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1.  Recent Progress in Development of Tnt1 Functional Genomics Platform for Medicago truncatula and Lotus japonicus in Bulgaria 
Current Genomics  2011;12(2):147-152.
Legumes, as protein-rich crops, are widely used for human food, animal feed and vegetable oil production. Over the past decade, two legume species, Medicago truncatula and Lotus japonicus, have been adopted as model legumes for genomics and physiological studies. The tobacco transposable element, Tnt1, is a powerful tool for insertional mutagenesis and gene inactivation in plants. A large collection of Tnt1-tagged lines of M. truncatula cv. Jemalong was generated during the course of the project ‘GLIP’: Grain Legumes Integrated Project, funded by the European Union (www.eugrainlegumes.org). In the project ‘IFCOSMO’: Integrated Functional and COmparative genomics Studies on the MOdel Legumes Medicago truncatula and Lotus japonicus, supported by a grant from the Ministry of Education, Youth and Science, Bulgaria, these lines are used for development of functional genomics platform of legumes in Bulgaria. This review presents recent advances in the evaluation of the M. truncatula Tnt1 mutant collection and outlines the steps that are taken in using the Tnt1-tagging for generation of a mutant collection of the second model legume L. japonicus. Both collections will provide a number of legume-specific mutants and serve as a resource for functional and comparative genomics research on legumes. Genomics technologies are expected to advance genetics and breeding of important legume crops (pea, faba bean, alfalfa and clover) in Bulgaria and worldwide.
doi:10.2174/138920211795564313
PMCID: PMC3129049  PMID: 21966253
Insertional mutagenesis; legume genomics; Medicago truncatula; Lotus japonicus; phenotyping; Tnt1 mutants.
2.  The Medicago Genome Initiative: a model legume database 
Nucleic Acids Research  2001;29(1):114-117.
The Medicago Genome Initiative (MGI) is a database of EST sequences of the model legume Medicago truncatula. The database is available to the public and has resulted from a collaborative research effort between the Samuel Roberts Noble Foundation and the National Center for Genome Resources to investigate the genome of M.truncatula. MGI is part of the greater integrated Medicago functional genomics program at the Noble Foundation (http://www.noble .org), which is taking a global approach in studying the genetic and biochemical events associated with the growth, development and environmental interactions of this model legume. Our approach will include: large-scale EST sequencing, gene expression profiling, the generation of M.truncatula activation-tagged and promoter trap insertion mutants, high-throughput metabolic profiling, and proteome studies. These multidisciplinary information pools will be interfaced with one another to provide scientists with an integrated, holistic set of tools to address fundamental questions pertaining to legume biology. The public interface to the MGI database can be accessed at http://www.ncgr.org/research/mgi.
PMCID: PMC29836  PMID: 11125064
3.  A community resource for high-throughput quantitative RT-PCR analysis of transcription factor gene expression in Medicago truncatula 
Plant Methods  2008;4:18.
Background
Medicago truncatula is a model legume species that is currently the focus of an international genome sequencing effort. Although several different oligonucleotide and cDNA arrays have been produced for genome-wide transcript analysis of this species, intrinsic limitations in the sensitivity of hybridization-based technologies mean that transcripts of genes expressed at low-levels cannot be measured accurately with these tools. Amongst such genes are many encoding transcription factors (TFs), which are arguably the most important class of regulatory proteins. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is the most sensitive method currently available for transcript quantification, and one that can be scaled up to analyze transcripts of thousands of genes in parallel. Thus, qRT-PCR is an ideal method to tackle the problem of TF transcript quantification in Medicago and other plants.
Results
We established a bioinformatics pipeline to identify putative TF genes in Medicago truncatula and to design gene-specific oligonucleotide primers for qRT-PCR analysis of TF transcripts. We validated the efficacy and gene-specificity of over 1000 TF primer pairs and utilized these to identify sets of organ-enhanced TF genes that may play important roles in organ development or differentiation in this species. This community resource will be developed further as more genome sequence becomes available, with the ultimate goal of producing validated, gene-specific primers for all Medicago TF genes.
Conclusion
High-throughput qRT-PCR using a 384-well plate format enables rapid, flexible, and sensitive quantification of all predicted Medicago transcription factor mRNAs. This resource has been utilized recently by several groups in Europe, Australia, and the USA, and we expect that it will become the 'gold-standard' for TF transcript profiling in Medicago truncatula.
doi:10.1186/1746-4811-4-18
PMCID: PMC2490690  PMID: 18611268
4.  Identification of a dominant gene in Medicago truncatula that restricts nodulation by Sinorhizobium meliloti strain Rm41 
BMC Plant Biology  2014;14:167.
Background
Leguminous plants are able to form a root nodule symbiosis with nitrogen-fixing soil bacteria called rhizobia. This symbiotic association shows a high level of specificity. Beyond the specificity for the legume family, individual legume species/genotypes can only interact with certain restricted group of bacterial species or strains. Specificity in this system is regulated by complex signal exchange between the two symbiotic partners and thus multiple genetic mechanisms could be involved in the recognition process. Knowledge of the molecular mechanisms controlling symbiotic specificity could enable genetic improvement of legume nitrogen fixation, and may also reveal the possible mechanisms that restrict root nodule symbiosis in non-legumes.
Results
We screened a core collection of Medicago truncatula genotypes with several strains of Sinorhizobium meliloti and identified a naturally occurring dominant gene that restricts nodulation by S. meliloti Rm41. We named this gene as Mt-NS1 (for M.truncatulanodulation specificity 1). We have mapped the Mt-NS1 locus within a small genomic region on M. truncatula chromosome 8. The data reported here will facilitate positional cloning of the Mt-NS1 gene.
Conclusions
Evolution of symbiosis specificity involves both rhizobial and host genes. From the bacterial side, specificity determinants include Nod factors, surface polysaccharides, and secreted proteins. However, we know relatively less from the host side. We recently demonstrated that a component of this specificity in soybeans is defined by plant NBS-LRR resistance (R) genes that recognize effector proteins delivered by the type III secretion system (T3SS) of the rhizobial symbionts. However, the lack of a T3SS in many sequenced S. meliloti strains raises the question of how the specificity is regulated in the Medicago-Sinorhizobium system beyond Nod-factor perception. Thus, cloning and characterization of Mt-NS1 will add a new dimension to our knowledge about the genetic control of nodulation specificity in the legume-rhizobial symbiosis.
doi:10.1186/1471-2229-14-167
PMCID: PMC4070093  PMID: 24934080
Legume; Medicago truncatula; Nodulation specificity; Nitrogen fixation
5.  Natural diversity in the model legume Medicago truncatula allows identifying distinct genetic mechanisms conferring partial resistance to Verticillium wilt 
Journal of Experimental Botany  2012;64(1):317-332.
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.
doi:10.1093/jxb/ers337
PMCID: PMC3528038  PMID: 23213135
biodiversity; Medicago truncatula; nodulation mutants; partial resistance; quantitative trait loci; root disease; vascular wilt; Verticillium albo-atrum.
6.  Comparative phylogenetic analysis of small GTP-binding genes of model legume plants and assessment of their roles in root nodules 
Journal of Experimental Botany  2008;59(14):3831-3844.
Small GTP-binding genes play an essential regulatory role in a multitude of cellular processes such as vesicle-mediated intracellular trafficking, signal transduction, cytoskeletal organization, and cell division in plants and animals. Medicago truncatula and Lotus japonicus are important model plants for studying legume-specific biological processes such as nodulation. The publicly available online resources for these plants from websites such as http://www.ncbi.nih.gov, http://www.medicago.org, http://www.tigr.org, and related sites were searched to collect nucleotide sequences that encode GTP-binding protein homologues. A total of 460 small GTPase sequences from several legume species including Medicago and Lotus, Arabidopsis, human, and yeast were phyletically analysed to shed light on the evolution and functional characteristics of legume-specific homologues. One of the main emphases of this study was the elucidation of the possible involvement of some members of small GTPase homologues in the establishment and maintenance of symbiotic associations in root nodules of legumes. A high frequency of vesicle-mediated trafficking in nodules led to the idea of a probable subfunctionalization of some members of this family in legumes. As a result of the analyses, a group of 10 small GTPases that are likely to be mainly expressed in nodules was determined. The sequences determined as a result of this study could be used in more detailed molecular genetic analyses such as creation of RNA inteference silencing mutants for further clarification of the role of GTPases in nodulation. This study will also assist in furthering our understanding of the evolutionary history of small GTPases in legume species.
doi:10.1093/jxb/ern223
PMCID: PMC2576638  PMID: 18849296
Arabidopsis thaliana; ARF; Lotus japonicus; Medicago truncatula; nodulation; phylogenetic; RAB; RAC; ROP; small GTPases
7.  Medicago truncatula transporter database: a comprehensive database resource for M. truncatula transporters 
BMC Genomics  2012;13:60.
Background
Medicago truncatula has been chosen as a model species for genomic studies. It is closely related to an important legume, alfalfa. Transporters are a large group of membrane-spanning proteins. They deliver essential nutrients, eject waste products, and assist the cell in sensing environmental conditions by forming a complex system of pumps and channels. Although studies have effectively characterized individual M. truncatula transporters in several databases, until now there has been no available systematic database that includes all transporters in M. truncatula.
Description
The M. truncatula transporter database (MTDB) contains comprehensive information on the transporters in M. truncatula. Based on the TransportTP method, we have presented a novel prediction pipeline. A total of 3,665 putative transporters have been annotated based on International Medicago Genome Annotated Group (IMGAG) V3.5 V3 and the M. truncatula Gene Index (MTGI) V10.0 releases and assigned to 162 families according to the transporter classification system. These families were further classified into seven types according to their transport mode and energy coupling mechanism. Extensive annotations referring to each protein were generated, including basic protein function, expressed sequence tag (EST) mapping, genome locus, three-dimensional template prediction, transmembrane segment, and domain annotation. A chromosome distribution map and text-based Basic Local Alignment Search Tools were also created. In addition, we have provided a way to explore the expression of putative M. truncatula transporter genes under stress treatments.
Conclusions
In summary, the MTDB enables the exploration and comparative analysis of putative transporters in M. truncatula. A user-friendly web interface and regular updates make MTDB valuable to researchers in related fields. The MTDB is freely available now to all users at http://bioinformatics.cau.edu.cn/MtTransporter/.
doi:10.1186/1471-2164-13-60
PMCID: PMC3298476  PMID: 22309450
8.  Ancient orphan crop joins modern era: gene-based SNP discovery and mapping in lentil 
BMC Genomics  2013;14:192.
Background
The genus Lens comprises a range of closely related species within the galegoid clade of the Papilionoideae family. The clade includes other important crops (e.g. chickpea and pea) as well as a sequenced model legume (Medicago truncatula). Lentil is a global food crop increasing in importance in the Indian sub-continent and elsewhere due to its nutritional value and quick cooking time. Despite this importance there has been a dearth of genetic and genomic resources for the crop and this has limited the application of marker-assisted selection strategies in breeding.
Results
We describe here the development of a deep and diverse transcriptome resource for lentil using next generation sequencing technology. The generation of data in multiple cultivated (L. culinaris) and wild (L. ervoides) genotypes together with the utilization of a bioinformatics workflow enabled the identification of a large collection of SNPs and the subsequent development of a genotyping platform that was used to establish the first comprehensive genetic map of the L. culinaris genome. Extensive collinearity with M. truncatula was evident on the basis of sequence homology between mapped markers and the model genome and large translocations and inversions relative to M. truncatula were identified. An estimate for the time divergence of L. culinaris from L. ervoides and of both from M. truncatula was also calculated.
Conclusions
The availability of the genomic and derived molecular marker resources presented here will help change lentil breeding strategies and lead to increased genetic gain in the future.
doi:10.1186/1471-2164-14-192
PMCID: PMC3635939  PMID: 23506258
9.  Characterisation of the legume SERK-NIK gene superfamily including splice variants: Implications for development and defence 
BMC Plant Biology  2011;11:44.
Background
SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) genes are part of the regulation of diverse signalling events in plants. Current evidence shows SERK proteins function both in developmental and defence signalling pathways, which occur in response to both peptide and steroid ligands. SERKs are generally present as small gene families in plants, with five SERK genes in Arabidopsis. Knowledge gained primarily through work on Arabidopsis SERKs indicates that these proteins probably interact with a wide range of other receptor kinases and form a fundamental part of many essential signalling pathways. The SERK1 gene of the model legume, Medicago truncatula functions in somatic and zygotic embryogenesis, and during many phases of plant development, including nodule and lateral root formation. However, other SERK genes in M. truncatula and other legumes are largely unidentified and their functions unknown.
Results
To aid the understanding of signalling pathways in M. truncatula, we have identified and annotated the SERK genes in this species. Using degenerate PCR and database mining, eight more SERK-like genes have been identified and these have been shown to be expressed. The amplification and sequencing of several different PCR products from one of these genes is consistent with the presence of splice variants. Four of the eight additional genes identified are upregulated in cultured leaf tissue grown on embryogenic medium. The sequence information obtained from M. truncatula was used to identify SERK family genes in the recently sequenced soybean (Glycine max) genome.
Conclusions
A total of nine SERK or SERK-like genes have been identified in M. truncatula and potentially 17 in soybean. Five M. truncatula SERK genes arose from duplication events not evident in soybean and Lotus. The presence of splice variants has not been previously reported in a SERK gene. Upregulation of four newly identified SERK genes (in addition to the previously described MtSERK1) in embryogenic tissue cultures suggests these genes also play a role in the process of somatic embryogenesis. The phylogenetic relationship of members of the SERK gene family to closely related genes, and to development and defence function is discussed.
doi:10.1186/1471-2229-11-44
PMCID: PMC3061892  PMID: 21385462
10.  Protection of Sinorhizobium against Host Cysteine-Rich Antimicrobial Peptides Is Critical for Symbiosis 
PLoS Biology  2011;9(10):e1001169.
A bacterial membrane protein, BacA, protects Sinorhizobium meliloti against the antimicrobial activity of host peptides, enabling the peptides to induce bacterial persistence rather than bacterial death.
Sinorhizobium meliloti differentiates into persisting, nitrogen-fixing bacteroids within root nodules of the legume Medicago truncatula. Nodule-specific cysteine-rich antimicrobial peptides (NCR AMPs) and the bacterial BacA protein are essential for bacteroid development. However, the bacterial factors central to the NCR AMP response and the in planta role of BacA are unknown. We investigated the hypothesis that BacA is critical for the bacterial response towards NCR AMPs. We found that BacA was not essential for NCR AMPs to induce features of S. meliloti bacteroids in vitro. Instead, BacA was critical to reduce the amount of NCR AMP-induced membrane permeabilization and bacterial killing in vitro. Within M. truncatula, both wild-type and BacA-deficient mutant bacteria were challenged with NCR AMPs, but this resulted in persistence of the wild-type bacteria and rapid cell death of the mutant bacteria. In contrast, BacA was dispensable for bacterial survival in an M. truncatula dnf1 mutant defective in NCR AMP transport to the bacterial compartment. Therefore, BacA is critical for the legume symbiosis by protecting S. meliloti against the bactericidal effects of NCR AMPs. Host AMPs are ubiquitous in nature and BacA proteins are essential for other chronic host infections by symbiotic and pathogenic bacteria. Hence, our findings suggest that BacA-mediated protection of bacteria against host AMPs is a critical stage in the establishment of different prolonged host infections.
Author Summary
Certain bacterial species have the unique capacity to enter into eukaryotic host cells and establish prolonged infections, which can be beneficial (e.g. bacterial-legume symbiosis) or detrimental (e.g. chronic disease) for the host. However, the mechanisms by which bacteria persist in host cells are poorly understood. Legume peptides and the bacterial BacA membrane protein play essential roles in enabling bacteria to establish prolonged legume infections. However, the biological function of BacA in persistent legume infections has eluded scientists for nearly two decades. In this article, we investigated a potential relationship between legume peptides and BacA in the establishment of prolonged bacterial-legume infections. We found that BacA was critical to protect bacteria against the antimicrobial action of legume peptides, thereby allowing the peptides to induce bacterial persistence within the legume rather than rapid bacterial death. Mammalian hosts also produce peptides in response to invading microorganisms and BacA proteins are critical for medically important bacterial pathogens such as Mycobacterium tuberculosis to form prolonged mammalian infections. Therefore, our results suggest that BacA-mediated protection against host peptides might be a conserved mechanism used by both symbiotic and pathogenic bacterial species to establish long-term host infections.
doi:10.1371/journal.pbio.1001169
PMCID: PMC3186793  PMID: 21990963
11.  The Medicago truncatula gene expression atlas web server 
BMC Bioinformatics  2009;10:441.
Background
Legumes (Leguminosae or Fabaceae) play a major role in agriculture. Transcriptomics studies in the model legume species, Medicago truncatula, are instrumental in helping to formulate hypotheses about the role of legume genes. With the rapid growth of publically available Affymetrix GeneChip Medicago Genome Array GeneChip data from a great range of tissues, cell types, growth conditions, and stress treatments, the legume research community desires an effective bioinformatics system to aid efforts to interpret the Medicago genome through functional genomics. We developed the Medicago truncatula Gene Expression Atlas (MtGEA) web server for this purpose.
Description
The Medicago truncatula Gene Expression Atlas (MtGEA) web server is a centralized platform for analyzing the Medicago transcriptome. Currently, the web server hosts gene expression data from 156 Affymetrix GeneChip® Medicago genome arrays in 64 different experiments, covering a broad range of developmental and environmental conditions. The server enables flexible, multifaceted analyses of transcript data and provides a range of additional information about genes, including different types of annotation and links to the genome sequence, which help users formulate hypotheses about gene function. Transcript data can be accessed using Affymetrix probe identification number, DNA sequence, gene name, functional description in natural language, GO and KEGG annotation terms, and InterPro domain number. Transcripts can also be discovered through co-expression or differential expression analysis. Flexible tools to select a subset of experiments and to visualize and compare expression profiles of multiple genes have been implemented. Data can be downloaded, in part or full, in a tabular form compatible with common analytical and visualization software. The web server will be updated on a regular basis to incorporate new gene expression data and genome annotation, and is accessible at: http://bioinfo.noble.org/gene-atlas/.
Conclusions
The MtGEA web server has a well managed rich data set, and offers data retrieval and analysis tools provided in the web platform. It's proven to be a powerful resource for plant biologists to effectively and efficiently identify Medicago transcripts of interest from a multitude of aspects, formulate hypothesis about gene function, and overall interpret the Medicago genome from a systematic point of view.
doi:10.1186/1471-2105-10-441
PMCID: PMC2804685  PMID: 20028527
12.  Construction of a comparative genetic map in faba bean (Vicia faba L.); conservation of genome structure with Lens culinaris 
BMC Genomics  2008;9:380.
Background
The development of genetic markers is complex and costly in species with little pre-existing genomic information. Faba bean possesses one of the largest and least studied genomes among cultivated crop plants and no gene-based genetic maps exist. Gene-based orthologous markers allow chromosomal regions and levels of synteny to be characterised between species, reveal phylogenetic relationships and chromosomal evolution, and enable targeted identification of markers for crop breeding. In this study orthologous codominant cross-species markers have been deployed to produce the first exclusively gene-based genetic linkage map of faba bean (Vicia faba), using an F6 population developed from a cross between the lines Vf6 (equina type) and Vf27 (paucijuga type).
Results
Of 796 intron-targeted amplified polymorphic (ITAP) markers screened, 151 markers could be used to construct a comparative genetic map. Linkage analysis revealed seven major and five small linkage groups (LGs), one pair and 12 unlinked markers. Each LG was comprised of three to 30 markers and varied in length from 23.6 cM to 324.8 cM. The map spanned a total length of 1685.8 cM. A simple and direct macrosyntenic relationship between faba bean and Medicago truncatula was evident, while faba bean and lentil shared a common rearrangement relative to M. truncatula. One hundred and four of the 127 mapped markers in the 12 LGs, which were previously assigned to M. truncatula genetic and physical maps, were found in regions syntenic between the faba bean and M. truncatula genomes. However chromosomal rearrangements were observed that could explain the difference in chromosome numbers between these three legume species. These rearrangements suggested high conservation of M. truncatula chromosomes 1, 5 and 8; moderate conservation of chromosomes 2, 3, 4 and 7 and no conservation with M. truncatula chromosome 6. Multiple PCR amplicons and comparative mapping were suggestive of small-scale duplication events in faba bean. This study also provides a preliminary indication for finer scale macrosynteny between M. truncatula, lentil and faba bean. Markers originally designed from genes on the same M. truncatula BACs were found to be grouped together in corresponding syntenic areas in lentil and faba bean.
Conclusion
Despite the large size of the faba bean genome, comparative mapping did not reveal evidence for polyploidisation, segmental duplication, or significant rearrangements compared to M. truncatula, although a bias in the use of single locus markers may have limited the detection of duplications. Non-coding repetitive DNA or transposable element content provides a possible explanation for the difference in genome sizes. Similar patterns of rearrangements in faba bean and lentil compared to M. truncatula support phylogenetic studies dividing these species into the tribes Viceae and Trifoliae. However, substantial macrosynteny was apparent between faba bean and M. truncatula, with the exception of chromosome 6 where no orthologous markers were found, confirming previous investigations suggesting chromosome 6 is atypical. The composite map, anchored with orthologous markers mapped in M. truncatula, provides a central reference map for future use of genomic and genetic information in faba bean genetic analysis and breeding.
doi:10.1186/1471-2164-9-380
PMCID: PMC2533332  PMID: 18691425
13.  Genome-wide determination of poly(A) sites in Medicago truncatula: evolutionary conservation of alternative poly(A) site choice 
BMC Genomics  2014;15(1):615.
Background
Alternative polyadenylation (APA) plays an important role in the post-transcriptional regulation of gene expression. Little is known about how APA sites may evolve in homologous genes in different plant species. To this end, comparative studies of APA sites in different organisms are needed. In this study, a collection of poly(A) sites in Medicago truncatula, a model system for legume plants, has been generated and compared with APA sites in Arabidopsis thaliana.
Results
The poly(A) tags from a deep-sequencing protocol were mapped to the annotated M. truncatula genome, and the identified poly(A) sites used to update the annotations of 14,203 genes. The results show that 64% of M. truncatula genes possess more than one poly(A) site, comparable to the percentages reported for Arabidopsis and rice. In addition, the poly(A) signals associated with M. truncatula genes were similar to those seen in Arabidopsis and other plants. The 3′-UTR lengths are correlated in pairs of orthologous genes between M. truncatula and Arabidopsis. Very little conservation of intronic poly(A) sites was found between Arabidopsis and M. truncatula, which suggests that such sites are likely to be species-specific in plants. In contrast, there is a greater conservation of CDS-localized poly(A) sites in these two species. A sizeable number of M. truncatula antisense poly(A) sites were found. A high percentage of the associated target genes possess Arabidopsis orthologs that are also associated with antisense sites. This is suggestive of important roles for antisense regulation of these target genes.
Conclusions
Our results reveal some distinct patterns of sense and antisense poly(A) sites in Arabidopsis and M. truncatula. In so doing, this study lends insight into general evolutionary trends of alternative polyadenylation in plants.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-615) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-615
PMCID: PMC4117952  PMID: 25048171
Alternative polyadenylation; RNA processing; Antisense; Evolutionary conservation; Legume; Medicago truncatula
14.  MediPlEx - a tool to combine in silico & experimental gene expression profiles of the model legume Medicago truncatula 
BMC Research Notes  2010;3:262.
Background
Expressed Sequence Tags (ESTs) are in general used to gain a first insight into gene activities from a species of interest. Subsequently, and typically based on a combination of EST and genome sequences, microarray-based expression analyses are performed for a variety of conditions. In some cases, a multitude of EST and microarray experiments are conducted for one species, covering different tissues, cell states, and cell types. Under these circumstances, the challenge arises to combine results derived from the different expression profiling strategies, with the goal to uncover novel information on the basis of the integrated datasets.
Findings
Using our new analysis tool, MediPlEx (MEDIcago truncatula multiPLe EXpression analysis), expression data from EST experiments, oligonucleotide microarrays and Affymetrix GeneChips® can be combined and analyzed, leading to a novel approach to integrated transcriptome analysis. We have validated our tool via the identification of a set of well-characterized AM-specific and AM-induced marker genes, identified by MediPlEx on the basis of in silico and experimental gene expression profiles from roots colonized with AM fungi.
Conclusions
MediPlEx offers an integrated analysis pipeline for different sets of expression data generated for the model legume Medicago truncatula. As expected, in silico and experimental gene expression data that cover the same biological condition correlate well. The collection of differentially expressed genes identified via MediPlEx provides a starting point for functional studies in plant mutants. MediPlEx can freely be used at http://www.cebitec.uni-bielefeld.de/mediplex.
doi:10.1186/1756-0500-3-262
PMCID: PMC2972298  PMID: 20958970
15.  The Mitochondrial Complexome of Medicago truncatula 
Legumes (Fabaceae, Leguminosae) are unique in their ability to carry out an elaborate endosymbiotic nitrogen fixation process with rhizobia proteobacteria. The symbiotic nitrogen fixation enables the host plants to grow almost independently of any other nitrogen source. Establishment of symbiosis requires adaptations of the host cellular metabolism, here foremost of the energy metabolism mainly taking place in mitochondria. Since the early 1990s, the galegoid legume Medicago truncatula Gaertn. is a well-established model for studying legume biology, but little is known about the protein complement of mitochondria from this species. An initial characterization of the mitochondrial proteome of M. truncatula (Jemalong A17) was published recently. In the frame of this study, mitochondrial protein complexes were characterized using Two-dimensional (2D) Blue native (BN)/SDS-PAGE. From 139 detected spots, the “first hit” (=most abundant) proteins of 59 spots were identified by mass spectrometry. Here, we present a comprehensive analysis of the mitochondrial “complexome” (the “protein complex proteome”) of M. truncatula via 2D BN/SDS-PAGE in combination with highly sensitive MS protein identification. In total, 1,485 proteins were identified within 158 gel spots, representing 467 unique proteins. Data evaluation by the novel GelMap annotation tool allowed recognition of protein complexes of low abundance. Overall, at least 36 mitochondrial protein complexes were found. To our knowledge several of these complexes were described for the first time in Medicago. The data set is accessible under http://www.gelmap.de/medicago/. The mitochondrial protein complex proteomes of Arabidopsis available at http://www.gelmap.de/arabidopsis/ and Medicago are compared.
doi:10.3389/fpls.2013.00084
PMCID: PMC3625726  PMID: 23596449
Medicago truncatula; mitochondrial complexome; 2D BN/SDS-PAGE; GelMap annotation tool; mitochondrial prohibitins
16.  The identification of novel loci required for appropriate nodule development in Medicago truncatula 
BMC Plant Biology  2013;13:157.
Background
The formation of functional symbiotic nodules is the result of a coordinated developmental program between legumes and rhizobial bacteria. Genetic analyses in legumes have been used to dissect the signaling processes required for establishing the legume-rhizobial endosymbiotic association. Compared to the early events of the symbiotic interaction, less attention has been paid to plant loci required for rhizobial colonization and the functioning of the nodule. Here we describe the identification and characterization of a number of new genetic loci in Medicago truncatula that are required for the development of effective nitrogen fixing nodules.
Results
Approximately 38,000 EMS and fast neutron mutagenized Medicago truncatula seedlings were screened for defects in symbiotic nitrogen fixation. Mutant plants impaired in nodule development and efficient nitrogen fixation were selected for further genetic and phenotypic analysis. Nine mutants completely lacking in nodule formation (Nod-) represented six complementation groups of which two novel loci have been identified. Eight mutants with ineffective nodules (Fix-) represented seven complementation groups, out of which five were new monogenic loci. The Fix- M. truncatula mutants showed symptoms of nitrogen deficiency and developed small white nodules. Microscopic analysis of Fix- nodules revealed that the mutants have defects in the release of rhizobia from infection threads, differentiation of rhizobia and maintenance of persistence of bacteria in nodule cells. Additionally, we monitored the transcriptional activity of symbiosis specific genes to define what transcriptional stage of the symbiotic process is blocked in each of the Fix- mutants. Based on the phenotypic and gene expression analysis a functional hierarchy of the FIX genes is proposed.
Conclusions
The new symbiotic loci of M. truncatula isolated in this study provide the foundation for further characterization of the mechanisms underpinning nodulation, in particular the later stages associated with bacterial release and nodule function.
doi:10.1186/1471-2229-13-157
PMCID: PMC3852326  PMID: 24119289
Medicago truncatula; Legume; Symbiosis; Mutant screen; Ineffective nitrogen fixation mutant
17.  Transcriptome sequencing of field pea and faba bean for discovery and validation of SSR genetic markers 
BMC Genomics  2012;13:104.
Background
Field pea (Pisum sativum L.) and faba bean (Vicia faba L.) are cool-season grain legume species that provide rich sources of food for humans and fodder for livestock. To date, both species have been relative 'genomic orphans' due to limited availability of genetic and genomic information. A significant enrichment of genomic resources is consequently required in order to understand the genetic architecture of important agronomic traits, and to support germplasm enhancement, genetic diversity, population structure and demographic studies.
Results
cDNA samples obtained from various tissue types of specific field pea and faba bean genotypes were sequenced using 454 Roche GS FLX Titanium technology. A total of 720,324 and 304,680 reads for field pea and faba bean, respectively, were de novo assembled to generate sets of 70,682 and 60,440 unigenes. Consensus sequences were compared against the genome of the model legume species Medicago truncatula Gaertn., as well as that of the more distantly related, but better-characterised genome of Arabidopsis thaliana L.. In comparison to M. truncatula coding sequences, 11,737 and 10,179 unique hits were obtained from field pea and faba bean. Totals of 22,057 field pea and 18,052 faba bean unigenes were subsequently annotated from GenBank. Comparison to the genome of soybean (Glycine max L.) resulted in 19,451 unique hits for field pea and 16,497 unique hits for faba bean, corresponding to c. 35% and 30% of the known gene space, respectively. Simple sequence repeat (SSR)-containing expressed sequence tags (ESTs) were identified from consensus sequences, and totals of 2,397 and 802 primer pairs were designed for field pea and faba bean. Subsets of 96 EST-SSR markers were screened for validation across modest panels of field pea and faba bean cultivars, as well as related non-domesticated species. For field pea, 86 primer pairs successfully obtained amplification products from one or more template genotypes, of which 59% revealed polymorphism between 6 genotypes. In the case of faba bean, 81 primer pairs displayed successful amplification, of which 48% detected polymorphism.
Conclusions
The generation of EST datasets for field pea and faba bean has permitted effective unigene identification and functional sequence annotation. EST-SSR loci were detected at incidences of 14-17%, permitting design of comprehensive sets of primer pairs. The subsets from these primer pairs proved highly useful for polymorphism detection within Pisum and Vicia germplasm.
doi:10.1186/1471-2164-13-104
PMCID: PMC3352077  PMID: 22433453
18.  Annual Medicago: From a Model Crop Challenged by a Spectrum of Necrotrophic Pathogens to a Model Plant to Explore the Nature of Disease Resistance 
Annals of Botany  2006;98(6):1117-1128.
• Background Annual Medicago spp., including M. truncatula, play an important agronomic role in dryland farming regions of the world where they are often an integral component of cropping systems, particularly in regions with a Mediterranean or Mediterranean-type climate where they grow as winter annuals that provide both nitrogen and disease breaks for rotational crops. Necrotrophic foliar and soil-borne pathogens dominate these regions and challenge the productivity of annual Medicago and crop legume species.
• Scope This review outlines some of the major and/or widespread diseases these necrotrophic pathogens cause on Medicago spp. It then explores the potential for using the spectrum of necrotrophic pathogen–host interactions, with annual Medicago as the host plant, to better understand and model pathosystems within the diseases caused by nectrotrophic pathogens across forage and grain legume crops.
• Conclusions Host resistance clearly offers the best strategy for cost-effective, long-term control of necrotrophic foliar and soil-borne pathogens, particularly as useful resistance to a number of these diseases has been identified. Recently and initially, the annual M. truncatula has emerged as a more appropriate and agronomically relevant substitute to Arabidopsis thaliana as a model plant for legumes, and is proving an excellent model to understand the mechanisms of resistance both to individual pathogens and more generally to most forage and grain legume necrotrophic pathogens.
doi:10.1093/aob/mcl132
PMCID: PMC3292268  PMID: 16803846
Fungal pathogens; medics; annual Medicago species; Medicago truncatula; Phoma medicaginis; Aphanomyces euteiches; Colletotrichum trifolii; Mycosphaerella pinodes; grain legumes
19.  Two euAGAMOUS Genes Control C-Function in Medicago truncatula 
PLoS ONE  2014;9(8):e103770.
C-function MADS-box transcription factors belong to the AGAMOUS (AG) lineage and specify both stamen and carpel identity and floral meristem determinacy. In core eudicots, the AG lineage is further divided into two branches, the euAG and PLE lineages. Functional analyses across flowering plants strongly support the idea that duplicated AG lineage genes have different degrees of subfunctionalization of the C-function. The legume Medicago truncatula contains three C-lineage genes in its genome: two euAG genes (MtAGa and MtAGb) and one PLENA-like gene (MtSHP). This species is therefore a good experimental system to study the effects of gene duplication within the AG subfamily. We have studied the respective functions of each euAG genes in M. truncatula employing expression analyses and reverse genetic approaches. Our results show that the M. truncatula euAG- and PLENA-like genes are an example of subfunctionalization as a result of a change in expression pattern. MtAGa and MtAGb are the only genes showing a full C-function activity, concomitant with their ancestral expression profile, early in the floral meristem, and in the third and fourth floral whorls during floral development. In contrast, MtSHP expression appears late during floral development suggesting it does not contribute significantly to the C-function. Furthermore, the redundant MtAGa and MtAGb paralogs have been retained which provides the overall dosage required to specify the C-function in M. truncatula.
doi:10.1371/journal.pone.0103770
PMCID: PMC4126672  PMID: 25105497
20.  Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds 
BMC Plant Biology  2010;10:183.
Background
Nitrogen is a crucial nutrient that is both essential and rate limiting for plant growth and seed production. Glutamine synthetase (GS), occupies a central position in nitrogen assimilation and recycling, justifying the extensive number of studies that have been dedicated to this enzyme from several plant sources. All plants species studied to date have been reported as containing a single, nuclear gene encoding a plastid located GS isoenzyme per haploid genome. This study reports the existence of a second nuclear gene encoding a plastid located GS in Medicago truncatula.
Results
This study characterizes a new, second gene encoding a plastid located glutamine synthetase (GS2) in M. truncatula. The gene encodes a functional GS isoenzyme with unique kinetic properties, which is exclusively expressed in developing seeds. Based on molecular data and the assumption of a molecular clock, it is estimated that the gene arose from a duplication event that occurred about 10 My ago, after legume speciation and that duplicated sequences are also present in closely related species of the Vicioide subclade. Expression analysis by RT-PCR and western blot indicate that the gene is exclusively expressed in developing seeds and its expression is related to seed filling, suggesting a specific function of the enzyme associated to legume seed metabolism. Interestingly, the gene was found to be subjected to alternative splicing over the first intron, leading to the formation of two transcripts with similar open reading frames but varying 5' UTR lengths, due to retention of the first intron. To our knowledge, this is the first report of alternative splicing on a plant GS gene.
Conclusions
This study shows that Medicago truncatula contains an additional GS gene encoding a plastid located isoenzyme, which is functional and exclusively expressed during seed development. Legumes produce protein-rich seeds requiring high amounts of nitrogen, we postulate that this gene duplication represents a functional innovation of plastid located GS related to storage protein accumulation exclusive to legume seed metabolism.
doi:10.1186/1471-2229-10-183
PMCID: PMC3095313  PMID: 20723225
21.  Transcriptional analysis of highly syntenic regions between Medicago truncatula and Glycine max using tiling microarrays 
Genome Biology  2008;9(3):R57.
The comparative transcriptional analysis of highly syntenic regions in six different organ types between Medicago truncatula (barrel medic) and Glycine max (soybean), using nucleotide tiling microarrays, provides insights into genome organization and transcriptional regulation in these legume plants.
Background
Legumes are the third largest family of flowering plants and are unique among crop species in their ability to fix atmospheric nitrogen. As a result of recent genome sequencing efforts, legumes are now one of a few plant families with extensive genomic and transcriptomic data available in multiple species. The unprecedented complexity and impending completeness of these data create opportunities for new approaches to discovery.
Results
We report here a transcriptional analysis in six different organ types of syntenic regions totaling approximately 1 Mb between the legume plants barrel medic (Medicago truncatula) and soybean (Glycine max) using oligonucleotide tiling microarrays. This analysis detected transcription of over 80% of the predicted genes in both species. We also identified 499 and 660 transcriptionally active regions from barrel medic and soybean, respectively, over half of which locate outside of the predicted exons. We used the tiling array data to detect differential gene expression in the six examined organ types and found several genes that are preferentially expressed in the nodule. Further investigation revealed that some collinear genes exhibit different expression patterns between the two species.
Conclusion
These results demonstrate the utility of genome tiling microarrays in generating transcriptomic data to complement computational annotation of the newly available legume genome sequences. The tiling microarray data was further used to quantify gene expression levels in multiple organ types of two related legume species. Further development of this method should provide a new approach to comparative genomics aimed at elucidating genome organization and transcriptional regulation.
doi:10.1186/gb-2008-9-3-r57
PMCID: PMC2397509  PMID: 18348734
22.  The molecular genetic linkage map of the model legume Medicago truncatula: an essential tool for comparative legume genomics and the isolation of agronomically important genes 
BMC Plant Biology  2002;2:1.
Background
The legume Medicago truncatula has emerged as a model plant for the molecular and genetic dissection of various plant processes involved in rhizobial, mycorrhizal and pathogenic plant-microbe interactions. Aiming to develop essential tools for such genetic approaches, we have established the first genetic map of this species. Two parental homozygous lines were selected from the cultivar Jemalong and from the Algerian natural population (DZA315) on the basis of their molecular and phenotypic polymorphism.
Results
An F2 segregating population of 124 individuals between these two lines was obtained using an efficient manual crossing technique established for M. truncatula and was used to construct a genetic map. This map spans 1225 cM (average 470 kb/cM) and comprises 289 markers including RAPD, AFLP, known genes and isoenzymes arranged in 8 linkage groups (2n = 16). Markers are uniformly distributed throughout the map and segregation distortion is limited to only 3 linkage groups. By mapping a number of common markers, the eight linkage groups are shown to be homologous to those of diploid alfalfa (M. sativa), implying a good level of macrosynteny between the two genomes. Using this M. truncatula map and the derived F3 populations, we were able to map the Mtsym6 symbiotic gene on linkage group 8 and the SPC gene, responsible for the direction of pod coiling, on linkage group 7.
Conclusions
These results demonstrate that Medicago truncatula is amenable to diploid genetic analysis and they open the way to map-based cloning of symbiotic or other agronomically-important genes using this model plant.
doi:10.1186/1471-2229-2-1
PMCID: PMC65051  PMID: 11825338
23.  An expression database for roots of the model legume Medicago truncatula under salt stress 
BMC Genomics  2009;10:517.
Background
Medicago truncatula is a model legume whose genome is currently being sequenced by an international consortium. Abiotic stresses such as salt stress limit plant growth and crop productivity, including those of legumes. We anticipate that studies on M. truncatula will shed light on other economically important legumes across the world. Here, we report the development of a database called MtED that contains gene expression profiles of the roots of M. truncatula based on time-course salt stress experiments using the Affymetrix Medicago GeneChip. Our hope is that MtED will provide information to assist in improving abiotic stress resistance in legumes.
Description
The results of our microarray experiment with roots of M. truncatula under 180 mM sodium chloride were deposited in the MtED database. Additionally, sequence and annotation information regarding microarray probe sets were included. MtED provides functional category analysis based on Gene and GeneBins Ontology, and other Web-based tools for querying and retrieving query results, browsing pathways and transcription factor families, showing metabolic maps, and comparing and visualizing expression profiles. Utilities like mapping probe sets to genome of M. truncatula and In-Silico PCR were implemented by BLAT software suite, which were also available through MtED database.
Conclusion
MtED was built in the PHP script language and as a MySQL relational database system on a Linux server. It has an integrated Web interface, which facilitates ready examination and interpretation of the results of microarray experiments. It is intended to help in selecting gene markers to improve abiotic stress resistance in legumes. MtED is available at http://bioinformatics.cau.edu.cn/MtED/.
doi:10.1186/1471-2164-10-517
PMCID: PMC2779821  PMID: 19906315
24.  Evolutionary history of mitogen-activated protein kinase (MAPK) genes in Lotus, Medicago, and Phaseolus 
Plant Signaling & Behavior  2013;8(11):e27189.
Mitogen-Activated Protein Kinase (MAPK) genes encode proteins that mediate various signaling pathways associated with biotic and abiotic stress responses in eukaryotes. The MAPK genes form a 3-tier signal transduction cascade between cellular stimuli and physiological responses. Recent identification of soybean MAPKs and availability of genome sequences from other legume species allowed us to identify their MAPK genes. The main objectives of this study were to identify MAPKs in 3 legume species, Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, and to assess their phylogenetic relationships. We used approaches in comparative genomics for MAPK gene identification and named the newly identified genes following Arabidopsis MAPK nomenclature model. We identified 19, 18, and 15 MAPKs and 7, 4, and 9 MAPKKs in the genome of Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, respectively. Within clade placement of MAPKs and MAPKKs in the 3 legume species were consistent with those in soybean and Arabidopsis. Among 5 clades of MAPKs, 4 founder clades were consistent to MAPKs of other plant species and orthologs of MAPK genes in the fifth clade-"Clade E" were consistent with those in soybean. Our results also indicated that some gene duplication events might have occurred prior to eudicot-monocot divergence. Highly diversified MAPKs in soybean relative to those in 3 other legume species are attributable to the polyploidization events in soybean. The identification of the MAPK genes in the legume species is important for the legume crop improvement; and evolutionary relationships and functional divergence of these gene members provide insights into plant genome evolution.
doi:10.4161/psb.27189
PMCID: PMC4091376  PMID: 24317362
Glycine max; Lotus japonicus; Medicago truncatula; Phaseolus vulgaris; Legume genomics; MAPK; MAPK evolution; MAPKK; functional divergence; gene duplication
25.  Auxin efflux transporter MtPIN10 regulates compound leaf and flower development in Medicago truncatula 
Plant Signaling & Behavior  2011;6(10):1537-1544.
Plant diversity in nature is to a large extent reflected by morphological diversity of their leaves. Both simple and dissected (with multiple blades or leaflets) leaves are initiated from shoot apical meristem (SAM) in a highly ordered fashion. Similarly, development of leaflets from leaf marginal meristem (marginal blastozone) is also highly ordered. How morphological diversity of plant leaves is regulated remains an important topic of studies on plant form evolution. Here, we describe isolation and characterization of loss-of-function mutants of auxin efflux transporter MtPIN10 of a legume species, Medicago truncatula. Mtpin10 mutants exhibit defects in diverse developmental processes including leaf and leaflet development. Cross species genetic complementation demonstrates that MtPIN10 and Arabidopsis PIN1 are functional orthologs. Double mutant analyses reveal complex genetic interactions between MtPIN10 and Medicago SINGLE LEAFLET1 (SGL1) and CUP-SHAPED COTYLEDON2 (MtCUC2), three regulatory genes involved in developmental processes including dissected leaf and flower development.
doi:10.4161/psb.6.10.17326
PMCID: PMC3256382  PMID: 21900740
auxin; auxin transport; compound leaf development; MtPIN10; SGL1; MtCUC2; Medicago truncatula

Results 1-25 (1153917)