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Key message The bs5 resistance gene against bacterial spot was identified by map-based cloning. Abstract The recessive bs5 gene of pepper (Capsicum annuum L.) conditions a non-hypersensitive resistance trait, characterized by a slightly swollen, pale green, photosynthetically active leaf tissue, following Xanthomonas euvesicatoria infection. The isolation of the bs5 gene by map-based cloning revealed that the bs5 protein was shorter by 2 amino acids as compared to the wild type Bs5 protein. The natural 2 amino acid deletion occurred in the cysteine-rich transmembrane domain of the tail-anchored (TA) protein, Ca_CYSTM1. The protein products of the wild type Bs5 and mutant bs5 genes were shown to be located in the cell membrane, indicating an unknown function in this membrane compartment. Successful infection of the Bs5 pepper lines was abolished by the 6 bp deletion in the TM encoding domain of the Ca_CYSTM1 gene in bs5 homozygotes, suggesting, that the resulting resistance might be explained by the lack of entry of the Xanthomonas specific effector molecules into the plant cells.

SummaryIn the nodules of Inverted Repeat-Lacking Clade legumes, includingM. truncatula, nitrogen-fixing rhizobia undergo terminal differentiation resulting in elongated and endoreduplicated bacteroids specialised for nitrogen fixation. This irreversible transition of rhizobia is mediated by host produced nodule-specific cysteine-rich (NCR) peptides, of which about 700 are encoded in theM. truncatulagenome. Some of these NCR peptides, NCR169, NCR211 and NCR247, are essential for nitrogen-fixing symbiosis.The analysis of bacteroid and symbiotic host cell differentiation revealed that the symbiotic phenotype ofM. truncatulamutants,Mtsym19,Mtsym20and NF-FN9363, were defective likewise in the formerly studiedncrmutants,Mtdnf4-1andMtdnf7-2. The incomplete differentiation of bacteroids triggered premature senescence of rhizobia in the nitrogen fixation zones of mutant nodules.Mtsym19andMtsym20mutants are defective in the same peptide NCR-new35 and the lack ofNCR343is responsible for the ineffective symbiosis of NF-FN9363.The activity ofNCR-new35is significantly lower and limited to the transition zone of the nodule compared with other crucialNCRs. The fluorescent protein-tagged version of NCR343 and NCR-new35 localize to the symbiotic compartment. Our discovery added two additional members to the group ofNCRgenes essential for nitrogen–fixing symbiosis inM. truncatula.

Legumes engage in root nodule symbioses with nitrogen-fixing soil bacteria known as rhizobia. In nodule cells, bacteria are enclosed in membrane-bound vesicles called symbiosomes and differentiate into bacteroids that are capable of converting atmospheric nitrogen into ammonia. Bacteroid differentiation and prolonged intracellular survival are essential for development of functional nodules. However, in the Medicago truncatula–Sinorhizobium meliloti symbiosis, incompatibility between symbiotic partners frequently occurs, leading to the formation of infected nodules defective in nitrogen fixation (Fix−). Here, we report the identification and cloning of the M. truncatula NFS2 gene that regulates this type of specificity pertaining to S. meliloti strain Rm41. We demonstrate that NFS2 encodes a nodule-specific cysteine-rich (NCR) peptide that acts to promote bacterial lysis after differentiation. The negative role of NFS2 in symbiosis is contingent on host genetic background and can be counteracted by other genes encoded by the host. This work extends the paradigm of NCR function to include the negative regulation of symbiotic persistence in host–strain interactions. Our data suggest that NCR peptides are host determinants of symbiotic specificity in M. truncatula and possibly in closely related legumes that form indeterminate nodules in which bacterial symbionts undergo terminal differentiation.

Hybrid tomato plants being heterozygous for certain induced loss-of-function mutations of the SP3D gene displayed fruit-yield heterosis. In order to investigate the possible occurrence of spontaneous mutations of that kind, the SP3D amplicon of 262 tomato seed bank accessions was deep sequenced at about 2000-fold coverage. Compared to the SL2.50 genome assembly, twenty-two novel SNPs in total were identified in the 652 bp upstream, 3817 bp coding and 613 bp downstream regions of the gene. Neither novel exonic mutations in the 262 accessions of this study nor possible loss-of-function mutations amongst the known exonic variants were identified in the SP3D gene of Solanum lycopersicum indicating that, very likely, no such spontaneous mutations occur in cultivated tomato. In wild tomato, however, there are missense variations in the coding region of SP3D, which might result in disabled SP3D proteins. This difference between cultivated and wild tomatoes might have reproductive-biological reasons, while in cultivated tomato missense SP3D mutations might have been eliminated during breeding because they could result in extremely late flowering phenotype. In the promoter region of the SP3D gene, two SNPs, of which one was novel, were identified in the − 62 and − 61 positions upstream of the ATG start codon. These positions were genotyped in the 262 accessions and, comparing our results with Ensembl Plants data, it could be concluded that the dinucleotide is a conserved CT in Solanum lycopersicum varieties, while it is either CA or TA in other Solanum species. This dinucleotide is in a predicted binding site of BELL homeodomain transcription factor(s), regulating meristem, fruit and inflorescence development, thus it might be an element of the regulatory cascade in which the SP3D gene is involved to regulate flowering in tomato. We presented a hypothesis that the SP3D and Single Flower Truss (SFT) genes might not be the same, as it is currently considered, and that SFT might be equivalent to the gene Solyc03g071710 encoding a putative Self-pruning interacting protein. This would logically explain both the absence of natural loss-of-function mutations in the SP3D gene and the absence of DNA lesions in the SP3D gene of sft mutant tomato.

Plant genomes contain two major classes of innate immune receptors to recognize different pathogens. The pattern recognition receptors perceive conserved pathogen‐associated molecular patterns and the resistance genes with nucleotide‐binding (NB) and leucine‐rich repeat (LRR) domains recognize specific pathogen effectors. The precise regulation of resistance genes is important since the unregulated expression of NB‐LRR genes can inhibit growth and may result in autoimmunity in the absence of pathogen infection. It was shown that a subset of miRNAs could target NB‐LRR genes and act as an important regulator of plant immunity in the absence of pathogens. Plants not only interact with pathogens, but they can also establish symbiotic interactions with microbes. Nitrogen‐fixing symbiotic interaction and nodule formation of legumes may also require the suppression of host defence to prevent immune responses. We found that upon symbiotic interactions, miRNAs repressing NB‐LRR expression are upregulated in the developing nodules of Medicago truncatula. Furthermore, we show that the suppression of the activity of the NB‐LRR genes targeted by these miRNAs is important during nodule development. Our results suggest that the downregulation of NB‐LRR resistance genes in the developing nodule produces a suitable niche that facilitates bacterial colonization and the development of an N‐fixing nodule., Nitrogen‐fixing symbiotic interaction and nodule formation of legumes require the suppression of host defenses by differential expression of NB‐LRR‐regulating miRNAs to prevent immune responses.

SummaryThe symbiotic relationship between legumes and rhizobium bacteria in root nodules has a high demand for iron. The host plant is known to provide iron to developing bacteroids, but questions remain regarding which transporters are involved. Here, we characterize two Vacuolar Iron Transporter-Like (VTL) proteins in Medicago truncatula that are specifically expressed during nodule development. VTL4 is mostly expressed during early infection and the protein was localized to membranes and the infection thread. vtl4 mutants were delayed in nodule development. VTL8 is closely related to SEN1 in Lotus japonicus and expressed in the late stages of bacteroid differentiation. The VTL8 protein was localized to the symbiosome membrane. A mutant line lacking the tandemly-arranged VTL4 – VTL8 genes, named 13U, was unable to develop functional nodules and failed to fix nitrogen, which was restored by expression of VTL8 alone. Using a newly developed lux reporter to monitor iron status of the bacteroids, a slight decrease in luminescence was observed in vtl4 mutants and a strong decrease in the 13U mutant. Iron transport capability of VTL4 and VTL8 was shown by yeast complementation. Taken together, these data indicate that VTL-type transporters are the main route for delivering iron to symbiotic rhizobia.

International audience; Plant systemic signaling pathways allow the integration and coordination of shoot and root organ metabolism and development at the whole-plant level depending on nutrient availability. In legumes, two systemic pathways have been reported in the Medicago truncatula model to regulate root nitrogen-fixing symbiotic nodulation. Both pathways involve leucine-rich repeat receptor-like kinases acting in shoots and proposed to perceive signaling peptides produced in roots depending on soil nutrient availability. In this study, we characterized in the M. truncatula Jemalong A17 genotype a mutant allelic series affecting the Compact Root Architecture2 (CRA2) receptor. These analyses revealed that this pathway acts systemically from shoots to positively regulate nodulation and is required for the activity of carboxyl-terminally encoded peptides (CEPs). In addition, we generated a double mutant to test genetic interactions of the CRA2 systemic pathway with the CLAVATA3/EMBRYO SURROUNDING REGION peptide (CLE)/Super Numeric Nodule (SUNN) receptor systemic pathway negatively regulating nodule number from shoots, which revealed an intermediate nodule number phenotype close to the wild type. Finally, we showed that the nitrate inhibition of nodule numbers was observed in cra2 mutants but not in sunn and cra2 sunn mutants. Overall, these results suggest that CEP/CRA2 and CLE/SUNN systemic pathways act independently from shoots to regulate nodule numbers.

Root nodule symbiosis in legumes is established following interaction of compatible rhizobia that activates an array of genes, commonly known as symbiotic-pathway, resulting in nodule development. In model legumes, bacterial entry mainly occurs through infection thread involving the expression of transcription factor CYCLOPS/IPD3. Here we show the functional analysis of AhCYCLOPS in Arachis hypogaea where bacteria invade roots through epidermal cracks. Exploiting significant cross-species domain conservation, trans-complementation experiments involving ectopic expression of AhCYCLOPS in transgenic hairy-roots of Medicago truncatula ipd3 mutants resulted in functional complementation of Medicago nodules. Moreover, native promoter of AhCYCLOPS was sufficient for this cross-species complementation irrespective of the different modes of infection of roots by rhizobia and nodule ontology. To unravel the role of AhCYCLOPS during 'crack-entry' nodulation in A. hypogaea, RNAi of AhCYCLOPS was performed which resulted in delayed nodule inception followed by drastic reduction in nodule number on transgenic hairy-roots. The infection zone of a significant number of RNAi nodules showed presence of infected cells with enlarged nucleus and rod shaped undifferentiated bacteria. Expression analysis showed downregulation of several nodulation responsible effectors endorsing the compromised condition of RNAi roots. Together, the results indicated that AhCYCLOPS plays an important role in A. hypogaea nodule development.

Nuclear magnetic resonance (NMR) spectroscopy profiling was used to provide an unbiased assessment of changes to the metabolite composition of seeds and to define genetic variation for a range of pea seed metabolites. Mature seeds from recombinant inbred lines, derived from three mapping populations for which there is substantial genetic marker linkage information, were grown in two environments/years and analyzed by non-targeted NMR. Adaptive binning of the NMR metabolite data, followed by analysis of quantitative variation among lines for individual bins, identified the main genomic regions determining this metabolic variability and the variability for selected compounds was investigated. Analysis by t-tests identified a set of bins with highly significant associations to genetic map regions, based on probability (p) values that were appreciably lower than those determined for randomized data. The correlation between bins showing high mean absolute deviation and those showing low p-values for marker association provided an indication of the extent to which the genetics of bin variation might be explained by one or a few loci. Variation in compounds related to aromatic amino acids, branched-chain amino acids, sucrose-derived metabolites, secondary metabolites and some unidentified compounds was associated with one or more genetic loci. The combined analysis shows that there are multiple loci throughout the genome that together impact on the abundance of many compounds through a network of interactions, where individual loci may affect more than one compound and vice versa. This work therefore provides a framework for the genetic analysis of the seed metabolome, and the use of genetic marker data in the breeding and selection of seeds for specific seed quality traits and compounds that have high commercial value.

International audience; Legumes form endosymbiotic interaction with host compatible rhizobia, resulting in the development of nitrogen-fixing root nodules. Within symbiotic nodules, rhizobia are intracellularly accommodated in plant-derived membrane compartments, termed symbiosomes. In mature nodule, the massively colonized cells tolerate the existence of rhizobia without manifestation of visible defense responses, indicating the suppression of plant immunity in the nodule in the favur of the symbiotic partner. Medicago truncatula DNF2 (defective in nitrogen fixation 2) and NAD1 (nodules with activated defense 1) genes are essential for the control of plant defense during the colonization of the nitrogen-fixing nodule and are required for bacteroid persistence. The previously identified nodule-specific NAD1 gene encodes a protein of unknown function. Herein, we present the analysis of novel NAD1 mutant alleles to better understand the function of NAD1 in the repression of immune responses in symbiotic nodules. By exploiting the advantage of plant double and rhizobial mutants defective in establishing nitrogen-fixing symbiotic interaction, we show that NAD1 functions following the release of rhizobia from the infection threads and colonization of nodule cells. The suppression of plant defense is self-dependent of the differentiation status of the rhizobia. The corresponding phenotype of nad1 and dnf2 mutants and the similarity in the induction of defense-associated genes in both mutants suggest that NAD1 and DNF2 operate close together in the same pathway controlling defense responses in symbiotic nodules.

Nitrogen is a limiting factor for plant growth. Most crops obtain their nitrogen through the use of nitrogen-based fertilizers, which is costly, and also causes environmental pollution. Legumes, however, have the unique ability to fix atmospheric nitrogen through symbioses with nitrogen-fixing bacteria. Although legumes can be nodulated by indigenous soil bacteria, nitrogen fixation efficiency differs significantly depending on host and bacterial genotypes. Understanding the genetic mechanisms that underlie this specificity will allow for optimizing symbiotic partnerships with improved symbiotic performance. We report that specific nodule-specific cysteine-rich (NCR) peptides negatively regulate symbiotic persistence in a strain-specific manner in Medicago truncatula. This finding offers a strategy to improve nitrogen fixation efficiency through selection or manipulation of NCR alleles that favor specific bacterial strains.

Legumes form endosymbiotic associations with nitrogen-fixing bacteria and arbuscular mycorrhizal (AM) fungi which facilitate nutrient uptake. Both symbiotic interactions require a molecular signal exchange between the plant and the symbiont, and this involves a conserved symbiosis (Sym) signaling pathway. In order to identify plant genes required for intracellular accommodation of nitrogen-fixing bacteria and AM fungi, we characterized Medicago truncatula symbiotic mutants defective for rhizobial infection of nodule cells and colonization of root cells by AM hyphae. Here, we describe mutants impaired in the interacting protein of DMI3 (IPD3) gene, which has been identified earlier as an interacting partner of the calcium/calmodulin-dependent protein, a member of the Sym pathway. The ipd3 mutants are impaired in both rhizobial and mycorrhizal colonization and we show that IPD3 is necessary for appropriate Nod-factor-induced gene expression. This indicates that IPD3 is a member of the common Sym pathway. We observed differences in the severity of ipd3 mutants that appear to be the result of the genetic background. This supports the hypothesis that IPD3 function is partially redundant and, thus, additional genetic components must exist that have analogous functions to IPD3. This explains why mutations in an essential component of the Sym pathway have defects at late stages of the symbiotic interactions.

The formation of a nitrogen-fixing nodule requires the coordinated development of rhizobial colonization and nodule organogenesis. Based on its mutant phenotype, lumpy infections (lin), LIN functions at an early stage of the rhizobial symbiotic process, required for both infection thread growth in root hair cells and the further development of nodule primordia. We show that spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase is independent of LIN; thus, LIN is not necessary for nodule organogenesis. From this, we infer that LIN predominantly functions during rhizobial colonization and that the abortion of this process in lin mutants leads to a suppression of nodule development. Here, we identify the LIN gene in Medicago truncatula and Lotus japonicus, showing that it codes for a predicted E3 ubiquitin ligase containing a highly conserved U-box and WD40 repeat domains. Ubiquitin-mediated protein degradation is a universal mechanism to regulate many biological processes by eliminating rate-limiting enzymes and key components such as transcription factors. We propose that LIN is a regulator of the component(s) of the nodulation factor signal transduction pathway and that its function is required for correct temporal and spatial activity of the target protein(s).

In this paper we describe the identification of a gene, MsDWF1 coding for a putative gibberellin 3-beta-hydroxylase (GA3ox), whose natural mutation is conditioning a dwarf growth phenotype in Medicago sativa. The dwarf phenotype could not be complemented with grafting, which indicates that the bioactive gibberellin compound necessary for shoot elongation is immobile. On the contrary, exogenously added gibberellic acid restored normal growth. The genetic position of the Msdwf1 gene was mapped to linkage group 2 (LG2) and the physical location was delimited by map-based cloning using Medicago truncatula genomic resources. Based on the similar appearance and behavior of the dwarf Medicago sativa plants to the pea stem length mutant (le) as well as the synthenic map position of the two genes it was postulated that MsDWF1 and pea Le are orthologs. The comparison of wild type and mutant allele sequences of MsGA3ox revealed an amino acid change in a conserved position in the mutant allele, which most probably impaired the function of the enzyme. Our results indicate that the dwarf phenotype was the consequence of this mutation.

Rhizobial bacteria activate the formation of nodules on the appropriate host legume plant, and this requires the bacterial signaling molecule Nod factor. Perception of Nod factor in the plant leads to the activation of a number of rhizobial-induced genes. Putative transcriptional regulators in the GRAS family are known to function in Nod factor signaling, but these proteins have not been shown to be capable of direct DNA binding. Here, we identify an ERF transcription factor, ERF Required for Nodulation (ERN), which contains a highly conserved AP2 DNA binding domain, that is necessary for nodulation. Mutations in this gene block the initiation and development of rhizobial invasion structures, termed infection threads, and thus block nodule invasion by the bacteria. We show that ERN is necessary for Nod factor–induced gene expression and for spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase, DMI3, which is a component of the Nod factor signaling pathway. We propose that ERN is a component of the Nod factor signal transduction pathway and functions downstream of DMI3 to activate nodulation gene expression.

Three cDNA clones coding for Medicago sativa Rop GTPases have been isolated. The represented genes could be assigned to various linkage groups by genetic mapping. They were expressed in all investigated plant organs, although at different level. Relative gene expression patterns in response to Sinorhizobium infection of roots as well as during somatic embryogenesis indicated their differential participation in these processes. DNA sequences coding for altogether six different Medicago sp. Rop GTPases could be identified in sequence databases. Based on their homology to each other and to their Arabidopsis counterparts, a unified nomenclature is suggested for Medicago Rop GTPases.

Rhizobial bacteria enter a symbiotic interaction with legumes, activating diverse responses in roots through the lipochito oligosaccharide signaling molecule Nod factor. Here, we show that NSP2 from Medicago truncatula encodes a GRAS protein essential for Nod-factor signaling. NSP2 functions downstream of Nod-factor–induced calcium spiking and a calcium/calmodulin-dependent protein kinase. We show that NSP2-GFP expressed from a constitutive promoter is localized to the endoplasmic reticulum/nuclear envelope and relocalizes to the nucleus after Nod-factor elicitation. This work provides evidence that a GRAS protein transduces calcium signals in plants and provides a possible regulator of Nod-factor–inducible gene expression.

Comparative genome analysis has been performed between alfalfa (Medicago sativa) and pea (Pisum sativum), species which represent two closely related tribes of the subfamily Papilionoideae with different basic chromosome numbers. The positions of genes on the most recent linkage map of diploid alfalfa were compared to those of homologous loci on the combined genetic map of pea to analyze the degree of co-linearity between their linkage groups. In addition to using unique genes, analysis of the map positions of multicopy (homologous) genes identified syntenic homologs (characterized by similar positions on the maps) and pinpointed the positions of non-syntenic homologs. The comparison revealed extensive conservation of gene order between alfalfa and pea. However, genetic rearrangements (due to breakage and reunion) were localized which can account for the difference in chromosome number (8 for alfalfa and 7 for pea). Based on these genetic events and our increasing knowledge of the genomic structure of pea, it was concluded that the difference in genome size between the two species (the pea genome is 5- to 10-fold larger than that of alfalfa) is not a consequence of genome duplication in pea. The high degree of synteny observed between pea and Medicago loci makes further map-based cloning of pea genes based on the genome resources now available for M. truncatula a promising strategy.

Leguminous plants are able to establish a nitrogen-fixing symbiosis with soil bacteria generally known as rhizobia. Metabolites exuded by the plant root activate the production of a rhizobial signal molecule, the Nod factor, which is essential for symbiotic nodule development1,2. This lipo-chitooligosaccharide signal is active at femtomolar concentrations, and its structure is correlated with host specificity of symbiosis3, suggesting the involvement of a cognate perception system in the plant host. Here we describe the cloning of a gene from Medicago sativa that is essential for Nod-factor perception in alfalfa, and by genetic analogy, in the related legumes Medicago truncatula and Pisum sativum. The identified ‘nodulation receptor kinase’, NORK, is predicted to function in the Nod-factor perception/transduction system (the NORK system) that initiates a signal cascade leading to nodulation. The family of ‘NORK extracellular-sequence-like’ (NSL) genes is broadly distributed in the plant kingdom, although their biological function has not been previously ascribed. We suggest that during the evolution of symbiosis an ancestral NSL system was co-opted for transduction of an external ligand, the rhizobial Nod factor, leading to development of the symbiotic root nodule.

An improved genetic map of diploid (2n=2x=16) alfalfa has been developed by analyzing the inheritance of more than 800 genetic markers on the F2 population of 137 plant individuals. The F2 segregating population derived from a self-pollinated F1 hybrid individual of the cross Medicago sativa ssp. quasifalcata ×Medicago sativa ssp. coerulea. This mapping population was the same one which had been used for the construction of our previous alfalfa genetic map. The genetic analyses were performed by using maximum-likelihood equations and related computer programs. The improved genetic map of alfalfa in its present form contains 868 markers (four morphological, 12 isozyme, 26 seed protein, 216 RFLP, 608 RAPD and two specific PCR markers) in eight linkage groups. Of the markers 80 are known genes, including 2 previously cytologically localized genes, the rDNA and the β-tubulin loci. The genetic map covers 754 centimorgans (cM) with an average marker density of 0.8/cM. The correlation between the physical and genetic distances is about 1000–1300 kilobase pairs per centiMorgan. In this map, the linkage relationships of some markers on linkage groups 6, 7, and 8 are different from the previously published one. The cause of this discrepancy was that the genetic linkage of markers displaying distorted segregation (characterized by an overwhelming number of heterozygous individuals) had artificially linked genetic regions that turned out to be unlinked. To overcome the disadvantageous influence of the excess number of heterozygous genotypes on the recombination fractions, we used recently described maximum-likelihood formulas and colormapping, which allowed us to exclude the misleading linkages and to estimate the genetic distances more precisely.

MnNC-1008(NN) (referred to as MN-1008) is a tetraploid alfalfa mutant with two recessive genes (nn 1 and nn 2 )conditioning the non-nodulating trait. The tetraploid level (2n=4x=32) of this Medicago sativa germ plasm was reduced to the diploid (2n=2x=16) level using the 4x-2x genetic cross originally described as a workable method for the induction of haploidy in alfalfa by T. E. Bingham. In our experiments more than 7000 emasculated flowers of a single non-nodulating MN-1008 mutant alfalfa plant with purple petals were cross-pollinated with pollen from a single, diploid, yellow-flowered alfalfa plant. Mature seeds from these crosses were collected and germinated, after which the plants were subjected to morphological and cytogenetic analyses as well as to DNA fingerprinting. Out of 26 viable progeny, 6 were hybrid plants, 19 proved to be self-mated derivatives of MN-1008, while one descendant turned out to be a diploid (2n=2x=16), purple flowered, non-nodulating plant denoted as M. sativa DN-1008. This diploid, non-nodulating alfalfa plant can serve as starting material to facilitate the comprehensive morphological, physiological and genetic analysis (gene mapping and cloning) of nodulation in order to learn more about the biology of the symbiotic root nodule development. To produce diploid, nodulating hybrid F1 plants, DN-1008 was crossed with a diploid, yellow-flowered M. sativa ssp. quasifalcata plant. An F2 population segregating the nn 1 and nn 2 genes in a diploid manner, in which the genetic analysis is more simple than in a tetraploid population, can be established by self-mating of the F1 plants.

The genetic map for alfalfa presented here has eight linkage groups representing the haploid chromosome set of the Medicago species. The genetic map was constructed by ordering the linkage values of 89 RFLP, RAPD, isozyme and morphological markers collected from a segregating population of 138 individuals. The segregating population is self-mated progeny of an F1 hybrid plant deriving from a cross between the diploid (2n = 2x = 16) yellow-flowered. Medicago sativa ssp. quasifalcata and the diploid (2n = 2x = 16) blue-flowered M. sativa ssp. coerulea. The inheritance of many traits displayed distorted segregation, indicating the presence of lethal loci in the heterozygotic parent plants. In spite of the lack of uniform segregation, linkage groups could be assigned and the order of the markers spanning659 centimorgans could be unambiguously determined. This value and the calculated haploid genome size for Medicago (1n = 1x = 1.0 x 10(9) bp) gives a ratio of1500 kb per centimorgan.

Rispail, N. et al., Faba bean is an excellent candidate crop to provide nitrogen input into temperate agricultural systems. However, its growth is hampered by several factors including environmental stresses and the presence of anti-nutritional factors. To solve these limitations, breeding programs have been initiated that were successful for monogenic traits but not so for multigenic traits. The large genome size of faba bean has slowed down breeding processes. Several other legumes have emerged as model legumes including Medicago truncatula, Lotus japonicus, Glycine max and Pisum sativum. The establishment of these models has already boosted our understanding of important processes such as the nitrogen-fixing symbiotic interaction. The high level of synteny and collinearity existing between legumes makes possible the transfer of key knowledge from model legumes to faba bean. Here we review the most recent knowledge gained from model legumes on grain quality, resistance to biotic and abiotic stresses, nitrogen-fixing symbiosis and how this knowledge can be employed for faba bean breeding. © 2009 Elsevier B.V., This work was funded in part by the European Union Grain Legumes Integrated Project (FOOD-CT-2004-506223), the Spanish Ministry of Education (AGL2005-0274/AGR, AGL2008-01239/AGR), the Australian Research Council for Centre of Excellence funding, UQ for strategic funds from the VC, DVCR and BACS Faculty.

Nodulation is tightly regulated in legumes to ensure appropriate levels of nitrogen fixation without excessive depletion of carbon reserves. This balance is maintained by intimately linking nodulation and its regulation with plant hormones. It has previously been shown that ethylene and jasmonic acid (JA) are able to regulate nodulation and Nod factor signal transduction. Here, we characterize the nature of abscisic acid (ABA) regulation of nodulation. We show that application of ABA inhibits nodulation, bacterial infection, and nodulin gene expression in Medicago truncatula. ABA acts in a similar manner as JA and ethylene, regulating Nod factor signaling and affecting the nature of Nod factor-induced calcium spiking. However, this action is independent of the ethylene signal transduction pathway. We show that genetic inhibition of ABA signaling through the use of a dominant-negative allele of ABSCISIC ACID INSENSITIVE1 leads to a hypernodulation phenotype. In addition, we characterize a novel locus of M. truncatula, SENSITIVITY TO ABA, that dictates the sensitivity of the plant to ABA and, as such, impacts the regulation of nodulation. We show that ABA can suppress Nod factor signal transduction in the epidermis and can regulate cytokinin induction of the nodule primordium in the root cortex. Therefore, ABA is capable of coordinately regulating the diverse developmental pathways associated with nodule formation and can intimately dictate the nature of the plants' response to the symbiotic bacteria.

The increased amount of data produced by large genome sequencing projects allows scientists to carry out important syntenic studies to a great extent. Detailed genetic maps and entirely or partially sequenced genomes are compared, and macro- and microsyntenic relations can be determined for different species. In our study, the syntenic relationships between key legume plants and two model plants, Arabidopsis thaliana and Populus trichocarpa were investigated. The comparison of the map position of 172 gene-based Medicago sativa markers to the organization of homologous A. thaliana genes could not identify any sign of macrosynteny between the two genomes. A 276 kb long section of chromosome 5 of the model legume Medicago truncatula was used to investigate potential microsynteny with the other legume Lotus japonicus, as well as with Arabidopsis and Populus. Besides the overall correlation found between the legume plants, the comparison revealed several microsyntenic regions in the two more distant plants with significant resemblance. Despite the large phylogenetic distance, clear microsyntenic regions between Medicago and Arabidopsis or Populus were detected unraveling new intragenomic evolutionary relations in Arabidopsis.

Legumes form symbiotic associations with both mycorrhizal fungi and nitrogen-fixing soil bacteria called rhizobia. Several of the plant genes required for transduction of rhizobial signals, the Nod factors, are also necessary for mycorrhizal symbiosis. Here, we describe the cloning and characterization of one such gene from the legume Medicago truncatula . The DMI1 (does not make infections) gene encodes a novel protein with low global similarity to a ligand-gated cation channel domain of archaea. The protein is highly conserved in angiosperms and ancestral to land plants. We suggest that DMI1 represents an ancient plant-specific innovation, potentially enabling mycorrhizal associations.

A core genetic map of the legume Medicago truncatula has been established by analyzing the segregation of 288 sequence-characterized genetic markers in an F2 population composed of 93 individuals. These molecular markers correspond to 141 ESTs, 80 BAC end sequence tags, and 67 resistance gene analogs, covering 513 cM. In the case of EST-based markers we used an intron-targeted marker strategy with primers designed to anneal in conserved exon regions and to amplify across intron regions. Polymorphisms were significantly more frequent in intron vs. exon regions, thus providing an efficient mechanism to map transcribed genes. Genetic and cytogenetic analysis produced eight well-resolved linkage groups, which have been previously correlated with eight chromosomes by means of FISH with mapped BAC clones. We anticipated that mapping of conserved coding regions would have utility for comparative mapping among legumes; thus 60 of the EST-based primer pairs were designed to amplify orthologous sequences across a range of legume species. As an initial test of this strategy, we used primers designed against M. truncatula exon sequences to rapidly map genes in M. sativa. The resulting comparative map, which includes 68 bridging markers, indicates that the two Medicago genomes are highly similar and establishes the basis for a Medicago composite map.

The genetic map of diploid alfalfa was generated based on the genetic analyses of genotypes of markers collected for the individuals of an F2 population originating from the cross of two diploid alfalfa subspecies, M. sativa ssp. quasifalcata and M. sativa ssp. coerulea (Kiss et al. 1993). The constructed linkage map composed of eight linkage groups which contain more than 1800 morphological, allozyme, and DNA-based markers, of which more than 300 represent loci with known function or sequences in the genome (Kalo et al. 2000). The markers cover approximately 760cM genetic distance. The average correlation between the physical and the genetic distance is about 1000kb/cM. This genetic system was used to map protein markers originating from nodule and seed tissue.

Roots of the non-nodulating Medicago sativa mutant MN-1008 neither undergo root-hair curling, cortical cell division nor any of the early molecular events that accompany nodule initiation and development following rhizobial infection or treatment with Nod factor. These observations suggested that the mutation(s) impaired a pivotal function in Nod factor perception or in the signal transduction pathway. In this paper we show that the genetic lesion conditioning the recessive non-nodulation phenotype in the tetraploid alfalfa mutant MN-1008 can be localized to a single region on LG5 of the M. sativa genetic map. This conclusion is based on genetic analyses conducted at the tetraploid level, involving both segregation analysis and genetic mapping of the trait with respect to molecular DNA markers. The genetic mapping of the Nod(-) phenotype was performed in a segregating tetraploid F2 population, taking advantage of the availability of an advanced genetic map for diploid alfalfa. Two tightly linked flanking markers have been identified which will facilitate the physical mapping and cloning of the gene(s) that underlie(s) the non-nodulation phenotype.

PsCyp15a is a gene that encodes a vacuolar cysteine protease expressed in wilt-induced shoots of Pisum sativum (pea) and in root nodules. To further the understanding of nodular PsCyp15a expression, a region 5′ to the coding sequence of the gene was cloned. Varying lengths of 5′ untranslated sequence were fused with the uidA coding region and introduced from Agrobacterium rhizogenes into “hairy roots” of Vicia hirsuta. In this transgenic root nodulation assay, a promoter sequence of 900 bp was sufficient to give an expression pattern indistinguishable from that obtained in pea nodules by in situ hybridization. An orthologue of PsCyp15a was cloned from nodule mRNA of Medicago sativa and a corresponding gene identified in M. truncatula was also shown to express strongly in nodules. With molecular mapping techniques, it was demonstrated that these genes map to a syntenic genome location in pea and Medicago spp., but the map positions of the Cyp15a genes cannot be correlated with existing nodulation mutants.

An improved genetic linkage map of diploid (2n=2x=16) alfalfa has been developed using more than 800 genetic markers and 137 F2 plant individuals. The F2 segregation population derived from the self pollinated F1/1 hybrid individual of the cross Medicago sativa ssp. quasifalcata x Medicago sativa ssp. coerulea. Some markers on linkage groups 6, 7, and 8 displayed extreme distorted segregation which miss-linked genetic regions. Systematic investigation of the genetic linkage of markers displaying distorted segregation (characterized by the overwhelming number of heterozygous individuals) demonstrated that the excess number of heterozygous genotypes exert a positive influence on the linkage values. The more individuals carrying heterozygotic marker pairs were present in the population the larger was the linkage value. To overcome this disadvantageous influence, the heterozygotic marker pairs were ignored in the subsequent two-point linkage analysis by which, genuine and false linkages could be distinguished. The fidelity of this method was also demonstrated by displaying the genotypes as a colormap. The improved genetic map of alfalfa in its present form contains 868 markers (four morphological, 12 isozyme, 27 seed protein, 213 RFLP, 608 RAPD and four specific PCR markers) on eight linkage groups. The genetic map covers 523 centimorgan (cM) with an average marker density of 0.6 cM. The correlation between the physical and genetic distance is about 1500 kilobase pairs per one cM. Among the 868 mapped markers 80 were known genes including two previously cytologically localized genes, the rDNA and the β-tubulin loci.

Symbiotic nitrogen fixation between Rhizobium bacteria and their host plants is established during a multistep process which is governed by symbiosis-specific genes of both partners. Our future goal is to isolate Medicago gene(s) playing role in the formation of symbiotic nodules by map-based cloning approach relying on the genetic map for diploid alfalfa constructed by our research group. For this purpose the tetraploid MnNC-1008(NN) Medicago sativa mutant was chosen in which the non-nodulating trait is assumed to be conditioned by two recessive genes, nn 1 and nn 2 (Peterson and Barnes, 1981). Neither root hair curling nor cortical cell division can be induced by effective Rhizobium meliloti bacteria on this plant, that is a very early stage in the nodule organogenesis is impaired, so the mutated genes must have indispensable function in the nodulation process.

Cyclins in association with the protein kinase p34cdc2 and related cyclin-dependent protein kinases (cdks) are key regulatory elements in controlling the cell division cycle. Here, we describe the identification and characterization of a full-length cDNA clone of alfalfa mitotic cyclin, termed CycIIIMs. Computer analysis of known plant cyclin gene sequences revealed that this cyclin belongs to the same structural group as the other known partial alfalfa cyclin sequences. Genetic segregation analysis based on DNA-DNA hybridization data showed that the CycIIIMs gene(s) locates in a single chromosomal region on linkage group 5 of the alfalfa genetic map between RFLP markers UO89A and CG13. The assignment of this cyclin to the mitotic cyclin class was based on its cDNA-derived sequence and its differential expression during G2/M cell cycle phase transition of a partially synchronized alfalfa cell culture. Sequence analysis indicated common motifs with both the A- and B-types of mitotic cyclins similarly to the newly described B3-type of animal cyclins.

In higher eukaryotic plants, the number of the genes is approximately 50, 000, therefore the plant genomes are fairly complex, consequently it is very hard to correlate genes with functions of an appropriate biological process, like symbiotic nitrogen fixation. Some techniques which are applicable for prokaryotic gene isolation (e.g. complementation of mutants) can not be used for most of the plant species. In order to correlate functions with cloned genes indirect biochemical approaches have been developed, like differential hybridization, construction of subtraction libraries, etc. Another laborious biochemical possibility is to identify a gene by its protein product. This approach involves the identification, purification and sequencing of a protein followed by the synthesis of specific oligonucleotides to use as hybridization probe or as primers for PCR. Fortunately there is an additional possibility by which specific genes can be identified: this is the genetic approach. In this case a mutant plant impaired in the appropriate biological process has to be isolated, then the gene suffered the mutation has to be genetically mapped using molecular markers, and finally the gene has to be cloned with the help of tightly linked molecular markers. This approach is the so-called map-based cloning strategy. Though the genetic strategy can not be used for every cases, nevertheless in those ones where applicable it provides an elegant solution to the problem. It has a unique advantage over the ambiguous biochemical strategies, that is one can be sure that the isolated gene is really involved and necessary in the biological process in question (see Csanadi et al,1994)

Background Recent genome sequencing enables mega-base scale comparisons between related genomes. Comparisons between animals, plants, fungi, and bacteria demonstrate extensive synteny tempered by rearrangements. Within the legume plant family, glimpses of synteny have also been observed. Characterizing syntenic relationships in legumes is important in transferring knowledge from model legumes to crops that are important sources of protein, fixed nitrogen, and health-promoting compounds. Results We have uncovered two large soybean regions exhibiting synteny with M. truncatula and with a network of segmentally duplicated regions in Arabidopsis. In all, syntenic regions comprise over 500 predicted genes spanning 3 Mb. Up to 75% of soybean genes are colinear with M. truncatula, including one region in which 33 of 35 soybean predicted genes with database support are colinear to M. truncatula. In some regions, 60% of soybean genes share colinearity with a network of A. thaliana duplications. One region is especially interesting because this 500 kbp segment of soybean is syntenic to two paralogous regions in M. truncatula on different chromosomes. Phylogenetic analysis of individual genes within these regions demonstrates that one is orthologous to the soybean region, with which it also shows substantially denser synteny and significantly lower levels of synonymous nucleotide substitutions. The other M. truncatula region is inferred to be paralogous, presumably resulting from a duplication event preceding speciation. Conclusion The presence of well-defined M. truncatula segments showing orthologous and paralogous relationships with soybean allows us to explore the evolution of contiguous genomic regions in the context of ancient genome duplication and speciation events.