinicians: from candidate genes to whole genome scans (technological advances).

Human genetics has progressed at an unprecedented pace during the past 10 years. DNA microarrays currently allow screening of the entire human genome with high level of coverage and we are now entering the era of high-throughput sequencing. These remarkable technical advances are influencing the way medical research is conducted and have boosted our understanding of the structure of the human genome as well as of disease biology. In this context, it is crucial for clinicians to understand the main concepts and limitations of modern genetics. This review will describe key concepts in genetics, including the different types of genetic markers in the human genome, review current methods to detect DNA variation, describe major online public databases in genetics, explain key concepts in statistical genetics and finally present commonly used study designs in clinical and epidemiological research. This review will therefore concentrate on human genetic variation analysis.

Structured RNAs in the ENCODE selected regions of the human genome

Functional RNA structures play an important role both in the context of noncoding RNA transcripts as well as regulatory elements in mRNAs. Here we present a computational study to detect functional RNA structures within the ENCODE regions of the human genome. Since structural RNAs in general lack characteristic signals in primary sequence, comparative approaches evaluating evolutionary conservation of structures are most promising. We have used three recently introduced programs based on either phylogenetic–stochastic context-free grammar (EvoFold) or energy directed folding (RNAz and AlifoldZ), yielding several thousand candidate structures (corresponding to ∼2.7% of the ENCODE regions). EvoFold has its highest sensitivity in highly conserved and relatively AU-rich regions, while RNAz favors slightly GC-rich regions, resulting in a relatively small overlap between methods. Comparison with the GENCODE annotation points to functional RNAs in all genomic contexts, with a slightly increased density in 3′-UTRs. While we estimate a significant false discovery rate of ∼50%–70% many of the predictions can be further substantiated by additional criteria: 248 loci are predicted by both RNAz and EvoFold, and an additional 239 RNAz or EvoFold predictions are supported by the (more stringent) AlifoldZ algorithm. Five hundred seventy RNAz structure predictions fall into regions that show signs of selection pressure also on the sequence level (i.e., conserved elements). More than 700 predictions overlap with noncoding transcripts detected by oligonucleotide tiling arrays. One hundred seventy-five selected candidates were tested by RT-PCR in six tissues, and expression could be verified in 43 cases (24.6%).

Structure and expression profile of the Arabidopsis PHO1 gene family indicates a broad role in inorganic phosphate homeostasis.

PHO1 has been recently identified as a protein involved in the loading of inorganic phosphate into the xylem of roots in Arabidopsis. The genome of Arabidopsis contains 11 members of the PHO1 gene family. The cDNAs of all PHO1 homologs have been cloned and sequenced. All proteins have the same topology and harbor a SPX tripartite domain in the N-terminal hydrophilic portion and an EXS domain in the C-terminal hydrophobic portion. The SPX and EXS domains have been identified in yeast (Saccharomyces cerevisiae) proteins involved in either phosphate transport or sensing or in sorting proteins to endomembranes. The Arabidopsis genome contains additional proteins of unknown function containing either a SPX or an EXS domain. Phylogenetic analysis indicated that the PHO1 family is subdivided into at least three clusters. Reverse transcription-PCR revealed a broad pattern of expression in leaves, roots, stems, and flowers for most genes, although two genes are expressed exclusively in flowers. Analysis of the activity of the promoter of all PHO1 homologs using promoter-beta-glucuronidase fusions revealed a predominant expression in the vascular tissues of roots, leaves, stems, or flowers. beta-Glucuronidase expression is also detected for several promoters in nonvascular tissue, including hydathodes, trichomes, root tip, root cortical/epidermal cells, and pollen grains. The expression pattern of PHO1 homologs indicates a likely role of the PHO1 proteins not only in the transfer of phosphate to the vascular cylinder of various tissues but also in the acquisition of phosphate into cells, such as pollen or root epidermal/cortical cells.

Genome-Wide Prediction Methods in Highly Diverse and Heterozygous Species: Proof-of-Concept through Simulation in Grapevine.

Nowadays, genome-wide association studies (GWAS) and genomic selection (GS) methods which use genome-wide marker data for phenotype prediction are of much potential interest in plant breeding. However, to our knowledge, no studies have been performed yet on the predictive ability of these methods for structured traits when using training populations with high levels of genetic diversity. Such an example of a highly heterozygous, perennial species is grapevine. The present study compares the accuracy of models based on GWAS or GS alone, or in combination, for predicting simple or complex traits, linked or not with population structure. In order to explore the relevance of these methods in this context, we performed simulations using approx 90,000 SNPs on a population of 3,000 individuals structured into three groups and corresponding to published diversity grapevine data. To estimate the parameters of the prediction models, we defined four training populations of 1,000 individuals, corresponding to these three groups and a core collection. Finally, to estimate the accuracy of the models, we also simulated four breeding populations of 200 individuals. Although prediction accuracy was low when breeding populations were too distant from the training populations, high accuracy levels were obtained using the sole core-collection as training population. The highest prediction accuracy was obtained (up to 0.9) using the combined GWAS-GS model. We thus recommend using the combined prediction model and a core-collection as training population for grapevine breeding or for other important economic crops with the same characteristics.