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Lipo-chitooligosaccharides (LCOs) were originally found as symbiotic signals called Nod Factors (Nod-LCOs) controlling the nodulation of legumes by rhizobia. More recently, LCOs were also found in symbiotic fungi and, more surprisingly, very widely in the kingdom Fungi, including in saprophytic and pathogenic fungi. The LCO-V(C18:1, fucosylated/methyl fucosylated), hereafter called Fung-LCOs, are the LCO structures most commonly found in fungi. This raises the question of how legume plants such as Medicago truncatula can discriminate between Nod-LCOs and Fung-LCOs. To address this question, we performed a genome-wide association study on 173 natural accessions of M. truncatula, using a root branching phenotype and a newly developed local score approach. Both Nod-LCOs and Fung-LCOs stimulated root branching in most accessions, but the root responses to these two types of LCO molecules were not correlated. In addition, the heritability of the root response was higher for Nod-LCOs than for Fung-LCOs. We identified 123 loci for Nod-LCO and 71 for Fung-LCO responses, of which only one was common. This suggests that Nod-LCOs and Fung-LCOs both control root branching but use different molecular mechanisms. The tighter genetic constraint of the root response to Fung-LCOs possibly reflects the ancestral origin of the biological activity of these molecules.

International audience; Cannabis sativa has a long history of domestication both for its bioactive compounds and its fibers. This has produced hundreds of varieties, usually characterized in the literature by chemotypes, with Δ9-THC and CBD content as the main markers. However, chemotyping could also be done based on minor compounds (phytocannabinoids and others). In this work, a workflow, which we propose to name cannabinomics, combines mass spectrometry of the whole metabolome and statistical analysis to help differentiate C. sativa varieties and deciphering their characteristic markers. By applying this cannabinomics approach to the data obtained from 20 varieties of C. sativa (classically classified as chemotype I, II, or III), we compared the results with those obtained by a targeted quantification of 11 phytocannabinoids. Cannabinomics can be considered as a complementary tool for phenotyping and genotyping, allowing the identification of minor compounds playing a key role as markers of differentiation.

International audience; Quantitative trait loci (QTL) with small effects, which are pervasive in quantitative phenotypic variation, are difficult to detect in genome-wide association studies (GWAS). To improve their detection, we propose to use a local score approach that accounts for the surrounding signal due to linkage disequilibrium, by accumulating association signals from contiguous single markers. Simulations revealed that, in a GWAS context with high marker density, the local score approach outperforms single SNP p-value-based tests for detecting minor QTL (heritability of 5-10%) and is competitive with regard to alternative methods, which also aggregate p-values. Using more than five million SNPs, this approach was applied to identify loci involved in Quantitative Disease Resistance (QDR) to different isolates of the plant root rot pathogen Aphanomyces euteiches, from a GWAS performed on a collection of 174 accessions of the model legume Medicago truncatula. We refined the position of a previously reported major locus, underlying MYB/NB-ARC/tyrosine kinase candidate genes conferring resistance to two closely related A. euteiches isolates belonging to pea pathotype I. We also discovered a diversity of minor resistance QTL, not detected using p-value-based tests, some of which being putatively shared in response to pea (pathotype I and III) and/or alfalfa (race 1 and 2) isolates. Candidate genes underlying these QTL suggest pathogen effector recognition and plant proteasome as key functions associated with M. truncatula resistance to A. euteiches. GWAS on any organism can benefit from the local score approach to uncover many weak-effect QTL.

SummaryLipo-chitooligosaccharides (LCOs) were originally found as symbiotic signals called Nod Factors (Nod-LCOs) controlling nodulation of legumes by rhizobia. More recently LCOs were also found in symbiotic fungi and, more surprisingly, very widely in the kingdom fungi including in saprophytic and pathogenic fungi. The LCO-V(C18:1, Fuc/MeFuc), hereafter called Fung-LCOs, are the LCO structures most commonly found in fungi. This raises the question of how legume plants, such asMedicago truncatula, can perceive and discriminate between Nod-LCOs and these Fung-LCOs.To address this question, we performed a Genome Wide Association Study on 173 natural accessions ofMedicago truncatula, using a root branching phenotype and a newly developed local score approach.Both Nod- and Fung-LCOs stimulated root branching in most accessions but there was very little correlation in the ability to respond to these types of LCO molecules. Moreover, heritability of root response was higher for Nod-LCOs than for Fung-LCOs. We identified 123 loci for Nod-LCO and 71 for Fung-LCO responses, but only one was common.This suggests that Nod- and Fung-LCOs both control root branching but use different molecular mechanisms. The tighter genetic constraint of the root response to Fung-LCOs possibly reflects the ancestral origin of the biological activity of these molecules.

The quest for signatures of selection using single nucleotide polymorphism (SNP) data has proven efficient to uncover genes involved in conserved and/or adaptive molecular functions, but none of the statistical methods were designed to identify interacting alleles as targets of selective processes. Here, we propose a statistical test aimed at detecting epistatic selection, based on a linkage disequilibrium (LD) measure accounting for population structure and heterogeneous relatedness between individuals. SNP-based ( $$T_{r_v}$$ ) and window-based ( $$T_{corPC1_v}$$ ) statistics fit a Student distribution, allowing to test the significance of correlation coefficients. As a proof of concept, we use SNP data from the Medicago truncatula symbiotic legume plant and uncover a previously unknown gene coadaptation between the MtSUNN (Super Numeric Nodule) receptor and the MtCLE02 (CLAVATA3-Like) signaling peptide. We also provide experimental evidence supporting a MtSUNN-dependent negative role of MtCLE02 in symbiotic root nodulation. Using human HGDP-CEPH SNP data, our new statistical test uncovers strong LD between SLC24A5 (skin pigmentation) and EDAR (hairs, teeth, sweat glands development) world-wide, which persists after correction for population structure and relatedness in Central South Asian populations. This result suggests that epistatic selection or coselection could have contributed to the phenotypic make-up in some human populations. Applying this approach to genome-wide SNP data will facilitate the identification of coadapted gene networks in model or non-model organisms.

The quest for genome-wide signatures of selection in populations using SNP data has proven efficient to uncover genes involved in conserved or adaptive molecular functions, but none of the statistical methods were designed to identify interacting genes as targets of selective processes. Here, we propose a straightforward statistical test aimed at detecting epistatic selection, based on a linkage disequilibrium (LD) measure accounting for population structure and heterogeneous relatedness between individuals. SNP-based (Trv) and window-based (TcorPC1v) statistics fit a Student distribution, allowing to easily and quickly test the significance of correlation coefficients in the frame of Genome-Wide Epistatic Selection Scans (GWESS) using candidate genes as baits. As a proof of concept, use of SNP data from theMedicago truncatulasymbiotic legume plant uncovered a previously unknown gene coadaptation between theMtSUNN(Super Numeric Nodule) receptor and theMtCLE02(CLAVATA3-Like) signalling peptide, and experimental evidence accordingly supported aMtSUNN-dependent negative role ofMtCLE02in symbiotic root nodulation. Using human HGDP-CEPH SNP data, our new statistical test uncovered strong LD betweenSLC24A5andEDARworldwide, which persists after correction for population structure and relatedness in Central South Asian populations. This result suggests adaptive genetic interaction or coselection between skin pigmentation and the ectodysplasin pathway involved in the development of ectodermal organs (hairs, teeth, sweat glands), in some human populations. Applying this approach to genome-wide SNP data will foster the identification of evolutionary coadapted gene networks.Author summaryPopulation genomic methods have allowed to identify many genes associated with adaptive processes in populations with complex histories. However, they are not designed to identify gene coadaptation between genes through epistatic selection, in structured populations. To tackle this problem, we developed a straightforward LD-based statistical test accounting for population structure and heterogeneous relatedness between individuals, using SNP-based (Trv) or windows-based (TcorPC1v) statistics. This allows easily and quickly testing for significance of correlation coefficients between polymorphic loci in the frame of Genome Wide Epistatic Selection Scans (GWESS). Following detection of gene coadaptation using SNP data from human and the model plantMedicago truncatula, we report experimental evidence of genetic interaction between two receptors involved in the regulation of root nodule symbiosis inMedicago truncatula. This test opens new avenues for exploring the evolution of genes as interacting units and thus paves the way to infer new networks based on evolutionary coadaptation between genes.

Using a genome-wide association mapping approach, we identified a new role for the GRAS transcription factor RAD1 in supporting infection by an oomycete root pathogen. Previously, RAD1 was exclusively implicated in symbiosis signalling., The roots of most land plants are colonized by symbiotic arbuscular mycorrhiza (AM) fungi. To facilitate this symbiosis, plant genomes encode a set of genes required for microbial perception and accommodation. However, the extent to which infection by filamentous root pathogens also relies on some of these genes remains an open question. Here, we used genome-wide association mapping to identify genes contributing to colonization of Medicago truncatula roots by the pathogenic oomycete Phytophthora palmivora. Single-nucleotide polymorphism (SNP) markers most significantly associated with plant colonization response were identified upstream of RAD1, which encodes a GRAS transcription regulator first negatively implicated in root nodule symbiosis and recently identified as a positive regulator of AM symbiosis. RAD1 transcript levels are up-regulated both in response to AM fungus and, to a lower extent, in infected tissues by P. palmivora where its expression is restricted to root cortex cells proximal to pathogen hyphae. Reverse genetics showed that reduction of RAD1 transcript levels as well as a rad1 mutant are impaired in their full colonization by AM fungi as well as by P. palmivora. Thus, the importance of RAD1 extends beyond symbiotic interactions, suggesting a general involvement in M. truncatula microbe-induced root development and interactions with unrelated beneficial and detrimental filamentous microbes.

International audience; Plant -specific lysin-motif receptor-like kinases (LysM-RLKs) are implicated in the perception of N-acetyl glucosamine-containing compounds, some of which are important signal molecules in plant-microbe interactions. Among these, both lipo-chitooligosaccharides (LCOs) and chitooligosaccharides (COs) are proposed as arbuscular mycorrhizal (AM) fungal symbiotic signals. COs can also activate plant defence, although there are scarce data about CO production by pathogens, especially nonfungal pathogens. We tested Medicago truncatula mutants in the LysM-RLK MtLYK9 for their abilities to interact with the AM fungus Rhizophagus irregularis and the oomycete pathogen Aphanomyces euteiches. This prompted us to analyse whether A. euteiches can produce COs. Compared with wild-type plants, Mtlyk9 mutants had fewer infection events and were less colonised by the AM fungus. By contrast, Mtlyk9 mutants were more heavily infected by A. euteiches and showed more disease symptoms. Aphanomyces euteiches was also shown to produce short COs, mainly CO II, but also CO III and CO IV, and traces of CO V, both ex planta and in planta. MtLYK9 thus has a dual role in plant immunity and the AM symbiosis, which raises questions about the functioning and the ancestral origins of such a receptor protein.

International audience; Natural variations help in identifying genetic mechanisms of morphologically and developmentally complex traits. Mountainous habitats provide an altitudinal gradient where one species encounters different abiotic conditions. We report the study of 341 individuals of Arabidopsis thaliana derived from 30 natural populations not belonging to the 1001 genomes, collected at increasing altitudes, between 200 and 1800 m in the Pyrenees. Class III peroxidases and ribosomal RNA sequences were used as markers to determine the putative genetic relationships among these populations along their altitudinal gradient. Using Bayesian-based statistics and phylogenetic analyses, these Pyrenean populations appear with significant divergence from the other regional accessions from 1001 genome (i.e., from north Spain or south France). Individuals of these populations exhibited varying phenotypic changes, when grown at sub-optimal temperature (22 vs. 15 • C). These phenotypic variations under controlled conditions reflected intraspecific morphological variations. This study could bring new information regarding the west European population structure of A. thaliana and its phenotypic variations at different temperatures. The integrative analysis combining genetic, phenotypic variation and environmental datasets is used to analyze the acclimation of population in response to temperature changes. Regarding their geographical proximity and environmental diversity, these populations represent a tool of choice for studying plant response to temperature variation. HIGHLIGHTS-Studying the natural diversity of A. thaliana in the Pyrenees mountains helps to understand European population structure and to evaluate the phenotypic trait variation in response to climate change.

Summary To gain an insight into the molecular mechanisms of quantitative disease resistance in Medicago truncatula to the root-infecting oomycete Aphanomyces euteiches, we selected two near-isogenic lines (NILs), NR and NS, partially resistant and susceptible, respectively, differing in the allelic state of the quantitative resistance locus (QRL) prAe1 (partially resistant to A. euteiches 1). Complementary molecular and cytological phenotyping methods showed that prAe1 alone confers quantitative resistance to A. euteiches. Root and stem tissues were colonized in NS plants and 80% of NS plants died by 21 days post-inoculation (dpi). In contrast, A. euteiches mycelium was restricted to the root cortex and the spread of symptoms was arrested in aerial parts of NR plants. A transcriptome analysis performed at 0, 1 and 6 dpi identified 1198 differentially expressed genes (DEGs) between NR and NS lines. More than 87% of the DEGs were significantly more expressed in NR. The highest number of DEGs was found in control conditions, with 723 genes over-expressed in NR versus 85 in NS. Genes belonging to secondary metabolism, pathogenesis-related (PR) proteins and kinases were significantly enriched. The significant role of the flavonoid pathway in resistance was corroborated by the detection of larger amounts of flavonoids in NR roots and the inhibition of A. euteiches zoospore germination by 2′-O-methyl-isoliquiritigenin, a compound synthesized by enzymes specifically induced in NR. Our study revealed that prAe1-dependent resistance relies mainly on the constitutive expression of defence-related pathways and signalling elements, which can be re-amplified in later time points of the infection.

Many genetic studies on catarrhines use microsatellite markers that were isolated from human DNA. A large number of these markers have been characterized in the great apes, macaques and baboons. However, there are few or no markers available for other members of this group. In this study, an extensive literature search was performed to find microsatellite markers that had been successfully amplified across a range of catarrhine species. These conserved loci can provide a valuable starting point for characterizing loci in other catarrhines. Finally, microsatellite markers were tested in a range of species that are not well represented in the literature.

This paper presents a comprehensive overview of the published literature on microsatellites studied in cetace- ans from 1989 to 2007. We inventoried 246 loci isolated from 18 and amplified in 51 cetacean species representing 11 families. The majority of loci (68%) were dinucleotide (CA) repeats, which were also shown to be on average more vari- able than tetranucleotide repeats. For each of these loci and the 1610 locus/species combinations, we present the species in which the markers were isolated and tested, specific diversity parameters (number of alleles, expected and observed het- erozygosities), together with the primer sequences, the size range of the PCR products and the GenBank accession num- ber. Simple analyses were performed on the assembled data and the widespread use of cross-species amplification, an im- portant source of microsatellites in cetaceans, is discussed using laboratory data from Stenella cœruleoalba. No significant ascertainment bias was detected when considering all dinucleotide or tetranucleotide loci. This comprehensive database should help to inform those working on population and conservation genetic studies in most cetacean species.

Rhesus macaque (Macaca mulatta) and long-tailed macaque (Macaca fascicularis) are the 2 most commonly used primate model species in biomedical sciences. Although morphological studies have revealed a weak hybridization at the interspecific contact zone, in the north of Indochina, a molecular study has suggested an ancient introgression from rhesus to long-tailed macaque into the Indo-Chinese peninsula. However, the gene flow between these 2 taxa has never been quantified using genetic data and theoretical models. In this study, we have examined genetic variation within and between the parapatric Chinese rhesus macaque and Indo-Chinese long-tailed macaque populations, using 13 autosomal, 5 sex-linked microsatellite loci and mitochondrial DNA sequence data. From these data, we assessed genetic structure and estimated gene flow using a Bayesian clustering approach and the "Isolation with Migration" model. Our results reveal a weak interspecific genetic differentiation at both autosomal and sex-linked loci, suggesting large population sizes and/or gene flow between populations. According to the Bayesian clustering, Chinese rhesus macaque is a highly homogeneous gene pool that contributes strongly to the current Indo-Chinese long-tailed macaque genetic makeup, whether or not current admixture is assumed. Coalescent simulations, which integrated the characteristics of the loci, pointed out 1) a higher effective population size in rhesus macaque, 2) no mitochondrial gene flow, and 3) unilateral and male-mediated nuclear gene flow of approximately 10 migrants per generation from rhesus to long-tailed macaque. These patterns of genetic structure and gene flow suggest extensive ancient introgression from Chinese rhesus macaque into the Indo-Chinese long-tailed macaque population.

Chimpanzees experienced a reduction of the allelic repertoire at the major histocompatibility complex (MHC) class I A and B loci, which may have been caused by a retrovirus belonging to the simian immunodeficiency virus (SIV) family. Extended MHC haplotypes were defined in a pedigreed chimpanzee colony. Comparison of genetic variation at microsatellite markers mapping inside and outside the Mhc region was carried out in humans and chimpanzees to investigate the genomic extent of the repertoire reduction. Multilocus demographic analyses underscored that chimpanzees indeed experienced a selective sweep that mainly targeted the chromosomal segment carrying the Mhc class I region. Probably due to genetic linkage, the sweep also affected other polymorphic loci, mapping in the close vicinity of the Mhc class I region genes. Nevertheless, although the allelic repertoire at particular Mhc class I and II loci appears to be limited, naturally occurring recombination events allowed the establishment of haplotype diversity after the sweep. However, recombination did not have sufficient time to erase the signal of the selective sweep.

Cynomolgus macaques (Macaca fascicularis) were introduced on the island of Mauritius between 400 and 500 years ago and underwent a strong population expansion after a probable initial founding event. However, in practice, little is known of the geographical origin of the individuals that colonized the island, on how many individuals were introduced, and of whether the following demographic expansion erased any signal of this putative bottleneck. In this study, we asked whether the current nuclear genome of the Mauritius population retained a signature that would allow us to answer these questions. Altogether, 21 polymorphic autosomal and sex-linked microsatellites were surveyed from 81 unrelated Mauritius individuals and 173 individuals from putative geographical sources in Southeast Asia: Java, the Philippines islands and the Indochinese peninsula. We found that (i) the Mauritius population was closer to different populations depending on the markers we used, which suggests a possible mixed origin with Java playing most probably a major role; and (ii) the level of diversity was lower than the other populations but there was no clear and consistent bottleneck signal using either summary statistics or full-likelihood methods. However, summary statistics strongly suggest that Mauritius is not at mutation-drift equilibrium and favours an expansion rather than a bottleneck. This suggests that on a short time scale, population decline followed by growth can be difficult to deduce from genetic data based on mutation-drift theory. We then used a simple Bayesian rejection algorithm to estimate the number of founders under different demographic models (exponential, logistic and logistic with lag) and pure genetic drift. This new method uses current population size estimates and expected heterozygosity of Mauritius and source population(s). Our results indicate that a simple exponential growth is unlikely and that, under the logistic models, the population may have expanded from an initial effective number of individuals of 10-15. The data are also consistent with a logistic growth with different lag values, indicating that we cannot exclude past population fluctuation.

AGAP : équipe GEpop; International audience; Local climatic conditions likely constitute an important selective pressure on genes underlying important fitness-related traits such as flowering time and in many species flowering phenology and climatic gradients strongly covary. To test whether climate shapes genetic variation on flowering time genes and to identify candidate flowering genes involved in the adaptation to environmental heterogeneity, we used a large M. truncatula core collection to examine the association between nucleotide polymorphisms at 224 candidate genes and both climate variables and flowering phenotypes. Unlike genome-wide studies, candidate gene approaches are expected to enrich for the number of meaningful trait associations because they specifically target genes that are known to affect the trait of interest. We found that flowering time mediates adaptation to climatic conditions mainly by variation at genes located upstream in the flowering pathways, close to the environmental stimuli. Variables related to the annual precipitation regime reflected selective constraints on flowering time genes better than the other variables tested (temperature, altitude, latitude or longitude). By comparing phenotype and climate associations, we identified 12 flowering genes as the most promising candidates responsible for phenological adaptation to climate. Four of these genes were located in the known flowering time QTL region on chromosome 7. However, climate and flowering associations also highlighted largely distinct gene sets, suggesting different genetic architectures for adaptation to climate and flowering onset.

Across a large distribution range, population-specific factors as well as pathogen-mediated selection may shape species genetic diversity in the major histocompatibility complex (MHC). We have studied genetic diversity and population differentiation in the MHC region of the Southeast Asian cynomolgus macaque (Macaca fascicularis fascicularis), a species with large and discontinuous range, in order to investigate the role of demography vs selection. Genetic variation was assessed at seven MHC microsatellites on 272 individuals from five populations (Indochina, Java, Borneo, Philippines, and Mauritius). A high genetic diversity was observed in all populations and the Philippines but also the Mauritius populations were the most genetically differentiated. The strength and extent of linkage disequilibrium (LD) (up to 4 Mb) varies across populations mainly because of demographic factors. In Indochina, the complete lack of LD could be the signature of ancient hybridization between cynomolgus and rhesus macaques in the Indochinese peninsula. With the additional support of seven autosomal microsatellites, tests for outlier loci based on intrapopulation diversity and interpopulation differentiation (using F-statistic) allowed to dissociate demographic from selective histories: (i) demographic history may itself explain levels of MHC variability in the Mauritius populations and (ii) positive selection could be responsible for the Philippines population differentiation, especially in the MHC class II region. Among various pathogens, Plasmodium knowlesi and Plasmodium coatneyi are two likely candidates to explain the higher frequency of some MHC haplotypes. Indeed, literature describes low parasitemia in the Philippines individuals, contrasting with fatal infections provoked by these parasites in other cynomolgus macaque populations.

Cross-amplification of 15 human microsatellites was performed successfully in cynomolgus (Macaca fascicularis) and rhesus (M. mulatta) macaques and 11 other Cercopithecidae species of biomedical and conservation relevance. To allow for quick, efficient, and high-throughput genotyping to assess intra- and interspecific genetic variation, we performed three multiplex sets, each comprised of five markers from different parts of the genome (i.e., autosomes, the MHC region, and the X-chromosome). These multiplex sets are likely to reveal allelic divergence between taxa, which could be used for their discrimination. Population studies on three regional populations of M. fascicularis and one of M. mulatta revealed that most of the loci, with the exception of one monomorphic locus, displayed polymorphisms (the expected heterozygosities were 0.48–0.91 for M. fascicularis, and 0.61–0.93 for M. mulatta), which makes them useful for population genetics. For the multiplex set M1, including the nonlinked autosomal markers, low probabilities of identity were observed: P(ID) values ranged from 8 × 10–7 to 3 × 1 0–5. This multiplex set is reliable for forensic applications, such as individual identification, parentage testing, and kinship analysis, in wild and captive populations. Am. J. Primatol. 67:385–391, 2005. © 2005 Wiley-Liss, Inc.

International audience; Medicago truncatula is a model legume species used to investigate plant-microorganism interactions, notably root symbioses. Massive population genomic and transcriptomic data now available for this species open the way for a comprehensive investigation of genomic variations associated with adaptation of M. truncatula to its environment. Here we performed a fine-scale genome scan of selective sweep signatures in M. truncatula using more than 15 million single nucleotide polymorphisms identified on 283 accessions from two populations (Circum and Far West), and exploited annotation and published transcriptomic data to identify biological processes associated with molecular adaptation. We identified 58 swept genomic regions with a 15 kb average length and comprising 3.3 gene models on average. The unimodal sweep state probability distribution in these regions enabled us to focus on the best single candidate gene per region. We detected two unambiguous species-wide selective sweeps, one of which appears to underlie morphological adaptation. Population genomic analyses of the remaining 56 sweep signatures indicate that sweeps identified in the Far West population are less population-specific and probably more ancient than those identified in the Circum population. Functional annotation revealed a predominance of immunity-related adaptations in the Circum population. Transcriptomic data from accessions of the Far West population allowed inference of four clusters of coregulated genes putatively involved in the adaptive control of symbiotic carbon flow and nodule senescence, as well as in other root adaptations upon infection with soil microorganisms. We demonstrate that molecular adaptations in M. truncatula were primarily triggered by selective pressures from root-associated microorganisms.

International audience; To gain an insight into the molecular mechanisms of quantitative disease resistance in Medicago truncatula to the root-infecting oomycete Aphanomyces euteiches, we selected two near-isogenic lines (NILs), NR and NS, partially resistant and susceptible, respectively, differing in the allelic state of the quantitative resistance locus (QRL) prAe1 (partially resistant to A.euteiches 1). Complementary molecular and cytological phenotyping methods showed that prAe1 alone confers quantitative resistance to A.euteiches. Root and stem tissues were colonized in NS plants and 80% of NS plants died by 21 days post-inoculation (dpi). In contrast, A.euteiches mycelium was restricted to the root cortex and the spread of symptoms was arrested in aerial parts of NR plants. A transcriptome analysis performed at 0, 1 and 6dpi identified 1198 differentially expressed genes (DEGs) between NR and NS lines. More than 87% of the DEGs were significantly more expressed in NR. The highest number of DEGs was found in control conditions, with 723 genes over-expressed in NR versus 85 in NS. Genes belonging to secondary metabolism, pathogenesis-related (PR) proteins and kinases were significantly enriched. The significant role of the flavonoid pathway in resistance was corroborated by the detection of larger amounts of flavonoids in NR roots and the inhibition of A.euteiches zoospore germination by 2-O-methyl-isoliquiritigenin, a compound synthesized by enzymes specifically induced in NR. Our study revealed that prAe1-dependent resistance relies mainly on the constitutive expression of defence-related pathways and signalling elements, which can be re-amplified in later time points of the infection.

International audience; Understanding how immune genetic variation is shaped by selective and neutral processes in wild populations is of prime importance in both evolutionary biology and epidemiology. The European roe deer (Capreolus capreolus) has considerably expanded its distribution range these last decades, notably by colonizing agricultural landscapes. This range shift is likely to have led to bottlenecks and increased roe deer exposure to a new range of pathogens that until recently predominantly infected humans and domestic fauna. We therefore investigated the historical and contemporary forces that have shaped variability in a panel of genes involved in innate and acquired immunity in roe deer, including Mhc-Drb and genes encoding cytokines or toll-like receptors (TLRs). Together, our results suggest that genetic drift is the main contemporary evolutionary force shaping immunogenetic variation within populations. However, in contrast to the classical view, we found that some innate immune genes involved in micropathogen recognition (e.g. Tlrs) continue to evolve dynamically in roe deer in response to pathogen-mediated positive selection. Most studied Tlrs (Tlr2, Tlr4 and Tlr5) had similarly high levels of amino acid diversity in the three studied populations including one recently established in southwestern France that showed a clear signature of genetic bottleneck. Tlr2 implicated in the recognition of Gram-positive bacteria in domestic ungulates, showed strong evidence of balancing selection. The high immunogenetic variation revealed here implies that roe deer are able to cope with a wide spectrum of pathogens and to respond rapidly to emerging infectious diseases.

Summary � The use of quantitative disease resistance (QDR) is a promising strategy for promoting durable resistance to plant pathogens, but genes involved in QDR are largely unknown. To identify genetic components and accelerate improvement of QDR in legumes to the root pathogen Aphanomyces euteiches, we took advantage of both the recently generated massive genomic data for Medicago truncatula and natural variation of this model legume. � A high-density (� 5.1 million single nucleotide polymorphisms (SNPs)) genome-wide association study (GWAS) was performed with both in vitro and glasshouse phenotyping data collected for 179 lines. � GWAS identified several candidate genes and pinpointed two independent major loci on the top of chromosome 3 that were detected in both phenotyping methods. Candidate SNPs in the most significant locus (r 2 = 23%) were in the promoter and coding regions of an F-box protein coding gene. Subsequent qRT-PCR and bioinformatic analyses performed on 20 lines demonstrated that resistance is associated with mutations directly affecting the interaction domain of the F-box protein rather than gene expression. � These results refine the position of previously identified QTL to specific candidate genes, suggest potential molecular mechanisms, and identify new loci explaining QDR against A. euteiches.

International audience; Plant LysM proteins control the perception of microbial-derived N-acetylglucosamine compounds for the establishment of symbiosis or activation of plant immunity. This raises questions about how plants, and notably legumes, can differentiate friends and foes using similar molecular actors and whether any receptors can intervene in both symbiosis and resistance. To study this question, nfp and lyk3 LysM-receptor like kinase mutants of Medicago truncatula that are affected in the early steps of nodulation, were analysed following inoculation with Aphanomyces euteiches, a root oomycete. The role of NFP in this interaction was further analysed by overexpression of NFP and by transcriptome analyses. nfp, but not lyk3, mutants were significantly more susceptible than wildtype plants to A.euteiches, whereas NFP overexpression increased resistance. Transcriptome analyses on A.euteiches inoculation showed that mutation in the NFP gene led to significant changes in the expression of c. 500 genes, notably involved in cell dynamic processes previously associated with resistance to pathogen penetration. nfp mutants also showed an increased susceptibility to the fungus Colletotrichum trifolii. These results demonstrate that NFP intervenes in M.truncatula immunity, suggesting an unsuspected role for NFP in the perception of pathogenic signals.

Detecting genetic signatures of selection is of great interest for many research issues. Common approaches to separate selective from neutral processes focus on the variance of FST across loci, as does the original Lewontin and Krakauer (LK) test. Modern developments aim to minimize the false positive rate and to increase the power, by accounting for complex demographic structures. Another stimulating goal is to develop straightforward parametric and computationally tractable tests to deal with massive SNP data sets. Here, we propose an extension of the original LK statistic (TLK), named TF–LK, that uses a phylogenetic estimation of the population's kinship (\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\mathscr{F}}}}\end{equation*}\end{document}) matrix, thus accounting for historical branching and heterogeneity of genetic drift. Using forward simulations of single-nucleotide polymorphisms (SNPs) data under neutrality and selection, we confirm the relative robustness of the LK statistic (TLK) to complex demographic history but we show that TF–LK is more powerful in most cases. This new statistic outperforms also a multinomial-Dirichlet-based model [estimation with Markov chain Monte Carlo (MCMC)], when historical branching occurs. Overall, TF–LK detects 15–35% more selected SNPs than TLK for low type I errors (P < 0.001). Also, simulations show that TLK and TF–LK follow a chi-square distribution provided the ancestral allele frequencies are not too extreme, suggesting the possible use of the chi-square distribution for evaluating significance. The empirical distribution of TF–LK can be derived using simulations conditioned on the estimated \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\mathscr{F}}}}\end{equation*}\end{document} matrix. We apply this new test to pig breeds SNP data and pinpoint outliers using TF–LK, otherwise undetected using the less powerful TLK statistic. This new test represents one solution for compromise between advanced SNP genetic data acquisition and outlier analyses.

We studied the mitochondrial DNA (mtDNA) polymorphism of 304 Macaca fascicularis fascicularis (M. f. fascicularis) individuals, representative of 4 cynomolgus macaque populations (Indochina, Indonesia, Philippines, and Mauritius). By sequencing a 590-bp fragment in the hypervariable II region of the D-loop region, we defined 70 haplotypes. The homologous region was also characterized in 22 Chinese Macaca mulatta and 2 Macaca sylvanus. The phylogenetic analysis confirms the monophyly of M. f. fascicularis and defines 2 haplotype groups inside the M. f. fascicularis clade: one ‘‘insular,’’ encompassing 6 Philippines, 2 Mauritius, and 31 Indonesian haplotypes, the other ‘‘continental’’ that contains all Indochinese and 6 Indonesian haplotypes. Continental and insular group divergence time was estimated to be approximately 10 6 years before present (BP). Among Indonesian haplotypes, some have a continental origin. This suggests either direct migration from mainland to Indonesia or that remnant lineages from an ancient population genetically close to the mainland (i.e., in the Sunda Shelf, ,550 000 years BP) were subsequently brought southward to Indonesia. The low nucleotide diversity in the Philippines population suggests a bottleneck following colonization by Indonesian individuals, around 110 000 years BP. mtDNA and further observations of nuclear genetic data corroborate the mixed origin (Indonesian/continental) hypothesis of Mauritius individuals and a population bottleneck.

Chimpanzee populations are diminishing as a consequence of human activities, and as a result this species is now endangered. In the context of conservation programmes, genetic data can add vital information, for instance on the genetic diversity and structure of threatened populations. Single nucleotide polymorphisms (SNP) are biallelic markers that are widely used in human molecular studies and can be implemented in efficient microarray systems. This technology offers the potential of robust, multiplexed SNP genotyping at low reagent cost in other organisms than humans, but it is not commonly used yet in wild population studies. Here, we describe the characterization of new SNPs in Y-chromosomal intronic regions in chimpanzees and also identify SNPs from mitochondrial genes, with the aim of developing a microarray system that permits the simultaneous study of both paternal and maternal lineages. Our system consists of 42 SNPs for the Y chromosome and 45 SNPs for the mitochondrial genome. We demonstrate the applicability of this microarray in a captive population where genotypes accurately reflected its large pedigree. Two wild-living populations were also analysed and the results show that the microarray will be a useful tool alongside microsatellite markers, since it supplies complementary information about population structure and ecology. SNP genotyping using microarray technology, therefore, is a promising approach and may become an essential tool in conservation genetics to help in the management and study of captive and wild-living populations. Moreover, microarrays that combine SNPs from different genomic regions could replace microsatellite typing in the future.

In humans, the single polymorphic B locus of the major histocompatibility complex is linked to the microsatellite MIB. In rhesus macaques, however, haplotypes are characterized by the presence of unique combinations of multiple B genes, which may display different levels of polymorphism. The aim of the study was to shed light on the evolutionary history of this highly complex region. First, the robustness of the microsatellite MIB-linked to almost half of the B genes in rhesus macaques (Mamu-B)-for accurate B haplotyping was studied. Based on the physical map of an established haplotype comprising 7 MIB loci, each located next to a certain Mamu-B gene, two MIB loci, MIB1 and MIB6, were investigated in a panel of MHC homozygous monkeys. MIB1 revealed a complex genotyping pattern, whereas MIB6 analysis resulted in the detection of one or no amplicon. Both patterns are specific for a given B haplotype, show Mendelian segregation, and even allow a more precise haplotype definition than do traditional typing methods. Second, a search was performed for retroelements that may have played a role in duplication processes as observed in the macaque B region. This resulted in the description of two types of duplicons. One basic unit comprises an expressed Mamu-B gene, adjacent to an HERV16 copy closely linked to MIB. The second type of duplicon comprises a Mamu-B (pseudo)gene, linked to a truncated HERV16 structure lacking its MIB segment. Such truncation seems to coincide with the loss of B gene transcription. Subsequent to the duplication processes, recombination between MIB and Mamu-B loci appears to have occurred, resulting in a hyperplastic B region. Thus, analysis of MIB in addition to B loci allows deciphering of the compound evolutionary history of the class I B region in Old World monkeys.

BackgroundIn sharp contrast to humans and great apes, the expandedMhc-Bregion of rhesus and cynomolgus macaques is characterized by the presence of differential numbers and unique combinations of polymorphic class IBgenes per haplotype. The MIB microsatellite is closely linked to the single class IBgene in human and in some great apes studied. The physical map of theMhcof a heterozygous rhesus monkey provides unique material to analyze MIB andMamu-Bcopy number variation and then allows one to decipher the compound evolutionary history of this region in primate species.ResultsIn silicoresearch pinpointed 12 MIB copies (duplicons), most of which are associated with expressedB-genes that cluster in a separate clade in the phylogenetic tree. Generic primers tested on homozygous rhesus and pedigreed cynomolgus macaques allowed the identification of eight to eleven MIB copies per individual. The number of MIB copies present per haplotype varies from a minimum of three to six in cynomolgus macaques and from five to eight copies in rhesus macaques. Phylogenetic analyses highlight a strong transpecific sharing of MIB duplicons. Using the physical map, we observed that, similar to MIB duplicons, highly divergentMamu-Bgenes can be present on the same haplotype. Haplotype variation as reflected by the copy number variation of class IBloci is best explained by recombination events, which are found to occur between MIBs andMamu-B.ConclusionThe data suggest the existence of highly divergent MIB andMamu-Blineages on a given haplotype, as well as variable MIB andBcopy numbers and configurations, at least in rhesus macaque. Recombination seems to occur between MIB andMamu-Bloci, and the resulting haplotypic plasticity at the individual level may be a strategy to better cope with pathogens. Therefore, evolutionary inferences based on the multiplicated MIB loci but also other markers close toB-genes appear to be promising for the study ofB-region organization and evolution in primates.

Adaptation by natural selection is central to the evolution of species. By targeting differences in survival and/or reproduction of individuals according to changes in the environment, selection filters genetic variants in populations. Extremely conserved genes are subjected to purifying selection which eliminates deleterious mutations, while other more polymorphic genes will carry positively selected mutations in a certain environmental context. For more than 20 years, modeling in population genetics and the emergence of sequencing technologies allowing the identification of genetic variants at the genome level (e.g. Single Nucleotide Polymorphisms - SNP) have allowed the development of numerous statistical methods analyzing polymorphism to identify genes or regions of the genome presenting selection signatures, while taking into account the other evolutionary forces (genetic drift, gene flow) influencing this polymorphism. However, they test the selection hypothesis independently at each locus and do not allow the interaction between the alleles of genes to be explored as a potential target for epistatic selection. Still, quantitative genetics and modern biology unquestionably show that genes are not functionally independent entities, but that they are interacting elements in larger networks allowing the expression of biological characteristics. The purpose of this thesis is to propose a new statistical test which makes it possible to identify epistatic selection signatures, therefore coadapted genes, on the basis of linkage disequilibrium (DL) using SNP markers. In the first part, we describe the proposed statistics, Trv and TcorPC1v, which compare pairs of SNPs or genomic regions. They are based on recent work showing that the correlation coefficient (r), strongly influenced by the genetic structure of populations and the degree of genetic similarity between individuals, must be corrected (rv) by the relationship matrix between individuals (V). Coupled with intensive calculations, simulations of genome-wide SNP data in structured populations have made it possible to demonstrate that Trv and TcorPC1v follow a Student distribution t(n-2), greatly reduce noisy DL background generated by non-selective evolutionary forces, and show good detection power. In a second part, we use the "Genome-Wide Epistatic Selection Scan" (GWESS) approach in the model plant Medicago truncatula, where a candidate gene is used as bait to calculate its correlation with all the other genes in the genome. Following the identification of an epistatic selection signature between MtSUNN and MtCLE02, coding respectively for a receptor and a signaling peptide, a proof of concept is provided by the experimental demonstration (collaboration) that MtCLE02 has a MtSUNN-dependent negative role on nodulation. The GWESS approach applied to SNP data in humans shows an epistatic selection signature between the SLC24A5 and EDAR genes, involved in skin pigmentation and the development of ectodermal organs (hair, teeth).[...]; L'adaptation par sélection naturelle est centrale dans l'évolution des espèces. En ciblant les différences de survie et/ou de reproduction des individus en fonction des changements de l'environnement, la sélection filtre les variants génétiques dans les populations. Les gènes extrêmement conservés sont soumis à la sélection purifiante qui élimine les mutations délétères, alors que d'autres gènes plus polymorphes porteront des mutations positivement sélectionnées dans un certain contexte environnemental. Depuis plus de 20 ans, la modélisation en génétique des populations et l'émergence de technologies de séquençage accélérant l'identification de variants génétiques à l'échelle du génome (e.g. les Single Nucleotide Polymorphisms - SNP) ont permis le développement de nombreuses méthodes statistiques analysant le polymorphisme pour identifier des gènes ou régions du génome présentant des signatures de sélection, tout en tenant compte des autres forces évolutives (dérive génétique, flux géniques) influençant ce polymorphisme. Cependant, elles testent l'hypothèse de sélection indépendamment sur chaque locus et ne permettent pas d'explorer l'interaction entre les allèles des gènes comme cible potentielle de la sélection épistatique. Or, la génétique quantitative et la biologie moderne montrent indiscutablement que les gènes ne sont pas des entités fonctionnellement indépendantes, mais qu'ils sont des éléments interagissant dans des réseaux plus vastes permettant l'expression des caractéristiques biologiques. Cette thèse a pour objectif de proposer un nouveau test statistique qui permet d'identifier des signatures de sélection épistatique, donc des gènes coadaptés, sur la base du déséquilibre de liaison (DL) à l'aide de marqueurs SNP. Dans une première partie, nous décrivons les statistiques proposées, Trv et TcorPC1v, qui comparent des paires de SNP ou de régions génomiques. Elles sont basées sur des travaux récents montrant que le coefficient de corrélation (r), fortement influencé par la structuration génétique des populations et l'apparentement des individus, doit être corrigé (rv) par la matrice d'apparentement entre les individus (V). Couplées à des calculs intensifs, des simulations de données SNP pangénomiques en populations structurées ont permis de démontrer que Trv et TcorPC1v suivent une distribution de Student t(n-2), réduisent fortement le bruit de fond de DL généré par les forces évolutives non sélectives, et ont une bonne puissance de détection. Dans une deuxième partie, nous utilisons l'approche de " Genome-Wide Epistatic Selection Scan " (GWESS) chez la plante modèle Medicago truncatula, où un gène candidat est utilisé comme appât pour calculer sa corrélation avec tous les autres gènes du génome. Suite à l'identification d'une signature de sélection épistatique entre MtSUNN et MtCLE02, codant respectivement pour un récepteur et un peptide de signalisation, une preuve de concept est apportée par la démonstration expérimentale (collaboration) que MtCLE02 a un rôle négatif sur la nodulation et dépendant de MtSUNN.[...]