Systems biology of plants : from intraspecific genome variation to computational morphodynamics

Recently, the introduction of second generation sequencing and further advance-ments in confocal microscopy have enabled system-level studies for the functional characterization of genes. The degree of complexity intrinsic to these approaches needs the development of bioinformatics methodologies and computational models for extracting meaningful biological knowledge from the enormous amount of experi¬mental data which is continuously generated. This PhD thesis presents several novel bioinformatics methods and computational models to address specific biological questions in Plant Biology by using the plant Arabidopsis thaliana as a model system. First, a spatio-temporal qualitative analysis of quantitative transcript and protein profiles is applied to show the role of the BREVIS RADIX (BRX) protein in the auxin- cytokinin crosstalk for root meristem growth. Core of this PhD work is the functional characterization of the interplay between the BRX protein and the plant hormone auxin in the root meristem by using a computational model based on experimental evidence. Hyphotesis generated by the modelled to the discovery of a differential endocytosis pattern in the root meristem that splits the auxin transcriptional response via the plasma membrane to nucleus partitioning of BRX. This positional information system creates an auxin transcriptional pattern that deviates from the canonical auxin response and is necessary to sustain the expression of a subset of BRX-dependent auxin-responsive genes to drive root meristem growth. In the second part of this PhD thesis, we characterized the genome-wide impact of large scale deletions on four divergent Arabidopsis natural strains, through the integration of Ultra-High Throughput Sequencing data with data from genomic hybridizations on tiling arrays. Analysis of the identified deletions revealed a considerable portion of protein coding genes affected and supported a history of genomic rearrangements shaped by evolution. In the last part of the thesis, we showed that VIP3 gene in Arabidopsis has an evo-lutionary conserved role in the 3' to 5' mRNA degradation machinery, by applying a novel approach for the analysis of mRNA-Seq data from random-primed mRNA. Altogether, this PhD research contains major advancements in the study of natural genomic variation in plants and in the application of computational morphodynamics models for the functional characterization of biological pathways essential for the plant. - Récemment, l'introduction du séquençage de seconde génération et les avancées dans la microscopie confocale ont permis des études à l'échelle des différents systèmes cellulaires pour la caractérisation fonctionnelle de gènes. Le degrés de complexité intrinsèque à ces approches ont requis le développement de méthodologies bioinformatiques et de modèles mathématiques afin d'extraire de la masse de données expérimentale générée, des information biologiques significatives. Ce doctorat présente à la fois des méthodes bioinformatiques originales et des modèles mathématiques pour répondre à certaines questions spécifiques de Biologie Végétale en utilisant la plante Arabidopsis thaliana comme modèle. Premièrement, une analyse qualitative spatio-temporelle de profiles quantitatifs de transcripts et de protéines est utilisée pour montrer le rôle de la protéine BREVIS RADIX (BRX) dans le dialogue entre l'auxine et les cytokinines, des phytohormones, dans la croissance du méristème racinaire. Le noyau de ce travail de thèse est la caractérisation fonctionnelle de l'interaction entre la protéine BRX et la phytohormone auxine dans le méristème de la racine en utilisant des modèles informatiques basés sur des preuves expérimentales. Les hypothèses produites par le modèle ont mené à la découverte d'un schéma différentiel d'endocytose dans le méristème racinaire qui divise la réponse transcriptionnelle à l'auxine par le partitionnement de BRX de la membrane plasmique au noyau de la cellule. Cette information positionnelle crée une réponse transcriptionnelle à l'auxine qui dévie de la réponse canonique à l'auxine et est nécessaire pour soutenir l'expression d'un sous ensemble de gènes répondant à l'auxine et dépendant de BRX pour conduire la croissance du méristème. Dans la seconde partie de cette thèse de doctorat, nous avons caractérisé l'impact sur l'ensemble du génome des délétions à grande échelle sur quatre souches divergentes naturelles d'Arabidopsis, à travers l'intégration du séquençage à ultra-haut-débit avec l'hybridation génomique sur puces ADN. L'analyse des délétions identifiées a révélé qu'une proportion considérable de gènes codant était affectée, supportant l'idée d'un historique de réarrangement génomique modelé durant l'évolution. Dans la dernière partie de cette thèse, nous avons montré que le gène VÏP3 dans Arabidopsis a conservé un rôle évolutif dans la machinerie de dégradation des ARNm dans le sens 3' à 5', en appliquant une nouvelle approche pour l'analyse des données de séquençage d'ARNm issue de transcripts amplifiés aléatoirement. Dans son ensemble, cette recherche de doctorat contient des avancées majeures dans l'étude des variations génomiques naturelles des plantes et dans l'application de modèles morphodynamiques informatiques pour la caractérisation de réseaux biologiques essentiels à la plante. - Le développement des plantes est écrit dans leurs codes génétiques. Pour comprendre comment les plantes sont capables de s'adapter aux changements environnementaux, il est essentiel d'étudier comment leurs gènes gouvernent leur formation. Plus nous essayons de comprendre le fonctionnement d'une plante, plus nous réalisons la complexité des mécanismes biologiques, à tel point que l'utilisation d'outils et de modèles mathématiques devient indispensable. Dans ce travail, avec l'utilisation de la plante modèle Arabidopsis thalicinci nous avons résolu des problèmes biologiques spécifiques à travers le développement et l'application de méthodes informatiques concrètes. Dans un premier temps, nous avons investigué comment le gène BREVIS RADIX (BRX) régule le développement de la racine en contrôlant la réponse à deux hormones : l'auxine et la cytokinine. Nous avons employé une analyse statistique sur des mesures quantitatives de transcripts et de produits de gènes afin de démontrer que BRX joue un rôle antagonisant dans le dialogue entre ces deux hormones. Lorsque ce-dialogue moléculaire est perturbé, la racine primaire voit sa longueur dramatiquement réduite. Pour comprendre comment BRX répond à l'auxine, nous avons développé un modèle informatique basé sur des résultats expérimentaux. Les simulations successives ont mené à la découverte d'un signal positionnel qui contrôle la réponse de la racine à l'auxine par la régulation du mouvement intracellulaire de BRX. Dans la seconde partie de cette thèse, nous avons analysé le génome entier de quatre souches naturelles d'Arabidopsis et nous avons trouvé qu'une grande partie de leurs gènes étaient manquant par rapport à la souche de référence. Ce résultat indique que l'historique des modifications génomiques conduites par l'évolution détermine une disponibilité différentielle des gènes fonctionnels dans ces plantes. Dans la dernière partie de ce travail, nous avons analysé les données du transcriptome de la plante où le gène VIP3 était non fonctionnel. Ceci nous a permis de découvrir le rôle double de VIP3 dans la régulation de l'initiation de la transcription et dans la dégradation des transcripts. Ce rôle double n'avait jusqu'alors été démontrée que chez l'homme. Ce travail de doctorat supporte le développement et l'application de méthodologies informatiques comme outils inestimables pour résoudre la complexité des problèmes biologiques dans la recherche végétale. L'intégration de la biologie végétale et l'informatique est devenue de plus en plus importante pour l'avancée de nos connaissances sur le fonctionnement et le développement des plantes.

Intraspecific genetic variation in arbuscular mycorrhizal fungi and its consequences for molecular biology, ecology, and development of inoculum

It has been known for some time that different arbuscular mycorrhizal fungal (AMF) taxa confer differences in plant growth. Although genetic variation within AMF species has been given less attention, it could potentially be an ecologically important source of variation. Ongoing studies on variability in AMF genes within Glomus intraradices indicate that at least for some genes, such as the BiP gene, sequence variability can be high, even in coding regions. This suggests that genetic variation within an AMF may not be selectively neutral. This clearly needs to be investigated in more detail for other coding regions of AMF genomes. Similarly, studies on AMF population genetics indicate high genetic variation in AMF populations, and a considerable amount of variation seen in phenotypes in the population can be attributed to genetic differences among the fungi. The existence of high within-species genetic variation could have important consequences for how investigations on AMF gene expression and function are conducted. Furthermore, studies of within-species genetic variability and how it affects variation in plant growth will help to identify at what level of precision ecological studies should be conducted to identify AMF in plant roots in the field. A population genetic approach to studying AMF genetic variability can also be useful for inoculum development. By knowing the amount of genetic variability in an AMF population, the maximum and minimum numbers of spores that will contain a given amount of genetic diversity can be estimated. This could be particularly useful for developing inoculum with high adaptability to different environments.

Intraseasonal variation in immune defence, body mass and hematocrit in adult house martins Delichon urbica

The intensity of parasite infections often increases during the reproductive season of the host as a result of parasite reproduction, increased parasite transmission and increased host susceptibility. We report within-individual variation in immune parameters, hematocrit and body mass in adult house martins Delichon urbica rearing nestlings in nests experimentally infested with house martin bugs Oeciacus hirundinis and birds rearing nestlings in initially parasite-free nests. From first to second broods body mass and hematocrit of breeding adult house martins decreased. In contrast leucocytes and immunoglobulins became more abundant. When their nests were infested with ectoparasites adults lost more weight compared with birds raising nestlings in nests treated with pyrethrin, whereas the decrease in hematocrit was more pronounced during infection with blood parasites. Neither experimental infestation with house martin bugs nor blood parasites had a significant effect on the amount of immune defences.

Phenotypic consequences of copy number variation: insights from Smith-Magenis and Potocki-Lupski syndrome mouse models.

A large fraction of genome variation between individuals is comprised of submicroscopic copy number variation of genomic DNA segments. We assessed the relative contribution of structural changes and gene dosage alterations on phenotypic outcomes with mouse models of Smith-Magenis and Potocki-Lupski syndromes. We phenotyped mice with 1n (Deletion/+), 2n (+/+), 3n (Duplication/+), and balanced 2n compound heterozygous (Deletion/Duplication) copies of the same region. Parallel to the observations made in humans, such variation in gene copy number was sufficient to generate phenotypic consequences: in a number of cases diametrically opposing phenotypes were associated with gain versus loss of gene content. Surprisingly, some neurobehavioral traits were not rescued by restoration of the normal gene copy number. Transcriptome profiling showed that a highly significant propensity of transcriptional changes map to the engineered interval in the five assessed tissues. A statistically significant overrepresentation of the genes mapping to the entire length of the engineered chromosome was also found in the top-ranked differentially expressed genes in the mice containing rearranged chromosomes, regardless of the nature of the rearrangement, an observation robust across different cell lineages of the central nervous system. Our data indicate that a structural change at a given position of the human genome may affect not only locus and adjacent gene expression but also "genome regulation." Furthermore, structural change can cause the same perturbation in particular pathways regardless of gene dosage. Thus, the presence of a genomic structural change, as well as gene dosage imbalance, contributes to the ultimate phenotype.

Indirect costs of parasitism are shaped by variation in the type of immune challenge and food availability

Parasites can inflict indirect fitness costs to their hosts by eliciting costly immune responses. These costs depend on the type and amount of immunostimulants presented to the host immune system but also on the amount of resources available to fuel host immune responses. Here, we investigated how the relative costs of two different types of immune challenge are modulated by variation in food availability. We injected nestling tawny owls (Strix aluco) with either 10 mu g of phytohaemagglutinin (PHA) or 20 mu g of lipopolysaccharide (LPS), and subsequently raised them under two different food regimes (food-restricted vs. ad libitum). After controlling for food consumption, we found that LPS-injected nestlings lost more body mass than PHA-injected ones only when food-restricted. We also found that body mass gain of owlets fed ad libitum decreased with the intensity of the skin swelling response against LPS, but not PHA. These experimental and correlative results suggest that nestling tawny owls suffered greater immune costs when treated with LPS than PHA, and that variation in the costs of two different types of immune challenge can be exacerbated under conditions of low food availability. Our study highlights the importance of taking into consideration the interplay between host immunity and nutrition in the study of indirect costs of parasitism.

Variation in habitat suitability does not always relate to variation in species' plant functional traits.

Habitat suitability models, which relate species occurrences to environmental variables, are assumed to predict suitable conditions for a given species. If these models are reliable, they should relate to change in plant growth and function. In this paper, we ask the question whether habitat suitability models are able to predict variation in plant functional traits, often assumed to be a good surrogate for a species' overall health and vigour. Using a thorough sampling design, we show a tight link between variation in plant functional traits and habitat suitability for some species, but not for others. Our contrasting results pave the way towards a better understanding of how species cope with varying habitat conditions and demonstrate that habitat suitability models can provide meaningful descriptions of the functional niche in some cases, but not in others.

Variation in the intensity of inbreeding depression among successive life-cycle stages and generations in gynodioecious Silene vulgaris (Caryophyllaceae).

Inbreeding depression is one of the hypotheses explaining the maintenance of females within gynodioecious plant populations. However, the measurement of fitness components in selfed and outcrossed progeny depends on life-cycle stage and the history of inbreeding. Comparative data indicate that strong inbreeding depression is more likely to occur at later life-cycle stages. We used hermaphrodite individuals of Silene vulgaris originating from three populations located in different valleys in the Swiss Alps to investigate the effect of two generations of self- and cross-fertilization on fitness components among successive stages of the life cycle in a glasshouse experiment. We detected significant inbreeding depression for most life-cycle stages including: the number of viable and aborted seeds per fruit, probability of germination, above ground biomass, probability of flowering, number of flowers per plant, flower size and pollen viability. Overall, the intensity of inbreeding depression increased among successive stages of the life cycle and cumulative inbreeding depression was significantly stronger in the first generation (delta approximately 0.5) compared with the second generation (delta approximately 0.35). We found no evidence for synergistic epistasis in our experiment. Our finding of more intense inbreeding depression during later stages of the life cycle may help to explain the maintenance of females in gynodioecious populations of S. vulgaris because purging of genetic load is less likely to occur.

Mating system and avpr1a promoter variation in primates.

It has been suggested that primate mating and social behaviours may be influenced by variation in promoter region repetitive DNA of the vasopressin receptor 1a gene (avpr1a). We show that male mating behaviour does not covary in a simple way with promoter repetitive DNA in 12 Old World primates. We found that one microsatellite (-553 bp upstream) was present in all species, irrespective of their behaviour. By contrast, two microsatellites (-3956 and -3625 bp upstream) were present only in some species, yet this variation did not correlate with behaviour. These findings agree with a recent comparative analysis of voles and show that the variation in repetitive DNA in the avpr1a promoter region does not generally explain variation in male mating behaviour. Phylogenetic analysis revealed a GAGTA motif that has been independently deleted three times and involved in another larger deletion. Importantly, the presence/absence of this GAGTA motif leads to changes in predicted transcription factor-binding sites. Given the repeated loss of this motif, we speculate that it might be of functional relevance. We suggest that such non-repetitive variation, either in indels or in sequence variation, are likely to be important in explaining interspecific variation in avpr1a expression.

Regional variation in sex ratios and sex allocation in androdioecious Mercurialis annua.

In androdioecious metapopulations, where males co-occur with hermaphrodites, the absence of males from certain populations or regions may be explained by locally high selfing rates, high hermaphrodite outcross siring success (e.g. due to high pollen production by hermaphrodites), or to stochastic processes (e.g. the failure of males to invade populations or regions following colonization or range expansion by hermaphrodites). In the Iberian Peninsula and Morocco, the presence of males with hermaphrodites in the wind-pollinated androdioecious plant Mercurialis annua (Euphorbiaceae) varies both among populations within relatively small regions and among regions, with some regions lacking males from all populations. The species is known to have expanded its range into the Iberian Peninsula from a southern refugium. To account for variation in male presence in M. annua, we test the following hypotheses: (1) that males are absent in areas where plant densities are lower, because selfing rates should be correspondingly higher; (2) that males are absent in areas where hermaphrodites produce more pollen; and (3) that males are absent in areas where there is an elevated proportion of populations in which plant density and hermaphrodite pollen production disfavour their invasion. We found support for predictions two and three in Morocco (the putative Pleistocene refugium for M. annua) but no support for any hypothesis in Iberia (the expanded range). Our results are partially consistent with a hypothesis of sex-allocation equilibrium for populations in Morocco; in Iberia, the absence of males from large geographical regions is more consistent with a model of sex-ratio evolution in a metapopulation with recurrent population turnover. Our study points to the role of both frequency-dependent selection and contingencies imposed by colonization during range expansions and in metapopulations.

Mate choice evolution, dominance effects, and the maintenance of genetic variation.

Female mate choice influences the maintenance of genetic variation by altering the mating success of males with different genotypes. The evolution of preferences themselves, on the other hand, depends on genetic variation present in the population. Few models have tracked this feedback between a choice gene and its effects on genetic variation, in particular when genes that determine offspring viability and attractiveness have dominance effects. Here we build a population genetic model that allows comparing the evolution of various choice rules in a single framework. We first consider preferences for good genes and show that focused preferences for homozygotes evolve more easily than broad preferences, which allow heterozygous males high mating success too. This occurs despite better maintenance of genetic diversity in the latter scenario, and we discuss why empirical findings of superior mating success of heterozygous males consequently do not immediately lead to a better understanding of the lek paradox. Our results thus suggest that the mechanisms that help maintain genetic diversity also have a flipside of making female choice an inaccurate means of producing the desired kind of offspring. We then consider preferences for heterozygosity per se, and show that these evolve only under very special conditions. Choice for compatible genotypes can evolve but its selective advantage diminishes quickly due to frequency-dependent selection. Finally, we show that our model reproduces earlier results on selfing, when the female choice strategy produces assortative mating. Overall, our model indicates that various forms of heterozygote-favouring (or variable) female choice pose a problem for the theory of sexual ornamentation based on indirect benefits, rather than a solution.

Identification of structural variation in mouse genomes.

Structural variation is variation in structure of DNA regions affecting DNA sequence length and/or orientation. It generally includes deletions, insertions, copy-number gains, inversions, and transposable elements. Traditionally, the identification of structural variation in genomes has been challenging. However, with the recent advances in high-throughput DNA sequencing and paired-end mapping (PEM) methods, the ability to identify structural variation and their respective association to human diseases has improved considerably. In this review, we describe our current knowledge of structural variation in the mouse, one of the prime model systems for studying human diseases and mammalian biology. We further present the evolutionary implications of structural variation on transposable elements. We conclude with future directions on the study of structural variation in mouse genomes that will increase our understanding of molecular architecture and functional consequences of structural variation.

Can we continue to neglect genomic variation in introgression rates when inferring the history of speciation? A case study in a Mytilus hybrid zone.

The use of molecular data to reconstruct the history of divergence and gene flow between populations of closely related taxa represents a challenging problem. It has been proposed that the long-standing debate about the geography of speciation can be resolved by comparing the likelihoods of a model of isolation with migration and a model of secondary contact. However, data are commonly only fit to a model of isolation with migration and rarely tested against the secondary contact alternative. Furthermore, most demographic inference methods have neglected variation in introgression rates and assume that the gene flow parameter (Nm) is similar among loci. Here, we show that neglecting this source of variation can give misleading results. We analysed DNA sequences sampled from populations of the marine mussels, Mytilus edulis and M. galloprovincialis, across a well-studied mosaic hybrid zone in Europe and evaluated various scenarios of speciation, with or without variation in introgression rates, using an Approximate Bayesian Computation (ABC) approach. Models with heterogeneous gene flow across loci always outperformed models assuming equal migration rates irrespective of the history of gene flow being considered. By incorporating this heterogeneity, the best-supported scenario was a long period of allopatric isolation during the first three-quarters of the time since divergence followed by secondary contact and introgression during the last quarter. By contrast, constraining migration to be homogeneous failed to discriminate among any of the different models of gene flow tested. Our simulations thus provide statistical support for the secondary contact scenario in the European Mytilus hybrid zone that the standard coalescent approach failed to confirm. Our results demonstrate that genomic variation in introgression rates can have profound impacts on the biological conclusions drawn from inference methods and needs to be incorporated in future studies.

Evolution of intraspecific variation : a comparative phylogenetic approach

Understanding the evolution of intraspecific variance is a major research question in evolutionary biology. While its importance to processes operating at individual and population levels is well-documented, much less is known about its role in macroevolutionary patterns. Nevertheless, both experimental and theoretical evidence suggest that the intraspecific variance is susceptible to selection, can transform into interspecific variation and, therefore, is crucial for macroevolutionary processes. The main objectives of this thesis were: (l) to investigate which factors impact evolution of intraspecific variation in Polygonaceae and determine if evolution of intraspecific variation influences species diversification; and (2) to develop a novel comparative phylogenetic method to model evolution of intraspecific variation. Using the buckwheat family, Polygonaceae, as a study system, I demonstrated which life-history and ecological traits are relevant to the evolution of intraspecific variation. I analyzed how differential intraspecific variation drives species diversification patterns. I showed with computer simulations the shortcomings of existing comparative methods with respect to intraspecific variation. I developed a novel comparative model that readily incorporates the intraspecific variance into phylogenetic comparative methods. The obtained results are complimentary, because they affect both empirical and methodological aspects of comparative analysis. Overall, I highlight that intraspecific variation is an important contributor to the macroevolutionary patterns and it should be explicitly considered in the comparative phylogenetic analysis. - En biologie évolutive comprendre l'évolution de la variance intraspécifique est un axe de recherche majeur. Bien que l'importance de cette variation soit bien documentée au niveau individuel et populationnel, on en sait beaucoup moins sur son rôle au niveau macroévolutif. Néanmoins, des preuves expérimentales et théoriques suggèrent que la variance intraspécifique est sensible à la sélection et peut se transformer en variation interspécifique. Par conséquent, elle est cruciale pour mieux comprendre les processus macroévolutifs. Les principaux objectifs de ma thèse étaient : (i) d'enquêter sur les facteurs qui affectent l'évolution de la variation intraspécifique chez les Polygonaceae et de déterminer si l'évolution de cette dernière influence la diversification des espèces, et (2) de développer une nouvelle méthode comparative permettant de modéliser l'évolution de la variation intraspécifique dans un cadre phylogénétique. En utilisant comme système d'étude la famille du sarrasin, les Polygonacées, je démontre que les traits d'histoire de vie sont pertinents pour comprendre l'évolution de la variation intraspécifique. J'ai également analysé l'influence de la variation intraspécifique au niveau de la diversification des espèces. J'ai ensuite démontré avec des données simulées les limites des méthodes comparatives existantes vis à vis de la variation intraspécifique. Finalement, j'ai développé un modèle comparatif qui intègre facilement la variance intraspécifique dans les méthodes comparatives phylogénétiques existantes. Les résultats obtenus lors de ma thèse sont complémentaires car ils abordent aspects empiriques et méthodologiques de l'analyse comparative. En conclusion, je souligne que la variation intraspécifique est un facteur important en macroévolution et qu'elle doit être explicitement considérée lors d'analyses comparatives phylogénétiques.

Isotopic variation across the Audubon's-myrtle warbler hybrid zone.

Differences in seasonal migratory behaviours are thought to be an important component of reproductive isolation in many organisms. Stable isotopes have been used with success in estimating the location and qualities of disjunct breeding and wintering areas. However, few studies have used isotopic data to estimate the movements of hybrid offspring in species that form hybrid zones. Here, we use stable hydrogen to estimate the wintering locations and migratory patterns of two common and widespread migratory birds, Audubon's (Setophaga auduboni) and myrtle (S. coronata) warblers, as well as their hybrids. These two species form a narrow hybrid zone with extensive interbreeding in the Rocky Mountains of British Columbia and Alberta, Canada, which has been studied for over four decades. Isotopes in feathers grown on the wintering grounds or early on migration reveal three important patterns: (1) Audubon's and myrtle warblers from allopatric breeding populations winter in isotopically different environments, consistent with band recovery data and suggesting that there is a narrow migratory transition between the two species, (2) most hybrids appear to overwinter in the south-eastern USA, similar to where myrtle warblers are known to winter, and (3) some hybrid individuals, particularly those along the western edge of the hybrid zone, show Audubon's-like isotopic patterns. These data suggest there is a migratory divide between these two species, but that it is not directly coincident with the centre of the hybrid zone in the breeding range. We interpret these findings and discuss them within the context of previous research on hybrid zones, speciation and migratory divides.

The case for resequencing studies of Arabidopsis thaliana accessions: mining the dark matter of natural genetic variation.

Ultra-high-throughput sequencing (UHTS) techniques are evolving rapidly and may soon become an affordable and routine tool for sequencing plant DNA, even in smaller plant biology labs. Here we review recent insights into intraspecific genome variation gained from UHTS, which offers a glimpse of the rather unexpected levels of structural variability among Arabidopsis thaliana accessions. The challenges that will need to be addressed to efficiently assemble and exploit this information are also discussed.