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We analysed the natural variation of drought response in 11 Tunisian populations ofMedicago truncatulasampled from environments that varied in soil composition, salinity and water availability. Plants were cultivated in a greenhouse under well-irrigated and water-deficit treatments (50% of field capacity), and a number of traits associated with drought response were measured. Variance analysis indicated that the variation of phenotypic traits was explained by the effects of population, line, treatment, and population × treatment and line × treatment interactions, with treatment being the one with the greatest effect. A large degree of phenotypic variation for most traits in the two water treatments was found within populations. Most of the measured characters showed higher broad-sense heritabilities (H2) in well-irrigated treatment than in water-deficit treatment. Furthermore, the largest population differentiation (QST) for most of the measured traits was observed under drought stress. Most of the correlations between measured traits under both treatments were positive. Four groups of lines differing in drought tolerance were identified, with 45 susceptible, 14 moderately susceptible, 31 tolerant and 23 most tolerant lines. The tolerant group experienced lowest reductions in the length of plagiotropic axes, length of stems, number of internodes and number of leaves. The large phenotypic variation ofM. truncatulain response to drought stress can be used to identify genes and alleles important for the complex trait of drought tolerance.

Seed vigour is important for successful establishment and high yield, especially under suboptimal environmental conditions. In legumes, raffinose oligosaccharide family (RFO) sugars have been proposed as an easily available energy reserve for seedling establishment. In this study, we investigated whether the composition or amount of soluble sugars (sucrose and RFO) is part of the genetic determinants of seed vigour of Medicago truncatula using two recombinant inbred line (RIL) populations. Quantitative trait loci (QTL) mapping for germination rate, hypocotyl and radicle growth under water deficit and nutritional stress, seed weight and soluble sugar content was performed using RIL populations LR1 and LR4. Seven of the 12 chromosomal regions containing QTL for germination rate or post-germinative radicle growth under optimal or stress conditions co-located with Suc/RFO QTL. A significant negative correlation was also found between seed vigour traits and Suc/RFO. In addition, one QTL that explained 80% of the variation in the ratio stachyose/verbascose co-located with a stachyose synthase gene whose expression profile in the parental lines could explain the variation in oligosaccharide composition. The correlation and co-location of Suc/RFO ratio with germination and radicle growth QTL suggest that an increased Suc/RFO ratio in seeds of M. truncatula might negatively affect seed vigour.

Summary Genome-wide association studies rely upon segregating natural genetic variation, particularly the patterns of polymorphism and correlation between adjacent markers. To facilitate association studies in the model legume Medicago truncatula, we present a genome-scale polymorphism scan using existing Affymetrix microarrays. We develop and validate a method that uses a simple information-criteria algorithm to call polymorphism from microarray data without reliance on a reference genotype. We genotype 12 inbred M. truncatula lines sampled from four wild Tunisian populations and find polymorphisms at approximately 7% of features, comprising 31 419 probes. Only approximately 3% of these markers assort by population, and of these only 10% differentiate between populations from saline and non-saline sites. Fifty-two differentiated probes with unique genome locations correspond to 18 distinct genome regions. Sanger resequencing was used to characterize a subset of maker loci and develop a single nucleotide polymorphism (SNP)-typing assay that confirmed marker assortment by habitat in an independent sample of 33 individuals from the four populations. Genome-wide linkage disequilibrium (LD) extends on average for approximately 10 kb, falling to background levels by approximately 500 kb. A similar range of LD decay was observed in the 18 genome regions that assort by habitat; these LD blocks delimit candidate genes for local adaptation, many of which encode proteins with predicted functions in abiotic stress tolerance and are targets for functional genomic studies. Tunisian M. truncatula populations contain substantial amounts of genetic variation that is structured in relatively small LD blocks, suggesting a history of migration and recombination. These populations provide a strong resource for genome-wide association studies.

We used 19 quantitative traits and 14 microsatellite markers (SSRs) to analyze the genetic variation in four natural populations of the model legume Medicago truncatula sampled in southern Tunisia. The greatest genetic variation of quantitative traits and molecular markers occurred within populations (>71%). In contrast to quantitative population differentiation (Q ST = 0.09), a high level of molecular differentiation (F ST = 0.23) was found among populations. The majority of quantitative traits exhibited Q ST values significantly less than F ST values, suggesting that selection may be acting to suppress differentiation for these traits. There was no significant correlation between genetic variation of quantitative traits and molecular markers within populations. On the other hand, significant correlations were found between measured quantitative characters and the site-of-origin environmental factors. The eco-geographical factors with the greatest influence on the variation of measured traits among populations were altitude, followed by soil texture, assimilated phosphorus (P2O5) and organic matter. Nevertheless, there were no consistent patterns of associations between gene diversity (He) and eco-geographical factors.

Increasing the amount of bioavailable micronutrients such as iron and zinc in plant foods for human consumption is an international goal, intended especially for developing countries where micronutrient deficiencies are an ongoing health risk. Legume seeds have the potential to provide the essential nutrients required by humans, but concentrations of several minerals are low when compared to other foods. In order to increase seed mineral concentrations, it is important to understand the genes and processes involved in mineral distribution within the plant. The main objectives of this study were to use a Medicago truncatula recombinant inbred population (Jemalong-6 x DZA 315.16) to determine loci governing seed mineral concentrations, seed mineral content, and average seed weight, and to use these loci to propose candidate genes whose expression might contribute to these traits. Ninety-three lines in 2004 and 169 lines in 2006 were grown for seed harvest and subsequent analysis of seed Ca, Cu, Fe, K, Mg, Mn, P, and Zn concentrations and content. Quantitative trait loci (QTL) cartographer was used to identify QTL using composite interval mapping (CIM). CIM identified 46 QTL for seed mineral concentration, 26 for seed mineral content, and 3 for average seed weight. At least one QTL was detected for each mineral trait, and colocation of QTL for several minerals was found in both years. Results comparing seed weight with seed mineral concentration and content QTL demonstrate that seed size can be an important determinant of seed mineral concentration. The identification, in this model legume, of transgressive segregation for nearly all the minerals suggests that allelic recombination of relevant mineral-related genes in agronomic legumes could be a successful strategy to increase seed mineral concentrations above current levels.

Pilet-Nayel, M.-L., Prosperi, J.-M., Hamon, C., Lesne, A., Lecointe, R., Le Goff, I., Herve, M., Deniot, G., Delalande, M., Huguet, T., Jacquet, C., and Baranger, A. 2009. AER1, a major gene conferring resistance to Aphanomyces euteiches in Medicago truncatula. Phytopathology 99:203208. Aphanomyces euteiches is a major soilborne oomycete pathogen that infects various legume species, including pea and alfalfa. The model legume Medicago truncatula has recently emerged as a valuable genetic system for understanding the genetic basis of resistance to A. euteiches in leguminous crops. The objective of this study was to identify genetic determinants of resistance to a broad host-range pea-infecting strain of A. euteiches in M. truncatula. Two M. truncatula segregating populations of 178 F5 recombinant inbred lines and 200 F3 families from the cross F83005.5 (susceptible) × DZA045.5 (resistant) were screened for resistance to A. euteiches. Phenotypic distributions observed suggested a dominant monogenic control of resistance. A major locus associated with resistance to A. euteiches, namely AER1, was mapped by bulk segregant analysis to a terminal end of chromosome 3 in M. truncatula and explained 88% of the phenotypic variation. AER1 was identified in a resistance-gene-rich region, where resistance gene analogs and genes associated with disease resistance phenotypes have been identified. Discovery of AER1 opens up new prospects for improving resistance to A. euteiches in cultivated legumes using a comparative genomics approach.

Medicago truncatula was used to characterize resistance to anthracnose and powdery mildew caused by Colletotrichum trifolii and Erysiphe pisi, respectively. Two isolates of E. pisi (Ep-p from pea and Ep-a from alfalfa) and two races of C. trifolii (races 1 and 2) were used in this study. The A17 genotype was resistant and displayed a hypersensitive response after inoculation with either pathogen, while lines F83005.5 and DZA315.16 were susceptible to anthracnose and powdery mildew, respectively. To identify the genetic determinants underlying resistance in A17, two F7 recombinant inbred line (RIL) populations, LR4 (A17 × DZA315.16) and LR5 (A17 × F83005.5), were phenotyped with E. pisi isolates and C. trifolii races, respectively. Genetic analyses showed that i) resistance to anthracnose is governed mainly by a single major locus to both races, named Ct1 and located on the upper part of chromosome 4; and ii) resistance to powdery mildew involves three distinct loci, Epp1 on chromosome 4 and Epa1 and Epa2 on chromosome 5. The use of a consensus genetic map for the two RIL populations revealed that Ct1 and Epp1, although located in the same genome region, were clearly distinct. In silico analysis in this region identified the presence of several clusters of nucleotide binding site leucine-rich repeat genes. Many of these genes have atypical resistance gene analog structures and display differential expression patterns in distinct stress-related cDNA libraries.

In this paper, we analyze the genetic variability in four Tunisian natural populations of Medicago ciliaris using 19 quantitative traits and six polymorphic microsatellite loci. We investigated the amplification transferability of 30 microsatellites developed in the model legume M. truncatula to M. ciliaris. Results revealed that about 56.66% of analyzed markers are valuable genetic markers for M. ciliaris. The most genetic diversity at quantitative traits and microsatellite loci was found to occur within populations (>80%). Low differentiations among populations at quantitative traits Q ST = 0.146 and molecular markers F ST = 0.18 were found. The majority of measured traits exhibited no significant difference in the level of Q ST and F ST . Furthermore, significant correlations established between these traits and eco-geographical factors suggested that natural selection should be invoked to explain the level of phenotypic divergence among populations rather than drift. There was no significant correlation between population differentiation at quantitative traits and molecular markers. Significant spatial genetic structure consistent with models of isolation by distance was detected within all studied populations. The site-of-origin environmental factors explain about 9.07% of total phenotypic genetic variation among populations. The eco-geographical factors that influence more the variation of measured traits among populations are the soil texture and altitude. Nevertheless, there were no consistent pattern of associations between gene diversity (He) and environmental factors.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; The identification of the molecular polymorphisms giving rise to phenotypic trait variability—both quantitative and qualitative—is a major goal of the present agronomic research. Various approaches such as positional cloning or transposon tagging, as well as the candidate gene strategy have been used to discover the genes underlying this variation in plants. The construction of functional maps, i.e. composed of genes of known function, is an important component of the candidate gene approach. In the present paper we report the development of 63 single nucleotide polymorphism markers and 15 single-stranded conformation polymorphism markers for genes encoding enzymes mainly involved in primary metabolism, and their genetic mapping on a composite map using two pea recombinant inbred line populations. The complete genetic map covers 1,458 cM and comprises 363 loci, including a total of 111 gene-anchored markers: 77 gene-anchored markers described in this study, 7 microsatellites located in gene sequences, 16 flowering time genes, the Tri gene, 5 morphological markers, and 5 other genes. The mean spacing between adjacent markers is 4 cM and 90% of the markers are closer than 10 cM to their neighbours. We also report the genetic mapping of 21 of these genes in Medicago truncatula and add 41 new links between the pea and M. truncatula maps. We discuss the use of this new composite functional map for future candidate gene approaches in pea.

The legume genus Medicago interacts with soil bacteria commonly referred to as rhizobia, in a nitrogen fixing symbiosis. We analysed the diversity of symbiotic association specificity among the two organisms, and its evolution in the plant genus. Nitrogen fixation tests and molecular phylogenetic reconstructions revealed that the genus Medicago includes more symbiotic specificity groups than previously suggested and that plant specificity is highly unstable and has repeatedly switched along the diversification of this genus. A phylogenetic analysis including geographical data shows that bacterial geographical diversity distribution has a strong influence on the geographic distribution of plant species and their ability to colonize new areas. Multiple other modifications of specificity occurred along the diversification of the genus, presumably due to selection for specialization to a single bacterial biovar. Codivergence between plants and bacteria may also have taken place.

The relationships between symbiotic performance and nodular antioxidant enzymes were studied for the associations between three Medicago truncatula lines and three Sinorhizobium meliloti strains. The results showed that the variability in symbiotic efficiency was dependent on the bacterial partner, host plant and their interaction. The contribution of each factor to the total amount of variance differed with the measured parameter. The aerial biomass production and nitrogen-fixing capacity were affected similarly by the three factors, whereas root and nodule biomass and catalase (CAT, E.C. 1.11.1.6), guaiacol peroxidase (POX, E.C. 1.11.1.7) and ascorbate peroxidase (APX, E.C. 1.11.1.11) antioxidant activities were mainly influenced by the M. truncatula line. The nodule number was dependent on the bacterial strain, and superoxide dismutase (SOD, E.C. 1.15.1.1) was dependent mainly on the plant-rhizobium interaction. A highly significant correlation was found between nitrogen-fixing activity, shoot biomass production, total nodule protein content and catalase activity. The other nodular antioxidant enzymes were differentially expressed between associations and showed no clear correlation with symbiotic efficiency.

Model plants are facilitating the genetic characterization and comparative mapping of a number of traditional crops. Medicago truncatula has been widely accepted as a model plant to this end as it provides the essential tools for multiple aspects of legume genetics and genomics. A large set of markers from highly conserved M. truncatula gene regions is being created and used to establish a worldwide framework for comparative genomic studies in legumes. We have investigated the potential for cross-species amplification of 209 expressed sequence tag (EST)-based and 33 bacterial artificial chromosome (BAC)-based microsatellites from M. truncatula in the three most important European legume pulses-pea, faba bean and chickpea-that might facilitate future comparative mapping. Our results revealed significant transferability of M. truncatula microsatellites to the three pulses (40% in faba bean, 36.3% in chickpea and 37.6% in pea). The percentage of M. truncatula EST-SSRs (simple sequence repeats) amplified in the three crops (39-43%) was twofold higher than that of the genomic SSRs (21-24%). Sequence analysis determined that the level of conservation in the microsatellite motif was very low, while the flanking regions were generally well conserved. The variations in the sequences were mainly due to changes in the number of repeat motifs in the microsatellite region combined with indel and base substitutions. None of the functional microsatellites showed direct polymorphism among the parental genotypes tested, consequently preventing their immediate use for mapping purposes.

The DMI1, DMI2, and DMI3 genes of Medicago truncatula, which are required for both nodulation and mycorrhization, control early steps of Nod factor signal transduction. Here, we have used diverse approaches to pave the way for the map-based cloning of these genes. Molecular amplification fragment length polymorphism markers linked to the three genes were identified by bulked segregant analysis. Integration of these markers into the general genetic map of M. truncatula revealed that DMI1, DMI2, and DMI3 are located on linkage groups 2, 5, and 8, respectively. Cytogenetic studies using fluorescent in situ hybridization (FISH) on mitotic and pachytene chromThe DMI1, DMI2, and DMI3 genes of Medicago truncatula, which are required for both nodulation and mycorrhization, control early steps of Nod factor signal transduction. Here, we have used diverse approaches to pave the way for the map-based cloning of these genes. Molecular amplification fragment length polymorphism markers linked to the three genes were identified by bulked segregant analysis. Integration of these markers into the general genetic map of M. truncatula revealed that DMI1, DMI2, and DMI3 are located on linkage groups 2, 5, and 8, respectively. Cytogenetic studies using fluorescent in situ hybridization (FISH) on mitotic and pachytene chromosomes confirmed the location of DMI1, DMI2, and DMI3 on chromosomes 2, 5, and 8. FISH-pachytene studies revealed that the three genes are in euchromatic regions of the genome, with a ratio of genetic to cytogenetic distances between 0.8 and 1.6 cM per ¿m in the DMI1, DMI2, and DMI3 regions. Through grafting experiments, we showed that the genetic control of the dmi1, dmi2, and dmi3 nodulation phenotypes is determined at the root level. This means that mutants can be transformed by Agrobacterium rhizogenes to accelerate the complementation step of map-based cloning projects for DMI1, DMI2, and DMI3.

Depuis une vingtaine d’annees, les nombreuses recherches focalisees sur la plante modele Arabidopsis thaliana ont permis des progres considerables dans notre connaissance des bases moleculaires de la biologie des plantes. Avec l’achevement du sequencage du genome d’Arabidopsis [1], la palette complete des outils de genetique et de genomique maintenant disponibles devrait encore accelerer le rythme des decouvertes. De son cote, le riz fait l’objet de rapides developpements genomiques en tant que modele pour les monocotyledones. Cependant, il est clair que ces deux especes ne suffisent pas pour representer le monde vegetal dans toute sa diversite biologique [2]. Ce fait est particulierement bien illustre par le cas des Legumineuses (Fabacees) qui representent l’un des taxons vegetaux les plus importants, tant du point de vue de la biologie et de l’ecologie fondamentales que du point de vue agronomique et environnemental.

A molecular cytogenetic map of Medicago truncatula (2n = 2x = 16) was constructed on the basis of a pachytene DAPI karyogram. Chromosomes at this meiotic prophase stage are 20 times longer than at mitotic metaphase, and display a well differentiated pattern of brightly fluorescing heterochromatin segments. We describe here a pachytene karyogram in which all chromosomes can be identified based on chromosome length, centromere position, heterochromatin patterns, and the positions of three repetitive sequences (5S rDNA, 45S rDNA and the MtR1 tandem repeat), visualized by fluorescence in situ hybridization (FISH). We determined the correlation between genetic linkage groups and chromosomes by FISH mapping of bacterial artificial chromosome (BAC) clones, with two to five BACs per linkage group. In the cytogenetic map, chromosomes were numbered according to their corresponding linkage groups. We determined the relative positions of the 20 BACs and three repetitive sequences on the pachytene chromosomes, and compared the genetic and cytological distances between markers. The mapping resolution was determined in a euchromatic part of chromosome 5 by comparing the cytological distances between FISH signals of clones of a BAC contig with their corresponding physical distance, and showed that resolution in this region is about 60 kb. The establishment of this FISH pachytene karyotype, with a far better mapping resolution and detection sensitivity compared to those in the highly condensed mitotic metaphase complements, has created the basis for the integration of molecular, genetic and cytogenetic maps in M. truncatula.

The international consensus on Medicago truncatula as a model system has lead to the development of powerful approaches for dissecting the genetic and molecular bases of legume nitrogen nutrition. However, such approaches now come up against a poor knowledge of the phenotypic traits that should be used for the large-scale screening of the genotypic variability associated with nitrogen nutrition. This issue was unravelled in a previous report, in which an ecophysiological approach allowed a better understanding of the relationships between plant nitrogen nutrition and plant growth traits, for the model symbiotic association between M. truncatula cv. Jemalong and Rhizobium meliloti strain 2011. From this analysis, phenotypic traits were identified as potentially relevant for the large-scale screening of the genotypic variability. Here, by the phenotyping of a recombinant inbred lines population, we show that the proposed methodology provides a valuable support for assisting the detection of genetic variants affected for nitrogen uptake. Especially, the relative expansion rate of plant leaf area is identified as a good proxy for ranking genotypes according to their ability to uptake nitrogen in given environmental conditions. As leaf area can be measured non-destructively, such finding should pave the way for a more efficient evaluation of the genotypic variability.

Alfalfa (Medicago sativa L.) is a forage legume of world-wide importance whose both allogamous and autotetraploid nature maximizes the genetic diversity within natural and cultivated populations. This genetic diversity makes difficult the discrimination between two related populations. We analyzed this genetic diversity by screening DNA from individual plants of eight cultivated and natural populations of M. sativa and M. falcata using the RAPD method. A high level of genetic variation was found within and between populations. Using five primers, 64 intense bands were scored as present or absent across all populations. Most of the loci were revealed to be highly polymorphic whereas very few population-specific polymorphisms were identified. From these observations, we adopted a method based on the Roger's genetic distance between populations using the observed frequency of bands to discriminate populations pairwise. Except for one case, the between-population distances were all significantly different from zero. We have also determined the minimal number of bands and individuals required to test for the significance of between-population distances.

International audience; About 40% (α=0.05) of the PCR-derived markers scored in a Medicago truncatula and M. tornata intraspecific cross departed from Mendelian expectations at α=0.05. This proportion is among the highest ever documented in the literature, notably for intraspecific crosses. Estimations of DNA amount were also implemented for the parental genotypes or parental lines, and significant variations were observed. Our results suggest that the parental genotypes have diverged for quite a while, and we propose that the level of distortion we documented is correlated with the genome size difference we measured.

We evaluated the responses to salt stress of 106 Medicago truncatula lines from 11 Tunisian natural populations collected from areas that varied in soil composition, salinity and water availability. Five references lines were also included in this study. Plants were cultivated in two treatments (0 and 50 mM of NaCl) during a period of 60 days. At harvest, we measured 14 quantitative traits of the aerial and root growth to identify genotypic variability in salt response. Analysis of variance showed that, the response to salt lines within populations was dependent on the effects of treatment, population, line within population and their interaction with maximum value recorded for treatment (93.78%). This study also analyzed heritability of the salt response index (SRI), defined as the ratio between the observed values with and without salt treatment. SRI of most measured traits had high broad-sense heritability (H²). Most of established correlations among SRI values of measured traits were positive. SRI means revealed that, Soliman and Bulla Regia are the most salt-tolerant populations. Based on Ward’s estimated distance, all lines were classified into 4 clusters showing similarity and dissimilarity in response to salt stress. The eco-geographical factors that influence more the variation of SRI of measured traits among populations are assimilated P 2 O 5 , % organic matter and carbon and mean annual rainfall. Findings from this study will provide the basis for identifying and breeding of salt-tolerant lines in M. truncatula . Key words : Environmental factors, lines, Medicago truncatula, NaCl stress, populations, quantitative traits.

International audience; Translational genomics, i.e., the transfer of genetic information from model species to cultivated crops, is on the brink of revolutionizing plant breeding. The recent publication of genomic sequences for several cultivated legumes is also accelerating this process. For pea, recent highthroughput RNA sequencing, and the prospect of a genome sequencing project, will further accelerate the transfer of information from the Medicago truncatula model to the cultivated crop. We have been using genomics approaches with Medicago as a tool to identify key genes determining seed yield and composition in closely related legumes. Analyses of the proteome and transcriptome of the component tissues of the developing seed revealed extensive compartmentalization of gene expression and metabolic activities. Using a TF (Transcription Factor) qRT-PCR platform and the Affymetrix Gene Chip, TFs specific for each seed tissue were identified, along with putative target genes. These TFs have been located on the M. truncatula genetic map and correlations between map positions of TF loci and QTLs for protein quantities and other seed phenotypes were detected. These correlations can be recently confirmed in numerous cases by the existence of similar QTLs at syntenic positions in pea. Two genes, both specifically expressed in the developing endosperm, have received particular attention. One of the genes encodes a DOF class transcription factor, whose mutant phenotype severely affects endosperm development. The second gene encodes an endosperm-specific subtilase (SBT1.1), which affects final seed weight. MiRNAs constitute another level of gene regulation whose importance in the developing seed is beginning to become apparent. We have recently started to compare the sRNA profiles of developing M. truncatula and pea seeds and preliminary results will be presented.

International audience; Ralstonia solanacearum is a major soilborne pathogen that attacks > 200 plant species, including major crops. To characterize MtQRRS1, a major quantitative trait locus (QTL) for resistance towards this bacterium in the model legume Medicago truncatula, genetic and functional approaches were combined. QTL analyses together with disease scoring of heterogeneous inbred families were used to define the locus. The candidate region was studied by physical mapping using a bacterial artificial chromosome (BAC) library of the resistant line, and sequencing. In planta bacterial growth measurements, grafting experiments and gene expression analysis were performed to investigate the mechanisms by which this locus confers resistance to R. solanacearum. The MtQRRS1 locus was localized to the same position in two recombinant inbred line populations and was narrowed down to a 64 kb region. Comparison of parental line sequences revealed 15 candidate genes with sequence polymorphisms, but no evidence of differential gene expression upon infection. A role for the hypocotyl in resistance establishment was shown. These data indicate that the quantitative resistance to bacterial wilt conferred by MtQRRS1, which contains a cluster of seven R genes, is shared by different accessions and may act through intralocus interactions to promote resistance.

International audience; Verticillium wilt is a major threat to alfalfa (Medicago sativa) and many other crops. The model legume Medicago truncatula was used as a host for studying resistance and susceptibility to Verticillium albo-atrum. In addition to presenting well-established genetic resources, this wild plant species enables to investigate biodiversity of the response to the pathogen and putative crosstalk between disease and symbiosis. Symptom scoring after root inoculation and modelling of disease curves allowed assessing susceptibility levels in recombinant lines of three crosses between susceptible and resistant lines, in a core collection of 32 lines, and in mutants affected in symbiosis with rhizobia. A GFP-expressing V. albo-atrum strain was used to study colonization of susceptible plants. Symptoms and colonization pattern in infected M. truncatula plants were typical of Verticillium wilt. Three distinct major quantitative trait loci were identified using a multicross, multisite design, suggesting that simple genetic mechanisms appear to control Verticillium wilt resistance in M. truncatula lines A17 and DZA45.5. The disease functional parameters varied largely in lines of the core collection. This biodiversity with regard to disease response encourages the development of association genetics and ecological approaches. Several mutants of the resistant line, impaired in different steps of rhizobial symbiosis, were affected in their response to V. albo-atrum, which suggests that mechanisms involved in the establishment of symbiosis or disease might have some common regulatory control points.

[i]Medicago truncatula[/i] is used as a model plant for exploring the genetic and molecular determinants of nitrogen (N) nutrition in legumes. In this study, our aim was to detect quantitative trait loci (QTL) controlling plant N nutrition using a simple framework of carbon/N plant functioning stemming from crop physiology. This framework was based on efficiency variables which delineated the plant's efficiency to take up and process carbon and N resources. A recombinant inbred line population (LR4) was grown in a glasshouse experiment under two contrasting nitrate concentrations. At low nitrate, symbiotic N-2 fixation was the main N source for plant growth and a QTL with a large effect located on linkage group (LG) 8 affected all the traits. Significantly, efficiency variables were necessary both to precisely localize a second QTL on LG5 and to detect a third QTL involved in epistatic interactions on LG2. At high nitrate, nitrate assimilation was the main N source and a larger number of QTL with weaker effects were identified compared to low nitrate. Only two QTL were common to both nitrate treatments: a QTL of belowground biomass located at the bottom of LG3 and another one on LG6 related to three different variables (leaf area, specific N uptake and aboveground:belowground biomass ratio). Possible functions of several candidate genes underlying QTL of efficiency variables could be proposed. Altogether, our results provided new insights into the genetic control of N nutrition in M. truncatula. For instance, a novel result for [i]M. truncatula[/i] was identification of two epistatic interactions in controlling plant N-2 fixation. As such this study showed the value of a simple conceptual framework based on efficiency variables for studying genetic determinants of complex traits and particularly epistatic interactions

International audience; Medicago truncatula, as a model species, is useful to study the genetic control of traits of agronomic interest in legumes species. Aerial morphogenesis is a key component of forage and seed yield. It was measured in four mapping populations originating from five parental lines. Single and multi-population quantitative trait locus (QTL) detections were carried out. A large variation was observed within populations and transgressive segregation was noted. Most traits showed high heritabilities in all seasons. Length of primary branches (LPB, cm) was positively correlated to branch elongation rate (BER, cm day(-1)) and aerial dry matter (ADM, g). Flowering time (FT, degrees C day(-1)) showed negative correlations with length of main stem (LMS, cm) and BER. One hundred and forty-one QTLs for BER, LMS, FT, LPB, diameter of primary branches (DPB), number of primary branches (NPB), number of nodes (NI) and ADM were identified and localized over all eight chromosomes. Single and multi-population analyses showed that the most important regions for aerial morphogenetic traits were chromosomes 1, 2, 7 and 8. Multi-population analysis revealed three regions of major QTLs affecting aerial morphogenetic traits (LPB, LMS, NPB, BER and FT). A region involved in flowering time variation was revealed on chromosome 6 on a single population. These results were used to identify candidate genes that could control variation for aerial morphogenesis traits in this species and in related crop legume species.

Article en libre accès; Enhancing the knowledge on the genetic basis of germination and heterotrophic growth at extreme temperatures is of major importance for improving crop establishment. A quantitative trait loci (QTL) analysis was carried out at sub- and supra-optimal temperatures at these early stages in the model Legume Medicago truncatula. On the basis of an ecophysiological model framework, two populations of recombinant inbred lines were chosen for the contrasting behaviours of parental lines: LR5 at sub-optimal temperatures (5 or 10°C) and LR4 at a supra-optimal temperature (20°C). Seed masses were measured in all lines. For LR5, germination rates and hypocotyl growth were measured by hand, whereas for LR4, imbibition and germination rates as well as early embryonic axis growth were measured using an automated image capture and analysis device. QTLs were found for all traits. The phenotyping framework we defined for measuring variables, distinguished stages and enabled identification of distinct QTLs for seed mass (chromosomes 1, 5, 7 and 8), imbibition (chromosome 4), germination (chromosomes 3, 5, 7 and 8) and heterotrophic growth (chromosomes 1, 2, 3 and 8). The three QTL identified for hypocotyl length at sub-optimal temperature explained the largest part of the phenotypic variation (60% together). One digenic interaction was found for hypocotyl width at sub-optimal temperature and the loci involved were linked to additive QTLs for hypocotyl elongation at low temperature. Together with working on a model plant, this approach facilitated the identification of genes specific to each stage that could provide reliable markers for assisting selection and improving crop establishment. With this aim in view, an initial set of putative candidate genes was identified in the light of the role of abscissic acid/gibberellin balance in regulating germination at high temperatures (e.g. ABI4, ABI5), the molecular cascade in response to cold stress (e.g. CBF1, ICE1) and hypotheses on changes in cell elongation (e.g. GASA1, AtEXPA11) with changes in temperatures based on studies at the whole plant scale.

To understand the complex inheritance of tolerance to salt stress in Medicago truncatula, quantitative trait loci (QTLs) analysis was performed using a set of recombinant inbred lines (RILs) derived from a cross between the tolerant line Jemalong A17 and susceptible line F83005.5. The RILs and parental lines were grown in individual pots filled with sterilized sand in a greenhouse under 0 and 50 mM NaCl. Plants were harvested after a period of 60 days. Fourteen quantitative traits related to aerial and root growths were measured. Broad-sense heritability of measured traits ranged from 0.21 to 0.83 and from 0.05 to 0.62 in control and in salt-stressed conditions, respectively. Established correlations between measured traits are dependent on treatment effect. We identified and mapped 10 QTLs in control conditions and 19 in salt stress. No major QTL was identified indicating that tolerance to salt stress is governed by several genes with low effects. The QTLs detected under control and under salt-stressed conditions almost did not share the same map locations suggesting that the loci that are not stable across treatments reflect adaptation to this constraint. The maximum of QTLs was observed on the chromosome 8. The usefulness of these QTLs, identified in greenhouse conditions, for marker-assisted selection should therefore be evaluated under field conditions, and validated in other genetic backgrounds.

Water shortage and heterogeneity in precipitation constitute the essential limiting factors for agricultural production in the Mediterranean basin. To understand the complex inheritance of tolerance to drought stress in Medicago truncatula, quantitative trait loci (QTLs) analysis was performed using a set of recombinant inbred lines (RILs) derived from a cross between the susceptible line Jemalong A17 and the tolerant line F83005.5. We conducted two experiments where the RILs and parental lines were grown in 0 mM and 75 mM of mannitol (D-) in tubes and in control and 33% of the field capacity (FC) in pots. In both experiments, line and line by water treatment interaction were significant. QTLs analysis showed that several putative genomic regions are involved in the variation of measured traits under the two water treatments in tubes and pots. A total of 21 QTLs on 8 linkage groups were detected for the measured traits in both experiments, 11 of them were in tubes conditions. Eight of these 11 QTLs were identified in control treatment and three in mannitol. While the number of QTLs identified in tubes under control conditions was more than under mannitol conditions, the highest number of QTLs was detected in water-stressed conditions in pots. The percentage of phenotypic variance explained by a single QTL (R 2) ranges from 11.7 to 26.3% in tubes and from 11.2 to 18.4% in pots depending on the trait, with 6 QTLs in each condition explaining

Genome-wide association studies rely upon segregating natural genetic variation, particularly the patterns of polymorphism and correlation between adjacent markers. To facilitate association studies in the model legume Medicago truncatula, we present a genome-scale polymorphism scan using existing Affymetrix microarrays. We develop and validate a method that uses a simple information-criteria algorithm to call polymorphism from microarray data without reliance on a reference genotype. We genotype 12 inbred M. truncatula lines sampled from four wild Tunisian populations and find polymorphisms at approximately 7% of features, comprising 31 419 probes. Only approximately 3% of these markers assort by population, and of these only 10% differentiate between populations from saline and non-saline sites. Fifty-two differentiated probes with unique genome locations correspond to 18 distinct genome regions. Sanger resequencing was used to characterize a subset of maker loci and develop a single nucleotide polymorphism (SNP)-typing assay that confirmed marker assortment by habitat in an independent sample of 33 individuals from the four populations. Genome-wide linkage disequilibrium (LD) extends on average for approximately 10 kb, falling to background levels by approximately 500 kb. A similar range of LD decay was observed in the 18 genome regions that assort by habitat; these LD blocks delimit candidate genes for local adaptation, many of which encode proteins with predicted functions in abiotic stress tolerance and are targets for functional genomic studies. Tunisian M. truncatula populations contain substantial amounts of genetic variation that is structured in relatively small LD blocks, suggesting a history of migration and recombination. These populations provide a strong resource for genome-wide association studies.

National audience

National audience; Alfalfa is the most important forage crop cultivated in semi-arid areas, both in rainfed and irrigated conditions. Water available for crops is a limited resource and improvement of water use efficiency (WUE) is an important goal for plant breeding. The objective of this study was to detect quantitative traits loci (QTL) for WUE in a mapping population of alfalfa. A F(1) mapping population was obtained and a genetic map based on 85 SSR markers was built. The F(1) plants and the parents were transplanted in soil columns, and WUE was measured during six regrowth cycles under well-watered conditions. QTL detection was carried out by single and multiple factor analysis of variance. The two parents significantly differed for WUE as expected. The F(1) population showed quantitative variation for this trait. In single factor ANOVA, nine markers/alleles had a significant effect on WUE variation. In multiple factor ANOVA, six markers/alleles had a significant effect and explained 31.0% of the variation. These QTL will be useful to better understand adaptation to water stress conditions in lucerne and to breed improved varieties.

Genetic variability in 10 natural Tunisian populations of Medicago laciniata were analysed using 19 quantitative traits and 12 polymorphic microsatellite loci. A large degree of genetic variability within-populations and among-populations was detected for both quantitative characters and molecular markers. High genetic differentiation among populations for quantitative traits was seen, with Q(ST) = 0.47, and F(ST) = 0.47 for microsatellite markers. Several quantitative traits displayed no statistical difference in the levels of Q(ST) and F(ST). Further, significant correlations between quantitative traits and eco-geographical factors suggest that divergence in the traits among populations may track environmental differences. There was no significant correlation between genetic variability at quantitative traits and microsatellite markers within populations. The site-of-origin of eco-geographical factors explain between 18.13% and 23.40% of genetic variance among populations at quantitative traits and microsatellite markers, respectively. The environmental factors that most influence variation in measured traits among populations are assimilated phosphorus (P(2)0(5)) and mean annual rainfall, followed by climate and soil texture, altitude and organic matter. Significant associations between eco-geographical factors and gene diversity, He, were established in five microsatellite loci suggesting that these simple sequence repeats (SSRs) are not necessarily biologically neutral.

Contact :thierry.huguet@ensat.fr; International audience; In this study, we investigated the genetic diversity of a collection of 136 Medicago truncatula lines from 10 Tunisian natural populations collected in well-defined locations and in various ecological conditions of soil, salinity and water availability. The genetic diversity was evaluated using a set of 18 microsatellites (SSRs), representing the 8 chromosomes of M. truncatula. A neutrality test showed that 7 SSRs were non-neutral with evidence of balancing selection. The 11 neutral SSRs revealed a geographical pooling with the Tunisian Dorsale axis restricting migration of alleles. The 7 non-neutral alleles demonstrate a correlation with rainfall, altitude and salinity environmental variables suggesting that these SSRs are linked to genes involved in water use efficiency, resistance to salinity or adaptation to altitude, and that there is local adaptation of M. truncatula to these variables. This demonstrates that the choice of so-called neutral markers should be carefully evaluated in population genetic studies. This study illustrates the genetic diversity occurring in natural Tunisian populations of M. truncatula and describes the first collection of this species dedicated to natural variation involved in adaptation to the environment

A pathosystem between Aphanomyces euteiches, the causal agent of pea root rot disease, and the model legume Medicago truncatula was developed to gain insights into mechanisms involved in resistance to this oomycete. The F83005.5 French accession and the A17-Jemalong reference line, susceptible and partially resistant, respectively, to A. euteiches, were selected for further cytological and genetic analyses. Microscopy analyses of thin root sections revealed that a major difference between the two inoculated lines occurred in the root stele, which remained pathogen free in A17. Striking features were observed in A17 roots only, including i) frequent pericycle cell divisions, ii) lignin deposition around the pericycle, and iii) accumulation of soluble phenolic compounds. Genetic analysis of resistance was performed on an F7 population of 139 recombinant inbred lines and identified a major quantitative trait locus (QTL) near the top of chromosome 3. A second study, with near-isogenic line responses to A. euteiches confirmed the role of this QTL in expression of resistance. Fine-mapping allowed the identification of a 135-kb sequenced genomic DNA region rich in proteasome-related genes. Most of these genes were shown to be induced only in inoculated A17. Novel mechanisms possibly involved in the observed partial resistance are proposed.

Contact: g.powell@imperial.ac.uk Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Plant resistance to pathogens is commonly associated with a hypersensitive response (HR), but the degree to which the HR is responsible for incompatibility is subject to debate. Resistance to aphids is likely to share features with resistance to pathogens but is less well understood. Here, we report effective resistance to the pea aphid Acyrthosiphon pisum in Medicago truncatula. Aphids lost weight and died rapidly (within two days) on the resistant genotype Jemalong, which developed necrotic lesions following infestation. Lesions were induced by nonvascular intracellular stylet punctures by aphids, remained localized to the site of stylet entry, stained for the presence of reactive oxygen species, and were similar to the HR induced by the bacterial pathogen Pseudomonas syringae pv. phaseolicola. The implication that aphid-induced lesions confer resistance was tested by quantitative trait loci analysis using recombinant inbred lines derived from a cross between Jemalong and the susceptible genotype DZA315.16. One major locus, RAP1, was identified that was sufficient to confer race-specific resistance against the pea aphid and was mapped to the middle of chromosome 3. Surprisingly, a separate locus, mapping to the top of chromosome 3, governed aphid-induced HR, indicating that the HR-like lesions are not required for RAP1-mediated aphid resistance

Two leghaemoglobin genes from the diploid, autogamous Medicago truncatula (Mtlb1 and Mtlb2) have been cloned and their nucleotide sequences determined. The deduced amino acid sequences encoded by these two genes differ significantly (18%), confirming that they belong to different sub-groups of Medicago leghaemoglobin genes. RNAse protection experiments have been used to show that both genes are transcriptionally active, and are expressed specifically in the nitrogen-fixing root nodule of M. truncatula. Whilst Mtlb1 mRNA is present at approximatively 3-fold higher steady-state levels than Mtlb2 mRNA, the transcription of both genes is triggered concomitantly during nodule development (5 days after inoculation with Rhizobium meliloti), and the ratio of the steady-state levels of the two mRNA species remains constant throughout nodule maturation. When the growth medium of nodulated M. truncatula is supplemented with 5 mM KNO3 over a period of 2-3 days there is a progressive drop in specific nitrogen fixation activity to only 20-25% of the original level. This is accompanied with a parallel and synchronous reduction in the quantities of mRNA corresponding to both Mtlb1 and Mtlb2. By contrast, the expression of the nodule parenchyma-specific gene ENOD2 is not significantly modified following nitrate treatment, clearly demonstrating differences in tissue-specific gene regulation in response to combined nitrogen.

Adaptation to the environment and reproduction are dependent on the date of flowering in the season. The objectives of this paper were to evaluate the effect of photoperiod on flowering date of the model species for legume crops, Medicago truncatula and to describe genetic architecture of this trait in multiple mapping populations. The effect of photoperiod (12 and 18 h) was analysed on eight lines. Quantitative variation in three recombinant inbred lines (RILs) populations involving four parental lines was evaluated, and QTL detection was carried out. Flowering occurred earlier in long than in short photoperiods. Modelling the rate of progression to flowering with temperature and photoperiod gave high R-2, with line-specific parameters that indicated differential responses of the lines to both photoperiod and temperature. QTL detection showed a QTL on chromosome 7 that was common to all populations and seasons. Taking advantage of the multiple mapping populations, it was condensed into a single QTL with a support interval of only 0.9 cM. In a bioanalysis, six candidate genes were identified in this interval. This design also indicated other genomic regions that were involved in flowering date variation more specifically in one population or one season. The analysis on three different mapping populations detected more QTLs than on a single population, revealed more alleles and gave a more precise position of the QTLs that were common to several populations and/or seasons. Identification of candidate genes was a result of integration of QTL analysis and genomics in M. truncatula.

International audience

Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.

The soilborne pathogen Ralstonia solanacearum is the causal agent of bacterial wilt and attacks more than 200 plant species, including some legumes and the model legume plant Medicago truncatula. We have demonstrated that M. truncatula accessions Jemalong A17 and F83005.5 are susceptible to R. solanacearum and, by screening 28 R. solana-cearum strains on the two M. truncatula lines, differential interactions were identified. R. solanacearum GMI1000 infected Jemalong A17 line, and disease symptoms were dependent upon functional hrp genes. An in vitro root inoculation method was employed to demonstrate that R. solanacearum colonized M. truncatula via the xylem and intercellular spaces. R. solanacearum multiplication was restricted by a factor greater than 1 × 105 in the resistant line F83005.5 compared with susceptible Jemalong A17. Genetic analysis of recombinant inbred lines from a cross between Jemalong A17 and F83005.5 revealed the presence of major quantitative trait loci for bacterial wilt resistance located on chromosome 5. The results indicate that the root pathosystem for M. truncatula will provide useful traits for molecular analyses of disease and resistance in this model plant species.

Rhizobial Nod factors are key symbiotic signals responsible for starting the nodulation process in host legume plants. Of the six Medicago truncatula genes controlling a Nod factor signaling pathway, Nod Factor Perception (NFP) was reported as a candidate Nod factor receptor gene. Here, we provide further evidence for this by showing that NFP is a lysine motif (LysM)-receptor-like kinase (RLK). NFP was shown both to be expressed in association with infection thread development and to be involved in the infection process. Consistent with deviations from conserved kinase domain sequences, NFP did not show autophosphorylation activity, suggesting that NFP needs to associate with an active kinase or has unusual functional characteristics different from classical kinases. Identification of nine new M. truncatula LysM-RLK genes revealed a larger family than in the nonlegumes Arabidopsis (Arabidopsis thaliana) or rice (Oryza sativa) of at least 17 members that can be divided into three subfamilies. Three LysM domains could be structurally predicted for all M. truncatula LysM-RLK proteins, whereas one subfamily, which includes NFP, was characterized by deviations from conserved kinase sequences. Most of the newly identified genes were found to be expressed in roots and nodules, suggesting this class of receptors may be more extensively involved in nodulation than was previously known.

In many legume crops, especially in forage legumes, aerial morphogenesis defined as growth and development of plant organs, is an essential trait as it determines plant and seed biomass as well as forage quality (protein concentration, dry matter digestibility). Medicago truncatula is a model species for legume crops. A set of 29 accessions of M. truncatula was evaluated for aerial morphogenetic traits. A recombinant inbred lines (RILs) mapping population was used for analysing quantitative variation in aerial morphogenetic traits and QTL detection. Genes described to be involved in aerial morphogenetic traits in other species were mapped to analyse co-location between QTLs and genes. A large variation was found for flowering date, morphology and dynamics of branch elongation among the 29 accessions and within the RILs population. Flowering date was negatively correlated to main stem and branch length. QTLs were detected for all traits, and each QTL explained from 5.2 to 59.2% of the phenotypic variation. A QTL explaining a large part of genetic variation for flowering date and branch growth was found on chromosome 7. The other chromosomes were also involved in the variation detected in several traits. Mapping of candidate genes indicates a co-location between a homologue of Constans gene or a flowering locus T (FT) gene and the QTL of flowering date on chromosome 7. Other candidate genes for several QTLs are described.

Alfalfa (Medicago sativa) is an autotetraploid, allogamous and heterozygous species whose cultivars are synthetic populations. The breeders apply selection pressure for some agronomic traits within a breeding pool to increase the frequency of favorable individuals. The objective of this study was to investigate the differentiation level among seven cultivars originating from one breeding program, and between these cultivars and the breeding pool, with eight SSR markers. These highly polymorphic and codominant markers, together with recent population genetic statistics extended to autotetraploids, offer tools to analyse genetic diversity in alfalfa. The number of alleles per locus varied between 3 and 24. All loci were at a panmictic equilibrium in the cultivars, except one, probably because of null alleles. With seven SSR loci, each cultivar was at panmictic equilibrium. The mean gene diversity was high, ranging from 0.665 to 0.717 in the cultivars. The parameter F ST indicated a low but significant diversity among cultivars. Among 21 pairs of cultivars, 15 were significantly different. The breeding pool also had a high diversity, and was significantly different from each cultivar except the most recent one. Considering the characteristics of the breeding program and the mode of cultivar elaboration, we found that they were unable to generate a large variety differentiation. Estimation of population genetics parameters at SSR loci can be applied for assessing the differences between cultivars or populations, either for variety distinction or the management of genetic resources.