Milano ER, Payne CE, Wolfrum E, Lovell JT, Jenkins J, Schmutz J, Juenger TE. Quantitative trait loci for cell wall composition traits measured using near-infrared spectroscopy in the model C4 perennial grass Panicum hallii. Biotechnology for Biofuels [Internet]. 2018;11 (25) : Publisher's VersionAbstract
Biofuels derived from lignocellulosic plant material are an important component of current renewable energy strategies. Improvement eforts in biofuel feedstock crops have been primarily focused on increasing biomass yield with less consideration for tissue quality or composition. Four primary components found in the plant cell wall contribute to the overall quality of plant tissue and conversion characteristics, cellulose and hemicellulose polysaccharides are the primary targets for fuel conversion, while lignin and ash provide structure and defense. We explore the genetic architecture of tissue characteristics using a quantitative trait loci (QTL) mapping approach in Panicum hallii, a model lignocellulosic grass system. Diversity in the mapping population was generated by crossing xeric and mesic varietals, comparative to northern upland and southern lowland ecotypes in switchgrass. We use near-infrared spectroscopy with a primary analytical method to create a P. hallii specifc calibration model to quickly quantify cell wall components.
Campitelli BE, Kenney AM, Hopkins R, Soule J, Lovell JT, Juenger TE. Genetic mapping reveals an anthocyanin biosynthesis pathway gene potentially influencingevolutionary divergence between two subspecies of scarlet gilia (Ipomopsis aggregata). Molecular Biology and Evolution [Internet]. 2017;(msx318) : Publisher's VersionAbstract
Immense floral trait variation has likely arisen as an adaptation to attract pollinators. Different pollinator syndromes—suites of floral traits that attract specific pollinator functional groups—are repeatedly observed across closely related taxa or divergent populations. The observation of these trait syndromes suggests that pollinators use floral cues to signal the underlying nectar reward, and that complex trait combinations may persist and evolve through genetic correlations. Here, we explore pollinator preferences and the genetic  architecture of floral divergence using an extensive genetic mapping study in the hybrid zone of two Ipomopsis aggregata subspecies that exhibit a hummingbird and a hawkmoth pollinator syndrome. We found that natural selection acts on several floral traits, and that hummingbirds and hawkmoths exhibited flower color preferences as predicted by their respective pollinator syndromes. Our quantitative trait loci (QTL) analyses revealed 46 loci affecting floral features, many of which co-localize across the genome. Two of these QTL have large effects explaining > 15 percent of the phenotypic variance. The strongest QTL was associated with flower color and localized to a SNP in the anthocyanin biosynthesis pathway (ABP) gene, dihydroflavonol-4-reductase (DFR). Further analysis revealed strong associations between DFR SNP variants, gene  expression and flower color across populations from the hybrid zone. Hence, DFR may be a target of pollinatormediated selection in the hybrid zone of these two subspecies. Together, our findings suggest that hummingbirds and hawkmoths exhibit contrasting flower color preferences, which may drive the divergence of several floral traits through correlated trait evolution.
Lemmon EM, Juenger TE. Geographic variation in hybridization across a reinforcementcontact zone of chorus frogs (Pseudacris). Ecology and Evolution [Internet]. 2017;7 (22) :9485–9502. Publisher's VersionAbstract

Reinforcement contact zones, which are secondary contact zones where species are diverging in reproductive behaviors due to selection against hybridization, represent natural laboratories for studying speciation-in-action. Here, we examined replicate localities across the entire reinforcement contact zone between North American chorus frogs Pseudacris feriarum and P. nigrita to investigate geographic variation in hybridization frequencies and to assess whether reinforcement may have contributed to increased genetic divergence within species. Previous work indicated these species have undergone reproductive character displacement (RCD) in male acoustic signals and female preferences due to reinforcement. We also examined acoustic signal variation
across the contact zone to assess whether signal characteristics reliably predict
hybrid index and to elucidate whether the degree of RCD predicts hybridization rate. Using microsatellites, mitochondrial sequences, and acoustic signal information from >1,000 individuals across >50 localities and ten sympatric focal regions, we demonstrate: (1) hybridization occurs and (2) varies substantially across the geographic range of the contact zone, (3) hybridization is asymmetric and in the direction predicted from observed patterns of asymmetric RCD, (4) in one species, genetic distance is higher between conspecific localities where one or both have been reinforced than between nonreinforced localities, after controlling for geographic distance, (5) acoustic signal characters strongly predict hybrid index, and (6) the degree of RCD does not strongly predict admixture levels. By showing that hybridization occurs in all sympatric localities,
this study provides the fifth and final line of evidence that reproductive character
displacement is due to reinforcement in the chorus frog contact zone.  Furthermore, this work suggests that the dual action of cascade reinforcement and partial geographic isolation is promoting genetic diversification within one of the reinforced species.

Zhang L, Juenger TE, Osorio JM, Behrman KD. Sensitivity Analysis of the APEX Model for AssessingSustainability of Switchgrass Grown for Biofuel Productionin Central Texas. Bioenerg. Res. [Internet]. 2017 : Publisher's VersionAbstract

Crop simulation models are increasingly being used to understand the feasibility of large-scale cellulosic biofuel production along with the multi-dimensional impacts on environmental sustainability. However, how the uncertainty in model parameters impacts model performance for sustainability is unclear. In this case study, sensitivity analyses were conducted for three switchgrass sustainability metrics: total biomass production, nitrogen loss, and soil carbon change using the APEX (Agricultural Policy/Environmental eXtender) model. Fifteen out of the 45 parameters (25 crop growth (CROP) parameters and 20 additional model parameters (PARM)) were identified as influential for the three sustainability metrics for three lowland genotypes (WBC, AP13, and KAN) across two locations (Temple, TX, and Austin, TX). Our sensitivity results showed that parameter importance was not dependent on the genotypes but depended on the variables of interest, and differed only slightly between locations. Influential belowground-related CROP and PARM parameters were identified for each sustainability metric, indicating that belowground-related parameters are just as important as commonly measured aboveground CROP parameters. Further investigation of the linear or non-linear relationships and the two-way interactions between each of the individual influential parameters with the three sustainability metrics reflected the functions and characteristics within the APEX model and the interrelations among different processes. Strong interactions between the most influential parameters for total biomass, nitrogen loss, and soil carbon change also highlighted the importance of accurately setting these parameters. Identification of influential model parameters for switchgrass sustainability may help guide field measurements and provide further understanding of the interrelated processes in the APEX model. Furthermore, future field experiments can be designed to measure these influential parameters and understand the non-linear relationships identified between influential parameters and response variables. More accurate model parameterization will help improve APEX model performance and our understanding of the possible underlying physiological mechanisms.

Gordon SP, Contreras-Moreira B, Woods DP, Des Marais DL, Burgess D, Shu S, Stritt C, Roulin AC, Schackwitz W, Tyler L, et al. Extensive gene content variation in theBrachypodium distachyon pan-genome correlateswith population structure. Nature Communications [Internet]. 2017;(2184) :doi:10.1038/s41467-017-02292-8. Publisher's VersionAbstract
While prokaryotic pan-genomes have been shown to contain many more genes than any individual organism, the prevalence and functional significance of differentially present genes in eukaryotes remains poorly understood. Whole-genome de novo assembly and annotation of 54 lines of the grass Brachypodium distachyon yield a pan-genome containing nearly twice the number of genes found in any individual genome. Genes present in all lines are enriched for essential biological functions, while genes present in only some lines are enriched for conditionally beneficial functions (e.g., defense and development), display faster evolutionary rates, lie closer to transposable elements and are less likely to be syntenic with orthologous genes in other grasses. Our data suggest that differentially present genes contribute substantially to phenotypic variation within a eukaryote species, these genes have a major influence in population genetics, and transposable elements play a key role in pan-genome evolution.
Razzaque S, Haque T, Elias SM, Rahman S, Biswas S, Schwartz S, Ismail AM, Walia H, Juenger TE, Seraj ZI. Reproductive stage physiologicaland transcriptional responsesto salinity stress in reciprocalpopulations derived from tolerant(Horkuch) and susceptible (IR29)rice. Science Reports [Internet]. 2017;7 (46138) :doi:10.1038/srep46138. Publisher's VersionAbstract
Global increase in salinity levels has made it imperative to identify novel sources of genetic variation
for tolerance traits, especially in rice. The rice landrace Horkuch, endemic to the saline coastal area of
Bangladesh, was used in this study as the source of tolerance in reciprocal crosses with the sensitive but
high-yielding IR29 variety for discovering transcriptional variation associated with salt tolerance in the
resulting populations. The cytoplasmic effect of the Horkuch background in leaves under stress showed
functional enrichment for signal transduction, DNA-dependent regulation and transport activities. In
roots the enrichment was for cell wall organization and macromolecule biosynthesis. In contrast, the
cytoplasmic effect of IR29 showed upregulation of apoptosis and downregulation of phosphorylation
across tissues relative to Horkuch. Differential gene expression in leaves of the sensitive population
showed downregulation of GO processes like photosynthesis, ATP biosynthesis and ion transport. Roots
of the tolerant plants conversely showed upregulation of GO terms like G-protein coupled receptor
pathway, membrane potential and cation transport. Furthermore, genes involved in regulating
membrane potentials were constitutively expressed only in the roots of tolerant individuals. Overall
our work has developed genetic resources and elucidated the likely mechanisms associated with the
tolerance response of the Horkuch genotype.
Kalladan R, Lasky JR, Chang TZ, Sharma S, Juenger TE, Verslues PE. Natural variation identifies genes affecting droughtinducedabscisic acid accumulation inArabidopsis thaliana. PNAS: Proceedings of the National Academy of Sciences [Internet]. 2017;114 (43) :11536-11541. Publisher's VersionAbstract
Accumulation of the stress hormone abscisic acid (ABA) in response
to drought and low water-potential controls many downstream
acclimation mechanisms. However, mechanisms controlling ABA
accumulation itself are less known. There was a 10-fold range of
variation in ABA levels among nearly 300 Arabidopsis thaliana accessions
exposed to the same low water-potential severity. Genomewide
association analysis (GWAS) identified genomic regions containing
clusters of ABA-associated SNPs. Candidate genes within
these regions included few genes with known stress or ABArelated
function. The GWAS data were used to guide reverse genetic
analysis, which found effectors of ABA accumulation. These included
plasma-membrane–localized signaling proteins such as receptor-like
kinases, aspartic protease, a putative lipid-binding START domain
protein, and other membrane proteins of unknown function as well
as a RING U-box protein and possible effect of tonoplast transport on
ABA accumulation. Putative loss-of-function polymorphisms within
the START domain protein were associated with climate factors at
accession sites of origin, indicating its potential involvement in
drought adaptation. Overall, using ABA accumulation as a basis for
a combined GWAS–reverse genetic strategy revealed the broad natural
variation in low-water-potential–induced ABA accumulation and
was successful in identifying genes that affect ABA levels and may
act in upstream drought-related sensing and signaling mechanisms.
ABA effector loci were identified even when each one was of incremental
effect, consistent with control of ABA accumulation being
distributed among the many branches of ABA metabolism or mediated
by genes with partially redundant function.
Des Marais DL, Lasky JR, Verslues PE, Chang TZ, Juenger TE. Interactive effects of water limitation and elevated temperatureon the physiology, development and fitness of diverse accessionsof Brachypodium distachyon. New Phytologist [Internet]. 2017;214 (1) :132-144. Publisher's VersionAbstract
An enduring question in plant physiology and evolution is how single genotypes of plants
optimize performance in diverse, often highly variable, environments.
We grew 35 natural accessions of the grass Brachypodium distachyon in four environments
in the glasshouse, contrasting soil water deficit, elevated temperature and their interaction.
We modeled treatment, genotype and interactive effects on leaf-level and whole-plant traits,
including fecundity. We also assessed the relationship between glasshouse-measured traits
and parameters related to climate at the place of origin.
We found abundant genetic variation in both constitutive and induced traits related to
plant–water relations. Most traits showed strong interaction between temperature and water
availability, and we observed genotype-by-environment interaction for several traits. Notably,
leaf free proline abundance showed a strong effect of genotype 9 temperature 9 water. We
found strong associations between phenology, biomass and water use efficiency (WUE) with
parameters describing climate of origin.
Plants respond to multiple stressors in ways not directly predictable from single stressors,
underscoring the complex and trait-specific mechanisms of environmental response. Climate–
trait correlations support a role for WUE and phenology in local adaptation to climate in
B. distachyon.
Milano ER, Lowry DB, Juenger TE. The genetic basis of upland/lowland ecotype divergence in switchgrass (Panicum virgatum). G3: Genes|Genomes|Genetics [Internet]. 2016. Publisher's VersionAbstract

The evolution of locally adapted ecotypes is a common phenomenon that generates diversity within plant species. However, we know surprisingly little about the genetic mechanisms underlying the locally adapted traits involved in ecotype formation. The genetic architecture underlying locally adapted traits dictates how an organism will respond to environmental selection pressures and has major implications for evolutionary ecology, conservation, and crop breeding. To understand the genetic architecture underlying the divergence of switchgrass (Panicum virgatum) ecotypes, we constructed a genetic mapping population through a four-way outbred cross between two northern upland and two southern lowland accessions. Trait segregation in this mapping population was largely consistent with multiple independent loci controlling the suite of traits that characterizes ecotype divergence. We assembled a joint linkage map using ddRADseq and mapped quantitative trait loci (QTL) for traits that are divergent between ecotypes, including flowering time, plant size, physiological processes, and disease resistance. Overall, we found that most QTL had small to intermediate effects. While we identified colocalizing QTLs for multiple traits, we did not find any large-effect QTLs that clearly controlled multiple traits through pleiotropy or tight physical linkage. These results indicate that ecologically important traits in switchgrass have a complex genetic basis and that similar loci may underlie divergence across the geographic range of the ecotypes.

Taylor SH, Lowry DB, Aspinwall MJ, Bonnette JE, Fay PA, Juenger TE. QTL and Drought Effects on Leaf Physiology in Lowland Panicum virgatum. BioEnergy Research [Internet]. 2016. Publisher's VersionAbstract

Switchgrass is a key component of plans to develop sustainable cellulosic ethanol production for bioenergy in the USA.We sought quantitative trait loci (QTL) for leaf structure and function, using the Albany full-sib mapping population, an F1 derived from lowland tetraploid parents. We also assessed both genotype × environment interactions (G×E) in response to drought and spatial trends within experimental plots, using the mapping population and check clones drawn from the parent cultivars. Phenotypes for leaf structure and
physiological performance were determined under wellwatered conditions in two consecutive years, and we applied drought to one of two replicates to test for G×E. Phenotypes for check clones varied with location in our plot and were impacted by drought, but there was limited evidence of G×E except in quantum yield (ΦPSII). Phenotypes of Albany were also influenced by plant location within our plot, and after correcting for experimental design factors and spatial effects,
we detected QTL for leaf size, tissue density (LMA), and stomatal conductance (gs). Clear evidence of G×E was detected at a QTL for intrinsic water use efficiency (iWUE) that was expressed only under drought. Loci influencing physiological traits had small additive effects, showed complex patterns of heritability, and did not co-localize with QTL for morphological traits. These insights into the genetic architecture of leaf structure and function set the stage for consideration of leaf physiological phenotypes as a component of switchgrass improvement for bioenergy purposes.

Milano ER, Kenney AM, Juenger TE. Adaptive differentiation in floral traits in the presence of high gene flow in scarlet gilia (Ipomopsis aggregata). Molecular Ecology [Internet]. 2016. Publisher's VersionAbstract

Plant-pollinator interactions are thought to be major drivers of floral trait diversity. However, the relative importance of divergent pollinator-mediated selection versus neutral processes in floral character evolution has rarely been explored. We tested for adaptive floral trait evolution by comparing differentiation at neutral genetic loci to differentiation at quantitative floral traits in a putative Ipomopsis aggregata hybrid zone. Typical I. aggregata subsp. candida displays slender white tubular flowers that are typical of flowers pollinated by hawkmoths and subsp. collina displays robust red tubular flowers typical of flowers pollinated by hummingbirds; yet hybrid flower morphs are abundant across the East Slope of the Colorado Rockies. We estimated genetic differentiation (FST ) for nuclear and chloroplast microsatellite loci and used a half-sib design to calculate quantitative trait divergence (QST ) from collection sites across the morphological hybrid zone. We found little evidence for population structure and estimated mean FST to be 0.032. QST values for several floral traits including corolla tube length and width, color, and nectar volume were large and significantly greater than mean FST . We performed multivariate comparisons of neutral loci to genetic correlations within and between populations and found a strong signal for divergent selection, suggesting that specific combinations of floral display and reward traits may be the targets of selection. Our results show little support for historical subspecies categories, yet floral traits are more diverged than expected due to drift alone. Non-neutral divergence for multivariate quantitative traits suggests that selection by pollinators is maintaining a correlation between display and reward traits.

Lovell JT, Schwartz S, Lowry DB, Shakirov EV, Bonnette JE, Weng X, Wang M, Johnson J, Sreedasyam A, Plott C, et al. Drought responsive gene expression regulatory divergence between upland and lowland ecotypesof a perennial C4 grass. Genome Research [Internet]. 2016. Publisher's VersionAbstract

Climatic adaptation is an example of a genotype-by-environment interaction (G×E) of fitness. Selection upon gene expression regulatory variation can contribute to adaptive phenotypic diversity; however, surprisingly few studies have examined how genome-wide patterns of gene expression G×E are manifested in response to environmental stress and other selective agents that cause climatic adaptation. Here, we characterize drought-responsive expression divergence between upland (drought-adapted) and lowland (mesic) ecotypes of the perennial C4 grass, Panicum hallii, in natural field conditions. Overall, we find that cis-regulatory elements contributed to gene expression divergence across 47% of genes, 7.2% of which exhibit drought-responsive G×E. While less well-represented, we observe 1294 genes (7.8%) with trans effects. Trans-by-environment
interactions are weaker and much less common than cis G×E, occurring in only 0.7% of trans-regulated genes. Finally, gene expression heterosis is highly enriched in expression phenotypes with significant G×E. As such, modes of inheritance that drive heterosis, such as dominance or overdominance, may be common among G×E genes. Interestingly, motifs specific to drought-responsive transcription factors are highly enriched in the promoters of genes exhibiting G×E and trans regulation, indicating that expression G×E and heterosis may result from the evolution of transcription factors or their binding sites. P. hallii serves as the genomic model for its close relative and emerging biofuel crop, switchgrass (Panicum virgatum). Accordingly, the results here not only aid in the discovery of the genetic mechanisms that underlie local adaptation but also provide a foundation to improve switchgrass yield under water-limited conditions.

Campitelli BE, Des Marais DL, Juenger TE. Ecological interactions and the fitness effect of water-use efficiency: Competition and drought alter the impact of natural MPK12 alleles in Arabidopsis. Ecology Letters [Internet]. 2016. Publisher's VersionAbstract

The presence of substantial genetic variation for water-use efficiency (WUE) suggests that natural selection plays a role in maintaining alleles that affect WUE. Soil water deficit can reduce plant survival, and is likely to impose selection to increase WUE, whereas competition for resources may select for decreased WUE to ensure water acquisition. We tested the fitness consequences of natural allelic variation in a single gene (MPK12) that influences WUE in Arabidopsis, using transgenic lines contrasting in MPK12 alleles, under four treatments; drought/competition, drought/no competition, well-watered/competition, well-watered/no competition. Results revealed an allele 9 environment interaction: Low WUE plants performed better in competition, resulting from increased resource consumption. Contrastingly, high WUE individuals performed better in no competition, irrespective of water availability, presumably from enhanced water conservation and nitrogen acquisition. Our findings suggest that selection can influence MPK12 evolution, and represents the first assessment of plant fitness resulting from natural allelic variation at a single locus affecting WUE.

Aspinwall MJ, Fay PA, Hawkes CV, Lowry DB, Khasanova A, Bonnette JE, Whitaker BK, Johnson N, Juenger TE. Intraspecific variation in precipitation responses of a widespread C4 grass depends on site water limitation. Journal of Plant Ecology [Internet]. 2016. Publisher's VersionAbstract

Variation in precipitation strongly influences plant growth, species distributions and genetic diversity. Intraspecific variation in phenotypic plasticity, the ability of a genotype to alter its growth, morphology or physiology in response to the environment, could influence species responses to changing precipitation and climate change. Despite this, the patterns and mechanisms of intraspecific variation in plasticity to variable precipitation, and the degree to which genotype responses to precipitation are influenced by variation in edaphic conditions, remain poorly understood. Thus, we determined whether genotypes of a widespread C4 grass (Panicum virgatum L., switchgrass) varied in aboveground productivity in response to changes in precipitation, and if site edaphic conditions modified genotype aboveground productivity responses to precipitation. We also determined if genotype productivity responses to precipitation are related to plasticity in underlying growth and phenological traits.
Nine P. virgatum genotypes originating from an aridity gradient were grown under four treatments spanning the 10th to the 90th percentiles of annual precipitation at two sites in central Texas: one site with deep, fine-textured soils and another site with shallow, coarse-textured soils. We measured volumetric soil water content (VWC), aboveground net primary productivity (ANPP), tiller production (tiller number), average tiller mass, canopy height, leaf area index (LAI) and flowering time on all plants at both sites and examined genotype responses to changes in precipitation.
Important Findings
Across precipitation treatments, VWC was 39% lower and more variable at the site with shallow, coarse-textured soils compared to the site with deep, fine-textured soils. ANPP averaged across genotypes and precipitation treatments was also 103% higher at the site with deep, fine-textured soils relative to the site with shallow, coarse-textured soils, indicating substantial differences in site water limitation. Where site water limitation was higher, ANPP of most genotypes increased with increasing precipitation. Where site water limitation was less, genotypes expressed variable plasticity in response to precipitation, from no change to almost a 5-fold increase in ANPP with increasing precipitation. Genotype ANPP increased with greater tiller mass, LAI and later flowering time at both sites, but not with tiller number at either site. Genotype ANPP plasticity increased with genotype tiller mass and LAI plasticity at the site with deep, fine-textured soils, and only with genotype tiller mass plasticity at the site with shallow, coarse-textured soils. Thus, variation in genotype ANPP plasticity was explained primarily by variation in tiller and leaf growth. Genotype ANPP plasticity was not associated with temperature or aridity at the genotype’s origin. Edaphic factors such as soil depth and texture may alter genotype ANPP responses to precipitation, and the underlying growth traits contributing to the ANPP response. Thus, edaphic factors may contribute to spatial variation in genotype performance and success under altered precipitation.

Fransz P, Linc G, Lee C-R, Aflitos SA, Lasky JR, Toomajian C, Ali H, Peters J, Van Dam P, Ji X, et al. Molecular, genetic and evolutionary analysis of a paracentric inversion in Arabidopsis thaliana. The Plant Journal [Internet]. 2016. Publisher's VersionAbstract

Chromosomal inversions can provide windows onto the cytogenetic, molecular, evolutionary and demographic histories of a species. Here we investigate a paracentric 1.17 Mb inversion on chromosome 4 of Arabidopsis thaliana with nucleotide precision of its borders. The inversion is created by Vandal transposon activity, splitting an F-box and relocating a pericentric heterochromatin segment in juxtaposition with euchromatin without affecting the epigenetic landscape. Examination of the RegMap panel and the 1001 Arabidopsis genomes revealed more than 170 inversion accessions in Europe and North America. The SNP patterns revealed historical recombinations from which we infer diverse haplotype patterns, ancient introgression events and phylogenetic relationships. We find a robust association between the inversion and fecundity under drought. We also find linkage disequilibrium (LD) between the inverted region and the early flowering Col-FRIGIDA allele. Finally, SNP analysis elucidates the origin of the inversion to South-Eastern Europe ~5,000 years ago and the FRI-Col allele to North-West Europe, and reveals the spreading of a single haplotype to North America during the 17th to 19th century. The “American haplotype” was identified from several European localities, potentially due to return migration.

Lovell JT, Shakirov EV, Schwartz S, Lowry DB, Aspinwall MJ, Taylor SH, Bonnette JE, Palacio-Mejía JD, Hawkes CV, Fay PA, et al. Promises and challenges of eco-physiological genomics in the field: tests of drought responses in switchgrass. Plant Physiology [Internet]. 2016. Publisher's VersionAbstract

Identifying the physiological and genetic basis of stress tolerance in plants has proven to be critical to understanding adaptation in both agricultural and natural systems. However, many discoveries were initially made in the controlled conditions of greenhouses or laboratories, not in the field. To test the comparability of drought responses across field and greenhouse environments, we undertook three independent experiments using the switchgrass reference genotype Alamo AP13. We analyzed physiological and gene-expression variation across four locations, two sampling times and three years. Relatively similar physiological responses and expression coefficients of variation across experiments masked highly dissimilar gene expression responses to drought. Critically, a drought experiment utilizing small pots in the greenhouse elicited nearly identical physiological changes as an experiment conducted in the field, but an order of magnitude more differentially expressed genes. However, we were able to define a suite of several hundred genes that were differentially expressed across all experiments. This list was strongly enriched in photosynthesis, water status and reactive oxygen species responsive genes. The strong across-experiment correlations between physiological plasticity—but not differential gene expression—highlight the complex and diverse genetic mechanisms that can produce phenotypically similar responses to various soil water deficits.

Des Marais DL, Razzaque S, Hernandez KM, Garvin DF, Juenger TE. Quantitative trait loci associated with natural diversity in water-useefficiency and response to soil drying in Brachypodium distachyon. Plant Science [Internet]. 2016. Publisher's VersionAbstract

All plants must optimize their growth with finite resources. Water use efficiency (WUE) measures the relationship between biomass acquisition and transpired water. In the present study, we performed two experiments to understand the genetic basis of WUE and other parameters of plant-water interaction under control and water-limited conditions. Our study used two inbred natural accessions of Brachy-podium distachyon, a model grass species with close phylogenetic affinity to temperate forage and cereal crops. First, we identify the soil water content which causes a reduction in leaf relative water content and an increase in WUE. Second, we present results from a large phenotyping experiment utilizing a recombinant inbred line mapping population derived from these same two natural accessions. We identify QTLs associated with environmentally-insensitive genetic variation in WUE, including a pair of epistatically interacting loci. We also identify QTLs associated with constitutive differences in biomass and a QTL describing an environmentally-sensitive difference in leaf carbon content. Finally, we present a new link-age map for this mapping population based on new SNP markers as well as updated genomic positions for previously described markers. Our studies provide an initial characterization of plant-water relations in B. distachyon and identify candidate genomic regions involved in WUE.

Ko DK, Rohozinski D, Song Q, Taylor SH, Juenger TE, Harmon FG, Chen JZ. Temporal Shift of Circadian-Mediated Gene Expression and Carbon Fixation Contributes to Biomass Heterosis in Maize Hybrids. PLOS Genetics [Internet]. 2016. Publisher's VersionAbstract

Heterosis has been widely used in agriculture, but the molecular mechanism for this remains largely elusive. In Arabidopsis hybrids and allopolyploids, increased photosynthetic and metabolic activities are linked to altered expression of circadian clock regulators, including CIRCADIAN CLOCK ASSOCIATED1 (CCA1). It is unknown whether a similar mechanism mediates heterosis in maize hybrids. Here we report that higher levels of carbon fixation and starch accumulation in the maize hybrids are associated with altered temporal gene expression. Two maize CCA1 homologs, ZmCCA1a and ZmCCA1b, are diurnally up-regulated in the hybrids. Expressing ZmCCA1 complements the cca1 mutant phenotype in Arabidopsis, and overexpressing ZmCCA1b disrupts circadian rhythms and biomass heterosis. Furthermore, overexpressing ZmCCA1b in maize reduced chlorophyll content and plant height. Reduced height stems from reduced node elongation but not total node number in both greenhouse and field conditions. Phenotypes are less severe in the field than in the greenhouse, suggesting that enhanced light and/or metabolic activities in the field can compensate for altered circadian regulation in growth vigor. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis reveals a temporal shift of ZmCCA1-binding targets to the early morning in the hybrids, suggesting that activation of morning-phased genes in the hybrids promotes photosynthesis and growth vigor. This temporal shift of ZmCCA1-binding targets correlated with nonadditive and additive gene expression in early and late stages of seedling development. These results could guide breeding better hybrid crops to meet the growing demand in food and bioenergy.

Des Marais DL, Juenger TE. Brachypodium and the Abiotic Environment. Plant Genetics and Genomics of Brachypodium [Internet]. 2015;18 (02 October 2015) :291-311. Publisher's VersionAbstract

Brachypodium distachyon and its congeners are found in diverse
environments throughout the temperate regions of the world. Brachypodium species also show considerable variation in life history strategy, with species representing short-season annual habits and other species persisting for multiple years. This variation in ecological setting and life history suggests the existence of considerable genetic diversity in adaptation to the abiotic environment, both in constitutive tolerance to local conditions and in the capacity of single genotypes to acclimate to changing or unpredictable conditions. We review the limited but growing empirical literature on the physiology, development, and molecular biology of the interaction of Brachypodium with the abiotic environment. We highlight how these findings inform studies of ecologically and agriculturally-important plant species, and identify areas of future research that will extend the utility of Brachypodium as a model genetic system for understanding plant-environment interactions.

Lovell JT, Mullen JL, Lowry DB, Awole K, Richards JH, Sen S, Verslues PE, Juenger TE, McKay JK. Exploiting Differential Gene Expression and Epistasis to Discover Candidate Genes for Drought-Associated QTLs in Arabidopsis thaliana. The Plant Cell [Internet]. 2015. Publisher's VersionAbstract

Soil water availability represents one of the most important selective agents for plants in nature and the single greatest abiotic determinant of agricultural productivity, yet the genetic bases of drought acclimation responses remain poorly understood. Here, we developed a systems-genetic approach to characterize quantitative trait loci (QTLs), physiological traits and genes that affect responses to soil moisture deficit in the TSUxKAS mapping population of Arabidopsis thaliana. To determine the effects of candidate genes underlying QTLs, we analyzed gene expression as a covariate within the QTL model in an effort to mechanistically link markers, RNA expression, and the phenotype. This strategy produced ranked lists of candidate genes for several drought-associated traits, including water use efficiency, growth, abscisic acid concentration (ABA), and proline concentration. As a proof of concept, we recovered known causal loci for several QTLs. For other traits, including ABA, we identified novel loci not previously associated with drought. Furthermore, we documented natural variation at two key steps in proline metabolism and demonstrated that the mitochondrial genome differentially affects genomic QTLs to influence proline accumulation. These findings demonstrate that linking genome, transcriptome, and phenotype data holds
great promise to extend the utility of genetic mapping, even when QTL effects are modest or complex.