MtQRRS1, an R-locus required for Medicago truncatula quantitative resistance to Ralstonia solanacearum.

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.

Natural diversity in the model legume Medicago truncatula allows identifying distinct genetic mechanisms conferring partial resistance to Verticillium wilt.

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.

QTL mapping of physiological traits associated with salt tolerance in Medicago truncatula Recombinant Inbred Lines.

In this study, QTL mapping of physiological traits in the model Legume (Medicago truncatula) was performed using a set of RILs derived from LR5. Twelve parameters associated with Na+ and K+ content in leaves, stems and roots were measured. Broad-sense heritability of these traits was ranged from 0.15 to 0.83 in control and from 0.14 to 0.61 in salt stress. Variation among RILs was dependent on line, treatment and line by treatment effect. We mapped 6 QTLs in control, 2 in salt stress and 5 for sensitivity index. No major QTL was identified indicating that tolerance to salt stress is governed by several genes with low effects. Detected QTL for leaf, stem and root traits did not share the same map locations, suggesting that genes controlling transport of Na+ and K+ may be different. The maximum of QTL was observed on chromosome 1, no QTL was detected on chromosomes 5 and 6.

Multi-population QTL detection for aerial morphogenetic traits in the model legume Medicago truncatula.

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, °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.

Using a physiological framework for improving the detection of quantitative trait loci related to nitrogen nutrition in Medicago truncatula.

Medicago truncatula 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 M. truncatula 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.

Population genomic analysis of Tunisian Medicago truncatula reveals candidates for local adaptation.

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.

Quantitative trait loci analysis reveals a correlation between the ratio of sucrose/raffinose family oligosaccharides and seed vigour in Medicago truncatula.

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.

QTL analysis of seed germination and pre-emergence growth at extreme temperatures in Medicago truncatula.

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.

The RAP1 gene confers effective, race-specific resistance to the pea aphid in Medicago truncatula independent of the hypersensitive reaction.

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.

Partial resistance of Medicago truncatula to Aphanomyces euteiches is associated with protection of the root stele and is controlled by a major QTL rich in proteasome-related genes.

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.

Identification of QTL affecting seed mineral concentrations and content in the model legume Medicago truncatula.

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.

api, A novel Medicago truncatula symbiotic mutant impaired in nodule primordium invasion.

Genetic approaches have proved to be extremely useful in dissecting the complex nitrogen-fixing Rhizobium-legume endosymbiotic association. Here we describe a novel Medicago truncatula mutant called api, whose primary phenotype is the blockage of rhizobial infection just prior to nodule primordium invasion, leading to the formation of large infection pockets within the cortex of noninvaded root outgrowths. The mutant api originally was identified as a double symbiotic mutant associated with a new allele (nip-3) of the NIP/LATD gene, following the screening of an ethylmethane sulphonate-mutagenized population. Detailed characterization of the segregating single api mutant showed that rhizobial infection is also defective at the earlier stage of infection thread (IT) initiation in root hairs, as well as later during IT growth in the small percentage of nodules which overcome the primordium invasion block. Neither modulating ethylene biosynthesis (with L-alpha-(2-aminoethoxyvinylglycine or 1-aminocyclopropane-1-carboxylic acid) nor reducing ethylene sensitivity in a skl genetic background alters the basic api phenotype, suggesting that API function is not closely linked to ethylene metabolism or signaling. Genetic mapping places the API gene on the upper arm of the M. truncatula linkage group 4, and epistasis analyses show that API functions downstream of BIT1/ERN1 and LIN and upstream of NIP/LATD and the DNF genes.

Genetic dissection of resistance to anthracnose and powdery mildew in Medicago truncatula.

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 x DZA315.16) and LR5 (A17 x 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.

Morphological and microsatellite diversity associated with ecological factors in natural populations of Medicago laciniata Mill. (Fabaceae).

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.

Characterization of Root-Knot Nematode Resistance in Medicago truncatula.

Root knot (Meloidogyne spp.) and cyst (Heterodera and Globodera spp.) nematodes infect all important crop species, and the annual economic loss due to these pathogens exceeds $90 billion. We screened the worldwide accession collection with the root-knot nematodes Meloidogyne incognita, M. arenaria and M. hapla, soybean cyst nematode (SCN-Heterodera glycines), sugar beet cyst nematode (SBCN-Heterodera schachtii) and clover cyst nematode (CLCN-Heterodera trifolii), revealing resistant and susceptible accessions. In the over 100 accessions evaluated, we observed a range of responses to the root-knot nematode species, and a non-host response was observed for SCN and SBCN infection. However, variation was observed with respect to infection by CLCN. While many cultivars including Jemalong A17 were resistant to H. trifolii, cultivar Paraggio was highly susceptible. Identification of M. truncatula as a host for root-knot nematodes and H. trifolii and the differential host response to both RKN and CLCN provide the opportunity to genetically and molecularly characterize genes involved in plant-nematode interaction. Accession DZA045, obtained from an Algerian population, was resistant to all three root-knot nematode species and was used for further studies. The mechanism of resistance in DZA045 appears different from Mi-mediated root-knot nematode resistance in tomato. Temporal analysis of nematode infection showed that there is no difference in nematode penetration between the resistant and susceptible accessions, and no hypersensitive response was observed in the resistant accession even several days after infection. However, less than 5% of the nematode population completed the life cycle as females in the resistant accession. The remainder emigrated from the roots, developed as males, or died inside the roots as undeveloped larvae. Genetic analyses carried out by crossing DZA045 with a susceptible French accession, F83005, suggest that one gene controls resistance in DZA045.

Characterization of the interaction between the bacterial wilt pathogen Ralstonia solanacearum and the model legume plant Medicago truncatula.

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. solanacearum 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 x 10(5) 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.

The typA gene is required for stress adaptation as well as for symbiosis of Sinorhizobium meliloti 1021 with certain Medicago truncatula lines.

In this article, we describe the typA gene of Sinorhizobium meliloti, the orthologue of typA/bipA genes found in a wide range of bacteria. We found that typA was required for survival of S. meliloti under certain stress conditions, such as growth at low temperature or low pH and in the presence of sodium dodecyl sulfate (SDS). The cold-sensitive phenotype of both Escherichia coli bipA and S. meliloti typA mutants were cross-complemented, indicating that the two genes are functionally equivalent. typA was indispensable for symbiosis on Medicago truncatula Jemalong and F83005.5 and contributes to the full efficiency of symbiosis on other host plant lines such as DZA315.16 or several cultivars of M. sativa. Hence, the symbiotic requirement for typA is host dependent. Interestingly, the symbiotic defect was different on Jemalong and F83005.5 plants, thus indicating that typA is required at a different stage of the symbiotic interaction.

Cytological, genetic, and molecular analysis to characterize compatible and incompatible interactions between Medicago truncatula and Colletotrichum trifolii.

In this study, a new pathosystem was established using the model plant Medicago truncatula and Colletotrichum trifolii, the causal agent of anthracnose on Medicago sativa. Screening of a few M. truncatula lines identified Jemalong and F83005.5 as resistant and susceptible to Colletotrichum trifolii race 1, respectively. Symptom analysis and cytological studies indicated that resistance of Jemalong was associated with a hypersensitive response of the plant. The two selected lines were crossed, and inoculations with C. trifolii were performed on the resulting F1 and F2 progenies. Examination of the disease phenotypes indicated that resistance was dominant and was probably due to a major resistance gene. Molecular components of the resistance were analyzed through macroarray experiments. Expression profiling of 126 expressed sequence tags corresponding to 92 genes, which were selected for their putative functions in plant defense or signal transduction, were compared in Jemalong and F83005.5 lines. A strong correlation was observed between the number of up-regulated genes and the resistance phenotype. Large differences appeared at 48 h postinoculation; more than 40% of the tested genes were up-regulated in the Jemalong line compared with only 10% in the susceptible line. Interestingly, some nodulin genes were also induced in the resistant line upon inoculation with C. trifolii.

Genetic and cytogenetic mapping of DMI1, DMI2, and DMI3 genes of Medicago truncatula involved in Nod factor transduction, nodulation, and mycorrhization.

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 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 microm 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.

Construction of two genetic linkage maps in cultivated tetraploid alfalfa (Medicago sativa) using microsatellite and AFLP markers.

BACKGROUND: Alfalfa (Medicago sativa) is a major forage crop. The genetic progress is slow in this legume species because of its autotetraploidy and allogamy. The genetic structure of this species makes the construction of genetic maps difficult. To reach this objective, and to be able to detect QTLs in segregating populations, we used the available codominant microsatellite markers (SSRs), most of them identified in the model legume Medicago truncatula from EST database. A genetic map was constructed with AFLP and SSR markers using specific mapping procedures for autotetraploids. The tetrasomic inheritance was analysed in an alfalfa mapping population. RESULTS: We have demonstrated that 80% of primer pairs defined on each side of SSR motifs in M. truncatula EST database amplify with the alfalfa DNA. Using a F1 mapping population of 168 individuals produced from the cross of 2 heterozygous parental plants from Magali and Mercedes cultivars, we obtained 599 AFLP markers and 107 SSR loci. All but 3 SSR loci showed a clear tetrasomic inheritance. For most of the SSR loci, the double-reduction was not significant. For the other loci no specific genotypes were produced, so the significant double-reduction could arise from segregation distortion. For each parent, the genetic map contained 8 groups of four homologous chromosomes. The lengths of the maps were 2649 and 3045 cM, with an average distance of 7.6 and 9.0 cM between markers, for Magali and Mercedes parents, respectively. Using only the SSR markers, we built a composite map covering 709 cM. CONCLUSIONS: Compared to diploid alfalfa genetic maps, our maps cover about 88-100% of the genome and are close to saturation. The inheritance of the codominant markers (SSR) and the pattern of linkage repulsions between markers within each homology group are consistent with the hypothesis of a tetrasomic meiosis in alfalfa. Except for 2 out of 107 SSR markers, we found a similar order of markers on the chromosomes between the tetraploid alfalfa and M. truncatula genomes indicating a high level of colinearity between these two species. These maps will be a valuable tool for alfalfa breeding and are being used to locate QTLs.