Leveraging orthology within maize and Arabidopsis QTL to identify genes affecting natural variation in gravitropism.

Plants typically orient their organs with respect to the Earth's gravity field by a dynamic process called gravitropism. To discover conserved genetic elements affecting seedling root gravitropism, we measured the process in a set of Zea mays (maize) recombinant inbred lines with machine vision and compared the results with those obtained in a similar study of Arabidopsis thaliana. Each of the several quantitative trait loci that we mapped in both species spanned many hundreds of genes, too many to test individually for causality. We reasoned that orthologous genes may be responsible for natural variation in monocot and dicot root gravitropism. If so, pairs of orthologous genes affecting gravitropism may be present within the maize and Arabidopsis QTL intervals. A reciprocal comparison of sequences within the QTL intervals identified seven pairs of such one-to-one orthologs. Analysis of knockout mutants demonstrated a role in gravitropism for four of the seven: CCT2 functions in phosphatidylcholine biosynthesis, ATG5 functions in membrane remodeling during autophagy, UGP2 produces the substrate for cellulose and callose polymer extension, and FAMA is a transcription factor. Automated phenotyping enabled this discovery of four naturally varying components of a conserved process (gravitropism) by making it feasible to conduct the same large-scale experiment in two species.

Mycorrhizal status and host genotype interact to shape plant nutrition in field grown maize (Zea mays ssp. mays).

Arbuscular mycorrhizal fungi (AMF) establish symbioses with the major cereal crops, providing plants with increased access to nutrients while enhancing their tolerance to toxic heavy metals. However, not all plant varieties benefit equally from this association. In this study, we used quantitative trait loci (QTL) mapping to evaluate the combined effect of host genotypic variation (G) and AMF across 141 genotypes on the concentration of 20 mineral elements in the leaves and grain of field grown maize (Zea mays spp. mays). Our mapping design included selective incorporation of a castor AMF-incompatibility mutation, allowing estimation of AMF, QTL and QTLxAMF effects by comparison of mycorrhizal and non-mycorrhizal plants. Overall, AMF compatibility was associated with higher concentrations of boron (B), copper (Cu), molybdenum (Mo), phosphorus (P), selenium (Se) and zinc (Zn) and lower concentrations of arsenic (As), iron (Fe), magnesium (Mg), manganese (Mn), potassium (K) and strontium (Sr). In addition to effects on individual elements, pairwise correlation matrices for element concentration differed between mycorrhizal and non-mycorrhizal plants. We mapped 22 element QTLs, including 18 associated with QTLxAMF effects that indicate plant genotype-specific differences in the impact of AMF on the host ionome. Although there is considerable interest in AMF as biofertilizers, it remains challenging to estimate the impact of AMF in the field. Our design illustrates an effective approach for field evaluation of AMF effects. Furthermore, we demonstrate the capacity of the ionome to reveal host genotype-specific variation in the impact of AMF on plant nutrition.

A genetic link between leaf carbon isotope composition and whole-plant water use efficiency in the C4 grass Setaria.

Genetic selection for whole-plant water use efficiency (yield per transpiration; WUEplant ) in any crop-breeding programme requires high-throughput phenotyping of component traits of WUEplant such as intrinsic water use efficiency (WUEi ; CO2 assimilation rate per stomatal conductance). Measuring WUEi by gas exchange measurements is laborious and time consuming and may not reflect an integrated WUEi over the life of the leaf. Alternatively, leaf carbon stable isotope composition (δ13 Cleaf ) has been suggested as a potential time-integrated proxy for WUEi that may provide a tool to screen for WUEplant . However, a genetic link between δ13 Cleaf and WUEplant in a C4 species has not been well established. Therefore, to determine if there is a genetic relationship in a C4 plant between δ13 Cleaf and WUEplant under well watered and water-limited growth conditions, a high-throughput phenotyping facility was used to measure WUEplant in a recombinant inbred line (RIL) population created between the C4 grasses Setaria viridis and S. italica. Three quantitative trait loci (QTL) for δ13 Cleaf were found and co-localized with transpiration, biomass accumulation, and WUEplant . Additionally, WUEplant for each of the δ13 Cleaf QTL allele classes was negatively correlated with δ13 Cleaf , as would be predicted when WUEi influences WUEplant . These results demonstrate that δ13 Cleaf is genetically linked to WUEplant , likely to be through their relationship with WUEi , and can be used as a high-throughput proxy to screen for WUEplant in these C4 species.

Hybridization, polyploidy and clonality influence geographic patterns of diversity and salt tolerance in the model halophyte seashore paspalum (Paspalum vaginatum).

In plant species, variation in levels of clonality, ploidy and interspecific hybridization can interact to influence geographic patterns of genetic diversity. These factors commonly vary in plants that specialize on saline habitats (halophytes) and may play a role in how they adapt to salinity variation across their range. One such halophyte is the turfgrass and emerging genomic model system seashore paspalum (Paspalum vaginatum Swartz). To investigate how clonal propagation, ploidy variation, and interspecific hybridization vary across ecotypes and local salinity levels in wild P. vaginatum, we employed genotyping-by-sequencing, cpDNA sequencing and flow cytometry in 218 accessions representing > 170 wild collections from throughout the coastal southern United States plus USDA germplasm. We found that the two morphologically distinct ecotypes of P. vaginatum differ in their adaptive strategies. The fine-textured ecotype is diploid and appears to reproduce in the wild both sexually and by clonal propagation; in contrast, the coarse-textured ecotype consists largely of clonally-propagating triploid and diploid genotypes. The coarse-textured ecotype appears to be derived from hybridization between fine-textured P. vaginatum and an unidentified Paspalum species. These clonally propagating hybrid genotypes are more broadly distributed than clonal fine-textured genotypes and may represent a transition to a more generalist adaptive strategy. Additionally, the triploid genotypes vary in whether they carry one or two copies of the P. vaginatum subgenome, indicating multiple evolutionary origins. This variation in subgenome composition shows associations with local ocean salinity levels across the sampled populations and may play a role in local adaptation.

Correlation and co-localization of QTL for stomatal density, canopy temperature, and productivity with and without drought stress in Setaria.

Mechanistic modeling indicates that stomatal conductance could be reduced to improve water use efficiency (WUE) in C4 crops. Genetic variation in stomatal density and canopy temperature was evaluated in the model C4 genus, Setaria. Recombinant inbred lines (RILs) derived from a Setaria italica×Setaria viridis cross were grown with ample or limiting water supply under field conditions in Illinois. An optical profilometer was used to rapidly assess stomatal patterning, and canopy temperature was measured using infrared imaging. Stomatal density and canopy temperature were positively correlated but both were negatively correlated with total above-ground biomass. These trait relationships suggest a likely interaction between stomatal density and the other drivers of water use such as stomatal size and aperture. Multiple quantitative trait loci (QTL) were identified for stomatal density and canopy temperature, including co-located QTL on chromosomes 5 and 9. The direction of the additive effect of these QTL on chromosome 5 and 9 was in accordance with the positive phenotypic relationship between these two traits. This, along with prior experiments, suggests a common genetic architecture between stomatal patterning and WUE in controlled environments with canopy transpiration and productivity in the field, while highlighting the potential of Setaria as a model to understand the physiology and genetics of WUE in C4 species.

Integrating Coexpression Networks with GWAS to Prioritize Causal Genes in Maize.

Genome-wide association studies (GWAS) have identified loci linked to hundreds of traits in many different species. Yet, because linkage equilibrium implicates a broad region surrounding each identified locus, the causal genes often remain unknown. This problem is especially pronounced in nonhuman, nonmodel species, where functional annotations are sparse and there is frequently little information available for prioritizing candidate genes. We developed a computational approach, Camoco, that integrates loci identified by GWAS with functional information derived from gene coexpression networks. Using Camoco, we prioritized candidate genes from a large-scale GWAS examining the accumulation of 17 different elements in maize (Zea mays) seeds. Strikingly, we observed a strong dependence in the performance of our approach based on the type of coexpression network used: expression variation across genetically diverse individuals in a relevant tissue context (in our case, roots that are the primary elemental uptake and delivery system) outperformed other alternative networks. Two candidate genes identified by our approach were validated using mutants. Our study demonstrates that coexpression networks provide a powerful basis for prioritizing candidate causal genes from GWAS loci but suggests that the success of such strategies can highly depend on the gene expression data context. Both the software and the lessons on integrating GWAS data with coexpression networks generalize to species beyond maize.

Increased temperatures may safeguard the nutritional quality of crops under future elevated CO2 concentrations.

Iron (Fe) and zinc (Zn) deficiencies are a global human health problem that may worsen by the growth of crops at elevated atmospheric CO2 concentration (eCO2 ). However, climate change will also involve higher temperature, but it is unclear how the combined effect of eCO2 and higher temperature will affect the nutritional quality of food crops. To begin to address this question, we grew soybean (Glycine max) in a Temperature by Free-Air CO2 Enrichment (T-FACE) experiment in 2014 and 2015 under ambient (400 µmol mol-1 ) and elevated (600 µmol mol-1 ) CO2 concentrations, and under ambient and elevated temperatures (+2.7°C day and +3.4°C at night). In our study, eCO2 significantly decreased Fe concentration in soybean seeds in both seasons (-8.7 and -7.7%) and Zn concentration in one season (-8.9%), while higher temperature (at ambient CO2 concentration) had the opposite effect. The combination of eCO2 with elevated temperature generally restored seed Fe and Zn concentrations to levels obtained under ambient CO2 and temperature conditions, suggesting that the potential threat to human nutrition by increasing CO2 concentration may not be realized. In general, seed Fe concentration was negatively correlated with yield, suggesting inherent limitations to increasing seed Fe. In addition, we confirm our previous report that the concentration of seed storage products and several minerals varies with node position at which the seeds developed. Overall, these results demonstrate the complexity of predicting climate change effects on food and nutritional security when various environmental parameters change in an interactive manner.

Time dependent genetic analysis links field and controlled environment phenotypes in the model C4 grass Setaria.

Vertical growth of plants is a dynamic process that is influenced by genetic and environmental factors and has a pronounced effect on overall plant architecture and biomass composition. We have performed six controlled growth trials of an interspecific Setaria italica x Setaria viridis recombinant inbred line population to assess how the genetic architecture of plant height is influenced by developmental queues, water availability and planting density. The non-destructive nature of plant height measurements has enabled us to monitor height throughout the plant life cycle in both field and controlled environments. We find that plant height is reduced under water limitation and high density planting and affected by growth environment (field vs. growth chamber). The results support a model where plant height is a heritable, polygenic trait and that the major genetic loci that influence plant height function independent of growth environment. The identity and contribution of loci that influence height changes dynamically throughout development and the reduction of growth observed in water limited environments is a consequence of delayed progression through the genetic program which establishes plant height in Setaria. In this population, alleles inherited from the weedy S. viridis parent act to increase plant height early, whereas a larger number of small effect alleles inherited from the domesticated S. italica parent collectively act to increase plant height later in development.

Components of Water Use Efficiency Have Unique Genetic Signatures in the Model C4 Grass Setaria.

Plant growth and water use are interrelated processes influenced by genetically controlled morphological and biochemical characteristics. Improving plant water use efficiency (WUE) to sustain growth in different environments is an important breeding objective that can improve crop yields and enhance agricultural sustainability. However, genetic improvement of WUE using traditional methods has proven difficult due to the low throughput and environmental heterogeneity of field settings. To overcome these limitations, this study utilizes a high-throughput phenotyping platform to quantify plant size and water use of an interspecific Setaria italica × Setaria viridis recombinant inbred line population at daily intervals in both well-watered and water-limited conditions. Our findings indicate that measurements of plant size and water use are correlated strongly in this system; therefore, a linear modeling approach was used to partition this relationship into predicted values of plant size given water use and deviations from this relationship at the genotype level. The resulting traits describing plant size, water use, and WUE all were heritable and responsive to soil water availability, allowing for a genetic dissection of the components of plant WUE under different watering treatments. Linkage mapping identified major loci underlying two different pleiotropic components of WUE. This study indicates that alleles controlling WUE derived from both wild and domesticated accessions can be utilized to predictably modulate trait values given a specified precipitation regime in the model C4 genus Setaria.

Grasses suppress shoot-borne roots to conserve water during drought.

Many important crops are members of the Poaceae family, which develop root systems characterized by a high degree of root initiation from the belowground basal nodes of the shoot, termed the crown. Although this postembryonic shoot-borne root system represents the major conduit for water uptake, little is known about the effect of water availability on its development. Here we demonstrate that in the model C4 grass Setaria viridis, the crown locally senses water availability and suppresses postemergence crown root growth under a water deficit. This response was observed in field and growth room environments and in all grass species tested. Luminescence-based imaging of root systems grown in soil-like media revealed a shift in root growth from crown-derived to primary root-derived branches, suggesting that primary root-dominated architecture can be induced in S. viridis under certain stress conditions. Crown roots of Zea mays and Setaria italica, domesticated relatives of teosinte and S. viridis, respectively, show reduced sensitivity to water deficit, suggesting that this response might have been influenced by human selection. Enhanced water status of maize mutants lacking crown roots suggests that under a water deficit, stronger suppression of crown roots actually may benefit crop productivity.

Co-ordinated Changes in the Accumulation of Metal Ions in Maize (Zea mays ssp. mays L.) in Response to Inoculation with the Arbuscular Mycorrhizal Fungus Funneliformis mosseae.

Arbuscular mycorrhizal symbiosis is an ancient interaction between plants and fungi of the phylum Glomeromycota. In exchange for photosynthetically fixed carbon, the fungus provides the plant host with greater access to soil nutrients via an extensive network of root-external hyphae. Here, to determine the impact of the symbiosis on the host ionome, the concentration of 19 elements was determined in the roots and leaves of a panel of 30 maize varieties, grown under phosphorus-limiting conditions, with or without inoculation with the fungus Funneliformis mosseae. Although the most recognized benefit of the symbiosis to the host plant is greater access to soil phosphorus, the concentration of a number of other elements responded significantly to inoculation across the panel as a whole. In addition, variety-specific effects indicated the importance of plant genotype to the response. Clusters of elements were identified that varied in a co-ordinated manner across genotypes, and that were maintained between non-inoculated and inoculated plants.

Phosphorus acquisition efficiency in arbuscular mycorrhizal maize is correlated with the abundance of root-external hyphae and the accumulation of transcripts encoding PHT1 phosphate transporters.

Plant interactions with arbuscular mycorrhizal fungi have long attracted interest for their potential to promote more efficient use of mineral resources in agriculture. Their use, however, remains limited by a lack of understanding of the processes that determine the outcome of the symbiosis. In this study, the impact of host genotype on growth response to mycorrhizal inoculation was investigated in a panel of diverse maize lines. A panel of 30 maize lines was evaluated with and without inoculation with arbuscular mycorrhizal fungi. The line Oh43 was identified to show superior response and, along with five other reference lines, was characterized in greater detail in a split-compartment system, using 33 P to quantify mycorrhizal phosphorus uptake. Changes in relative growth indicated variation in host capacity to profit from the symbiosis. Shoot phosphate content, abundance of root-internal and -external fungal structures, mycorrhizal phosphorus uptake, and accumulation of transcripts encoding plant PHT1 family phosphate transporters varied among lines. Superior response in Oh43 is correlated with extensive development of root-external hyphae, accumulation of specific Pht1 transcripts and high phosphorus uptake by mycorrhizal plants. The data indicate that host genetic factors influence fungal growth strategy with an impact on plant performance.

Integration of Experiments across Diverse Environments Identifies the Genetic Determinants of Variation in Sorghum bicolor Seed Element Composition.

Seedling establishment and seed nutritional quality require the sequestration of sufficient element nutrients. The identification of genes and alleles that modify element content in the grains of cereals, including sorghum (Sorghum bicolor), is fundamental to developing breeding and selection methods aimed at increasing bioavailable element content and improving crop growth. We have developed a high-throughput work flow for the simultaneous measurement of multiple elements in sorghum seeds. We measured seed element levels in the genotyped Sorghum Association Panel, representing all major cultivated sorghum races from diverse geographic and climatic regions, and mapped alleles contributing to seed element variation across three environments by genome-wide association. We observed significant phenotypic and genetic correlation between several elements across multiple years and diverse environments. The power of combining high-precision measurements with genome-wide association was demonstrated by implementing rank transformation and a multilocus mixed model to map alleles controlling 20 element traits, identifying 255 loci affecting the sorghum seed ionome. Sequence similarity to genes characterized in previous studies identified likely causative genes for the accumulation of zinc, manganese, nickel, calcium, and cadmium in sorghum seeds. In addition to strong candidates for these five elements, we provide a list of candidate loci for several other elements. Our approach enabled the identification of single-nucleotide polymorphisms in strong linkage disequilibrium with causative polymorphisms that can be evaluated in targeted selection strategies for plant breeding and improvement.

Genome-wide association study of 107 phenotypes in Arabidopsis thaliana inbred lines.

Although pioneered by human geneticists as a potential solution to the challenging problem of finding the genetic basis of common human diseases, genome-wide association (GWA) studies have, owing to advances in genotyping and sequencing technology, become an obvious general approach for studying the genetics of natural variation and traits of agricultural importance. They are particularly useful when inbred lines are available, because once these lines have been genotyped they can be phenotyped multiple times, making it possible (as well as extremely cost effective) to study many different traits in many different environments, while replicating the phenotypic measurements to reduce environmental noise. Here we demonstrate the power of this approach by carrying out a GWA study of 107 phenotypes in Arabidopsis thaliana, a widely distributed, predominantly self-fertilizing model plant known to harbour considerable genetic variation for many adaptively important traits. Our results are dramatically different from those of human GWA studies, in that we identify many common alleles of major effect, but they are also, in many cases, harder to interpret because confounding by complex genetics and population structure make it difficult to distinguish true associations from false. However, a-priori candidates are significantly over-represented among these associations as well, making many of them excellent candidates for follow-up experiments. Our study demonstrates the feasibility of GWA studies in A. thaliana and suggests that the approach will be appropriate for many other organisms.

Should we treat the ionome as a combination of individual elements, or should we be deriving novel combined traits?

It has been more than 10 years since the concept of the ionome, all of the mineral nutrients in a cell tissue or organism, was introduced. In the intervening years, ionomics, high throughput elemental profiling, has been used to analyse over 400,000 samples from at least 10 different organisms. There are now multiple published examples where an ionomics approach has been used to find genes of novel function, find lines or environments that produce foods with altered nutritional profiles, or define gene by environmental effects on elemental accumulation. In almost all of these studies, the ionome has been treated as a collection of independent elements, with the analysis repeated on each measured element. However, many elements share chemical properties, are known to interact with each other, or have been shown to have similar interactions with biological molecules. Accordingly, there is strong evidence from ionomic studies that the elements of the ionome do not behave independently and that combinations of elements should be treated as the phenotypes of interest. In this review, I will consider the evidence that we have for the interdependence of the ionome, some of its causes, methods for incorporating this interdependence into analyses and the benefits, drawbacks, and challenges of taking these approaches.

Genome-wide association studies identify heavy metal ATPase3 as the primary determinant of natural variation in leaf cadmium in Arabidopsis thaliana.

Understanding the mechanism of cadmium (Cd) accumulation in plants is important to help reduce its potential toxicity to both plants and humans through dietary and environmental exposure. Here, we report on a study to uncover the genetic basis underlying natural variation in Cd accumulation in a world-wide collection of 349 wild collected Arabidopsis thaliana accessions. We identified a 4-fold variation (0.5-2 µg Cd g(-1) dry weight) in leaf Cd accumulation when these accessions were grown in a controlled common garden. By combining genome-wide association mapping, linkage mapping in an experimental F2 population, and transgenic complementation, we reveal that HMA3 is the sole major locus responsible for the variation in leaf Cd accumulation we observe in this diverse population of A. thaliana accessions. Analysis of the predicted amino acid sequence of HMA3 from 149 A. thaliana accessions reveals the existence of 10 major natural protein haplotypes. Association of these haplotypes with leaf Cd accumulation and genetics complementation experiments indicate that 5 of these haplotypes are active and 5 are inactive, and that elevated leaf Cd accumulation is associated with the reduced function of HMA3 caused by a nonsense mutation and polymorphisms that change two specific amino acids.

Population genetics of Setaria viridis, a new model system.

An extensive survey of the standing genetic variation in natural populations is among the priority steps in developing a species into a model system. In recent years, green foxtail (Setaria viridis), along with its domesticated form foxtail millet (S. italica), has rapidly become a promising new model system for C4 grasses and bioenergy crops, due to its rapid life cycle, large amount of seed production and small diploid genome, among other characters. However, remarkably little is known about the genetic diversity in natural populations of this species. In this study, we survey the genetic diversity of a worldwide sample of more than 200 S. viridis accessions, using the genotyping-by-sequencing technique. Two distinct genetic groups in S. viridis and a third group resembling S. italica were identified, with considerable admixture among the three groups. We find the genetic variation of North American S. viridis correlates with both geography and climate and is representative of the total genetic diversity in this species. This pattern may reflect several introduction/dispersal events of S. viridis into North America. We also modelled demographic history and show signal of recent population decline in one subgroup. Finally, we show linkage disequilibrium decay is rapid (<45 kb) in our total sample and slow in genetic subgroups. These results together provide an in-depth understanding of the pattern of genetic diversity of this new model species on a broad geographic scale. They also provide key guidelines for on-going and future work including germplasm preservation, local adaptation, crossing designs and genomewide association studies.