A generalist-specialist trade-off between switchgrass cytotypes impacts climate adaptation and geographic range.

Polyploidy results from whole-genome duplication and is a unique form of heritable variation with pronounced evolutionary implications. Different ploidy levels, or cytotypes, can exist within a single species, and such systems provide an opportunity to assess how ploidy variation alters phenotypic novelty, adaptability, and fitness, which can, in turn, drive the development of unique ecological niches that promote the coexistence of multiple cytotypes. Switchgrass, Panicum virgatum, is a widespread, perennial C4 grass in North America with multiple naturally occurring cytotypes, primarily tetraploids (4×) and octoploids (8×). Using a combination of genomic, quantitative genetic, landscape, and niche modeling approaches, we detect divergent levels of genetic admixture, evidence of niche differentiation, and differential environmental sensitivity between switchgrass cytotypes. Taken together, these findings support a generalist (8×)­specialist (4×) trade-off. Our results indicate that the 8× represent a unique combination of genetic variation that has allowed the expansion of switchgrass' ecological niche and thus putatively represents a valuable breeding resource.

Genetic dissection of natural variation in oilseed traits of camelina by whole-genome resequencing and QTL mapping.

Camelina [Camelina sativa (L.) Crantz] is an oilseed crop in the Brassicaceae family that is currently being developed as a source of bioenergy and healthy fatty acids. To facilitate modern breeding efforts through marker-assisted selection and biotechnology, we evaluated genetic variation among a worldwide collection of 222 camelina accessions. We performed whole-genome resequencing to obtain single nucleotide polymorphism (SNP) markers and to analyze genomic diversity. We also conducted phenotypic field evaluations in two consecutive seasons for variations in key agronomic traits related to oilseed production such as seed size, oil content (OC), fatty acid composition, and flowering time. We determined the population structure of the camelina accessions using 161,301 SNPs. Further, we identified quantitative trait loci (QTL) and candidate genes controlling the above field-evaluated traits by genome-wide association studies (GWAS) complemented with linkage mapping using a recombinant inbred line (RIL) population. Characterization of the natural variation at the genome and phenotypic levels provides valuable resources to camelina genetic studies and crop improvement. The QTL and candidate genes should assist in breeding of advanced camelina varieties that can be integrated into the cropping systems for the production of high yield of oils of desired fatty acid composition.

Geographic patterns of genomic diversity and structure in the C4 grass Panicum hallii across its natural distribution.

Geographic patterns of within-species genomic diversity are shaped by evolutionary processes, life history and historical and contemporary factors. New genomic approaches can be used to infer the influence of such factors on the current distribution of infraspecific lineages. In this study, we evaluated the genomic and morphological diversity as well as the genetic structure of the C4 grass Panicum hallii across its complex natural distribution in North America. We sampled extensively across the natural range of P. hallii in Mexico and the USA to generate double-digestion restriction-associated DNA (ddRAD) sequence data for 423 individuals from 118 localities. We used these individuals to study the divergence between the two varieties of P. hallii, P. hallii var. filipes and P. hallii var. hallii as well as the genetic diversity and structure within these groups. We also examined the possibility of admixture in the geographically sympatric zone shared by both varieties, and assessed distribution shifts related with past climatic fluctuations. There is strong genetic and morphological divergence between the varieties and consistent genetic structure defining seven genetic clusters that follow major ecoregions across the range. South Texas constitutes a hotspot of genetic diversity with the co-occurrence of all genetic clusters and admixture between the two varieties. It is likely a recolonization and convergence point of populations that previously diverged in isolation during fragmentation events following glaciation periods.

Genomic mechanisms of climate adaptation in polyploid bioenergy switchgrass.

Long-term climate change and periodic environmental extremes threaten food and fuel security1 and global crop productivity2-4. Although molecular and adaptive breeding strategies can buffer the effects of climatic stress and improve crop resilience5, these approaches require sufficient knowledge of the genes that underlie productivity and adaptation6-knowledge that has been limited to a small number of well-studied model systems. Here we present the assembly and annotation of the large and complex genome of the polyploid bioenergy crop switchgrass (Panicum virgatum). Analysis of biomass and survival among 732 resequenced genotypes, which were grown across 10 common gardens that span 1,800 km of latitude, jointly revealed extensive genomic evidence of climate adaptation. Climate-gene-biomass associations were abundant but varied considerably among deeply diverged gene pools. Furthermore, we found that gene flow accelerated climate adaptation during the postglacial colonization of northern habitats through introgression of alleles from a pre-adapted northern gene pool. The polyploid nature of switchgrass also enhanced adaptive potential through the fractionation of gene function, as there was an increased level of heritable genetic diversity on the nondominant subgenome. In addition to investigating patterns of climate adaptation, the genome resources and gene-trait associations developed here provide breeders with the necessary tools to increase switchgrass yield for the sustainable production of bioenergy.

A genome assembly and the somatic genetic and epigenetic mutation rate in a wild long-lived perennial Populus trichocarpa.

BACKGROUND: Plants can transmit somatic mutations and epimutations to offspring, which in turn can affect fitness. Knowledge of the rate at which these variations arise is necessary to understand how plant development contributes to local adaption in an ecoevolutionary context, particularly in long-lived perennials. RESULTS: Here, we generate a new high-quality reference genome from the oldest branch of a wild Populus trichocarpa tree with two dominant stems which have been evolving independently for 330 years. By sampling multiple, age-estimated branches of this tree, we use a multi-omics approach to quantify age-related somatic changes at the genetic, epigenetic, and transcriptional level. We show that the per-year somatic mutation and epimutation rates are lower than in annuals and that transcriptional variation is mainly independent of age divergence and cytosine methylation. Furthermore, a detailed analysis of the somatic epimutation spectrum indicates that transgenerationally heritable epimutations originate mainly from DNA methylation maintenance errors during mitotic rather than during meiotic cell divisions. CONCLUSION: Taken together, our study provides unprecedented insights into the origin of nucleotide and functional variation in a long-lived perennial plant.

Enhancing microRNA167A expression in seed decreases the α-linolenic acid content and increases seed size in Camelina sativa.

Despite well established roles of microRNAs in plant development, few aspects have been addressed to understand their effects in seeds especially on lipid metabolism. In this study, we showed that overexpressing microRNA167A (miR167OE) in camelina (Camelina sativa) under a seed-specific promoter changed fatty acid composition and increased seed size. Specifically, the miR167OE seeds had a lower α-linolenic acid with a concomitantly higher linoleic acid content than the wild-type. This decreased level of fatty acid desaturation corresponded to a decreased transcriptional expression of the camelina fatty acid desaturase3 (CsFAD3) in developing seeds. MiR167 targeted the transcription factor auxin response factor (CsARF8) in camelina, as had been reported previously in Arabidopsis. Chromatin immunoprecipitation experiments combined with transcriptome analysis indicated that CsARF8 bound to promoters of camelina bZIP67 and ABI3 genes. These transcription factors directly or through the ABI3-bZIP12 pathway regulate CsFAD3 expression and affect α-linolenic acid accumulation. In addition, to decipher the miR167A-CsARF8 mediated transcriptional cascade for CsFAD3 suppression, transcriptome analysis was conducted to implicate mechanisms that regulate seed size in camelina. Expression levels of many genes were altered in miR167OE, including orthologs that have previously been identified to affect seed size in other plants. Most notably, genes for seed coat development such as suberin and lignin biosynthesis were down-regulated. This study provides valuable insights into the regulatory mechanism of fatty acid metabolism and seed size determination, and suggests possible approaches to improve these important traits in camelina.

Genome-Wide Association Study in Pseudo-F2 Populations of Switchgrass Identifies Genetic Loci Affecting Heading and Anthesis Dates.

Switchgrass (Panicum virgatum) is a native prairie grass and valuable bio-energy crop. The physiological change from juvenile to reproductive adult can draw important resources away from growth into producing reproductive structures, thereby limiting the growth potential of early flowering plants. Delaying the flowering of switchgrass is one approach by which to increase total biomass. The objective of this research was to identify genetic variants and candidate genes for controlling heading and anthesis in segregating switchgrass populations. Four pseudo-F2 populations (two pairs of reciprocal crosses) were developed from lowland (late flowering) and upland (early flowering) ecotypes, and heading and anthesis dates of these populations were collected in Lafayette, IN and DeKalb, IL in 2015 and 2016. Across 2 years, there was a 34- and 73-day difference in heading and a 52- and 75-day difference in anthesis at the Lafayette and DeKalb locations, respectively. A total of 37,901 single nucleotide polymorphisms obtained by exome capture sequencing of the populations were used in a genome-wide association study (GWAS) that identified five significant signals at three loci for heading and two loci for anthesis. Among them, a homolog of FLOWERING LOCUS T on chromosome 5b associated with heading date was identified at the Lafayette location across 2 years. A homolog of ARABIDOPSIS PSEUDO-RESPONSE REGULATOR 5, a light modulator in the circadian clock associated with heading date was detected on chromosome 8a across locations and years. These results demonstrate that genetic variants related to floral development could lend themselves to a long-term goal of developing late flowering varieties of switchgrass with high biomass yield.

Genome-wide associations with flowering time in switchgrass using exome-capture sequencing data.

Flowering time is a major determinant of biomass yield in switchgrass (Panicum virgatum), a perennial bioenergy crop, because later flowering allows for an extended period of vegetative growth and increased biomass production. A better understanding of the genetic regulation of flowering time in switchgrass will aid the development of switchgrass varieties with increased biomass yields, particularly at northern latitudes, where late-flowering but southern-adapted varieties have high winter mortality. We use genotypes derived from recently published exome-capture sequencing, which mitigates challenges related to the large, highly repetitive and polyploid switchgrass genome, to perform genome-wide association studies (GWAS) using flowering time data from a switchgrass association panel in an effort to characterize the genetic architecture and genes underlying flowering time regulation in switchgrass. We identify associations with flowering time at multiple loci, including in a homolog of FLOWERING LOCUS T and in a locus containing TIMELESS, a homolog of a key circadian regulator in animals. Our results suggest that flowering time variation in switchgrass is due to variation at many positions across the genome. The relationship of flowering time and geographic origin indicates likely roles for genes in the photoperiod and autonomous pathways in generating switchgrass flowering time variation.

Genotypic diversity effects on biomass production in native perennial bioenergy cropping systems.

The perennial grass species that are being developed as biomass feedstock crops harbor extensive genotypic diversity, but the effects of this diversity on biomass production are not well understood. We investigated the effects of genotypic diversity in switchgrass (Panicum virgatum) and big bluestem (Andropogon gerardii) on perennial biomass cropping systems in two experiments conducted over 2008-2014 at a 5.4-ha fertile field site in northeastern Illinois, USA. We varied levels of switchgrass and big bluestem genotypic diversity using various local and nonlocal cultivars - under low or high species diversity, with or without nitrogen inputs - and quantified establishment, biomass yield, and biomass composition. In one experiment ('agronomic trial'), we compared three switchgrass cultivars in monoculture to a switchgrass cultivar mixture and three different species mixtures, with or without N fertilization. In another experiment ('diversity gradient'), we varied diversity levels in switchgrass and big bluestem (1, 2, 4, or 6 cultivars per plot), with one or two species per plot. In both experiments, cultivar mixtures produced yields equivalent to or greater than the best cultivars. In the agronomic trial, the three switchgrass mixture showed the highest production overall, though not significantly different than best cultivar monoculture. In the diversity gradient, genotypic mixtures had one-third higher biomass production than the average monoculture, and none of the monocultures were significantly higher yielding than the average mixture. Year-to-year variation in yields was lowest in the three-cultivar switchgrass mixtures and Cave-In-Rock (the southern Illinois cultivar) and also reduced in the mixture of switchgrass and big bluestem relative to the species monocultures. The effects of genotypic diversity on biomass composition were modest relative to the differences among species and genotypes. Our findings suggest that local genotypes can be included in biomass cropping systems without compromising yields and that genotypic mixtures could help provide high, stable yields of high-quality biomass feedstocks.

Thermal Density Functional Theory: Time-Dependent Linear Response and Approximate Functionals from the Fluctuation-Dissipation Theorem.

The van Leeuwen proof of linear-response time-dependent density functional theory (TDDFT) is generalized to thermal ensembles. This allows generalization to finite temperatures of the Gross-Kohn relation, the exchange-correlation kernel of TDDFT, and fluctuation dissipation theorem for DFT. This produces a natural method for generating new thermal exchange-correlation approximations.

Examining the efficacy of a genotyping-by-sequencing technique for population genetic analysis of the mushroom Laccaria bicolor and evaluating whether a reference genome is necessary to assess homology.

Given the diversity and ecological importance of Fungi, there is a lack of population genetic research on these organisms. The reason for this can be explained in part by their cryptic nature and difficulty in identifying genets. In addition the difficulty (relative to plants and animals) in developing molecular markers for fungal population genetics contributes to the lack of research in this area. This study examines the ability of restriction-site associated DNA (RAD) sequencing to generate SNPs in Laccaria bicolor. Eighteen samples of morphologically identified L. bicolor from the United States and Europe were selected for this project. The RAD sequencing method produced anywhere from 290 000 to more than 3 000 000 reads. Mapping these reads to the genome of L. bicolor resulted in 84 000-940 000 unique reads from individual samples. Results indicate that incorporation of non-L. bicolor taxa into the analysis resulted in a precipitous drop in shared loci among samples, suggests the potential of these methods to identify cryptic species. F-statistics were easily calculated, although an observable "noise" was detected when using the "All Loci" treatment versus filtering loci to those present in at least 50% of the individuals. The data were analyzed with tests of Hardy-Weinburg equilibrium, population genetic statistics (FIS and FST), and population structure analysis using the program Structure. The results provide encouraging feedback regarding the potential utility of these methods and their data for population genetic analysis. We were unable to draw conclusions of life history of L. bicolor populations from this dataset, given the small sample size. The results of this study indicate the potential of these methods to address population genetics and general life history questions in the Agaricales. Further research is necessary to explore the specific application of these methods in the Agaricales or other fungal groups.

Population genomic variation reveals roles of history, adaptation and ploidy in switchgrass.

Geographic patterns of genetic variation are shaped by multiple evolutionary processes, including genetic drift, migration and natural selection. Switchgrass (Panicum virgatum L.) has strong genetic and adaptive differentiation despite life history characteristics that promote high levels of gene flow and can homogenize intraspecific differences, such as wind-pollination and self-incompatibility. To better understand how historical and contemporary factors shape variation in switchgrass, we use genotyping-by-sequencing to characterize switchgrass from across its range at 98 042 SNPs. Population structuring reflects biogeographic and ploidy differences within and between switchgrass ecotypes and indicates that biogeographic history, ploidy incompatibilities and differential adaptation each have important roles in shaping ecotypic differentiation in switchgrass. At one extreme, we determine that two Panicum taxa are not separate species but are actually conspecific, ecologically divergent types of switchgrass adapted to the extreme conditions of coastal sand dune habitats. Conversely, we identify natural hybrids among lowland and upland ecotypes and visualize their genome-wide patterns of admixture. Furthermore, we determine that genetic differentiation between primarily tetraploid and octoploid lineages is not caused solely by ploidy differences. Rather, genetic diversity in primarily octoploid lineages is consistent with a history of admixture. This suggests that polyploidy in switchgrass is promoted by admixture of diverged lineages, which may be important for maintaining genetic differentiation between switchgrass ecotypes where they are sympatric. These results provide new insights into the mechanisms shaping variation in widespread species and provide a foundation for dissecting the genetic basis of adaptation in switchgrass.

Adaptations between ecotypes and along environmental gradients in Panicum virgatum.

Determining the patterns and mechanisms of natural selection in the wild is of fundamental importance to understanding the differentiation of populations and the evolution of new species. However, it is often unknown the extent to which adaptive genetic variation is distributed among ecotypes between distinct habitats versus along large-scale geographic environmental gradients, such as those that track latitude. Classic studies of selection in the wild in switchgrass, Panicum virgatum, tested for adaptation at both of these levels of natural variation. Here we review what these field experiments and modern agronomic field trials have taught us about natural variation and selection at both the ecotype and environmental gradient levels in P. virgatum. With recent genome sequencing efforts in P. virgatum, it is poised to become an excellent system for understanding the adaptation of grassland species across the eastern half of North America. The identification of genetic loci involved in different types of adaptations will help to understand the evolutionary mechanisms of diversification within P. virgatum and provide useful information for the breeding of high-yielding cultivars for different ecoregions.

Molecular dynamics simulations of classical stopping power.

Molecular dynamics can provide very accurate tests of classical kinetic theory; for example, unambiguous comparisons can be made for classical particles interacting via a repulsive 1/r potential. The plasma stopping power problem, of great interest in its own right, provides an especially stringent test of a velocity-dependent transport property. We have performed large-scale (~10(4)-10(6) particles) molecular dynamics simulations of charged-particle stopping in a classical electron gas that span the weak to moderately strong intratarget coupling regimes. Projectile-target coupling is varied with projectile charge and velocity. Comparisons are made with disparate kinetic theories (both Boltzmann and Lenard-Balescu classes) and fully convergent theories to establish regimes of validity. We extend these various stopping models to improve agreement with the MD data and provide a useful fit to our results.

Wave packet spreading and localization in electron-nuclear scattering.

The wave packet molecular dynamics (WPMD) method provides a variational approximation to the solution of the time-dependent Schrödinger equation. Its application in the field of high-temperature dense plasmas has yielded diverging electron width (spreading), which results in diminishing electron-nuclear interactions. Electron spreading has previously been ascribed to a shortcoming of the WPMD method and has been counteracted by various heuristic additions to the models used. We employ more accurate methods to determine if spreading continues to be predicted by them and how WPMD can be improved. A scattering process involving a single dynamic electron interacting with a periodic array of statically screened protons is used as a model problem for comparison. We compare the numerically exact split operator Fourier transform method, the Wigner trajectory method, and the time-dependent variational principle (TDVP). Within the framework of the TDVP, we use the standard variational form of WPMD, the single Gaussian wave packet (WP), as well as a sum of Gaussian WPs, as in the split WP method. Wave packet spreading is predicted by all methods, so it is not the source of the unphysical diminishing of electron-nuclear interactions in WPMD at high temperatures. Instead, the Gaussian WP's inability to correctly reproduce breakup of the electron's probability density into localized density near the protons is responsible for the deviation from more accurate predictions. Extensions of WPMD must include a mechanism for breakup to occur in order to yield dynamics that lead to accurate electron densities.

Molecular dynamics simulations and generalized Lenard-Balescu calculations of electron-ion temperature equilibration in plasmas.

We study the problem of electron-ion temperature equilibration in plasmas. We consider pure H at various densities and temperatures and Ar-doped H at temperatures high enough so that the Ar is fully ionized. Two theoretical approaches are used: classical molecular dynamics (MD) with statistical two-body potentials and a generalized Lenard-Balescu (GLB) theory capable of treating multicomponent weakly coupled plasmas. The GLB is used in two modes: (1) with the quantum dielectric response in the random-phase approximation (RPA) together with the pure Coulomb interaction and (2) with the classical (ℏ→0) dielectric response (both with and without local-field corrections) together with the statistical potentials. We find that the MD results are described very well by classical GLB including the statistical potentials and without local-field corrections (RPA only); worse agreement is found when static local-field effects are included, in contradiction to the classical pure-Coulomb case with like charges. The results of the various approaches are all in excellent agreement with pure-Coulomb quantum GLB when the temperature is high enough. In addition, we show that classical calculations with statistical potentials derived from the exact quantum two-body density matrix produce results in far better agreement with pure-Coulomb quantum GLB than classical calculations performed with older existing statistical potentials.

Genomic diversity in switchgrass (Panicum virgatum): from the continental scale to a dune landscape.

Connecting broad-scale patterns of genetic variation and population structure to genetic diversity on a landscape is a key step towards understanding historical processes of migration and adaptation. New genomic approaches can be used to increase the resolution of phylogeographic studies while reducing locus sampling effects and circumventing ascertainment bias. Here, we use a novel approach based on high-throughput sequencing to characterize genetic diversity in complete chloroplast genomes and >10,000 nuclear loci in switchgrass, at continental and landscape scales. Switchgrass is a North American tallgrass species, which is widely used in conservation and perennial biomass production, and shows strong ecotypic adaptation and population structure across the continental range. We sequenced 40.9 billion base pairs from 24 individuals from across the species' range and 20 individuals from the Indiana Dunes. Analysis of plastome sequence revealed 203 variable SNP sites that define eight haplogroups, which are differentiated by 4-127 SNPs and confirmed by patterns of indel variation. These include three deeply divergent haplogroups, which correspond to the previously described lowland-upland ecotypic split and a novel upland haplogroup split that dates to the mid-Pleistocene. Most of the plastome haplogroup diversity present in the northern switchgrass range, including in the Indiana Dunes, originated in the mid- or upper Pleistocene prior to the most recent postglacial recolonization. Furthermore, a recently colonized landscape feature (approximately 150 ya) in the Indiana Dunes contains several deeply divergent upland haplogroups. Nuclear markers also support a deep lowland-upland split, followed by limited gene flow, and show extensive gene flow in the local population of the Indiana Dunes.

A distance-weighted interaction map reveals a previously uncharacterized layer of the Bacillus subtilis spore coat.

Bacillus subtilis spores are encased in a protein assembly called the spore coat that is made up of at least 70 different proteins. Conventional electron microscopy shows the coat to be organized into two distinct layers. Because the coat is about as wide as the theoretical limit of light microscopy, quantitatively measuring the localization of individual coat proteins within the coat is challenging. We used fusions of coat proteins to green fluorescent protein to map genetic dependencies for coat assembly and to define three independent subnetworks of coat proteins. To complement the genetic data, we measured coat protein localization at subpixel resolution and integrated these two data sets to produce a distance-weighted genetic interaction map. Using these data, we predict that the coat comprises at least four spatially distinct layers, including a previously uncharacterized glycoprotein outermost layer that we name the spore crust. We found that crust assembly depends on proteins we predicted to localize to the crust. The crust may be conserved in all Bacillus spores and may play critical functions in the environment.

The coat morphogenetic protein SpoVID is necessary for spore encasement in Bacillus subtilis.

Endospores formed by Bacillus subtilis are encased in a tough protein shell known as the coat, which consists of at least 70 different proteins. We investigated the process of spore coat morphogenesis using a library of 40 coat proteins fused to green fluorescent protein and demonstrate that two successive steps can be distinguished in coat assembly. The first step, initial localization of proteins to the spore surface, is dependent on the coat morphogenetic proteins SpoIVA and SpoVM. The second step, spore encasement, requires a third protein, SpoVID. We show that in spoVID mutant cells, most coat proteins assembled into a cap at one side of the developing spore but failed to migrate around and encase it. We also found that SpoIVA directly interacts with SpoVID. A domain analysis revealed that the N-terminus of SpoVID is required for encasement and is a structural homologue of a virion protein, whereas the C-terminus is necessary for the interaction with SpoIVA. Thus, SpoVM, SpoIVA and SpoVID are recruited to the spore surface in a concerted manner and form a tripartite machine that drives coat formation and spore encasement.

The Bacillus subtilis spore coat protein interaction network.

Bacterial spores are surrounded by a morphologically complex, mechanically flexible protein coat, which protects the spore from toxic molecules. The interactions among the over 50 proteins that make up the coat remain poorly understood. We have used cell biological and protein biochemical approaches to identify novel coat proteins in Bacillus subtilis and describe the network of their interactions, in order to understand coat assembly and the molecular basis of its protective functions and mechanical properties. Our analysis characterizes the interactions between 32 coat proteins. This detailed view reveals a complex interaction network. A key feature of the network is the importance of a small subset of proteins that direct the assembly of most of the coat. From an analysis of the network topology, we propose a model in which low-affinity interactions are abundant in the coat and account, to a significant degree, for the coat's mechanical properties as well as structural variation between spores.