Unraveling coevolutionary dynamics using ecological genomics.

Coevolutionary interactions, from the delicate co-dependency in mutualistic interactions to the antagonistic relationship of hosts and parasites, are a ubiquitous driver of adaptation. Surprisingly, little is known about the genomic processes underlying coevolution in an ecological context. However, species comprise genetically differentiated populations that interact with temporally variable abiotic and biotic environments. We discuss the recent advances in coevolutionary theory and genomics as well as shortcomings, to identify coevolving genes that take into account this spatial and temporal variability of coevolution, and propose a practical guide to understand the dynamic of coevolution using an ecological genomics lens.

A combination of conserved and diverged responses underlies Theobroma cacao's defense response to Phytophthora palmivora.

BACKGROUND: Plants have complex and dynamic immune systems that have evolved to resist pathogens. Humans have worked to enhance these defenses in crops through breeding. However, many crops harbor only a fraction of the genetic diversity present in wild relatives. Increased utilization of diverse germplasm to search for desirable traits, such as disease resistance, is therefore a valuable step towards breeding crops that are adapted to both current and emerging threats. Here, we examine diversity of defense responses across four populations of the long-generation tree crop Theobroma cacao L., as well as four non-cacao Theobroma species, with the goal of identifying genetic elements essential for protection against the oomycete pathogen Phytophthora palmivora. RESULTS: We began by creating a new, highly contiguous genome assembly for the P. palmivora-resistant genotype SCA 6 (Additional file 1: Tables S1-S5), deposited in GenBank under accessions CP139290-CP139299. We then used this high-quality assembly to combine RNA and whole-genome sequencing data to discover several genes and pathways associated with resistance. Many of these are unique, i.e., differentially regulated in only one of the four populations (diverged 40 k-900 k generations). Among the pathways shared across all populations is phenylpropanoid biosynthesis, a metabolic pathway with well-documented roles in plant defense. One gene in this pathway, caffeoyl shikimate esterase (CSE), was upregulated across all four populations following pathogen treatment, indicating its broad importance for cacao's defense response. Further experimental evidence suggests this gene hydrolyzes caffeoyl shikimate to create caffeic acid, an antimicrobial compound and known inhibitor of Phytophthora spp. CONCLUSIONS: Our results indicate most expression variation associated with resistance is unique to populations. Moreover, our findings demonstrate the value of using a broad sample of evolutionarily diverged populations for revealing the genetic bases of cacao resistance to P. palmivora. This approach has promise for further revealing and harnessing valuable genetic resources in this and other long-generation plants.

Horizontal gene transfer of the Mer operon is associated with large effects on the transcriptome and increased tolerance to mercury in nitrogen-fixing bacteria.

BACKGROUND: Mercury (Hg) is highly toxic and has the potential to cause severe health problems for humans and foraging animals when transported into edible plant parts. Soil rhizobia that form symbiosis with legumes may possess mechanisms to prevent heavy metal translocation from roots to shoots in plants by exporting metals from nodules or compartmentalizing metal ions inside nodules. Horizontal gene transfer has potential to confer immediate de novo adaptations to stress. We used comparative genomics of high quality de novo assemblies to identify structural differences in the genomes of nitrogen-fixing rhizobia that were isolated from a mercury (Hg) mine site that show high variation in their tolerance to Hg. RESULTS: Our analyses identified multiple structurally conserved merA homologs in the genomes of Sinorhizobium medicae and Rhizobium leguminosarum but only the strains that possessed a Mer operon exhibited 10-fold increased tolerance to Hg. RNAseq analysis revealed nearly all genes in the Mer operon were significantly up-regulated in response to Hg stress in free-living conditions and in nodules. In both free-living and nodule environments, we found the Hg-tolerant strains with a Mer operon exhibited the fewest number of differentially expressed genes (DEGs) in the genome, indicating a rapid and efficient detoxification of Hg from the cells that reduced general stress responses to the Hg-treatment. Expression changes in S. medicae while in bacteroids showed that both rhizobia strain and host-plant tolerance affected the number of DEGs. Aside from Mer operon genes, nif genes which are involved in nitrogenase activity in S. medicae showed significant up-regulation in the most Hg-tolerant strain while inside the most Hg-accumulating host-plant. Transfer of a plasmid containing the Mer operon from the most tolerant strain to low-tolerant strains resulted in an immediate increase in Hg tolerance, indicating that the Mer operon is able to confer hyper tolerance to Hg. CONCLUSIONS: Mer operons have not been previously reported in nitrogen-fixing rhizobia. This study demonstrates a pivotal role of the Mer operon in effective mercury detoxification and hypertolerance in nitrogen-fixing rhizobia. This finding has major implications not only for soil bioremediation, but also host plants growing in mercury contaminated soils.

Genomic structural variants constrain and facilitate adaptation in natural populations of Theobroma cacao, the chocolate tree.

Genomic structural variants (SVs) can play important roles in adaptation and speciation. Yet the overall fitness effects of SVs are poorly understood, partly because accurate population-level identification of SVs requires multiple high-quality genome assemblies. Here, we use 31 chromosome-scale, haplotype-resolved genome assemblies of Theobroma cacao-an outcrossing, long-lived tree species that is the source of chocolate-to investigate the fitness consequences of SVs in natural populations. Among the 31 accessions, we find over 160,000 SVs, which together cover eight times more of the genome than single-nucleotide polymorphisms and short indels (125 versus 15 Mb). Our results indicate that a vast majority of these SVs are deleterious: they segregate at low frequencies and are depleted from functional regions of the genome. We show that SVs influence gene expression, which likely impairs gene function and contributes to the detrimental effects of SVs. We also provide empirical support for a theoretical prediction that SVs, particularly inversions, increase genetic load through the accumulation of deleterious nucleotide variants as a result of suppressed recombination. Despite the overall detrimental effects, we identify individual SVs bearing signatures of local adaptation, several of which are associated with genes differentially expressed between populations. Genes involved in pathogen resistance are strongly enriched among these candidates, highlighting the contribution of SVs to this important local adaptation trait. Beyond revealing empirical evidence for the evolutionary importance of SVs, these 31 de novo assemblies provide a valuable resource for genetic and breeding studies in Tcacao.

Discordant population structure among rhizobium divided genomes and their legume hosts.

Symbiosis often occurs between partners with distinct life history characteristics and dispersal mechanisms. Many bacterial symbionts have genomes comprising multiple replicons with distinct rates of evolution and horizontal transmission. Such differences might drive differences in population structure between hosts and symbionts and among the elements of the divided genomes of bacterial symbionts. These differences might, in turn, shape the evolution of symbiotic interactions and bacterial evolution. Here we use whole genome resequencing of a hierarchically structured sample of 191 strains of Sinorhizobium meliloti collected from 21 locations in southern Europe to characterize population structures of this bacterial symbiont, which forms a root nodule symbiosis with the host plant Medicago truncatula. S. meliloti genomes showed high local (within-site) variation and little isolation by distance. This was particularly true for the two symbiosis elements, pSymA and pSymB, which have population structures that are similar to each other, but distinct from both the bacterial chromosome and the host plant. Given limited recombination on the chromosome, compared to the symbiosis elements, distinct population structures may result from differences in effective gene flow. Alternatively, positive or purifying selection, with little recombination, may explain distinct geographical patterns at the chromosome. Discordant population structure between hosts and symbionts indicates that geographically and genetically distinct host populations in different parts of the range might interact with genetically similar symbionts, potentially minimizing local specialization.

Combining GWAS and population genomic analyses to characterize coevolution in a legume-rhizobia symbiosis.

The mutualism between legumes and rhizobia is clearly the product of past coevolution. However, the nature of ongoing evolution between these partners is less clear. To characterize the nature of recent coevolution between legumes and rhizobia, we used population genomic analysis to characterize selection on functionally annotated symbiosis genes as well as on symbiosis gene candidates identified through a two-species association analysis. For the association analysis, we inoculated each of 202 accessions of the legume host Medicago truncatula with a community of 88 Sinorhizobia (Ensifer) meliloti strains. Multistrain inoculation, which better reflects the ecological reality of rhizobial selection in nature than single-strain inoculation, allows strains to compete for nodulation opportunities and host resources and for hosts to preferentially form nodules and provide resources to some strains. We found extensive host by symbiont, that is, genotype-by-genotype, effects on rhizobial fitness and some annotated rhizobial genes bear signatures of recent positive selection. However, neither genes responsible for this variation nor annotated host symbiosis genes are enriched for signatures of either positive or balancing selection. This result suggests that stabilizing selection dominates selection acting on symbiotic traits and that variation in these traits is under mutation-selection balance. Consistent with the lack of positive selection acting on host genes, we found that among-host variation in growth was similar whether plants were grown with rhizobia or N-fertilizer, suggesting that the symbiosis may not be a major driver of variation in plant growth in multistrain contexts.

Pleiotropy facilitates local adaptation to distant optima in common ragweed (Ambrosia artemisiifolia).

Pleiotropy, the control of multiple phenotypes by a single locus, is expected to slow the rate of adaptation by increasing the chance that beneficial alleles also have deleterious effects. However, a prediction arising from classical theory of quantitative trait evolution states that pleiotropic alleles may have a selective advantage when phenotypes are distant from their selective optima. We examine the role of pleiotropy in regulating adaptive differentiation among populations of common ragweed (Ambrosia artemisiifolia); a species that has recently expanded its North American range due to human-mediated habitat change. We employ a phenotype-free approach by using connectivity in gene networks as a proxy for pleiotropy. First, we identify loci bearing footprints of local adaptation, and then use genotype-expression mapping and co-expression networks to infer the connectivity of the genes. Our results indicate that the putatively adaptive loci are highly pleiotropic, as they are more likely than expected to affect the expression of other genes, and they reside in central positions within the gene networks. We propose that the conditionally advantageous alleles at these loci avoid the cost of pleiotropy by having large phenotypic effects that are beneficial when populations are far from their selective optima. We further use evolutionary simulations to show that these patterns are in agreement with a model where populations face novel selective pressures, as expected during a range expansion. Overall, our results suggest that highly connected genes may be targets of positive selection during environmental change, even though they likely experience strong purifying selection in stable selective environments.

Host-Associated Rhizobial Fitness: Dependence on Nitrogen, Density, Community Complexity, and Legume Genotype.

The environmental context of the nitrogen-fixing mutualism between leguminous plants and rhizobial bacteria varies over space and time. Variation in resource availability, population density, and composition likely affect the ecology and evolution of rhizobia and their symbiotic interactions with hosts. We examined how host genotype, nitrogen addition, rhizobial density, and community complexity affected selection on 68 rhizobial strains in the Sinorhizobium meliloti-Medicago truncatula mutualism. As expected, host genotype had a substantial effect on the size, number, and strain composition of root nodules (the symbiotic organ). The understudied environmental variable of rhizobial density had a stronger effect on nodule strain frequency than the addition of low nitrogen levels. Higher inoculum density resulted in a nodule community that was less diverse and more beneficial but only in the context of the more selective host genotype. Higher density resulted in more diverse and less beneficial nodule communities with the less selective host. Density effects on strain composition deserve additional scrutiny as they can create feedback between ecological and evolutionary processes. Finally, we found that relative strain rankings were stable across increasing community complexity (2, 3, 8, or 68 strains). This unexpected result suggests that higher-order interactions between strains are rare in the context of nodule formation and development. Our work highlights the importance of examining mechanisms of density-dependent strain fitness and developing theoretical predictions that incorporate density dependence. Furthermore, our results have translational relevance for overcoming establishment barriers in bioinoculants and motivating breeding programs that maintain beneficial plant-microbe interactions across diverse agroecological contexts. IMPORTANCE Legume crops establish beneficial associations with rhizobial bacteria that perform biological nitrogen fixation, providing nitrogen to plants without the economic and greenhouse gas emission costs of chemical nitrogen inputs. Here, we examine the influence of three environmental factors that vary in agricultural fields on strain relative fitness in nodules. In addition to manipulating nitrogen, we also use two biotic variables that have rarely been examined: the rhizobial community's density and complexity. Taken together, our results suggest that (i) breeding legume varieties that select beneficial strains despite environmental variation is possible, (ii) changes in rhizobial population densities that occur routinely in agricultural fields could drive evolutionary changes in rhizobial populations, and (iii) the lack of higher-order interactions between strains will allow the high-throughput assessments of rhizobia winners and losers during plant interactions.

Gene Expression Modularity Reveals Footprints of Polygenic Adaptation in Theobroma cacao.

Separating footprints of adaptation from demography is challenging. When selection has acted on a single locus with major effect, this issue can be alleviated through signatures left by selective sweeps. However, as adaptation is often driven by small allele frequency shifts at many loci, studies focusing on single genes are able to identify only a small portion of genomic variants responsible for adaptation. In face of this challenge, we utilize coexpression information to search for signals of polygenetic adaptation in Theobroma cacao, a tropical tree species that is the source of chocolate. Using transcriptomics and a weighted correlation network analysis, we group genes with similar expression patterns into functional modules. We then ask whether modules enriched for specific biological processes exhibit cumulative effects of differential selection in the form of high FST and dXY between populations. Indeed, modules putatively involved in protein modification, flowering, and water transport show signs of polygenic adaptation even though individual genes that are members of those groups do not bear strong signatures of selection. Modeling of demography, background selection, and the effects of genomic features reveal that these patterns are unlikely to arise by chance. We also find that specific modules are enriched for signals of strong or relaxed purifying selection, with one module bearing signs of adaptive differentiation and an excess of deleterious mutations. Our results provide insight into polygenic adaptation and contribute to understanding of population structure, demographic history, and genome evolution in T. cacao.

Comparative genomics reveals high rates of horizontal transfer and strong purifying selection on rhizobial symbiosis genes.

Horizontal transfer (HT) alters the repertoire of symbiosis genes in rhizobial genomes and may play an important role in the on-going evolution of the rhizobia-legume symbiosis. To gain insight into the extent of HT of symbiosis genes with different functional roles (nodulation, N-fixation, host benefit and rhizobial fitness), we conducted comparative genomic and selection analyses of the full-genome sequences from 27 rhizobial genomes. We find that symbiosis genes experience high rates of HT among rhizobial lineages but also bear signatures of purifying selection (low Ka : Ks). HT and purifying selection appear to be particularly strong in genes involved in initiating the symbiosis (e.g. nodulation) and in genome-wide association candidates for mediating benefits provided to the host. These patterns are consistent with rhizobia adapting to the host environment through the loss and gain of symbiosis genes, but not with host-imposed positive selection driving divergence of symbiosis genes through recurring bouts of positive selection.

Individual-based eco-evolutionary models for understanding adaptation in changing seas.

As climate change threatens species' persistence, predicting the potential for species to adapt to rapidly changing environments is imperative for the development of effective conservation strategies. Eco-evolutionary individual-based models (IBMs) can be useful tools for achieving this objective. We performed a literature review to identify studies that apply these tools in marine systems. Our survey suggested that this is an emerging area of research fuelled in part by developments in modelling frameworks that allow simulation of increasingly complex ecological, genetic and demographic processes. The studies we identified illustrate the promise of this approach and advance our understanding of the capacity for adaptation to outpace climate change. These studies also identify limitations of current models and opportunities for further development. We discuss three main topics that emerged across studies: (i) effects of genetic architecture and non-genetic responses on adaptive potential; (ii) capacity for gene flow to facilitate rapid adaptation; and (iii) impacts of multiple stressors on persistence. Finally, we demonstrate the approach using simple simulations and provide a framework for users to explore eco-evolutionary IBMs as tools for understanding adaptation in changing seas.

A Select and Resequence Approach Reveals Strain-Specific Effects of Medicago Nodule-Specific PLAT-Domain Genes.

Genetic studies of legume symbiosis with nitrogen-fixing rhizobial bacteria have traditionally focused on nodule and nitrogen-fixation phenotypes when hosts are inoculated with a single rhizobial strain. These approaches overlook the potential effect of host genes on rhizobial fitness (i.e. how many rhizobia are released from host nodules) and strain-specific effects of host genes (i.e. genome × genome interactions). Using Medicago truncatula mutants in the recently described nodule-specific PLAT domain (NPD) gene family, we show how inoculating plants with a mixed inoculum of 68 rhizobial strains (Ensifer meliloti) via a select-and-resequence approach can be used to efficiently assay host mutants for strain-specific effects of late-acting host genes on interacting bacteria. The deletion of a single NPD gene (npd2) or all five members of the NPD gene family (npd1-5) differentially altered the frequency of rhizobial strains in nodules even though npd2 mutants had no visible nodule morphology or N-fixation phenotype. Also, npd1-5 nodules were less diverse and had larger populations of colony-forming rhizobia despite their smaller size. Lastly, NPD mutations disrupt a positive correlation between strain fitness and wild-type host biomass. These changes indicate that the effects of NPD proteins are strain dependent and that NPD family members are not redundant with regard to their effects on rhizobial strains. Association analyses of the rhizobial strains in the mixed inoculation indicate that rhizobial genes involved in chromosome segregation, cell division, GABA metabolism, efflux systems, and stress tolerance play an important role in the strain-specific effects of NPD genes.

Select and resequence reveals relative fitness of bacteria in symbiotic and free-living environments.

Assays to accurately estimate relative fitness of bacteria growing in multistrain communities can advance our understanding of how selection shapes diversity within a lineage. Here, we present a variant of the "evolve and resequence" approach both to estimate relative fitness and to identify genetic variants responsible for fitness variation of symbiotic bacteria in free-living and host environments. We demonstrate the utility of this approach by characterizing selection by two plant hosts and in two free-living environments (sterilized soil and liquid media) acting on synthetic communities of the facultatively symbiotic bacterium Ensifer meliloti We find (i) selection that hosts exert on rhizobial communities depends on competition among strains, (ii) selection is stronger inside hosts than in either free-living environment, and (iii) a positive host-dependent relationship between relative strain fitness in multistrain communities and host benefits provided by strains in single-strain experiments. The greatest changes in allele frequencies in response to plant hosts are in genes associated with motility, regulation of nitrogen fixation, and host/rhizobia signaling. The approach we present provides a powerful complement to experimental evolution and forward genetic screens for characterizing selection in bacterial populations, identifying gene function, and surveying the functional importance of naturally occurring genomic variation.

Context Dependence of Local Adaptation to Abiotic and Biotic Environments: A Quantitative and Qualitative Synthesis.

Understanding how spatially variable selection shapes adaptation is an area of long-standing interest in evolutionary ecology. Recent meta-analyses have quantified the extent of local adaptation, but the relative importance of abiotic and biotic factors in driving population divergence remains poorly understood. To address this gap, we combined a quantitative meta-analysis and a qualitative metasynthesis to (1) quantify the magnitude of local adaptation to abiotic and biotic factors and (2) characterize major themes that influence the motivation and design of experiments that seek to test for local adaptation. Using local-foreign contrasts as a metric of local adaptation (or maladaptation), we found that local adaptation was greater in the presence than in the absence of a biotic interactor, especially for plants. We also found that biotic environments had stronger effects on fitness than abiotic environments when ignoring whether those environments were local versus foreign. Finally, biotic effects were stronger at low latitudes, and abiotic effects were stronger at high latitudes. Our qualitative analysis revealed that the lens through which local adaptation has been examined differs for abiotic and biotic factors. It also revealed biases in the design and implementation of experiments that make quantitative results challenging to interpret and provided directions for future research.

Climate change is predicted to disrupt patterns of local adaptation in wild and cultivated maize.

Climate change is one of the most important threats to biodiversity and crop sustainability. The impact of climate change is often evaluated on the basis of expected changes in species' geographical distributions. Genomic diversity, local adaptation, and migration are seldom integrated into future species projections. Here, we examine how climate change will impact populations of two wild relatives of maize, the teosintes Zea mays ssp. mexicana and Z. mays ssp. parviglumis. Despite high levels of genetic diversity within populations and widespread future habitat suitability, we predict that climate change will alter patterns of local adaptation and decrease migration probabilities in more than two-thirds of present-day teosinte populations. These alterations are geographically heterogeneous and suggest that the possible impacts of climate change will vary considerably among populations. The population-specific effects of climate change are also evident in maize landraces, suggesting that climate change may result in maize landraces becoming maladapted to the climates in which they are currently cultivated. The predicted alterations to habitat distribution, migration potential, and patterns of local adaptation in wild and cultivated maize raise a red flag for the future of populations. The heterogeneous nature of predicted populations' responses underscores that the selective impact of climate change may vary among populations and that this is affected by different processes, including past adaptation.

Sex-biased gene expression in flowers, but not leaves, reveals secondary sexual dimorphism in Populus balsamifera.

Because sexual dimorphism in plants is often less morphologically conspicuous than in animals, studies of sex-biased gene expression may provide a quantitative metric to better address their commonality, molecular pathways, consistency across tissues and taxa, and evolution. The presence of sex-biased gene expression in tissues other than the androecium or gynoecium, termed secondary sexual characters, suggests that these traits arose after the initial evolution of dioecy. Patterns of sequence evolution may provide evidence of positive selection that drove sexual specialization. We compared gene expression in male and female flowers and leaves of Populus balsamifera to assess the extent of sex-biased expression, and tested whether sex-biased genes exhibit elevated rates of protein evolution. Sex-biased expression was pervasive in floral tissue, but nearly absent in leaf tissue. Female-biased genes in flowers were associated with photosynthesis, whereas male-biased genes were associated with mitochondrial function. Sex-biased genes did not exhibit elevated rates of protein evolution, contrary to results from other studies in animals and plants. Our results suggest that the ecological and physiological constraints associated with the energetics of flowering, rather than sexual conflict, have probably shaped the differences in male and female gene expression in P. balsamifera.

Does adaptation to historical climate shape plant responses to future rainfall patterns? A rainfall manipulation experiment with common ragweed.

Climate change is affecting both the volume and distribution of precipitation, which in turn is expected to affect the growth and reproduction of plant populations. The near ubiquity of local adaptation suggests that adaptive differentiation may have important consequences for how populations are affected by and respond to changing precipitation. Here, we manipulated rainfall in a common garden to examine how differentiation among populations of common ragweed, Ambrosia artemisiifolia (Asteraceae) affects responses to water availability expected under climate change. We collected seeds from 26 populations along gradients of historical rainfall and used event-based rainout shelters and watering additions to simulate drier summer conditions and more extreme rainfall events, respectively. Ambrosia artemisiifolia had higher fitness on average under reduced rainfall, suggesting it may spread and become more abundant in areas projected to become hotter and drier during the summer months. We also found strong evidence for phenotypic and fitness clines across both latitude and longitude, and that phenological responses and fitness effects of altered rainfall depended on seed source or historical climate. The effect of rainfall treatment on female fitness was highest in western and mid longitudes, but there was little effect on eastern populations. Across latitude, the effect of rainfall treatment on male fitness was highest in southern populations. These phenology and fitness clines suggest that adaptive differentiation across the species' range has the potential to shape future responses of A. artemisiifolia populations to climate change, particularly altered patterns of rainfall.

Little plant, big city: a test of adaptation to urban environments in common ragweed (Ambrosia artemisiifolia).

A full understanding of how cities shape adaptation requires characterizing genetically-based phenotypic and fitness differences between urban and rural populations under field conditions. We used a reciprocal transplant experiment with the native plant common ragweed, (Ambrosia artemisiifolia), and found that urban and rural populations have diverged in flowering time, a trait that strongly affects fitness. Although urban populations flowered earlier than rural populations, plants growing in urban field sites flowered later than plants in rural field sites. This counter-gradient variation is consistent adaptive divergence between urban and rural populations. Also consistent with local adaptation, both urban and rural genotypes experienced stronger net selection in the foreign than in the local habitat, but this pattern was not significant for male fitness. Despite the evidence for local adaptation, rural populations had higher lifetime fitness at all sites, suggesting that selection has been stronger or more uniform in rural than urban populations. We also found that inter-population differences in both flowering time and fitness tended to be greater among urban than rural populations, which is consistent with greater drift or spatial variation in selection within urban environments. In summary, our results are consistent with adaptive divergence of urban and rural populations, but also suggest there may be greater environmental heterogeneity in urban environments which also affects evolution in urban landscapes.

Validating Genome-Wide Association Candidates Controlling Quantitative Variation in Nodulation.

Genome-wide association (GWA) studies offer the opportunity to identify genes that contribute to naturally occurring variation in quantitative traits. However, GWA relies exclusively on statistical association, so functional validation is necessary to make strong claims about gene function. We used a combination of gene-disruption platforms (Tnt1 retrotransposons, hairpin RNA-interference constructs, and CRISPR/Cas9 nucleases) together with randomized, well-replicated experiments to evaluate the function of genes that an earlier GWA study in Medicago truncatula had identified as candidates contributing to variation in the symbiosis between legumes and rhizobia. We evaluated ten candidate genes found in six clusters of strongly associated single nucleotide polymorphisms, selected on the basis of their strength of statistical association, proximity to annotated gene models, and root or nodule expression. We found statistically significant effects on nodule production for three candidate genes, each validated in two independent mutants. Annotated functions of these three genes suggest their contributions to quantitative variation in nodule production occur through processes not previously connected to nodulation, including phosphorous supply and salicylic acid-related defense response. These results demonstrate the utility of GWA combined with reverse mutagenesis technologies to discover and validate genes contributing to naturally occurring variation in quantitative traits. The results highlight the potential for GWA to complement forward genetics in identifying the genetic basis of ecologically and economically important traits.

ODG: Omics database generator - a tool for generating, querying, and analyzing multi-omics comparative databases to facilitate biological understanding.

BACKGROUND: Rapid generation of omics data in recent years have resulted in vast amounts of disconnected datasets without systemic integration and knowledge building, while individual groups have made customized, annotated datasets available on the web with few ways to link them to in-lab datasets. With so many research groups generating their own data, the ability to relate it to the larger genomic and comparative genomic context is becoming increasingly crucial to make full use of the data. RESULTS: The Omics Database Generator (ODG) allows users to create customized databases that utilize published genomics data integrated with experimental data which can be queried using a flexible graph database. When provided with omics and experimental data, ODG will create a comparative, multi-dimensional graph database. ODG can import definitions and annotations from other sources such as InterProScan, the Gene Ontology, ENZYME, UniPathway, and others. This annotation data can be especially useful for studying new or understudied species for which transcripts have only been predicted, and rapidly give additional layers of annotation to predicted genes. In better studied species, ODG can perform syntenic annotation translations or rapidly identify characteristics of a set of genes or nucleotide locations, such as hits from an association study. ODG provides a web-based user-interface for configuring the data import and for querying the database. Queries can also be run from the command-line and the database can be queried directly through programming language hooks available for most languages. ODG supports most common genomic formats as well as generic, easy to use tab-separated value format for user-provided annotations. CONCLUSIONS: ODG is a user-friendly database generation and query tool that adapts to the supplied data to produce a comparative genomic database or multi-layered annotation database. ODG provides rapid comparative genomic annotation and is therefore particularly useful for non-model or understudied species. For species for which more data are available, ODG can be used to conduct complex multi-omics, pattern-matching queries.