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The current data revolution is characterized by the proliferation of massive data in all areas of economic activity and in sciences as well. In particular, this scientific data revolution concerns molecular biology. The study of gene expression in an organism is the key example in this thesis. Gene expression data are typically characterized by a high number of descriptive variables for a limited number of observations. Identifying the relevant variables is a crucial step for data exploitation and interpretation. This thesis focuses on the issue of variable selection in the statistical framework of high-dimensional Gaussian linear regression. The core of our analysis is based on introducing new penalty functions for the least squares adjustment criterion. These penalties depend on constants, considered as hyperparameters to be calibrated on the available data set. The originality of our approach lies in introducing the False Discovery Rate (FDR) to perform this calibration. First, we establish theoretical lower and upper bounds of the FDR when the variables are ordered. Then we set up a calibration algorithm of the hyperparameter to satisfy a trade-off between the predictive risk control and the FDR one. To select non-ordered variables in the high-dimension setting, we revisit the topic of variable selection via the minimization of a convex criterion such as the Lasso. We propose an approach to select the variables, which are ordered by the regularization path, via an adaptive penalization method. Intensive simulations show the interest in resampling and in non-asymptotic penalties. We generalize the adaptive penalty calibration method called "the slope heuristics" to calibrate two hyperparameters simultaneously and in the context of a random model collection. Finally, our new algorithm and some variable selection procedures are applied to a transcriptomic dataset of Arabidopsis thaliana. The biological problem here consists in identifying transcription factors of target genes., La révolution des données que nous connaissons aujourd'hui se caractérise par la prolifération de données massives dans tous les domaines d'activités économiques, mais aussi dans les sciences. Cette révolution des données scientifiques concerne en particulier la biologique moléculaire. L'étude de l'expression des gènes d'un organisme est l'exemple clé mis en avant dans cette thèse. Les données d'expression de gènes sont typiquement caractérisées par un nombre élevé de variables descriptives pour un nombre d'observations restant limité. Identifier les variables pertinentes constitue une étape cruciale pour l'exploitation des données ainsi que leur interprétation. Cette thèse est centrée sur la question de la sélection de variables dans le cadre statistique de la régression linéaire gaussienne en grande dimension. Le cœur de notre analyse repose sur l'introduction de nouvelles fonctions de pénalité pour le critère d'ajustement des moindres carrés. Celles-ci dépendent de constantes, que nous voyons comme des hyperparamètres à calibrer sur le jeu de données d'étude. L'originalité de notre approche réside en l'introduction du False Discovery Rate (FDR) pour réaliser cette calibration. Dans un premier temps, nous prouvons un encadrement théorique du FDR lorsque les variables sont ordonnées, puis nous mettons en place un algorithme de calibration de l'hyperparamètre pour satisfaire un compromis entre le contrôle du risque prédictif et celui du FDR. Pour sélectionner des variables non-ordonnées en grande dimension, nous revisitons le thème de la sélection de variables via la minimisation d'un critère convexe de type Lasso. Nous proposons une approche qui consiste à choisir les variables, ordonnées par le chemin de régularisation, via une méthode de pénalisation adaptative. Des simulations intensives mettent en évidence l'intérêt du ré-échantillonage et des pénalités non-asymptotiques. Nous généralisons la méthode de calibration adaptative de pénalité dite "de l'heuristique de pente" à la calibration de deux hyperparamètres simultanément ainsi qu'au contexte d'une collection de modèles aléatoires qui est ici le nôtre. Enfin, notre nouvel algorithme, ainsi que certaines procédures de sélection de variables, sont appliqués sur un jeu de données transcriptomiques d'Arabidopsis thaliana. L'identification des facteurs de transcription de gènes cibles constitue ici la problématique biologique.

Session : Génétique et Sélection; Masculinisation spontanée chez la truite arc-en-ciel : de nouvelles régions du génome identifiées. 6. Journées de la Recherche Filière Piscicole

BackgroundThe genus Oryzias is constituted of 35 medaka-fish species each exhibiting various ecological, morphological and physiological peculiarities and adaptations. Beyond of being a comprehensive phylogenetic group for studying intra-genus evolution of several traits like sex determination, behaviour, morphology or adaptation through comparative genomic approaches, all medaka species share many advantages of experimental model organisms including small size and short generation time, transparent embryos and genome editing tools for reverse and forward genetic studies. The Java medaka, Oryzias javanicus, is one of the two species of medaka perfectly adapted for living in brackish/sea-waters. Being an important component of the mangrove ecosystem, O. javanicus is also used as a valuable marine test-fish for ecotoxicology studies. Here, we sequenced and assembled the whole genome of O. javanicus, and anticipate this resource will be catalytic for a wide range of comparative genomic, phylogenetic and functional studies.FindingsComplementary sequencing approaches including long-read technology and data integration with a genetic map allowed the final assembly of 908 Mbp of the O. javanicus genome. Further analyses estimate that the O. javanicus genome contains 33% of repeat sequences and has a heterozygosity of 0.96%. The achieved draft assembly contains 525 scaffolds with a total length of 809.7 Mbp, a N50 of 6.3 Mbp and a L50 of 37 scaffolds. We identified 21454 expressed transcripts for a total transcriptome size of 57, 146, 583 bps.ConclusionsWe provide here a high-quality draft genome assembly of the euryhaline Javafish medaka, and give emphasis on the evolutionary adaptation to salinity.

International audience; In plants, a key class of genes comprising most of disease resistance (R) genes encodes Nucleotide-binding leucine-rich repeat (NL) proteins. Access to common bean (Phaseolus vulgaris) genome sequence provides unparalleled insight into the organization and evolution of this large gene family (similar to 400 NL) in this important crop. As observed in other plant species, most common bean NL are organized in cluster of genes. However, a particularity of common bean is that these clusters are often located in subtelomeric regions close to terminal knobs containing the satellite DNA khipu. Phylogenetically related NL are spread between different chromosome ends, suggesting frequent exchanges between non-homologous chromosomes. NL peculiar location, in proximity to heterochromatic regions, led us to study their DNA methylation status using a whole-genome cytosine methylation map. In common bean, NL genes displayed an unusual body methylation pattern since half of them are methylated in the three contexts, reminiscent of the DNA methylation pattern of repeated sequences. Moreover, 90 NL were also abundantly targeted by 24 nt siRNA, with 90% corresponding to methylated NL genes. This suggests the existence of a transcriptional gene silencing mechanism of NL through the RdDM (RNA-directed DNA methylation) pathway in common bean that has not been described in other plant species.

Maternal-effect genes and egg quality: usual suspects and new players. 6. International Workshop on the Biology of Fish Gametes

The oestrogen receptor-related receptors (ERRs) are orphan nuclear receptors that were originally identified on the basis of their close homology to the oestrogen receptors. The three mammalian ERR genes participate in the regulation of vital physiological processes including reproduction, development and metabolic homeostasis. Although unique ERRs have been found in insects, data on the function and regulation of these receptors remain sparse. In the present study, a 2095-bp full-length cDNA encoding an ERR, termed AiERR, was isolated from males of the moth Agrotis ipsilon and deposited in the GenBank database under the accession number KT944662. The predicted AiERR protein shared an overall identity of 47–82% with other known insect and mammalian ERR homologues. AiERR exhibited a broad tissue expression pattern with the detection of one transcript of approximately 2 kb in the primary olfactory centres, the antennal lobes (AL). In adult males, the amount of AiERR mRNA in the AL increased concomitantly with age and responses to the female-emitted sex pheromone. Moreover, AiERR knockdown induced an inhibition in the sex pheromone-orientated flight of male. Using A. ipsilon as a model, our study demonstrates that the insect ERR is critical for the performance of male sexual behaviour, probably by acting on central pheromone processing.

International audience; We previously reported that, in multiple sclerosis (MS) patients with a progressive form of the disease, spinal cord periplaques extend distance away from plaque borders and are characterized by the co-occurrence of partial demyelination, astrocytosis and low-grade inflammation. However, transcriptomic analyses did not allow providing a comprehensive view of molecular events in astrocytes vs. oligodendrocytes. Here, we re-assessed our transcriptomic data and performed co-expression analyses to characterize astrocyte vs. oligodendrocyte molecular signatures in periplaques. We identified an astrocytosis-related co-expression module whose central hub was the astrocyte gene Cx43/GJA1 (connexin-43, also named gap junction protein α-1). Such a module comprised GFAP (glial fibrillary acidic protein) and a unique set of transcripts forming a TGFB/SMAD1/SMAD2 (transforming growth factor β/SMAD family member 1/SMAD family member 2) genomic signature. Partial demyelination was characterized by a co-expression network whose central hub was the oligodendrocyte gene NDRG1 (N-myc downstream regulated 1), a gene previously shown to be specifically silenced in the normal-appearing white matter (NAWM) of MS patients. Surprisingly, besides myelin genes, the NDRG1 co-expression module comprised a highly significant number of translation/elongation-related genes. To identify a putative cause of NDRG1 downregulation in periplaques, we then sought to identify the cytokine/chemokine genes whose mRNA levels inversely correlated with those of NDRG1. Following this approach, we found five candidate immune-related genes whose upregulation associated with NDRG1 downregulation: TGFB1(transforming growth factor β 1), PDGFC (platelet derived growth factor C), IL17D (interleukin 17D), IL33 (interleukin 33), and IL12A (interleukin 12A). From these results, we propose that, in the spinal cord periplaques of progressive MS patients, TGFB1 may limit acute inflammation but concurrently induce astrocytosis and an alteration of the translation/elongation of myelin genes in oligodendrocytes.

Chrysomela tremula is a polyvoltine oligophagous leaf beetle responsible for massive attacks on poplar trees. This beetle is an important model for understanding mechanisms of resistance to Bacillus thuringiensis (Bt) insecticidal toxins, because a resistant C. tremula strain has been found that can survive and reproduce on transgenic poplar trees expressing high levels of the Cry3Aa Bt toxin. Resistance to Cry3Aa in this strain is recessive and is controlled by a single autosomal locus. We used a larval midgut transcriptome for C. tremula to search for candidate resistance genes. We discovered a mutation in an ABC protein, member of the B subfamily homologous to P-glycoprotein, which is genetically linked to Cry3Aa resistance in C. tremula. Cultured insect cells heterologously expressing this ABC protein swell and lyse when incubated with Cry3Aa toxin. In light of previous findings in Lepidoptera implicating A subfamily ABC proteins as receptors for Cry2A toxins and C subfamily proteins as receptors for Cry1A and Cry1C toxins, this result suggests that ABC proteins may be targets of insecticidal three-domain Bt toxins in Coleoptera as well.

The Art of Artemisia As the malaria parasite, which is transmitted through mosquito vectors, develops resistance, previously useful control mechanisms are beginning to fail. Combination therapies based on the plant product artemisinin are a promising alternative. Graham et al. (p. 328 ; see the Perspective by Milhous and Weina ) have now developed a genetic map of the plant Artemisia annua from which artemisinin is derived. The results lay the foundation for improving agricultural productivity of this natural product, which is becoming increasingly important in the fight against malaria.

International audience

Genome-wide transcription profiling is a powerful technique for studying the enormous complexity of cellular states. Moreover, when applied to disease tissue it may reveal quantitative and qualitative alterations in gene expression that give information on the context or underlying basis for the disease and may provide a new diagnostic approach. However, the data obtained from high-density microarrays is highly complex and poses considerable challenges in data mining. The data requires care in both pre-processing and the application of data mining techniques. This paper addresses the problem of dealing with microarray data that come from two known classes (Alzheimer and normal). We have applied three separate techniques to discover genes associated with Alzheimer disease (AD). The 67 genes identified in this study included a total of 17 genes that are already known to be associated with Alzheimer's or other neurological diseases. This is higher than any of the previously published Alzheimer's studies. Twenty known genes, not previously associated with the disease, have been identified as well as 30 uncharacterized expressed sequence tags (ESTs). Given the success in identifying genes already associated with AD, we can have some confidence in the involvement of the latter genes and ESTs. From these studies we can attempt to define therapeutic strategies that would prevent the loss of specific components of neuronal function in susceptible patients or be in a position to stimulate the replacement of lost cellular function in damaged neurons. Although our study is based on a relatively small number of patients (four AD and five normal), we think our approach sets the stage for a major step in using gene expression data for disease modeling (i.e. classification and diagnosis). It can also contribute to the future of gene function identification, pathology, toxicogenomics, and pharmacogenomics.

Production of organic acids by microorganisms is of great importance for obtaining building-block chemicals from sustainable biomass. Extracellular accumulation of organic acids involved a series of transporters, which play important roles in the accumulation of specific organic acid while lack of systematic demonstration in eukaryotic microorganisms. To circumvent accumulation of by-product, efforts have being orchestrated to carboxylate transport mechanism for potential clue in Yarrowia lipolytica WSH-Z06. Six endogenous putative transporter genes, YALI0B19470g, YALI0C15488g, YALI0C21406g, YALI0D24607g, YALI0D20108g and YALI0E32901g, were identified. Transport characteristics and substrate specificities were further investigated using a carboxylate-transport-deficient Saccharomyces cerevisiae strain. These transporters were expressed in Y. lipolytica WSH-Z06 to assess their roles in regulating extracellular keto acids accumulation. In a Y. lipolytica T1 line over expressing YALI0B19470g, α-ketoglutarate accumulated to 46.7 g·L−1, whereas the concentration of pyruvate decreased to 12.3 g·L−1. Systematic identification of these keto acids transporters would provide clues to further improve the accumulation of specific organic acids with higher efficiency in eukaryotic microorganisms.

Session : Génétique et qualitéSession : Génétique et qualité; National audience; Selection for high prolificacy and lean growth rate in swine has been associated with a substantial increase in piglet mortality. The first 24‐48 hours after birth represent a critical period for survival. A major determinant for early survival is piglet maturity at birth which relies greatly on the process of tissue maturation during the last month of gestation. The objective of this study was to compare the progenies from Large White (LW) and Meishan (MS) breeds which differ for piglet survival and neonatal mortality.This project proposes multi‐disciplinary and multi‐omic approaches to identify the molecular and genetic basis related to maturity and perinatal survival. The metabolome was analyzed on plasma, urine, and amniotic liquid on 612 fetuses: it displayed higher heterogeneity at the end of gestation. The transcriptome of adrenal glands, muscle, liver, subcutaneous adipose tissue, and small intestine and the proteome of muscle and adipose subcutaneous tissue were analyzed on 64 fetuses. The first analyses identified the biological processes involved in development and maturity, and key genes that may explain the differences observed between LW and MS.

Workshop : Poultry; absent

Large efforts have been devoted to Medicago truncatula genomics. They offer data and tools to identify M.truncatula genes involved in genetic variation for traits related to agronomic value in crop species. This strategy is based on the hypothesis that the same genes are involved in trait variation in both species. These tools are now used to develop alfalfa genomics in order to carry out genomic studies in the target species. Identification of genes or markers in the crop species will contribute to develop tools and theory to implement marker assisted selection in alfalfa.

International audience; Potato cyst nematodes induce changes in plant host gene expression following root invasion. For an accurate comparison of gene expression by reverse transcription quantitative real-time PCR (RT-qPCR), internal reference genes are necessary for transcript normalization. Very few experimental data on suitable reference genes are available for interactions between plant and root pathogens. In this study, we tested eight potential candidate reference genes for normalizing levels of potato gene transcripts by RT-qPCR after inoculation by nematodes. The ranking of candidate reference genes was performed using RefFinder WEB-based software. Four reference genes, RPN7 (26S proteasome regulatory subunit), UBP22 (ubiquitin-specific protease 22), OXA1 (OXA1 protein), and MST2 (mercaptopyruvate sulfurtransferase), were stably expressed in roots of susceptible or resistant potato plants, infected or uninfected by Globodera pallida. A normalization factor based on data from these four genes, highly homologous between potato and tomato, was used to normalize the expression of a chitinase gene, which was induced by nematodes in roots of potatoes carrying the resistance allele at a low-effect QTL, GpaXIspl. .

Root-knot nematodes are globally the most aggressive and damaging plant-parasitic nematodes. Chemical nematicides have so far constituted the most efficient control measures against these agricultural pests. Because of their toxicity for the environment and danger for human health, these nematicides have now been banned from use. Consequently, new and more specific control means, safe for the environment and human health, are urgently needed to avoid worldwide proliferation of these devastating plant-parasites. Mining the genomes of root-knot nematodes through an evolutionary and comparative genomics approach, we identified and analyzed 15,952 nematode genes conserved in genomes of plant-damaging species but absent from non target genomes of chordates, plants, annelids, insect pollinators and mollusks. Functional annotation of the corresponding proteins revealed a relative abundance of putative transcription factors in this parasite-specific set compared to whole proteomes of root-knot nematodes. This may point to important and specific regulators of genes involved in parasitism. Because these nematodes are known to secrete effector proteins in planta, essential for parasitism, we searched and identified 993 such effector-like proteins absent from non-target species. Aiming at identifying novel targets for the development of future control methods, we biologically tested the effect of inactivation of the corresponding genes through RNA interference. A total of 15 novel effector-like proteins and one putative transcription factor compatible with the design of siRNAs were present as non-redundant genes and had transcriptional support in the model root-knot nematode Meloidogyne incognita. Infestation assays with siRNA-treated M. incognita on tomato plants showed significant and reproducible reduction of the infestation for 12 of the 16 tested genes compared to control nematodes. These 12 novel genes, showing efficient reduction of parasitism when silenced, constitute promising targets for the development of more specific and safer control means., Author Summary Plant-parasitic nematodes are annually responsible for more than $100 billion crop yield loss worldwide and those considered as causing most of the damages are root-knot nematodes. These nematodes used to be controlled by chemicals that are now banned from use because of their poor specificity and high toxicity for the environment and human health. In the absence of sustainable alternative solutions, new control means, more specifically targeted against these nematodes and safe for the environment are needed. We searched in root-knot nematode genomes, genes conserved in various plant-damaging species while otherwise absent from the genomes of non target species such as those of chordates, plants, annelids, insect pollinators and mollusks. These genes are probably important for plant parasitism and their absence from non-target species make them interesting candidates for the development of more specific and safer control means. Further bioinformatics pruning of this set of genes yielded 16 novel candidates that could be biologically tested. Using RNA interference, we knocked down each of these 16 genes in a root-knot nematode and tested the effect on plant parasitism efficiency. Out of the 16 tested genes, 12 showed a significant and reproducible diminution of infestation when silenced and are thus particularly promising.

International audience

International audience; Aquaporins (AQPs) belong to the Major Intrinsic Protein family that conducts water and other small solutes across biological membranes. This study aimed to identify and characterize AQP genes in the primary root axis of two oak species, Quercus petraea and Quercus robur. Nine putative AQP genes were cloned, and their expression was profiled in different developmental root zones by real-time PCR. A detailed examination of the predicted amino acid sequences and subsequent phylogenetic analysis showed that the isolated AQPs could be divided into two subfamilies, which included six plasma membrane intrinsic proteins (PIPs) and three tonoplast intrinsic proteins (TIPs). We characterized the anatomical features of the roots and defined three developmental root zones: the immature, transition and mature zones. Expression analysis of the AQPs was performed according to these root developmental stages. Our results showed that the expression of PIP2;3 and TIP1 was significantly higher in Quercus petraea compared with Quercus robur in the three root zones. However, PIP2;1 and TIP2;1 were found to be differentially expressed in the mature zone of the two oak species. Of the nine AQP genes identified and analyzed, we highlighted four genes that might facilitate a deeper understanding of how these two closely related tree species adapted to different environments.

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Bacterial wilt, caused by the soilborne betaproteobacterium Ralstonia solanacearum, is a major disease of solanaceous crops, throughout tropical and subtropical areas, and emerging in temperate regions. If genetic resistance is still considered as the most promising control measure, breeding for resistance to Ralstonia solanacearum (Rsol}, has been hindered for decades by the scarcity of high level resistance sources, strong genotype x environment interactions, and the huge genomic and phenotypic plasticity of the pathogen. Ralstonia solanacearum is a species complex composed of four phylotypes related to geographical origins (1: Africa-Asia, Il: American, III: Africa, IV: Indonesia). Solanaceae-pathogenic strains belong to ail four phylotypes, but most epidemiologically active strains belong to phylotype l, liA, IIB, and III, phylotype 1being the most widespread lineage. Extensive screenings of resistant accessions of tomate, eggplant and pepper towards a worldwide R.sol core-collection, lead to formalize grain Solanaceae-Rsol interactions into six main virulence profiles, and allowed to identify highly resistant accessions, mainly in eggplant and pepper. Among the rn, the eggplant MM960 was demonstrated to carry a phylotype I-resistance major dominant gene (Ersl) and two partial resistance QTLs with strain-specific expression. We identified patterns of strain -specificity, but found no evidence of phvlotvpe-specificltv.'phvlotvpe 1containing four of the six pathopprofiles. Searching for durable resistance implies both the identification of bacterial genes responsible for avirulence and virulence, and assessment of the pathogen evolutionary potential. Considering "immune" accessions (five eggplants, two peppers, one tomato) giving true incompatible interactions (no wilt and no colonization), we then searched for genetic factors associated with bacterial virulence and avirulence using comparative genomics hybridization (CGH) data. We thus identified 61 type III effectors fairly to highly associated to avirulence to eggplants Dingras, SM6, Ceylan, Surya, and MM960, peppers CA8 and Perennial, and tomate HW7996. Interestingly, some of these effectors were elsewhere shown to contribute to bacterial fitness. Focusing on the accessions MM960 and Dingras, less than 10 genes were highly associated with avirulence, among which popP2, avirulence gene interacting with the RRS1-Rresistance gene in Arabidopsis thaliana. We inferred evolutionary dynamics of the R.solanacearum species complex (RSSC) at a global scale, performing multi-Iocus sequence analysis (MLSA) and gene genealogies on a worldwide collection. Although recombination was found ubiquitous within the RSSC, we observed phylotype-specific recombination rates and demographic histories. Phylotype 1 (and, to a lesser extent, phylotype III) is highly recombinogenic, and carries molecular signatures of a recent and rapid demographic expansion. Phylotype Il is actually composed of two evolutionary lineages, one (liA) being moderately recombinogenic and expanding, and the other (IIB) being clonai and poorly expanding. Taken together, these findings strongly suggest that phylotype 1 is the most prone to adapt rapidly to new hosts and varieties, thank to its recombination and mutation rates, capacity to disseminate, and large virulence range. To decipher evolutionary dynamics leading to adaptation at the local, field scale, an experimental evolution trial is in course to monitor the impact of Ersl-carrying eggplant MM960 successive craps on the population structure and putative avirulence genes evolution.

Interest in the genomics of Eucalyptus has skyrocketed thanks to the recent sequencing of the genome of Eucalyptus grandis and to a growing number of large-scale transcriptomic studies. Quantitative reverse transcription-PCR (RT-PCR) is the method of choice for gene expression analysis and can now also be used as a high-throughput method. The selection of appropriate internal controls is becoming of utmost importance to ensure accurate expression results in Eucalyptus. To this end, we selected 21 candidate reference genes and used high-throughput microfluidic dynamic arrays to assess their expression among a large panel of developmental and environmental conditions with a special focus on wood-forming tissues. We analyzed the expression stability of these genes by using three distinct statistical algorithms (geNorm, NormFinder and ΔCt), and used principal component analysis to compare methods and rankings. We showed that the most stable genes identified depended not only on the panel of biological samples considered but also on the statistical method used. We then developed a comprehensive integration of the rankings generated by the three methods and identified the optimal reference genes for 17 distinct experimental sets covering 13 organs and tissues, as well as various developmental and environmental conditions. The expression patterns of Eucalyptus master genes EgMYB1 and EgMYB2 experimentally validated our selection. Our findings provide an important resource for the selection of appropriate reference genes for accurate and reliable normalization of gene expression data in the organs and tissues of Eucalyptus trees grown in a range of conditions including abiotic stresses.

Expanding genomic data on plant pathogens open new perspectives for the development of specific and environment friendly pest management strategies based on the inhibition of parasitism genes that are essential for the success of infection. Identifying such genes relies on accurate reverse genetics tools and the screening of pathogen knock-down phenotypes. Root-knot nematodes are major cosmopolitan crop pests that feed on a wide range of host plants. Small interfering RNAs (siRNAs) would provide a powerful tool for reverse genetics of nematode parasitism genes provided that they could (1) target genes expressed in inner tissues of infective nematodes and (2) target genes expressed during parasitism. In this study, we show that siRNAs can access inner tissues of the infective juveniles during soaking and accumulate in the esophagus, amphidial pouches and related neurons of the nematode. We provide evidence that siRNAs can trigger knock-down of the parasitism gene Mi-CRT, a calreticulin gene expressed in the esophageal glands of Meloidogyne incognita. Mi-CRT knock-down in infective juveniles affected nematode virulence. However, Mi-CRT knock-down was not persistent after plant infection, indicating that siRNA-mediated RNAi is best suited for functional analysis of genes involved in pre-parasitic stages or in the early steps of infection.

Cette publication est également parue dans la revue "Journal of Animal Science", Vol.89, E-Suppl.1.; Identification of the main esterase involved in lipolysis by Propionibacterium freudenreichii. ADSA-ASAS Annual Meeting

La rouille foliaire causée par le champignon Melampsora larici-populina (Mlp) est l’une des principales maladies affectant les peupleraies cultivées en Europe. Dans une population en ségrégation Populus deltoides x P. trichocarpa, un QTL majeur (RUS) a été caractérisé. Ce locus, hérité de P. trichocarpa, contrôle la taille des sores lors de l’infection, paramètre quantitatif de la résistance à Mlp. Ce locus avait été cartographié sur l’extrémité du chromosome 19. Cette région génomique regroupe plusieurs dizaines de gènes (cluster) qui codent des protéines avec les domaines Nucleotide Binding Site (NBS) et Leucine Rich Repeats (LRR). Cette étude porte sur la cartographie génétique et physique du locus RUS, avec l’aide du génome de P. trichocarpa. La première tâche a été d’ordonner correctement les séquences de l’extrémité du chromosome 19. Les cartes physiques locales pour chacun des haplotypes autour du locus RUS ont été construites à partir d’une banque d’ADN de grand fragment (BAC) de l’individu parent P. trichocarpa. Elles regroupent la plus grande partie du cluster de gènes NBS-LRR. La carte génétique fine a été construite avec une population en ségrégation de 2 144 individus. Le locus RUS a été cartographié sur une dans un intervalle de 0,14 cM. Le séquençage de clones BAC a permis d’identifier un seul gène NBS-LRR candidat. Dans la même population, un second locus (R1) de résistance à Mlp, a été caractérisé sur la même extrémité du chromosome 19. Il contrôle une résistance qualitative, hérité de P. deltoides. La microsynthénie entre les deux espèces a été utilisée pour cartographier finement ce locus dans un intervalle de 0,66 cM. Cette cartographie comparée a permis aussi de déterminer que R1 et RUS sont deux loci indépendants., Leaf rust caused by the fungus Melampsora larici-populina (Mlp) is one of the major diseases affecting poplar cultivation in Europe. In a segregating population Populus deltoides x P. trichocarpa, a major QTL (RUS) has been characterized. This locus inherited from P. trichocarpa, controls the size of uredinia during the infection, a parameter of quantitative resistance to Mlp. This locus was mapped on the top of chromosome 19. This genomic region contains several tens of genes arranged in cluster that encode proteins with domains Nucleotide Binding Site (NBS) and Leucine Rich Repeats (LRR). This study focuses on genetic and physical mapping of the RUS locus, with the help of the genome of P. trichocarpa. The first task was to order the correct sequence of the top of chromosome 19. Local physical maps for each haplotypes around the RUS locus were constructed from a DNA library of large fragment (BAC) of the individual parent P. trichocarpa. They comprise the largest part of the cluster of NBS-LRR genes. The fine genetic map was constructed with a segregating population of 2 144 individuals. The locus was mapped on RUS in an interval of 0.14 cM. The sequencing of BAC clones identified a single NBS-LRR candidate gene. In the same population, a second locus (R1) of Mlp resistance was characterized at the same extremity of chromosome 19. It controls a qualitative resistance inherited from P. deltoides. Microsynteny between the two species was used to finely map this locus in an interval of 0.66 cM. This comparative mapping has also determined that R1 and RUS are two independent loci.

Because of its mixed mating system and high potential gene flow, Melampsora larici-populina belongs to the group of pathogens with the highest evolutionary risk. Indeed, all qualitative resistances that have been selected by breeders during the last decades have been defeated. Although quantitative resistance (QR) is often considered more durable, a multidisciplinary approach is being conducted at INRA not only to identify the genes controlling QR but also to predict potential adaptation of the pathogen. Starting with a single Populus deltoides × P. trichocarpa F 1 pedigree, this work led to the identification of several QTLs with contrasted levels of effect and strain-specificity. Much attention is being paid to two major loci, R 1 and R US , that both map on LG XIX. Dominant allele R 1 is inherited from P. deltoides. It controls a qualitative resistance to M. larici-populina, and it was found to be statistically associated with QR levels in several P. deltoides × P. trichocarpa F 1 progenies. Dominant allele R US is inherited from P. trichocarpa. It has major effect of uredinia size and other QR components in both intraspecific and interspecific F 1 progenies. Fine mapping of R US is being conducted using a 1300 genotype F 1 host progeny, and a BAC library was constructed to generate a physical map of 2000 kb around R US . A promising BAC clone has been identified and sequenced. In parallel, an expressional approach based on the analysis of R US and r US infected material generated transcriptional and translational candidates. On the pathogen side, one strain was found to be able to circumvent R US , thus offering the opportunity to study the frequency and the mechanisms of adaptation to QR. Several populations of M. larici-populina that underwent contrasted selection pressures by R US are being characterized for their phenotypes and for their genotypes at several microsatellite loci. The first results demonstrate the existence of preadaptation in “wild” pathogen Populations and a clear selective pressure by the host.

Association studies aim at linking phenotypic variation with allelic variation in candidate genes and benefit from several generations of recombination in natural populations to identify causative polymorphisms. Such association studies are under development on Populus nigra L., with emphasis on nucleotide polymorphism associated to quantitative resistance to Melampsora larici-populina Kleb (Mlp), the causal agent of leaf rust. Populus nigra, the natural host of Mlp, is widely represented in European riparian ecosystems, where the occurrence and impact of the fungus are highly variable. Geographical patterns of nucleotide diversity in genes involved in the host-pathogen interaction is expected from local co-evolution. As a first step, Single Nucleotide Polymorphisms (SNPs) have been identified in a discovery panel of twenty-one P. nigra genotypes covering French natural range. Sequencing was done on both functional and positional candidate resistance genes (closeness to mapped QTLs and resistance genes). An automated procedure for primer design allowed us to obtain successful amplification in P. nigra for 90% of gene fragments. Average nucleotide variation in P. nigra is high (1 SNP/97 pb). A collection of more than 1100 P. nigra genotypes collected in different natural populations mainly in France but also in Germany, Netherlands, Italy and Spain was evaluated for rust resistance under natural inoculation in a clonal test in France and under artificial inoculations with different strains in leaf-disk bioassays. Even if most of the genetic variation for rust resistant could be observed at the individual (genotype) level, significant differentiation between natural populations was detected. The high level of genetic diversity observed at both gene and phenotypic levels, the rapid linkage disequilibrium decay between SNP is offering a favourable situation to identify interesting resistance alleles and to avoid false positive associations.

International audience; We present theoretical and practical advances on the Guided Genome Halving problem, a combinatorial optimisation problem which aims at proposing ancestral configurations of extant genomes when one of them has undergone a whole genome duplication. We provide a lower bound on the optimal solution, devise a heuristic algorithm based on subgraph identification, and apply it to yeast gene order data. On some instances, the computation of the bound yields a proof that the obtained solutions are optimal. We analyse a set of optimal solutions, compare them with a manually curated standard ancestor, showing that on yeast data, results coming from different methodologies are largely convergent: the optimal solutions are distant of at most one rearrangement from the reference.

Diplôme : HDR

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We provide here an exhaustive overview of the glutathione (GSH) peroxidase (Gpx) family of poplar (Populus trichocarpa). Although these proteins were initially defined as GSH dependent, in fact they use only reduced thioredoxin (Trx) for their regeneration and do not react with GSH or glutaredoxin, constituting a fifth class of peroxiredoxins. The two chloroplastic Gpxs display a marked selectivity toward their electron donors, being exclusively specific for Trxs of the y type for their reduction. In contrast, poplar Gpxs are much less specific with regard to their electron-accepting substrates, reducing hydrogen peroxide and more complex hydroperoxides equally well. Site-directed mutagenesis indicates that the catalytic mechanism and the Trx-mediated recycling process involve only two (cysteine [Cys]-107 and Cys-155) of the three conserved Cys, which form a disulfide bridge with an oxidation-redox midpoint potential of −295 mV. The reduction/formation of this disulfide is detected both by a shift on sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by measuring the intrinsic tryptophan fluorescence of the protein. The six genes identified coding for Gpxs are expressed in various poplar organs, and two of them are localized in the chloroplast, with one colocalizing in mitochondria, suggesting a broad distribution of Gpxs in plant cells. The abundance of some Gpxs is modified in plants subjected to environmental constraints, generally increasing during fungal infection, water deficit, and metal stress, and decreasing during photooxidative stress, showing that Gpx proteins are involved in the response to both biotic and abiotic stress conditions.

We report the draft genome of the black cottonwood tree, Populus trichocarpa . Integration of shotgun sequence assembly with genetic mapping enabled chromosome-scale reconstruction of the genome. More than 45,000 putative protein-coding genes were identified. Analysis of the assembled genome revealed a whole-genome duplication event; about 8000 pairs of duplicated genes from that event survived in the Populus genome. A second, older duplication event is indistinguishably coincident with the divergence of the Populus and Arabidopsis lineages. Nucleotide substitution, tandem gene duplication, and gross chromosomal rearrangement appear to proceed substantially more slowly in Populus than in Arabidopsis. Populus has more protein-coding genes than Arabidopsis , ranging on average from 1.4 to 1.6 putative Populus homologs for each Arabidopsis gene. However, the relative frequency of protein domains in the two genomes is similar. Overrepresented exceptions in Populus include genes associated with lignocellulosic wall biosynthesis, meristem development, disease resistance, and metabolite transport.

Depuis une dizaine d'années, les peupleraies cultivées en Europe ont un problème récurrent dû à Melampsora larici-populina (Mlp), agent d’une rouille foliaire. Les méthodes de culture "intensives" des peupleraies, les effets de mode des cultivars couplés à une sélection basée sur des résistances qualitatives ont provoqué l'apparition de races de Mlp qui contournent toutes ces résistances. Les sélectionneurs se tournent à présent sur la recherche de gènes ayant une pression de sélection moindre sur l'agent pathogène ; les gènes de résistance quantitative sont une bonne alternative. La recherche des déterminants génétiques de la résistance quantitative a mis en évidence un facteur génétique majeur hérité de Populus trichocarpa (RUS) qui contrôle la taille des urédosores de Mlp. Pour permettre le clonage positionnel de ce facteur génétique il faut déterminer la localisation précise de ce facteur contrôlant la résistance quantitative à Mlp chez le peuplier.[br/] Pour ce faire, nous avons densifié la zone du génome contenant RUS par un criblage d'amorces AFLP1 en utilisant la technique BSA2. Les marqueurs les plus intéressants ont été clonés et séquencés. Les séquences nucléotidiques obtenues ont ensuite été ancrées sur la séquence du génome de P. trichocarpa Nisqually-1. En parallèle, nous avons construit des amorces spécifiques STS3 pour cartographier finement la région de RUS.[br/] Huit bandes s'avèrent discriminantes sur 62 couples d'amorces testées. L'ancrage de 9 marqueurs sur la séquence de Nisqually-1 confirme que le facteur RUS se situe sur le groupe de liaison XIX. Nous avons réussi à développer 3 marqueurs STS valides pour la cartographie fine du locus de RUS. Nous avons cartographié à l’aide de 1429 individus les marqueurs STS E1M4-1S, E6M5-1S et E21M18-1S respectivement à 8.1, 30.3 et 9.3 cM de RUS. Cependant il reste encore à améliorer les amorces STS des autres marqueurs qui ne ségrègeaient pas sur les descendants. Cela permettra d’améliorer la précision de la carte génétique à cet endroit précis et d’obtenir des marqueurs suffisamment proches pour faciliter le criblage d’une banque BAC développée par ailleurs., Since ten years, poplars cultivated in Europe have a recurring problem due to Melampsora larici-populina (Mlp), agent of a leaf rust. The "intensive" methods of poplars cultivation, the fashion effects of cultivars coupled with a selection based on qualitative resistances provoked the appearance of races of Mlp which by-pass all resistances. Breeders turn toward the research for genes having a lesser selection pressure on the pathogenic agent: the quantitative resistance genes are a good alternative. The research for genetic factors of quantitative resistance put in evidence a major genetic factor inherited from Populus trichocarpa (RUS) which controls the size of Mlp uredinia. To allow positionnal cloning of RUS it is necessary to determine the precise localization of this factor controlling the quantitative resistance to Mlp to the poplar.[br/] To do it, we have densified the zone of the genome containing RUS by a screening of AFLP primers using the BSA technique. The most interesting markers were cloned and sequenced. Nucleotidic sequences obtained were then anchored on the sequence of the genome of P. trichocarpa Nisqually-1. In parallel, we built specific STS primers in order to map finely the region of RUS.[br/] Eight bands were discriminating on 62 primers combinations. The anchoring of 9 markers on the sequence of Nisqually-1 confirms that the factor RUS is located on the linkage group XIX. We managed to develop 3 valid STS markers for fine mapping of the locus RUS. We mapped, with 1429 individuals, our STS markers, E1M4-1S, E6M5-1S and E21M18- 1S respectively at 8.1, 30.3 and 9.3 cM of RUS. However, it still necessary to improve the other STS markers which not segregate in the progenies. It will allow to improve the precision of the genetic map in this place and to obtain markers enough close to facilitate the screening of a BAC library developed elsewhere

Large-scale identification of genes expressed in roots of the model plant Arabidopsis was performed by serial analysis of gene expression (SAGE), on a total of 144,083 sequenced tags, representing at least 15,964 different mRNAs. For tag to gene assignment, we developed a computational approach based on 26,620 genes annotated from the complete sequence of the genome. The procedure selected warrants the identification of the genes corresponding to the majority of the tags found experimentally, with a high level of reliability, and provides a reference database for SAGE studies in Arabidopsis. This new resource allowed us to characterize the expression of more than 3,000 genes, for which there is no expressed sequence tag (EST) or cDNA in the databases. Moreover, 85% of the tags were specific for one gene. To illustrate this advantage of SAGE for functional genomics, we show that our data allow an unambiguous analysis of most of the individual genes belonging to 12 different ion transporter multigene families. These results indicate that, compared with EST-based tag to gene assignment, the use of the annotated genome sequence greatly improves gene identification in SAGE studies. However, more than 6,000 different tags remained with no gene match, suggesting that a significant proportion of transcripts present in the roots originate from yet unknown or wrongly annotated genes. The root transcriptome characterized in this study markedly differs from those obtained in other organs, and provides a unique resource for investigating the functional specificities of the root system. As an example of the use of SAGE for transcript profiling in Arabidopsis, we report here the identification of 270 genes differentially expressed between roots of plants grown either with NO3 - or NH4NO3 as N source.

The purpose of this study was to develop a method for identifying useful patterns in gene expression time-series data. We have developed a novel data mining approach that identifies interesting patterns. The method consists of a combination of data pre-processing as well as unsupervised and supervised learning techniques. To evaluate our approach, we have analyzed three time series data sets which investigate the temporal transcriptome changes that occur during: 1) the cell cycle of budding yeast (S. cerevisiae) [3], 2) the epithelial to mesenchymal transition induced by Transforming Growth Factor-?1 in mouse mammary epithelial BRI-JM01 cells, and 3) the program of differentiation induced by retinoic acid in human embryonal teratocarcinoma NT-2 cells. We present the results from all of our experiments, discuss the patterns discovered through the use of our approach and briefly explain future plans and directions for improving our method., The 16th European Conference on Artificial Intelligence (ECAI 2004), August 22-27, 2004, Valencia, Spain

*INRA Orléans, Documentation, Ardon 45166 Olivet Diffusion du document : INRA Orléans, Documentation, Ardon 45166 Olivet Diplôme : Licence Professionnelle

The inheritance of resistance to Ascochyta blight, an economically important foliar disease of field pea ( Pisum sativum L.) worldwide, was investigated. Breeding resistant pea varieties to this disease, caused by Mycosphaerella pinodes, is difficult due to the availability of only partial resistance. We mapped and characterized quantitative trait loci (QTLs) for resistance to M. pinodes in pea. A population of 135 recombinant inbred lines (RILs), derived from the cross between DP (partially resistant) and JI296 (susceptible), was genotyped with morphological, RAPD, SSR and STS markers. A genetic map was elaborated, comprising 206 markers distributed over eight linkage groups and covering 1,061 cM. The RILs were assessed under growth chamber and field conditions at the seedling and adult plant stages, respectively. Six QTLs were detected at the seedling stage, which together explained up to 74% of the variance. Ten QTLs were identified at the adult plant stage in the field, and together these explained 56.6-67.1% of the variance, depending on the resistance criteria and the organ considered. Four QTLs were detected under both growth chamber and field conditions, suggesting they were not plant-stage dependent. Three QTLs for flowering date and three QTLs for plant height were also identified in the RIL population, some of which co-located with QTLs for resistance. The relationship between QTLs for resistance to M. pinodes, plant height and flowering date is discussed.

309 ref. * INRA d'Orléans, Documentation, Ardon 45166 Olivet Diffusion du document : INRA d'Orléans, Documentation, Ardon 45166 Olivet Diplôme : Dr. d'Université

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International audience; Comparative QTL mapping for adaptive traits among populations and species can lead to the identification of genomic regions conserved over a long evolutionary period and therefore potentially harboring genes of great importance for adaptive processes. At the same time, non-conserved QTLs could be responsible for local adaptation or interspecific differentiation. The availability of Populus as a model system for tree and woody perennial plant biology has largely been driven by the rapid development of genomic and molecular biology resources for this genus, culminating in the completion of a draft sequence of the Populus trichocarpa (black cottonwood) genome. Numerous genetic maps are available in different Populus and Salix species and QTLs for adaptive traits (such as phenology and disease resistance) and sex have been mapped. All these maps showed a very good synteny with the P. trichocarpa genome; however, very few markers are currently shared between these maps. The objective of our study is to take advantage of the information on the P. trichocarpa genome sequence to perform QTL projection and identify candidate genes. Genetic maps from 7 pedigrees, belonging to 4 Populus spp. were aligned with the help of common simple sequence repeat and gene markers. Three additional published maps from Salix spp. were partially anchored to the P. trichocarpa genome sequence. QTL meta-analysis and projection of QTL intervals on the genome sequence were performed when sufficient genome anchoring markers were available. These analyses allowed identifying large genome regions containing several hundred of candidate genes. Thanks to the availability of biological knowledge accumulated in public data bases (e.g. Gene Ontology) and high-throughput enrichment tools, we have explored these gene lists by functional analysis if QTL regions are statistically enriched in some functional categories compared to the entire genome.