Genome assembly of Medicago truncatula accession SA27063 provides insight into spring black stem and leaf spot disease resistance.

Medicago truncatula, model legume and alfalfa relative, has served as an essential resource for advancing our understanding of legume physiology, functional genetics, and crop improvement traits. Necrotrophic fungus, Ascochyta medicaginicola, the causal agent of spring black stem (SBS) and leaf spot is a devasting foliar disease of alfalfa affecting stand survival, yield, and forage quality. Host resistance to SBS disease is poorly understood, and control methods rely on cultural practices. Resistance has been observed in M. truncatula accession SA27063 (HM078) with two recessively inherited quantitative-trait loci (QTL), rnpm1 and rnpm2, previously reported. To shed light on host resistance, we carried out a de novo genome assembly of HM078. The genome, referred to as MtHM078 v1.0, is comprised of 23 contigs totaling 481.19 Mbp. Notably, this assembly contains a substantial amount of novel centromere-related repeat sequences due to deep long-read sequencing. Genome annotation resulted in 98.4% of BUSCO fabales proteins being complete. The assembly enabled sequence-level analysis of rnpm1 and rnpm2 for gene content, synteny, and structural variation between SBS-resistant accession SA27063 (HM078) and SBS-susceptible accession A17 (HM101). Fourteen candidate genes were identified, and some have been implicated in resistance to necrotrophic fungi. Especially interesting candidates include loss-of-function events in HM078 because they fit the inverse gene-for-gene model, where resistance is recessively inherited. In rnpm1, these include a loss-of-function in a disease resistance gene due to a premature stop codon, and a 10.85 kbp retrotransposon-like insertion disrupting a ubiquitin conjugating E2. In rnpm2, we identified a frameshift mutation causing a loss-of-function in a glycosidase, as well as a missense and frameshift mutation altering an F-box family protein. This study generated a high-quality genome of HM078 and has identified promising candidates, that once validated, could be further studied in alfalfa to enhance disease resistance.

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.

Alfalfa (Medicago sativa L.) pho2 mutant plants hyperaccumulate phosphate.

In this article, we describe a set of novel alfalfa (Medicago sativa L.) plants that hyper-accumulate Phosphate ion (Pi) at levels 3- to 6-fold higher than wild-type. This alfalfa germplasm will have practical applications reclaiming Pi from contaminated or enriched soil or be used in conservation buffer strips to protect waterways from Pi run-off. Hyper-accumulating alfalfa plants were generated by targeted mutagenesis of PHOSPHATE2 (PHO2) using newly created CRISPR/Cas9 reagents and an improved mutant screening strategy. PHO2 encodes a ubiquitin conjugating E2 enzyme (UBC24) previously characterized in Arabidopsis thaliana, Medicago truncatula, and Oryza sativa. Mutations of PHO2 disrupt Pi homeostasis resulting in Pi hyper-accumulation. Successful CRISPR/Cas9 editing of PHO2 demonstrates that this is an efficient mutagenesis tool in alfalfa despite its complex autotetraploid genome structure. Arabidopsis and M. truncatula ortholog genes were used to identify PHO2 haplotypes in outcrossing tetraploid M. sativa with the aim of generating heritable mutations in both PHO2-like genes (PHO2-B and PHO2-C). After delivery of the reagent and regeneration from transformed leaf explants, plants with mutations in all haplotypes of PHO2-B and PHO2-C were identified. These plants were evaluated for morphology, Pi accumulation, heritable transmission of targeted mutations, segregation of mutant haplotypes and removal of T-DNA(s). The Agrobacterium-mediated transformation assay and gene editing reagents reported here were also evaluated for further optimization for future alfalfa functional genomic studies.

The genome of a wild Medicago species provides insights into the tolerant mechanisms of legume forage to environmental stress.

BACKGROUND: Medicago ruthenica, a wild and perennial legume forage widely distributed in semi-arid grasslands, is distinguished by its outstanding tolerance to environmental stress. It is a close relative of commonly cultivated forage of alfalfa (Medicago sativa). The high tolerance of M. ruthenica to environmental stress makes this species a valuable genetic resource for understanding and improving traits associated with tolerance to harsh environments. RESULTS: We sequenced and assembled genome of M. ruthenica using an integrated approach, including PacBio, Illumina, 10×Genomics, and Hi-C. The assembled genome was 904.13 Mb with scaffold N50 of 99.39 Mb, and 50,162 protein-coding genes were annotated. Comparative genomics and transcriptomic analyses were used to elucidate mechanisms underlying its tolerance to environmental stress. The expanded FHY3/FAR1 family was identified to be involved in tolerance of M. ruthenica to drought stress. Many genes involved in tolerance to abiotic stress were retained in M. ruthenica compared to other cultivated Medicago species. Hundreds of candidate genes associated with drought tolerance were identified by analyzing variations in single nucleotide polymorphism using accessions of M. ruthenica with varying tolerance to drought. Transcriptomic data demonstrated the involvements of genes related to transcriptional regulation, stress response, and metabolic regulation in tolerance of M. ruthenica. CONCLUSIONS: We present a high-quality genome assembly and identification of drought-related genes in the wild species of M. ruthenica, providing a valuable resource for genomic studies on perennial legume forages.

The antagonistic MYB paralogs RH1 and RH2 govern anthocyanin leaf markings in Medicago truncatula.

Patterned leaf coloration in plants generates remarkable diversity in nature, but the underlying mechanisms remain largely unclear. Here, using Medicago truncatula leaf marking as a model, we show that the classic M. truncatula leaf anthocyanin spot trait depends on two R2R3 MYB paralogous regulators, RED HEART1 (RH1) and RH2. RH1 mainly functions as an anthocyanin biosynthesis activator that specifically determines leaf marking formation depending on its C-terminal activation motif. RH1 physically interacts with the M. truncatula bHLH protein MtTT8 and the WDR family member MtWD40-1, and this interaction facilitates RH1 function in leaf anthocyanin marking formation. RH2 has lost transcriptional activation activity, due to a divergent C-terminal domain, but retains the ability to interact with the same partners, MtTT8 and MtWD40-1, as RH1, thereby acting as a competitor in the regulatory complex and exerting opposite effects. Moreover, our results demonstrate that RH1 can activate its own expression and that RH2-mediated competition can repress RH1 expression. Our findings reveal the molecular mechanism of the antagonistic gene paralogs RH1 and RH2 in determining anthocyanin leaf markings in M. truncatula, providing a multidimensional paralogous-antagonistic regulatory paradigm for fine-tuning patterned pigmentation.

Genome-wide association study and genomic selection for tolerance of soybean biomass to soybean cyst nematode infestation.

Soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is one of the most devastating pathogens affecting soybean production in the U.S. and worldwide. The use of SCN-resistant soybean cultivars is one of the most affordable strategies to cope with SCN infestation. Because of the limited sources of SCN resistance and changes in SCN virulence phenotypes, host resistance in current cultivars has increasingly been overcome by the pathogen. Host tolerance has been recognized as an additional tool to manage the SCN. The objectives of this study were to conduct a genome-wide association study (GWAS), to identify single nucleotide polymorphism (SNP) markers, and to perform a genomic selection (GS) study for SCN tolerance in soybean based on reduction in biomass. A total of 234 soybean genotypes (lines) were evaluated for their tolerance to SCN in greenhouse using four replicates. The tolerance index (TI = 100 × Biomass of a line in SCN infested / Biomass of the line without SCN) was used as phenotypic data of SCN tolerance. GWAS was conducted using a total of 3,782 high quality SNPs. GS was performed based upon the whole set of SNPs and the GWAS-derived SNPs, respectively. Results showed that (1) a large variation in soybean TI to SCN infection among the soybean genotypes was identified; (2) a total of 35, 21, and 6 SNPs were found to be associated with SCN tolerance using the models SMR, GLM (PCA), and MLM (PCA+K) with 6 SNPs overlapping between models; (3) GS accuracy was SNP set-, model-, and training population size-dependent; and (4) genes around Glyma.06G134900, Glyma.15G097500.1, Glyma.15G100900.3, Glyma.15G105400, Glyma.15G107200, and Glyma.19G121200.1 (Table 4). Glyma.06G134900, Glyma.15G097500.1, Glyma.15G100900.3, Glyma.15G105400, and Glyma.19G121200.1 are best candidates. To the best of our knowledge, this is the first report highlighting SNP markers associated with tolerance index based on biomass reduction under SCN infestation in soybean. This research opens a new approach to use SCN tolerance in soybean breeding and the SNP markers will provide a tool for breeders to select for SCN tolerance.

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.

Genome-wide association study and genomic selection for soybean chlorophyll content associated with soybean cyst nematode tolerance.

BACKGROUND: Soybean cyst nematode (SCN), Heterodera glycines Ichinohe, has been one of the most devastating pathogens affecting soybean production. In the United States alone, SCN damage accounted for more than $1 billion loss annually. With a narrow genetic background of the currently available SCN-resistant commercial cultivars, high risk of resistance breakdown can occur. The objectives of this study were to conduct a genome-wide association study (GWAS) to identify QTL, SNP markers, and candidate genes associated with soybean leaf chlorophyll content tolerance to SCN infection, and to carry out a genomic selection (GS) study for the chlorophyll content tolerance. RESULTS: A total of 172 soybean genotypes were evaluated for the effect of SCN HG Type 1.2.3.5.6.7 (race 4) on soybean leaf chlorophyll. The soybean lines were genotyped using a total of 4089 filtered and high-quality SNPs. Results showed that (1) a large variation in SCN tolerance based on leaf chlorophyll content indices (CCI); (2) a total of 22, 14, and 16 SNPs associated with CCI of non-SCN-infected plants, SCN-infected plants, and reduction of CCI SCN, respectively; (3) a new locus of chlorophyll content tolerance to SCN mapped on chromosome 3; (4) candidate genes encoding for Leucine-rich repeat protein, plant hormone signaling molecules, and biomolecule transporters; and (5) an average GS accuracy ranging from 0.31 to 0.46 with all SNPs and varying from 0.55 to 0.76 when GWAS-derived SNP markers were used across five models. This study demonstrated the potential of using genome-wide selection to breed chlorophyll-content-tolerant soybean for managing SCN. CONCLUSIONS: In this study, soybean accessions with higher CCI under SCN infestation, and molecular markers associated with chlorophyll content related to SCN were identified. In addition, a total of 15 candidate genes associated with chlorophyll content tolerance to SCN in soybean were also identified. These candidate genes will lead to a better understanding of the molecular mechanisms that control chlorophyll content tolerance to SCN in soybean. Genomic selection analysis of chlorophyll content tolerance to SCN showed that using significant SNPs obtained from GWAS could provide better GS accuracy.

Nodule-specific PLAT domain proteins are expanded in the Medicago lineage and required for nodulation.

Symbiotic nitrogen fixation in legumes is mediated by an interplay of signaling processes between plant hosts and rhizobial symbionts. In legumes, several secreted protein families have undergone expansions and play key roles in nodulation. Thus, identifying lineage-specific expansions (LSEs) of nodulation-associated genes can be a strategy to discover candidate gene families. Using bioinformatic tools, we identified 13 LSEs of nodulation-related secreted protein families, each unique to either Glycine, Arachis or Medicago lineages. In the Medicago lineage, nodule-specific Polycystin-1, Lipoxygenase, Alpha Toxin (PLAT) domain proteins (NPDs) expanded to five members. We examined NPD function using CRISPR/Cas9 multiplex genome editing to create Medicago truncatula NPD knockout lines, targeting one to five NPD genes. Mutant lines with differing combinations of NPD gene inactivations had progressively smaller nodules, earlier onset of nodule senescence, or ineffective nodules compared to the wild-type control. Double- and triple-knockout lines showed dissimilar nodulation phenotypes but coincided in upregulation of a DHHC-type zinc finger and an aspartyl protease gene, possible candidates for the observed disturbance of proper nodule function. By postulating that gene family expansions can be used to detect candidate genes, we identified a family of nodule-specific PLAT domain proteins and confirmed that they play a role in successful nodule formation.

Complete Genome Sequence of Sinorhizobium meliloti Bacteriophage HMSP1-Susan.

Sinorhiozbium bacteriophage HMSP1-Susan has a genome of 51,963 bp in size, with a GC content of 52.5%. It contains 97 putative coding sequences; 83% of these coding sequences (CDS) encode proteins classified as hypothetical or having unknown functions. HMSP1 has limited homology to previously reported viruses and likely represents a new phage that infects this nitrogen-fixing bacterium.

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.

Transcriptomic basis of genome by genome variation in a legume-rhizobia mutualism.

In the legume-rhizobia mutualism, the benefit each partner derives from the other depends on the genetic identity of both host and rhizobial symbiont. To gain insight into the extent of genome × genome interactions on hosts at the molecular level and to identify potential mechanisms responsible for the variation, we examined host gene expression within nodules (the plant organ where the symbiosis occurs) of four genotypes of Medicago truncatula grown with either Ensifer meliloti or E. medicae symbionts. These host × symbiont combinations show significant variation in nodule and biomass phenotypes. Likewise, combinations differ in their transcriptomes: host, symbiont and host × symbiont affected the expression of 70%, 27% and 21%, respectively, of the approximately 27,000 host genes expressed in nodules. Genes with the highest levels of expression often varied between hosts and/or symbiont strain and include leghemoglobins that modulate oxygen availability and hundreds of Nodule Cysteine-Rich (NCR) peptides involved in symbiont differentiation and viability in nodules. Genes with host × symbiont-dependent expression were enriched for functions related to resource exchange between partners (sulphate/iron/amino acid transport and dicarboxylate/amino acid synthesis). These enrichments suggest mechanisms for host control of the currencies of the mutualism. The transcriptome of M. truncatula accession HM101 (A17), the reference genome used for most molecular research, was less affected by symbiont identity than the other hosts. These findings underscore the importance of assessing the molecular basis of variation in ecologically important traits, particularly those involved in biotic interactions, in multiple genetic contexts.

Strategies for optimizing BioNano and Dovetail explored through a second reference quality assembly for the legume model, Medicago truncatula.

BACKGROUND: Third generation sequencing technologies, with sequencing reads in the tens- of kilo-bases, facilitate genome assembly by spanning ambiguous regions and improving continuity. This has been critical for plant genomes, which are difficult to assemble due to high repeat content, gene family expansions, segmental and tandem duplications, and polyploidy. Recently, high-throughput mapping and scaffolding strategies have further improved continuity. Together, these long-range technologies enable quality draft assemblies of complex genomes in a cost-effective and timely manner. RESULTS: Here, we present high quality genome assemblies of the model legume plant, Medicago truncatula (R108) using PacBio, Dovetail Chicago (hereafter, Dovetail) and BioNano technologies. To test these technologies for plant genome assembly, we generated five assemblies using all possible combinations and ordering of these three technologies in the R108 assembly. While the BioNano and Dovetail joins overlapped, they also showed complementary gains in continuity and join numbers. Both technologies spanned repetitive regions that PacBio alone was unable to bridge. Combining technologies, particularly Dovetail followed by BioNano, resulted in notable improvements compared to Dovetail or BioNano alone. A combination of PacBio, Dovetail, and BioNano was used to generate a high quality draft assembly of R108, a M. truncatula accession widely used in studies of functional genomics. As a test for the usefulness of the resulting genome sequence, the new R108 assembly was used to pinpoint breakpoints and characterize flanking sequence of a previously identified translocation between chromosomes 4 and 8, identifying more than 22.7 Mb of novel sequence not present in the earlier A17 reference assembly. CONCLUSIONS: Adding Dovetail followed by BioNano data yielded complementary improvements in continuity over the original PacBio assembly. This strategy proved efficient and cost-effective for developing a quality draft assembly compared to traditional reference assemblies.

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.

Hybrid assembly with long and short reads improves discovery of gene family expansions.

BACKGROUND: Long-read and short-read sequencing technologies offer competing advantages for eukaryotic genome sequencing projects. Combinations of both may be appropriate for surveys of within-species genomic variation. METHODS: We developed a hybrid assembly pipeline called "Alpaca" that can operate on 20X long-read coverage plus about 50X short-insert and 50X long-insert short-read coverage. To preclude collapse of tandem repeats, Alpaca relies on base-call-corrected long reads for contig formation. RESULTS: Compared to two other assembly protocols, Alpaca demonstrated the most reference agreement and repeat capture on the rice genome. On three accessions of the model legume Medicago truncatula, Alpaca generated the most agreement to a conspecific reference and predicted tandemly repeated genes absent from the other assemblies. CONCLUSION: Our results suggest Alpaca is a useful tool for investigating structural and copy number variation within de novo assemblies of sampled populations.

Exploring structural variation and gene family architecture with De Novo assemblies of 15 Medicago genomes.

BACKGROUND: Previous studies exploring sequence variation in the model legume, Medicago truncatula, relied on mapping short reads to a single reference. However, read-mapping approaches are inadequate to examine large, diverse gene families or to probe variation in repeat-rich or highly divergent genome regions. De novo sequencing and assembly of M. truncatula genomes enables near-comprehensive discovery of structural variants (SVs), analysis of rapidly evolving gene families, and ultimately, construction of a pan-genome. RESULTS: Genome-wide synteny based on 15 de novo M. truncatula assemblies effectively detected different types of SVs indicating that as much as 22% of the genome is involved in large structural changes, altogether affecting 28% of gene models. A total of 63 million base pairs (Mbp) of novel sequence was discovered, expanding the reference genome space for Medicago by 16%. Pan-genome analysis revealed that 42% (180 Mbp) of genomic sequences is missing in one or more accession, while examination of de novo annotated genes identified 67% (50,700) of all ortholog groups as dispensable - estimates comparable to recent studies in rice, maize and soybean. Rapidly evolving gene families typically associated with biotic interactions and stress response were found to be enriched in the accession-specific gene pool. The nucleotide-binding site leucine-rich repeat (NBS-LRR) family, in particular, harbors the highest level of nucleotide diversity, large effect single nucleotide change, protein diversity, and presence/absence variation. However, the leucine-rich repeat (LRR) and heat shock gene families are disproportionately affected by large effect single nucleotide changes and even higher levels of copy number variation. CONCLUSIONS: Analysis of multiple M. truncatula genomes illustrates the value of de novo assemblies to discover and describe structural variation, something that is often under-estimated when using read-mapping approaches. Comparisons among the de novo assemblies also indicate that different large gene families differ in the architecture of their structural variation.

Genome-wide association studies with proteomics data reveal genes important for synthesis, transport and packaging of globulins in legume seeds.

Improving nutritional seed quality is an important challenge in grain legume breeding. However, the genes controlling the differential accumulation of globulins, which are major contributors to seed nutritional value in legumes, remain largely unknown. We combined a search for protein quantity loci with genome-wide association studies on the abundance of 7S and 11S globulins in seeds of the model legume species Medicago truncatula. Identified genomic regions and genes carrying polymorphisms linked to globulin variations were then cross-compared with pea (Pisum sativum), leading to the identification of candidate genes for the regulation of globulin abundance in this crop. Key candidates identified include genes involved in transcription, chromatin remodeling, post-translational modifications, transport and targeting of proteins to storage vacuoles. Inference of a gene coexpression network of 12 candidate transcription factors and globulin genes revealed the transcription factor ABA-insensitive 5 (ABI5) as a highly connected hub. Characterization of loss-of-function abi5 mutants in pea uncovered a role for ABI5 in controlling the relative abundance of vicilin, a sulfur-poor 7S globulin, in pea seeds. This demonstrates the feasibility of using genome-wide association studies in M. truncatula to reveal genes that can be modulated to improve seed nutritional value.

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.

A Guide to Genome-Wide Association Mapping in Plants.

Genome-wide association studies (GWAS) have developed into a valuable approach for identifying the genetic basis of phenotypic variation. In this article, we provide an overview of the design, analysis, and interpretation of GWAS. First, we present results from simulations that explore key elements of experimental design as well as considerations for collecting the relevant genomic and phenotypic data. Next, we outline current statistical methods and tools used for GWA analyses and discuss the inclusion of covariates to account for population structure and the interpretation of results. Given that many false positive associations will occur in any GWA analysis, we highlight strategies for prioritizing GWA candidates for further statistical and empirical validation. While focused on plants, the material we cover is also applicable to other systems. © 2017 by John Wiley & Sons, Inc.

Adaptation to climate through flowering phenology: a case study in Medicago truncatula.

Local climatic conditions likely constitute an important selective pressure on genes underlying important fitness-related traits such as flowering time, and in many species, flowering phenology and climatic gradients strongly covary. To test whether climate shapes the genetic variation on flowering time genes and to identify candidate flowering genes involved in the adaptation to environmental heterogeneity, we used a large Medicago truncatula core collection to examine the association between nucleotide polymorphisms at 224 candidate genes and both climate variables and flowering phenotypes. Unlike genome-wide studies, candidate gene approaches are expected to enrich for the number of meaningful trait associations because they specifically target genes that are known to affect the trait of interest. We found that flowering time mediates adaptation to climatic conditions mainly by variation at genes located upstream in the flowering pathways, close to the environmental stimuli. Variables related to the annual precipitation regime reflected selective constraints on flowering time genes better than the other variables tested (temperature, altitude, latitude or longitude). By comparing phenotype and climate associations, we identified 12 flowering genes as the most promising candidates responsible for phenological adaptation to climate. Four of these genes were located in the known flowering time QTL region on chromosome 7. However, climate and flowering associations also highlighted largely distinct gene sets, suggesting different genetic architectures for adaptation to climate and flowering onset.