Sequence and gene expression variability in cultivars of oat (Avena sativa L.)

Many traits of economic importance in crop plants are quantitative, complicating the selection for desirable characteristics. Recent studies suggest a complex relationship between genotype and phenotype, with genetic variability often appearing as differences in gene expression rather than structural changes in proteins. In oat (Avena sativa L.), lipid and protein content are economically important traits. In the first of four studies reported here, partial sequences for eight genes involved in lipid or protein biosynthesis were obtained from ten oat cultivars with varying lipid and protein content. Phylogenetic analysis showed that these sequences clustered into families possibly corresponding to homeologous genes. Some cultivar- and family-specific polymorphisms were identified. In the second study, we surveyed differential gene expression between developing kernels of cultivars Kanota and Ogle by constructing reciprocal subtractive libraries. Of the 195 contig sequences obtained, only a minority had homology to characterized sequences. Grouping these sequences in categories based on gene ontology of their BLAST hits showed different profiles of expression for each cultivar. In the third study, we tested a method for transforming macroarray data consisting of dividing spot signal by the median array background. This reduced variation due to array exposure time. In the fourth study, gene expression levels were considered as quantitative traits in the Kanota x Ogle mapping population. Macroarrays featuring oat clones differentially expressed between both parents were hybridized with cDNA from the population lines. Among the 33 significant expression quantitative trait loci detected, most clustered to linkage group 29--43, a possible "hot-spot" of gene expression regulation

Using MapReduce Streaming for Distributed Life Simulation on the Cloud

Distributed software simulations are indispensable in the study of large-scale life models but often require the use of technically complex lower-level distributed computing frameworks, such as MPI. We propose to overcome the complexity challenge by applying the emerging MapReduce (MR) model to distributed life simulations and by running such simulations on the cloud. Technically, we design optimized MR streaming algorithms for discrete and continuous versions of Conway’s life according to a general MR streaming pattern. We chose life because it is simple enough as a testbed for MR’s applicability to a-life simulations and general enough to make our results applicable to various lattice-based a-life models. We implement and empirically evaluate our algorithms’ performance on Amazon’s Elastic MR cloud. Our experiments demonstrate that a single MR optimization technique called strip partitioning can reduce the execution time of continuous life simulations by 64%. To the best of our knowledge, we are the first to propose and evaluate MR streaming algorithms for lattice-based simulations. Our algorithms can serve as prototypes in the development of novel MR simulation algorithms for large-scale lattice-based a-life models.https://digitalcommons.chapman.edu/scs_books/1014/thumbnail.jp

Genotüpiseerimiskiibi andmete uudsed rakendused Euroopa geneetilise struktuuri analüüsil ning geneetilistes assotsiatsioonuuringutes

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Inimese iga fenotüüpiline tunnus kujuneb elukestvalt genotüübi ning keskkonna vahelise koosmõju tagajärjel. Inimesegeneetika üks peamisi eesmärke on mõista komplekshaiguste geneetilisi põhjuseid ning läbi selle muuta haiguste diagnoosimist täpsemaks ja ravi tõhusamaks. Inimese genoomi täisjärjestuse avaldamine on viinud genotüpiseerimistehnoloogiate kiirele arengule ning teinud võimalikuks tuvastada sadades DNA proovides samaaegselt miljoneid järjestusvariatsioone. Laiapõhjaliste genoomiuuringute tulemusena on rohkem kui 3000 DNA järjestusvariatsiooni seostatud enam kui 600 erineva komplekstunnusega. Kuna üksikud järjestusvariatsioonid kirjeldavad enamasti ära vähem kui 1% tunnuse pärilikust komponendist on ülegenoomsetes assotsiatsioonuuringutes vaja kombineerida paljude kohortide andmestikke, et oleks võimalik formuleerida statistiliselt usaldusväärseid järeldusi. Üheks selliseks populatsioonipõhiseks kohortidiks on Tartu Ülikooli Eesti Geenivaramu biobank, mis sisaldab informatsiooni 51 000 eestlase elustiili ja põetud haiguste kohta ning samas talletab ka geenidoonorite bioloogilist materjali. Käesolevas töös uuriti Kirde-Euroopa populatsioonide, sh eestlaste, paiknemist Euroopa alleelisageduste geneetilise struktuuri kaardil. Selgus, et soomlased distantseeruvad nii rootslastest kui ka teistest Loode-Euroopa populatsioonidest, samas kui eestlased paiknevad lähestikku lätlaste, leedulaste ning loode-venelastega. Antud tulemus on võimaldanud kaasata Geenivaramu andmestikku rohkem kui kaheksakümnesse rahvusvahelisse geneetilisse assotsiatsioonuuringusse. Käesolevas töös on kirjeldatud kahte uut komplekstunnusseoselist DNA järjestusvarianti. Tuginedes antud uuringutele võib väita, et ülegenoomsed genotüpiseerimiskiibi andmed võimaldavad tuvastada valimi geneetilist struktureeritust ja leida haigusriski suurendavaid DNA järjestusvariatsioone.Human complex traits arise from the new mutations as well as from the interplay between the existing genetic variants and exposure to environmental conditions. The ultimate goals of human genetics are to understand the genetic architecture of complex traits and transfer the genetic findings into medical field in order to improve disease diagnosis and treatment. The draft sequence of the human genome has lead to development of high-throughput genotyping techniques that enable cost-effective detection of millions of DNA sequence variants in large numbers of samples. These advances have made it possible to increase the number of validated complex human trait-associated loci to 3,000 independent genetic variants related to more than 600 distinct traits and diseases. A single sequence variant usually explains less than 1% of the phenotypic variance or genetic predispositions and hundreds of thousands of samples must be pooled in GWAS meta-analyses to detect statistically sound associations. The Estonian Biobank, which includes a wide range of health information, biological samples, and high-resolution genomics data for more than 50,000 samples from the Estonian population, is a valuable resource for human genetic research. The aim of the current dissertation was to fill in the gaps in the knowledge of the genetic structure of northeastern European popu¬lations using the whole-genome SNP allele frequency data. The results revealed that Finns positioned distantly from Swedes and the other northeastern Europeans, while Estonians clustered next to their geographical neighbors (Latvians, Lithuanians and northwestern Russians), and demonstrated, that the Estonian Biobank samples could be analyzed together with the other cohorts of European ancestry in large-scale gene discovery studies. Two novel associations between a complex trait and a DNA sequence variant were established. In summary, the results of this study demonstrated the importance of the whole-genome genotyping array data in detecting the genetic structure and seeking the DNA sequence variants that underlie the dynamic complexity of human phenotypes

Genetic Diversity Analysis and QTL Mapping in Pearl Millet (Pennisetum glaucum) using Diversity Arrays Technology (DArT)

It is concluded that the present study is the second report of the use of DArT technology in pearl millet and DArT markers were mapped for the first time in this species. The study has been proved to be useful for diversity assessment of inbred lines and for rapid development of reasonably-well saturated genetic linkage maps of RIL populations that can be used for precise and fine QTL mapping. It is anticipated that this DArT array will also prove useful for background genotyping in marker-assisted backcrossing programs to speed up recovery of elite recurrent parent genetic background on genomic regions outside that targeted for introgression of donor parent alleles. The rust resistance locus identified on LG 1 is a novel report and will be useful for providing a framework for marker-assisted selection and cloning of resistance genes

Social Evolution and Regulatory Architecture of Pseudomonas aeruginosa Quorum Sensing

Cell-cell communication in bacteria is understood to facilitate the coordination of population-wide cooperative behavior in the form of concerted gene expression. The opportunistic pathogen Pseudomonas aeruginosa uses such a communication mechanism to regulate a large group of genes important to virulence strategies in this bacterium. This general mechanism of communication is termed quorum sensing (QS) and restricts activation of target genes to high cell density when cooperation is beneficial. QS in P. aeruginosa, like many Gram-negative Proteobacteria, is mediated through the synthesis of diffusible N-acyl-homoserine lactone (AHL) signals by LuxI-type synthases, and recognition by LuxR-type receptors that function as transcriptional regulators. P. aeruginosa harbors two complete AHL QS synthase receptor pairs termed LasI R and RhlI R. Here we use P. aeruginosa QS as a model system to investigate mechanisms that help maintain cooperative, QS-dependent secretion in the face of non-cooperating cheater mutants, and that define the cell density threshold that triggers the activation of QS target gene expression.We begin with analysis of an in vitro evolution system in which P. aeruginosa must express QS-controlled extracellular proteases in order to grow. In this system, QS-deficient cheater mutants evolve over time. They take advantage of protease production by the QS-proficient wild-type. Curiously, QS-deficient cheaters onlyreach a frequency of about 25% during the duration of the experiment. They do not enrich to levels that would cause a collapse of the population, generally referred to as a “tragedy of the commons”. Genomic sequence analysis revealed a previously unknown mutation in this system in the transcriptional regulator PsdR. Mutations in the gene coding for PsdR derepress growth rate limiting nutrient uptake and metabolism, a non-social adaptation. Combining mutational analysis with phenotypic assays and measurements of relative fitness, we show that rapid fixation of PsdR mutation in evolving populations serves to preserve cooperation and prevent a tragedy of the commons.Next, we focus on the mechanisms that determine the threshold of QS induction in P. aeruginosa. We constructed a set of isogenic mutant strains deficient in one, two, or three anti-activator proteins that serve to delay QS activation: QteE, QscR, and QslA. While these anti-activator proteins are understood to bind LasR and RhlR QS receptors, it is yet unclear why multiple anti-activators are needed, and how they work in concert to achieve the QS threshold. Using phenotypic assays, QS gene activation kinetics, and transcriptomic profiling, we found additive effects in the deletion of multiple anti-activator genes with largely overlapping sets of anti-activator-affected genes. Progressive deletion of anti-activators advances the induction threshold and increases expression levels. Our results suggest some anti-activators may even co-associate with R-proteins in exerting their effect.Together, these studies contribute new mechanistic understanding of how P. aeruginosa uses QS to coordinate cooperative behaviors to specific conditions, and how this cooperative communication system may be safeguarded against social exploitation.Keywords: social interactions, evolution, gene regulation, social evolution, quorum sensin

Geeniekspressiooni andmete integreerimine teiste ‘oomika’ andmetega kirjeldamaks endomeetriumi retseptiivsuse bioloogilisi mehhanisme

Väitekirja elektrooniline versioon ei sisalda publikatsiooneMaailma Terviseorganisatsiooni statistika väidab, et umbes 10% püsisuhetes olevatest naistest on ühel või teisel põhjusel viljatud. Naise viljakust mõjutab välja palju erinevaid faktoreid ning setõttu on viljatuse põhjuste leidmine tihti väga keeruline. Viljatust põhjustavateks faktoriteks võivad olla üldine terviseseisund, erinevad haigused, geneetiline taust, väliskeskkonna ja eluviisiga seotud tegurid. Ühe näitena võib tuua embrüo pesastumist (implantatsioon) emaka limaskesta (endomeetriumi), mis võib toimuda ainult kindla lühikese perioodi vältel (implantatsiooni aken), kui endomeetrium on embrüo suhtes kõige vastuvõtlikum. Implantatsiooni akna periood on aga iga naise jaoks erinev, ning on määratud erinevate bioloogiliste protsesside poolt. Kunstliku viljastamise (IVF) läbiviimise jaoks on kriitiline teada täpset implantatsiooni akna aega, sellega seotud mehhanisme ja nende vastastikust mõju. Selleks, et uurida mehhanismide omavahelisi seoseid, panime paariviisiliselt kokku erinevaid geneetilise regulatsiooni andmekihte, milleks olid RNA, mikroRNA ja DNA metülatsiooni admed, ja mida koos nimetatakse ‘oomika’ andmekihtideks. Kokkuvõtvalt näitavad antud töö tulemused, et, võrreldes ühe ‘oomika’ andmekihi uurimisega, ‘oomika’ andmekihtide kombineerimine aitab paremini mõista endomeetriumi retseptiivsusega seotud bioloogilisi protsesse ning vältida valepositiivseid tulemusi. Antud tööga me rõhutame süsteemibioloogia ning paljude andmekihtide samaaegse kasutamise olulisust naise reproduktiivsuse bioloogiliste mehhanismide uurimisel.According to the World Health Organization, over 10% of females in a stable relationship are suffering from involuntary infertility/subfertility worldwide. Untangling the reasons for this is difficult because female reproduction is a sophisticated matter and can be affected by many factors such as health, accompanying diseases, genetic background, environment, and lifestyle. As a specific example, embryo implantation – its attachment to the uterine lining (endometrium) – occurs only during a relatively short period of time, called the window of implantation (WOI), when the endometrium is most receptive to an embryo. This is critical for a commonly used fertility treatment of in vitro fertilizaton (IVF) – and to make matters more complex, the WOI is not the same for everyone, but adjusted by an interlocking system of biological regulation mechanisms. Thus, to provide successful IVF, it is important to know these exact regulation mechanisms – and, since they interact with one another, to understand how they work together, not just individually. We used pairwise integration of data from different layers of genetic regulation, such as RNA, microRNA, and DNA methylation, called together the ‘omics’ layers, and showed the advantage of the data integration approach over the usage of just a single ‘omics’ layer. As a result, we obtained the lists of novel potential biomarkers that could regulate WOI, validated some previously known receptivity biomarkers, and showed that integration of different ‘omics’ layers helps to avoid false-positive results. With our work, we encourage other researchers in the female reproduction field to integrate several data layers for further studieshttps://www.ester.ee/record=b535138

Functional genomics in symbiotic interactions

Functional genomics provide information beyond simple genotype-phenotype relationships. Whole genome and transcriptome sequencing is used to compare existing results to sequencing data from newly sequenced species and to thereby infer functions. With this approach, also analysis of organisms that are difficult to culture, or for which no transformation methods are available, is feasible. This applies e.g. to the Glomeromycota fungi, which are obligate symbionts and their hyphae contain thousands of nuclei in a common cytoplasm. The work presented here shows the different stages of functional genomics in three parts. In the first part, a complete and gap-less assembly of the bacterial genome of Rhizobium leguminosarum Norway was created by using different 2nd and 3rd generation sequencing techniques. This assembly shows the advantages of using short read assemblies in combination with long reads. This is particularly relevant to bridge for example long repeat regions and retain base accuracy, which is also applicable for larger genomes. In the second part, the assembly and analysis of the transcriptome of the early diverging glomeromycotan fungus Geosiphon pyriformis was performed. The results of functional annotation of the non-redundant virtual transcripts show higher similarity to Asco-, Basidio-, and Zygomycota than to Glomeromycota. This indicates that the different symbiotic life-style of G. pyriformis, compared to other Glomeromycota, also demands a different setup of expressed genes. On the other hand, the absence of the “Missing Glomeromycota Core Genes” set in this transcriptome suggests that there was a common obligate symbiotic ancestor, depending on either cyanobacteria or algae. In the course of this analysis three transcripts were identified that show similarities to genes encoding amyloid proteins and pose promising candidates for functional characterization. The third part regards the functional characterization of effector candidate genes that had been identified in Rhizophagus irregularis through a genome comparison approach. Using host induced gene silencing one effector candidate gene was downregulated and showed a strong phenotype in colonization and arbuscules. Further analyses indicate that the protein encoded by this gene is involved in trehalose biosynthesis and is not an effector. The phenotype of downregulation shows that trehalose biosynthesis plays an important role in the establishment or maintenance of a functional symbiosis. In conclusion, this work demonstrates the possibilities of functional genomics: creating and analyzing complete gap-less genomes, using the acquired data to find candidate genes for specific functions, and functionally characterizing newly identified genes in vitro.Das Feld “Functional Genomics” ermöglicht Einblicke über einfache Genotyp-Phänotyp-Beziehungen hinaus. Die vollständige Genom- und Transkriptom-Sequenzierung wird verwendet, um bestehende Ergebnisse mit Sequenzierungsdaten von neu sequenzierten Arten zu vergleichen und damit auf Funktionen zu schließen. Mit diesem Ansatz ist auch eine Analyse von Organismen möglich, die schwierig zu kultivieren sind oder für die keine Transformationsmethoden verfügbar sind. Dies gilt z.B. für Glomeromycota-Pilze, welche obligate Symbionten sind und in deren Hyphen Tausende von Kernen in einem gemeinsamen Zytoplasma enthalten sind. Die hier vorgestellte Arbeit zeigt die verschiedenen Stadien der funktionellen Genomik in drei Teilen. Im ersten Teil wurde ein vollständiges und lückenloses Bakteriengenom von Rhizobium leguminosarum Norway mit verschiedenen Sequenzierungstechniken der 2. und 3. Generation erstellt. Dieses Assembly zeigt welche Vorteile die Verwendung einer Kombination aus kurzen und langen Sequenzier-Reads mit sich bringt, wie zum Beispiel die Möglichkeit lange “repeat regions” zu überbrücken, so dass sich diese Kombination auch gut auf größere Genome anwenden lässt. Im zweiten Teil wurde das Transkriptom des früh divergierenden Glomeromycota-Pilzes Geosiphon pyriformis assembliert und analysiert. Die Ergebnisse der funktionellen Annotation der “non-redundant virtual transcripts” zeigen eine höhere Ähnlichkeit zu Asco-, Basidio- und Zygomycota als zu Glomeromycota. Dies deutet darauf hin, dass der unterschiedliche symbiotische Lebensstil von G. pyriformis im Vergleich zu anderen Glomeromycota auch ein verändertes Genexpressionsprofil erfordert. Andererseits legt die Abwesenheit der "Missing Glomeromycota Core Genes" in diesem Transkriptom nahe, dass es einen gemeinsamen obligat symbiotischen Vorfahren gab, abhängig entweder von Cyanobakterien oder Algen. Im Zuge dieser Analyse wurden drei Transkripte identifiziert, die strukturell Ähnlichkeiten zu Genen aufweisen, welche für Amyloidproteinen kodieren, und vielversprechende Kandidaten für die funktionelle Charakterisierung darstellen. Der dritte Teil befasst sich mit der funktionellen Charakterisierung von Effektorkandidaten, die in Rhizophagus irregularis durch einen Genomvergleich identifiziert wurden. Unter Verwendung von "Host Induced Gene Silencing" wurde ein Kandidatengen herunterreguliert und zeigte in der Folge einen starken Phänotyp in Kolonisierung und der Form und Größe der Arbuskeln. Weitere Analysen zeigen, dass das von diesem Gen kodierte Protein an der Trehalose-Biosynthese beteiligt ist und kein Effektor ist. Der Phänotyp der Herunterregulation zeigt, dass die Trehalose-Biosynthese eine wichtige Rolle bei der Bildung oder Aufrechterhaltung einer funktionellen Symbiose spielt. Zusammenfassend zeigt diese Arbeit die umfangreichen Möglichkeiten von “functional genomics” auf: Erstellung und Analyse vollständiger lückenloser Genome, Verwendung der gewonnenen Daten, um Kandidatengene für spezifische Funktionen zu finden, und funktionelle Charakterisierung dieser so identifizierten Gene in vitro