Dataset Information

Whole-genome nucleotide diversity, recombination, and linkage disequilibrium in the model legume Medicago truncatula.

ABSTRACT: Medicago truncatula is a model for investigating legume genetics, including the genetics and evolution of legume-rhizobia symbiosis. We used whole-genome sequence data to identify and characterize sequence polymorphisms and linkage disequilibrium (LD) in a diverse collection of 26 M. truncatula accessions. Our analyses reveal that M. truncatula harbors both higher diversity and less LD than soybean (Glycine max) and exhibits patterns of LD and recombination similar to Arabidopsis thaliana. The population-scaled recombination rate is approximately one-third of the mutation rate, consistent with expectations for a species with a high selfing rate. Linkage disequilibrium, however, is not extensive, and therefore, the low recombination rate is likely not a major constraint to adaptation. Nucleotide diversity in 100-kb windows was negatively correlated with gene density, which is expected if diversity is shaped by selection acting against slightly deleterious mutations. Among putative coding regions, members of four gene families harbor significantly higher diversity than the genome-wide average. Three of these families are involved in resistance against pathogens; one of these families, the nodule-specific, cysteine-rich gene family, is specific to the galegoid legumes and is involved in control of rhizobial differentiation. The more than 3 million SNPs that we detected, approximately one-half of which are present in more than one accession, are a valuable resource for genome-wide association mapping of genes responsible for phenotypic diversity in legumes, especially traits associated with symbiosis and nodulation.

SUBMITTER: Branca A

PROVIDER: S-EPMC3198318 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Whole-genome nucleotide diversity, recombination, and linkage disequilibrium in the model legume Medicago truncatula.

Branca Antoine A Paape Timothy D TD Zhou Peng P Briskine Roman R Farmer Andrew D AD Mudge Joann J Bharti Arvind K AK Woodward Jimmy E JE May Gregory D GD Gentzbittel Laurent L Ben Cécile C Denny Roxanne R Sadowsky Michael J MJ Ronfort Joëlle J Bataillon Thomas T Young Nevin D ND Tiffin Peter P

Proceedings of the National Academy of Sciences of the United States of America 20110926 42

Medicago truncatula is a model for investigating legume genetics, including the genetics and evolution of legume-rhizobia symbiosis. We used whole-genome sequence data to identify and characterize sequence polymorphisms and linkage disequilibrium (LD) in a diverse collection of 26 M. truncatula accessions. Our analyses reveal that M. truncatula harbors both higher diversity and less LD than soybean (Glycine max) and exhibits patterns of LD and recombination similar to Arabidopsis thaliana. The p ...[more]

PMID: 21949378

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Project description:BACKGROUND: Medicago truncatula, a close relative of alfalfa, is a preeminent model for studying nitrogen fixation, symbiosis, and legume genomics. The Medicago sequencing project began in 2003 with the goal to decipher sequences originated from the euchromatic portion of the genome. The initial sequencing approach was based on a BAC tiling path, culminating in a BAC-based assembly (Mt3.5) as well as an in-depth analysis of the genome published in 2011. RESULTS: Here we describe a further improved and refined version of the M. truncatula genome (Mt4.0) based on de novo whole genome shotgun assembly of a majority of Illumina and 454 reads using ALLPATHS-LG. The ALLPATHS-LG scaffolds were anchored onto the pseudomolecules on the basis of alignments to both the optical map and the genotyping-by-sequencing (GBS) map. The Mt4.0 pseudomolecules encompass ~360 Mb of actual sequences spanning 390 Mb of which ~330 Mb align perfectly with the optical map, presenting a drastic improvement over the BAC-based Mt3.5 which only contained 70% sequences (~250 Mb) of the current version. Most of the sequences and genes that previously resided on the unanchored portion of Mt3.5 have now been incorporated into the Mt4.0 pseudomolecules, with the exception of ~28 Mb of unplaced sequences. With regard to gene annotation, the genome has been re-annotated through our gene prediction pipeline, which integrates EST, RNA-seq, protein and gene prediction evidences. A total of 50,894 genes (31,661 high confidence and 19,233 low confidence) are included in Mt4.0 which overlapped with ~82% of the gene loci annotated in Mt3.5. Of the remaining genes, 14% of the Mt3.5 genes have been deprecated to an "unsupported" status and 4% are absent from the Mt4.0 predictions. CONCLUSIONS: Mt4.0 and its associated resources, such as genome browsers, BLAST-able datasets and gene information pages, can be found on the JCVI Medicago web site (http://www.jcvi.org/medicago). The assembly and annotation has been deposited in GenBank (BioProject: PRJNA10791). The heavily curated chromosomal sequences and associated gene models of Medicago will serve as a better reference for legume biology and comparative genomics.

Project description:One of the major issues in healthcare today is antibiotic resistance. Antimicrobial peptides (AMPs), a subclass of host defense peptides, have been suggested as a viable solution for the multidrug resistance problem. Legume plants express more than 700 nodule-specific cysteine-rich (NCR) peptides. Three NCR peptides (NCR094, NCR888, and NCR992) were predicted to have antimicrobial activity using in silico AMP prediction programs. This study focused on investigating the roles of the NCRs in antimicrobial activity and antibiofilm activity, followed by in vitro toxicity profiling. Different variants were synthesized, i.e., mutated and truncated derivatives. The effect on the growth of Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA) was monitored post-treatment, and survived cells were counted using an in vitro and ex vivo killing assay. The antibiofilm assay was conducted using subinhibitory concentrations of the NCRs and monitoring K. pneumoniae biomass, followed by crystal violet staining. The cytotoxicity profile was evaluated using erythrocyte hemolysis and leukemia (K562) cell line toxicity assays. Out of the NCRs, NCR094 and NCR992 displayed mainly in vitro and ex vivo bactericidal activity on K. pneumoniae. NCR094 wild type (WT) and NCR992 eradicated K. pneumoniae at different potency; NCR094 and NCR992 killed K. pneumoniae completely at 25 and 50 µM, respectively. However, both peptides in the wild type showed negligible bactericidal effect on MRSA in vitro and ex vivo. NCR094 and its derivatives relatively retained the antimicrobial activity on K. pneumoniae in vitro and ex vivo. NCR992 WT lost its antimicrobial activity on K. pneumoniae ex vivo, yet the different truncated and mutated variants retained some of the antimicrobial role ex vivo. All the different variants of NCR094 had no effect on MRSA in vitro and ex vivo. Similarly, NCR992's variants had a negligible bactericidal role on MRSA in vitro, yet the truncated variants had a significantly high bactericidal effect on MRSA ex vivo. NCR094.3 (cystine replacement variant) and NCR992.1 displayed significant antibiofilm activity more than 90%. NCR992.3 and NCR992.2 displayed more than 50% of antibiofilm activity. All the NCR094 forms had no toxicity, except NCR094.1 (49.38%, SD ± 3.46) and all NCR992 forms (63%-93%), which were above the cutoff (20%). Only NCR992.2 showed low toxicity on K562 (24.8%, SD ± 3.40), yet above the 20% cutoff. This study provided preliminary antimicrobial and safety data for the potential use of these peptides for therapeutical applications.IMPORTANCEThe discovery of new antibiotics is urgently needed, given the global expansion of antibiotic-resistant bacteria and the rising mortality rate. One of the initial lines of defense against microbial infections is antimicrobial peptides (AMPs). Plants can express hundreds of such AMPs as defensins and defensin-like peptides. The nodule-specific cysteine-rich (NCR) peptides are a class of defensin-like peptides that have evolved in rhizobial-legume symbioses. This study screened the antimicrobial activity of a subset of NCR sequences using online computational AMP prediction algorithms. Two novel NCRs, NCR094 and NCR992, with different variants were identified to exhibit antimicrobial activity with various potency on two problematic pathogens, K. pneumoniae and MRSA, using in vitro and ex vivo killing assays. Yet, one variant, NCR094.3, had no toxicity toward human cells and displayed antibiofilm activity, which make it a promising lead for antimicrobial drug development.

Project description:BACKGROUND: Medicago truncatula Gaertn. (barrel medic) is cultivated as a pasture legume for its high protein content and ability to improve soils through nitrogen fixation. Toxic concentrations of the micronutrient Boron (B) in agricultural soils hamper the production of cereal and leguminous crops. In cereals, the genetic analysis of B tolerance has led to the development of molecular selection tools to introgress and maintain the B tolerance trait in breeding lines. There is a comparable need for selection tools in legumes that grow on these toxic soils, often in rotation with cereals. RESULTS: Genetic variation for B tolerance in Medicago truncatula was utilised to generate two F2 populations from crosses between tolerant and intolerant parents. Phenotyping under B stress revealed a close correlation between B tolerance and biomass production and a segregation ratio explained by a single dominant locus. M. truncatula homologues of the Arabidopsis major intrinsic protein (MIP) gene AtNIP5;1 and the efflux-type transporter gene AtBOR1, both known for B transport, were identified and nearby molecular markers screened across F2 lines to verify linkage with the B-tolerant phenotype. Most (95%) of the phenotypic variation could be explained by the SSR markers h2_6e22a and h2_21b19a, which flank a cluster of five predicted MIP genes on chromosome 4. Three CAPS markers (MtBtol-1,-2,-3) were developed to dissect the region further. Expression analysis of the five predicted MIPs indicated that only MtNIP3 was expressed when leaf tissue and roots were assessed. MtNIP3 showed low and equal expression in the roots of tolerant and intolerant lines but a 4-fold higher expression level in the leaves of B-tolerant cultivars. The expression profile correlates closely with the B concentration measured in the leaves and roots of tolerant and intolerant plants. Whereas no significant difference in B concentration exists between roots of tolerant and intolerant plants, the B concentration in the leaves of tolerant plants is less than half that of intolerant plants, which further supports MtNIP3 as the best candidate for the tolerance trait-defining gene in Medicago truncatula. CONCLUSION: The close linkage of the MtNIP3 locus to B toxicity tolerance provides a source of molecular selection tools to pasture breeding programs. The economical importance of the locus warrants further investigation of the individual members of the MIP gene cluster in other pasture and in grain legumes.

Dataset Information

Whole-genome nucleotide diversity, recombination, and linkage disequilibrium in the model legume Medicago truncatula.

Publications

Whole-genome nucleotide diversity, recombination, and linkage disequilibrium in the model legume Medicago truncatula.

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