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International audience; The Gram-negative bacterium Ralstonia solanacearum, the causal agent of bacterial wilt, is a worldwide major crop pathogen whose virulence strongly relies on a type III secretion system (T3SS). This extracellular apparatus allows the translocation of proteins, called type III effectors (T3Es), directly into the host cells. To date, very few data are available in plant-pathogenic bacteria concerning the role played by type III secretion (T3S) regulators at the posttranslational level. We have demonstrated that HpaP, a putative T3S substrate specificity switch protein of R. solanacearum, controls T3E secretion. To better understand the role of HpaP on T3S control, we analyzed the secretomes of the GMI1000 wild-type strain as well as the hpaP mutant using a mass spectrometry experiment (liquid chromatography tandem mass spectrometry). The secretomes of both strains appeared to be very similar and highlighted the modulation of the secretion of few type III substrates. Interestingly, only one type III-associated protein, HrpJ, was identified as specifically secreted by the hpaP mutant. HrpJ appeared to be an essential component of the T3SS, essential for T3S and pathogenicity. We further showed that HrpJ is specifically translocated in planta by the hpaP mutant and that HrpJ can physically interact with HpaP. Moreover, confocal microscopy experiments demonstrated a cytoplasmic localization for HrpJ once in planta. When injected into Arabidopsis thaliana leaves, HrpJ is able to trigger a necrosis on 16 natural accessions. A genome-wide association mapping revealed a major association peak with 12 highly significant single-nucleotide polymorphisms located on a plant acyl-transferase.

International audience; Microbial communities are influenced by a complex system of host effects, including traits involved in physical barriers, immunity, hormones, metabolisms and nutrient homeostasis. Variation of host control within species is governed by many genes of small effect and is sensitive to biotic and abiotic environments. On the flip side, these host impacts seem targeted on particular microbial species, with that impact percolating through the microbial community. There is not yet evidence that the nature and strength of these interactions differs between fungal and bacterial communities, or among different compartments of the plant. The challenge of deciphering how systems of host traits impact systems of microbial associates is vast but holds promise for developing novel strategies to improve plant health.

Plants are daily challenged by multiple abiotic and biotic stresses. A major biotic constraint corresponds to competition with other plant species. Although plants simultaneously interact with multiple neighboring species throughout their life cycle, there is still very limited information about the genetics of the competitive response in the context of plurispecific interactions. Using a local mapping population of Arabidopsis thaliana, we set up a genome wide association study (GWAS) to estimate the extent of genetic variation of competitive response in 12 plant species assemblages, based on three competitor species (Poa annua, Stellaria media, and Veronica arvensis). Based on five phenotypic traits, we detected strong crossing reaction norms not only between the three bispecific neighborhoods but also among the plurispecific neighborhoods. The genetic architecture of competitive response was highly dependent on the identity and the relative abundance of the neighboring species. In addition, most of the enriched biological processes underlying competitive responses largely differ among neighborhoods. While the RNA related processes might confer a broad range response toolkit for multiple traits in diverse neighborhoods, some processes, such as signaling and transport, might play a specific role in particular assemblages. Altogether, our results suggest that plants can integrate and respond to different species assemblages depending on the identity and number of each neighboring species, through a large range of candidate genes associated with diverse and unexpected processes leading to developmental and stress responses.

Associate Editor: Stephen Wright; International audience; Despite the importance of plant-plant interactions on plant community dynamics and crop yield, our understanding of the adaptive genetics underlying these interactions is still limited and deserves to be investigated in the context of complex and diffuse interactions occurring in plant assemblages. Here, based on 145 natural populations of Arabidopsis thaliana located in south-west of France and characterized for plant communities, we conducted a Genome-Environment Association analysis to finely map adaptive genomic regions of A. thaliana associated with plant community descriptors. To control for correlated abiotic environment effects, we also characterized the populations for a set of biologically meaningful climate and soil variables. A nonnegligible fraction of top single nucleotide polymorphisms was associated with both plant community descriptors and abiotic variables, highlighting the importance of considering the actual abiotic drivers of plant communities to disentangle genetic variants for biotic adaptation from genetic variants for abiotic adaptation. The adaptive loci associated with species abundance were highly dependent on the identity of the neighboring species suggesting a high degree of biotic specialization of A. thaliana to members of its plant interaction network. Moreover, the identification of adaptive loci associated with a-diversity and composition of plant communities supports the ability of A. thaliana to interact simultaneously with multiple plant neighbors, which in turn can help to understand the role of community-wide selection. Altogether, our study highlights that dissecting the genetic basis underlying plant-plant interactions at a regional scale while controlling for abiotic confounding factors can help understanding the adaptive mechanisms modulating natural plant assemblages.

A current challenge in microbial pathogenesis is to identify biological control agents that may prevent and/or limit host invasion by microbial pathogens. In natura, hosts are often infected by multiple pathogens. However, most of the current studies have been performed under laboratory controlled conditions and by taking into account the interaction between a single commensal species and a single pathogenic species. The next step is therefore to explore the relationships between host-microbial communities (microbiota) and microbial members with potential pathogenic behavior (pathobiota) in a realistic ecological context. In the present study, we investigated such relationships within root and leaf associated bacterial communities of 163 ecologically contrasted Arabidopsis thaliana populations sampled across two seasons in South-West of France. In agreement with the theory of the invasion paradox, we observed a significant humped-back relationship between microbiota and pathobiota α-diversity that was robust between both seasons and plant organs. In most populations, we also observed a strong dynamics of microbiota composition between seasons. Accordingly, the potential pathobiota composition was explained by combinations of season-specific microbiota OTUs. This result suggests that the potential biomarkers controlling pathogen’s invasion are highly dynamic.

Herbivorous insects exhibit strong feeding preferences when choosing among plant genotypes, yet experiments to map loci mediating plant susceptibility to herbivory rarely incorporate host choice. To address this gap, we applied genome-wide association (GWA) mapping to uncover genetic polymorphisms mediating damage from foraging insects (two populations ofScaptomyza flava) across a mixture ofArabidopsis thalianagenotypes in experimental enclosures. The effect of chemical defenses (glucosinolates) on herbivory depended on herbivore genotype. Unlike many studies that minimize the effects of host choice behavior, we also found a large effect of plant size on herbivory—likely through its effect on plant apparency—that was independent of herbivore genotype. These herbivory-associated loci are polymorphic at fine spatial scales, and thus have potential to shape variation in herbivory within natural populations. We also show that the polymorphism with the largest effect on herbivory underlies adaptive latitudinal variation inArabidopsisplant size across Europe. Overall, our results provide genetic support for ecological observations that variation in both chemical defenses and non-canonical defense traits (e.g., plant size and phenology) jointly shapes plant-herbivore interactions.

In recent years Arabidopsis thaliana has become a model species for genomic variability and adaptation studies. Although impressive quantities of data have been gathered on the nuclear genomic diversity of this species, little has been published regarding its cytoplasmic diversity. We analyzed the diversity of plastid (pt) and mitochondrial (mt) genomes among 95 accessions, covering most Arabidopsis geographic origins. Four intergenic regions of the pt genome were sequenced, and a total of 68 polymorphisms and 65 pt haplotypes were identified. Several strategies were developed to identify mt polymorphisms among a subset of 14 accessions. Fifteen polymorphisms were further developed as PCR-based markers and used to analyze the whole set of 95 accessions. Using statistical parsimony, we built pt and mt phylogenetic networks of haplotype groups. To root the pt network, the pt intergenic regions of two related Arabidopsis species, Arabidopsis lyrata and Arabidopsis arenosa, were also sequenced. The mt and pt phylogenies are highly congruent and could be combined into a single cytoplasmic phylogeny. To estimate whether co-adaptation between nuclear and cytoplasmic genomes exists in A. thaliana, we tested the germination capacity in challenging conditions of 27 pairs of reciprocal F(2) families. We found that the cytoplasm donor had a significant effect on the germination capacity of some F(2) families.

International audience; The advantage of the resistance conferred by a mutation can sometimes be offset by a high fitness-cost penalty. This balance will affect possible fate of the resistance allele. Few studies have explored the impact of the genetic background on the expression of the resistance fitness cost and none has attempted to measure the variation in fitness-cost dominance. However, both the fitness penalty and its dominance may modify evolutionary trajectory and outcome. Here the impact of Arabidopsis thaliana intraspecific genetic diversity on fitness cost and its associated dominance was investigated by analysing 12 quantitative traits in crosses between a mutant conferring resistance to the herbicide 2,4-D and nine different natural genetic backgrounds. Fitness cost values were found to be more affected by intraspecific genetic diversity than fitness cost dominance, even though this effect depends on the quantitative trait measured. This observation has implications for the choice of the best strategy for preventing herbicide resistance development. In addition, our results pinpoint a potential compensatory improvement of the resistance fitness cost and its associated dominance by the genetic diversity locally present within a species.

The evolution of resistance in response to pesticide selection is expected to be delayed if fitness costs are associated with resistance genes. The estimate of fitness costs usually involves comparing major growth traits of resistant versus susceptible individuals in the absence of pesticide. Ideally, a measure of changes in resistance allele frequency over several generations would allow the best estimate of the overall fitness cost of a resistance gene. In greenhouse conditions, we monitored the dynamics of the evolution of the frequencies of six herbicide-resistant mutations (acetolactate synthase, cellulose synthase, and auxin-induced target genes) in the model species Arabidopsis thaliana in a multigenerational study covering five to seven nonoverlapping generations. The microevolutionary dynamics in experimental populations indicated a mean fitness cost of 38%, 73%, and 94% for the ixr1-2, axr1-3, and axr2-1 resistances, respectively; no fitness cost for the csr1-1, and ixr2-1 resistances; and a transient advantage for the aux1-7 resistance. The result for the csr1-1 resistance contrasts with a cost of 37% based on total seed number in a previous study, demonstrating that single generation studies could have limitation for detecting cost. A positive frequency dependence for the fitness cost was also detected for the ixr1-2 resistance. The results are discussed in relation to the maintenance of polymorphism at resistance loci.

International audience; The "mustard oil bomb" is a major defense mechanism in the Brassicaceae, which includes crops such as canola and the model plant Arabidopsis thaliana. These plants produce and store blends of amino acid-derived secondary metabolites called glucosinolates. Upon tissue rupture by natural enemies, the myrosinase enzyme hydrolyses glucosinolates, releasing defense molecules. Brassicaceae display extensive variation in the mixture of glucosinolates that they produce. To investigate the genetics underlying natural variation in glucosinolate profiles, we conducted a large genome-wide association study of 22 methionine-derived glucosinolates using A. thaliana accessions from across Europe. We found that 36% of among accession variation in overall glucosinolate profile was explained by genetic differentiation at only three known loci from the glucosinolate pathway. Glucosinolate-related SNPs were up to 490-fold enriched in the extreme tail of the genome-wide F-ST scan, indicating strong selection on loci controlling this pathway. Glucosinolate profiles displayed a striking longitudinal gradient with alkenyl and hydroxyalkenyl glucosinolates enriched in the West. We detected a significant contribution of glucosinolate loci toward general herbivore resistance and lifetime fitness in common garden experiments conducted in France, where accessions are enriched in hydroxyalkenyls. In addition to demonstrating the adaptive value of glucosinolate profile variation, we also detected long-distance linkage disequilibrium at two underlying loci, GS-OH and GS-ELONG. Locally cooccurring alleles at these loci display epistatic effects on herbivore resistance and fitness in ecologically realistic conditions. Together, our results suggest that natural selection has favored a locally adaptive configuration of physically unlinked loci in Western Europe.

We extensively phenotyped 6000 Arabidopsis plants with experimentally perturbed DNA methylomes as well as a diverse panel of natural accessions in a common garden. We found that alterations in DNA methylation not only caused heritable phenotypic diversity but also produced heritability patterns closely resembling those of the natural accessions. Our findings indicate that epigenetically induced and naturally occurring variation in complex traits share part of their polygenic architecture and may offer complementary adaptation routes in ecological settings.

Long-term plant resistance polymorphism does not require obligate association but instead is maintained in the face of diffuse ecological interactions. Despite the prevalence of generalist pathogens, ancient balanced polymorphisms in resistance (R) genes are ubiquitous in plants such as Arabidopsis thaliana, although they rarely provide adequate defence in an agricultural context. The mechanisms involved in maintaining such polymorphisms are unknown. Here Joy Bergelson and colleagues identify a naturally interacting R gene (in A. thaliana) and effector (in A. thaliana's pathogen Pseudomonas syringae) pair. The protein product of this R gene, RPS5, recognizes AvrPphB2 protein in P. syringae and exhibits a balanced polymorphism that has been maintained for more than two million years. The presence of P. syringae carrying avrPphB seems insufficient to explain the RPS5 polymorphism. Rather, selection for RPS5 may involve multiple non-homologous effectors and multiple pathogen species, suggesting that this R gene polymorphism is maintained through complex and diffuse community-wide interactions. Plant resistance (R) genes are a crucial component in plant defence against pathogens1. Although R genes often fail to provide durable resistance in an agricultural context, they frequently persist as long-lived balanced polymorphisms in nature2,3,4. Standard theory explains the maintenance of such polymorphisms through a balance of the costs and benefits of resistance and virulence in a tightly coevolving host–pathogen pair5,6. However, many plant–pathogen interactions lack such specificity7. Whether, and how, balanced polymorphisms are maintained in diffusely interacting species8 is unknown. Here we identify a naturally interacting R gene and effector pair in Arabidopsis thaliana and its facultative plant pathogen, Pseudomonas syringae. The protein encoded by the R gene RPS5 recognizes an AvrPphB homologue (AvrPphB2) and exhibits a balanced polymorphism that has been maintained for over 2 million years (ref. 3). Consistent with the presence of an ancient balanced polymorphism, the R gene confers a benefit when plants are infected with P. syringae carrying avrPphB2 but also incurs a large cost in the absence of infection. RPS5 alleles are maintained at intermediate frequencies in populations globally, suggesting ubiquitous selection for resistance. However, the presence of P. syringae carrying avrPphB is probably insufficient to explain the RPS5 polymorphism. First, avrPphB homologues occur at very low frequencies in P. syringae populations on A. thaliana. Second, AvrPphB only rarely confers a virulence benefit to P. syringae on A. thaliana. Instead, we find evidence that selection for RPS5 involves multiple non-homologous effectors and multiple pathogen species. These results and an associated model suggest that the R gene polymorphism in A. thaliana may not be maintained through a tightly coupled interaction involving a single coevolved R gene and effector pair. More likely, the stable polymorphism is maintained through complex and diffuse community-wide interactions.

International audience; Often used as a proxy for the transition to reproduction, flowering time (FT) is an integrative trait of two successive biological processes, i.e. bolting time (BT) and the interval between bolting and flowering time (INT). In this study, we aimed to identify candidate genes associated with these composite traits in Arabidopsis thaliana using a field experiment. Genome-wide association (GWA) mapping was performed on BT, INT and FT based on a sample of 179 worldwide natural accessions genotyped for 216,509 SNPs. The high resolution conferred by GWA mapping indicates that FT is an integrative trait at the genetic level, with distinct genetics for BT and INT. BT is shaped largely by genes involved in the circadian clock whereas INT is shaped by genes involved in both the hormone pathways and cold acclimation. Finally, the florigen TSF appears to be the main integrator of environmental and internal signals in ecologically realistic conditions. Based on FT scored in a previous field experiment, we also studied the genetics underlying reaction norms across two years. Only four genes were common to both years, emphasizing the need to repeat field experiments. The gene regulation model appeared as the main genetic model for genotype × year interactions.

The failure of gene-for-gene resistance traits to provide durable and broad-spectrum resistance in an agricultural context has led to the search for genes underlying quantitative resistance in plants. Such genes have been identified in only a few cases, all for fungal or nematode resistance, and encode diverse molecular functions. However, an understanding of the molecular mechanisms of quantitative resistance variation to other enemies and the associated evolutionary forces shaping this variation remain largely unknown. We report the identification, map-based cloning and functional validation of QRX3 (RKS1, Resistance related KinaSe 1), conferring broad-spectrum resistance to Xanthomonas campestris (Xc), a devastating worldwide bacterial vascular pathogen of crucifers. RKS1 encodes an atypical kinase that mediates a quantitative resistance mechanism in plants by restricting bacterial spread from the infection site. Nested Genome-Wide Association mapping revealed a major locus corresponding to an allelic series at RKS1 at the species level. An association between variation in resistance and RKS1 transcription was found using various transgenic lines as well as in natural accessions, suggesting that regulation of RKS1 expression is a major component of quantitative resistance to Xc. The co-existence of long lived RKS1 haplotypes in A. thaliana is shared with a variety of genes involved in pathogen recognition, suggesting common selective pressures. The identification of RKS1 constitutes a starting point for deciphering the mechanisms underlying broad spectrum quantitative disease resistance that is effective against a devastating and vascular crop pathogen. Because putative RKS1 orthologous have been found in other Brassica species, RKS1 provides an exciting opportunity for plant breeders to improve resistance to black rot in crops., Author Summary During the evolution of plant-pathogen interactions, plants have evolved the capability to defend themselves from pathogen infection by different overlapping mechanisms. Disease resistance is constituted by an elaborate, multilayered system of defense. Among these responses, quantitative resistance is a prevalent form of resistance in crops and natural plant populations, for which the genetic and molecular bases remain largely unknown. Thus, identification of the genes underlying quantitative resistance constitutes a major challenge in plant breeding and evolutionary biology, and might have enormous practical implications for human health by increasing crop yield and quality. Our work contributes to understanding the molecular bases of quantitative resistance to the vascular pathogen Xanthomonas campestris (Xc), which is responsible for black rot, an important disease of crucifers worldwide. By multiple approaches, we demonstrate that RKS1 is a quantitative resistance gene in Arabidopsis thaliana conferring broad-spectrum resistance to Xc and that this resistance mechanism in plants is associated with regulation of RKS1 expression. We also provide evidence that RKS1 allelic variation is a major component of quantitative resistance to Xc at the species level. Finally, the long-lived polymorphism associated with RKS1 suggests that evolutionary stable broad-spectrum resistance to Xc may be achieved in natural populations of A. thaliana.

International audience; Despite the increasing number of genomic tools, identifying the genetics underlying adaptive complex traits remains challenging in the model species Arabidopsis thaliana. This is due, at least in part, to the lack of data on the geographical scale of adaptive phenotypic variation. The aims of this study were (i) to tease apart the historical roles of adaptive and nonselective processes in shaping phenological variation in A. thaliana in France and (ii) to gain insights into the spatial scale of adaptive variation by identifying the putative selective agents responsible for this selection. Forty-nine natu- ral stands from four climatically contrasted French regions were characterized (i) phe- nologically for six traits, (ii) genetically using 135 SNP markers and (iii) ecologically for 42 variables. Up to 63% of phenological variation could be explained by neutral genetic diversity. The remaining phenological variation displayed stronger associations with ecological variation within regions than among regions, suggesting the impor- tance of local selective agents in shaping adaptive phenological variation. Although climatic conditions have often been suggested as the main selective agents acting on phenology in A. thaliana, both edaphic conditions and interspecific competition appear to be strong selective agents in some regions. In a first attempt to identify the genetics of phenological variation at different geographical scales, we phenotyped worldwide accessions and local polymorphic populations from the French RegMap in a genome- wide association (GWA) mapping study. The genomic regions associated with pheno- logical variation depended upon the geographical scale considered, stressing the need to account for the scale of adaptive phenotypic variation when choosing accession pan- els for GWAS.

International audience; Phenological traits often show variation within and among natural populations of annual plants. Nevertheless, the adaptive value of post-anthesis traits is seldom tested. In this study, we estimated the adaptive values of pre- and post-anthesis traits in two stressful environments (water stress and interspecific competition), using the selfing annual species Arabidopsis thaliana. By estimating seed production and by performing laboratory natural selection (LNS), we assessed the strength and nature (directional, disruptive and stabilizing) of selection acting on phenological traits in A. thaliana under the two tested stress conditions, each with four intensities. Both the type of stress and its intensity affected the strength and nature of selection, as did genetic constraints among phenological traits. Under water stress, both experimental approaches demonstrated directional selection for a shorter life cycle, although bolting time imposes a genetic constraint on the length of the interval between bolting and anthesis. Under interspecific competition, results from the two experimental approaches showed discrepancies. Estimation of seed production predicted directional selection toward early pre-anthesis traits and long post-anthesis periods. In contrast, the LNS approach suggested neutrality for all phenological traits. This study opens questions on adaptation in complex natural environment where many selective pressures act simultaneously.

International audience; Background: The non conventional RTM (Restricted Tobacco etch virus Movement) resistance which restricts long distance movement of some plant viruses in Arabidopsis thaliana is still poorly understood. Though at least three RTM genes have been identified, their precise role(s) in the process as well as whether other genes are involved needs to be elucidated.Methodology/Principal Findings: In this study, the natural variation of the RTM genes was analysed at the amino acid level in relation with their functionality to restrict the long distance movement of Lettuce mosaic potyvirus (LMV). We identified non-functional RTM alleles in LMV-susceptible Arabidopsis accessions as well as some of the mutations leading to the nonfunctionality of the RTM proteins. Our data also indicate that more than 40% of the resistant accessions to LMV are controlled by the RTM genes. In addition, two new RTM loci were genetically identified. Conclusions/Significance: Our results show that the RTM resistance seems to be a complex biological process which would involves at least five different proteins. The next challenges will be to understand how the different RTM protein domains are involved in the resistance mechanism and to characterise the new RTM genes for a better understanding of the blocking of the long distance transport of plant viruses.

Plants exhibit a number of adaptive defence traits that endow resistance to past and current abiotic and biotic stresses. It is generally accepted that these adaptations will incur a cost when plants are not challenged by the stress to which they have become adaptedthe so-called cost of adaptation. The need to minimise or account for allelic variation at other fitness-related loci (genetic background control) is frequently overlooked when assessing resistance costs associated with plant defence traits. We provide a synthesis of the various experimental protocols that accomplish this essential requirement. We also differentiate those methods that enable the identification of the trait-specific or mechanistic basis of costs (direct methods) from those that provide an estimate of the impact of costs by examining the evolutionary trajectories of resistance allele frequencies at the population level (indirect methods). The advantages and disadvantages for each proposed experimental design are discussed. We conclude that plant resistance systems provide an ideal model to address fundamental questions about the cost of adaptation to stress. We also propose some ways to expand the scope of future studies for further fundamental and applied insight into the significance of adaptation costs. Fil: Vila Aiub, Martin Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura; Argentina Fil: Neve, P.. University of Warwick; Reino Unido Fil: Roux, F.. Centre National de la Recherche Scientifique. Institut de Biologie Moléculaire des Plantes; Francia

International audience; Understanding the genetic bases and modes of adaptation to current climatic conditions is essential to accurately predict responses to future environmental change. We conducted a genome-wide scan to identify climate-adaptive genetic loci and pathways in the plant Arabidopsis thaliana. Amino acid-changing variants were significantly enriched among the loci strongly correlated with climate, suggesting that our scan effectively detects adaptive alleles. Moreover, from our results, we successfully predicted relative fitness among a set of geographically diverse A. thaliana accessions when grown together in a common environment. Our results provide a set of candidates for dissecting the molecular bases of climate adaptations, as well as insights about the prevalence of selective sweeps, which has implications for predicting the rate of adaptation.

International audience; Although pioneered by human geneticists as a potential solution to the challenging problem of finding the genetic basis of common human diseases1,2, genome-wide association (GWA) studies have, owing to advances in genotyping and sequencing technology, become an obvious general approach for studying the genetics of natural variation and traits of agricultural importance. They are particularly useful when inbred lines are available, because once these lines have been genotyped they can be phenotyped multiple times, making it possible (as well as extremely cost effective) to study many different traits in many different environments, while replicating the phenotypic measurements to reduce environmental noise. Here we demonstrate the power of this approach by carrying out a GWA study of 107 phenotypes in Arabidopsis thaliana, a widely distributed, predominantly self-fertilizing model plant known to harbour considerable genetic variation for many adaptively important traits3. Our results are dramatically different from those of human GWA studies, in that we identify many common alleles of major effect, but they are also, in many cases, harder to interpret because confounding by complex genetics and population structure make it difficult to distinguish true associations from false. However, a-priori candidates are significantly over-represented among these associations as well, making many of them excellent candidates for follow-up experiments. Our study demonstrates the feasibility of GWA studies in A. thaliana and suggests that the approach will be appropriate for many other organisms.

Flowering time is a key life-history trait in the plant life cycle. Most studies to unravel the genetics of flowering time in Arabidopsis thaliana have been performed under greenhouse conditions. Here, we describe a study about the genetics of flowering time that differs from previous studies in two important ways: first, we measure flowering time in a more complex and ecologically realistic environment; and, second, we combine the advantages of genome-wide association (GWA) and traditional linkage (QTL) mapping. Our experiments involved phenotyping nearly 20,000 plants over 2 winters under field conditions, including 184 worldwide natural accessions genotyped for 216,509 SNPs and 4,366 RILs derived from 13 independent crosses chosen to maximize genetic and phenotypic diversity. Based on a photothermal time model, the flowering time variation scored in our field experiment was poorly correlated with the flowering time variation previously obtained under greenhouse conditions, reinforcing previous demonstrations of the importance of genotype by environment interactions in A. thaliana and the need to study adaptive variation under natural conditions. The use of 4,366 RILs provides great power for dissecting the genetic architecture of flowering time in A. thaliana under our specific field conditions. We describe more than 60 additive QTLs, all with relatively small to medium effects and organized in 5 major clusters. We show that QTL mapping increases our power to distinguish true from false associations in GWA mapping. QTL mapping also permits the identification of false negatives, that is, causative SNPs that are lost when applying GWA methods that control for population structure. Major genes underpinning flowering time in the greenhouse were not associated with flowering time in this study. Instead, we found a prevalence of genes involved in the regulation of the plant circadian clock. Furthermore, we identified new genomic regions lacking obvious candidate genes., Author Summary Dissecting the genetic bases of adaptive traits is of primary importance in evolutionary biology. In this study, we combined a genome-wide association (GWA) study with traditional linkage mapping in order to detect the genetic bases underlying natural variation in flowering time in ecologically realistic conditions in the plant Arabidopsis thaliana. Our study involved phenotyping nearly 20,000 plants over 2 winters under field conditions in a temperate climate. We show that combined linkage and association mapping clearly outperforms each method alone when it comes to identifying true associations. This highlights the utility of combining different methods to localize genes involved in complex trait natural variation. Most candidate genes found in this study are involved in the regulation of the plant circadian clock and, surprisingly, were not associated with flowering time scored under greenhouse conditions. While rapid advances have been made in high-throughput genotyping and sequencing, high-throughput phenotyping of complex traits under natural conditions will be the next challenge for dissecting the genetic bases of adaptive variation in “laboratory” model organisms.

International audience; A major challenge in evolutionary biology and plant breeding is to identify the genetic basis of complex quantitative traits, including those that contribute to adaptive variation. Here we review the development of new methods and resources to fine-map intraspecific genetic variation that underlies natural phenotypic variation in plants. In particular, the analysis of 107 quantitative traits reported in the first genome-wide association mapping study in Arabidopsis thaliana sets the stage for an exciting time in our understanding of plant adaptation. We also argue for the need to place phenotype–genotype association studies in an ecological context if one is to predict the evolutionary trajectories of plant species.

International audience; Agricultural weeds evolve in response to crop cultivation. Nevertheless, the central importance of evolutionary ecology for understanding weed invasion, persistence and management in agroecosystems is not widely acknowledged. This paper calls for more evolutionarily-enlightened weed management, in which management principles are informed by evolutionary biology to prevent or minimize weed adaptation and spread. As a first step, a greater knowledge of the extent, structure and significance of genetic variation within and between weed populations is required to fully assess the potential for weed adaptation. The evolution of resistance to herbicides is a classic example of weed adaptation. Even here, most research focuses on describing the physiological and molecular basis of resistance, rather than conducting studies to better understand the evolutionary dynamics of selection for resistance. We suggest approaches to increase the application of evolutionary- thinking to herbicide resistance research. Weed population dynamics models are increasingly important tools in weed management, yet these models often ignore intrapopulation and interpopulation variability, neglecting the potential for weed adaptation in response to management. Future agricultural weed management can benefit from greater integration of ecological and evolutionary principles to predict the long-term responses of weed populations to changing weed management, agricultural environments and global climate.

International audience; • It is often assumed that pathogen infection decreases plant fitness, thereby driving the evolution of plant resistance (R) genes. However, the impact of bacterial pathogens on fitness has been shown to be relatively subtle, ranging from positive to negative. • In this study, we focus on the Rps5-mediated resistance in Arabidopsis thaliana and examine the fitness effects of resistance by experimentally infecting resistant (R) and susceptible (S) plants with a natural avirulent Pseudomonas syringae strain at each of three initial infection dosage levels. Our methodology ensured control of the plant genetic backgrounds; within each of two natural accessions we created isolines varying in the presence or absence of Rps5. • In terms of lifetime fitness, R plants outperformed their S controls by 9.6–32% when infected by a pathogen carrying an associated Avr gene, depending on the initial dosage levels and genetic backgrounds. • We also found that the naturally R line, Col-0, is more tolerant than the naturally S accession, Ga-0. The negative impact of infection on fitness was 20% less in Col-0 than Ga-0. We found no effect of Rps5 itself on the tolerance of either accession. We therefore failed to find evidence for a trade-off between tolerance and resistance.

BACKGROUND: Environmental heterogeneity in space or time can drive the evolutionary trajectory of an adaptive trait. This concept could be of practical significance in pesticide resistance management that aims to delay the evolution of a resistance allele. Using a population genetics model, the dynamics of herbicide resistance in a weed species was simulated in a heterogeneous environment with alternation of two unrelated herbicides in time, in space or in both time and space. The level of the environmental heterogeneity (habitat grain) was simulated by a variation in the size of the herbicide-treated areas. RESULTS: The model confirms that several strategies based on habitat heterogeneity efficiently slow down and even prevent resistance evolution. For a recessive resistant trait in outcrossing species, a medium level of environmental heterogeneity (intermediate habitat grain) was found to be the best for delaying herbicide resistance, as previously observed for insecticide resistance management. In selfing species or for a dominant resistant trait in outcrossing species, a low level of environmental heterogeneity (coarse-grained habitat) was more efficient in delaying resistance evolution when heterogeneity in both space and time were considered. CONCLUSION: This model suggests that the choice of optimal tactics for delaying herbicide resistance by enhanced heterogeneity in space or time firstly depends on the interactions between the breeding system and the dominance of the resistance allele in the presence of herbicide, then on the value of the fitness cost and lastly on the dominance of this fitness cost. Copyright © 2007 Society of Chemical Industry

Various management strategies aim at maintaining pesticide resistance frequency under a threshold value by taking advantage of the benefit of the fitness penalty (the cost) expressed by the resistance allele outside the treated area or during the pesticide selection “off years.” One method to estimate a fitness cost is to analyze the resistance allele frequency along transects across treated and untreated areas. On the basis of the shape of the cline, this method gives the relative contributions of both gene flow and the fitness difference between genotypes in the treated and untreated areas. Taking advantage of the properties of such migration–selection balance, an artificial cline was built up to optimize the conditions where the fitness cost of two herbicide-resistant mutants (acetolactate synthase and auxin-induced target genes) in the model species Arabidopsis thaliana could be more accurately measured. The analysis of the microevolutionary dynamics in these experimental populations indicated mean fitness costs of ∼15 and 92% for the csr1-1 and axr2-1 resistances, respectively. In addition, negative frequency dependence for the fitness cost was also detected for the axr2-1 resistance. The advantages and disadvantages of the cline approach are discussed in regard to other methods of cost estimation. This comparison highlights the powerful ability of an experimental cline to measure low fitness costs and detect sensibility to frequency-dependent variations.

International audience; The application of high-throughput SNP genotyping is a great challenge for many research projects in the plant genetics domain. The GOOD assay for mass spectrometry, Amplifluor® and TaqMan® are three methods that rely on different principles for allele discrimination and detection, specifically, primer extension, allele-specific PCR and hybridization, respectively. First, with the goal of assessing allele frequencies by means of SNP genotyping, we compared these methods on a set of three SNPs present in the herbicide resistance genes CSR, AXR1 and IXR1 of Arabidopsis thaliana. In this comparison, we obtained the best results with TaqMan® based on PCR specificity, flexibility in primer design and success rate. We also used mass spectrometry for genotyping polyploid species. Finally, a combination of the three methods was used for medium- to high-throughput genotyping in a number of different plant species. Here, we show that all three genotyping technologies are successful in discriminating alleles in various plant species and discuss the factors that must be considered in assessing which method to use for a given application.

International audience; In wild and cultivated annual plant species, flowering time is an important life-history trait that coordinates the life cycle with local environmental conditions. Extensive studies on the genetic basis of flowering time in the model species Arabidopsis thaliana have revealed a complex genetic network that can detect environmental and internal signals. Based on this knowledge and on known pleiotropic effects associated with flowering time genes, we suggest that a natural shift towards an early-flowering life cycle might involve only particular functional regions in a limited number of genes. Our predictions are supported by genetic theories of adaptation and by recent data about genes associated with natural variation. We analyse the extent to which these predictions can also apply to crop species.

The type of interactions among deleterious mutations is considered to be crucial in numerous areas of evolutionary biology, including the evolution of sex and recombination, the evolution of ploidy, the evolution of selfing, and the conservation of small populations. Because the herbicide resistance genes could be viewed as slightly deleterious mutations in the absence of the pesticide selection pressure, the epistatic interactions among three herbicide resistance genes (acetolactate synthase CSR, cellulose synthase IXR1, and auxin-induced AXR1 target genes) were estimated in both the homozygous and the heterozygous states, giving 27 genotype combinations in the model plant Arabidopsis thaliana. By analyzing eight quantitative traits in a segregating population for the three herbicide resistances in the absence of herbicide, we found that most interactions in both the homozygous and the heterozygous states were best explained by multiplicative effects (each additional resistance gene causes a comparable reduction in fitness) rather than by synergistic effects (each additional resistance gene causes a disproportionate fitness reduction). Dominance coefficients of the herbicide resistance cost ranged from partial dominance to underdominance, with a mean dominance coefficient of 0.07. It was suggested that the csr1-1, ixr1-2, and axr1-3 resistance alleles are nearly fully recessive for the fitness cost. More interestingly, the dominance of a specific resistance gene in the absence of herbicide varied according to, first, the presence of the other resistance genes and, second, the quantitative trait analyzed. These results and their implications for multiresistance evolution are discussed in relation to the maintenance of polymorphism at resistance loci in a heterogeneous environment.

Resistance evolution depends upon the balance between advantage and disadvantage (cost) conferred in treated and untreated areas. By analyzing morphological characters and simple fitness components, the cost associated with each of eight herbicide resistance alleles (acetolactate synthase, cellulose synthase, and auxin-induced target genes) was studied in the model plant Arabidopsis thaliana. The use of allele-specific PCR to discriminate between heterozygous and homozygous plants was used to provide insights into the dominance of the resistance cost, a parameter rarely described. Morphological characters appear more sensitive than fitness (seed production) because 6 vs. 4 differences between resistant and sensitive homozygous plants were detected, respectively. Dominance levels for the fitness cost ranged from recessivity (csr1-1, ixr1-2, and axr1-3) to dominance (axr2-1) to underdominance (aux1-7). Furthermore, the dominance level of the herbicide resistance trait did not predict the dominance level of the cost of resistance. The relationship of our results to theoretical predictions of dominance and the consequences of fitness cost and its dominance in resistance management are discussed.

FRIGIDA (FRI) is a major gene involved in the regulation of flowering time in Arabidopsis thaliana. Nucleotide variation at this gene was investigated by sequencing 25 field ecotypes collected from western Europe. Genetic diversity at FRI was characterized by a high number of haplotypes and an excess of low-frequency polymorphisms. A large excess of intraspecific nonsynonymous variation associated with low synonymous variation was detected along the first exon in the FRI gene. In contrast, no excess of nonsynonymous divergence was detected between A. thaliana and A. lyrata. The Tajima and McDonald and Kreitman tests, however, suggested that this gene has evolved in a nonneutral fashion. Nonsynonymous variation included eight loss-of-function mutations that have probably arisen recently and independently in several locations. A phenotypic evaluation of the sequenced ecotypes confirmed that these loss-of-function mutations were associated with an early-flowering phenotype. Taken together, our results suggest that DNA polymorphism at the FRI gene in A. thaliana from western Europe has been shaped by recent positive selection for earliness in a set of isolated populations.

Proceedings Paper : Meeting on Current Knowledge and Management of Genetic Resources TOULOUSE, FRANCE, OCT 09-11, 2000; International audience; Two experiments were set up to investigate how to maintain or create genetic diversity in artificial or managed populations of plants. Using Arabidopsis thaliana, we established 18 metapopulations of 20 populations each, all with the same initial genetic composition. We tested the effects of the population size, the artificial selection regime and the extinction/recolonisation regime. We report, the results of the first four generations of evolution for a trait under selection (precocity) and for allozyme diversity. As expected, overall diversity decreased in each metapopulation, and differentiation among populations increased. As expected, the differentiation was weaker for larger population sizes and in the treatment with extinction and recolonisation with no bottleneck. Artificial selection was effective because the life cycle duration was much reduced. However, most of the reduction occurred during the first generation. We observed all increase of one allele at the LAP-2 locus in all metapopulations, breaching neutral assumptions for this locus. Finally, the selection regime made little difference for small population sizes, whereas large metapopulations were more differentiated when artificial selection was heterogeneous among populations. Altogether, our results agree with theoretical exportations, and provide some new results, which could not have been anticipated. In particular, the overall decrease in genetic diversity was very large (of the order of 20% in 4 generations) even for metapopulations of 2000 individuals.

The population structure of an organism reflects its evolutionary history and influences its evolutionary trajectory. It constrains the combination of genetic diversity and reveals patterns of past gene flow. Understanding it is a prerequisite for detecting genomic regions under selection, predicting the effect of population disturbances, or modeling gene flow. This paper examines the detailed global population structure of Arabidopsis thaliana. Using a set of 5,707 plants collected from around the globe and genotyped at 149 SNPs, we show that while A. thaliana as a species self-fertilizes 97% of the time, there is considerable variation among local groups. This level of outcrossing greatly limits observed heterozygosity but is sufficient to generate considerable local haplotypic diversity. We also find that in its native Eurasian range A. thaliana exhibits continuous isolation by distance at every geographic scale without natural breaks corresponding to classical notions of populations. By contrast, in North America, where it exists as an exotic species, A. thaliana exhibits little or no population structure at a continental scale but local isolation by distance that extends hundreds of km. This suggests a pattern for the development of isolation by distance that can establish itself shortly after an organism fills a new habitat range. It also raises questions about the general applicability of many standard population genetics models. Any model based on discrete clusters of interchangeable individuals will be an uneasy fit to organisms like A. thaliana which exhibit continuous isolation by distance on many scales., Author Summary Much of the modern field of population genetics is premised on particular models of what an organism's population structure is and how it behaves. The classic models generally start with the idea of a single randomly mating population that has reached an evolutionary equilibrium. Many models relax some of these assumptions, allowing for phenomena such as assortative mating, discrete sub-populations with migration, self-fertilization, and sex-ratio distortion. Virtually all models, however, have as their core premise the notion that there exist classes of exchangeable individuals each of which represents an identical, independent sample from that class' distribution. For certain organisms, such as Drosophila melanogaster, these models do an excellent job of describing how populations work. For other organisms, such as humans, these models can be reasonable approximations but require a great deal of care in assembling samples and can begin to break down as sampling becomes locally dense. For the vast majority of organisms the applicability of these models has never been investigated.