The reference genome and abiotic stress responses of the model perennial grass Brachypodium sylvaticum.

Perennial grasses are important forage crops and emerging biomass crops and have the potential to be more sustainable grain crops. However, most perennial grass crops are difficult experimental subjects due to their large size, difficult genetics, and/or their recalcitrance to transformation. Thus, a tractable model perennial grass could be used to rapidly make discoveries that can be translated to perennial grass crops. Brachypodium sylvaticum has the potential to serve as such a model because of its small size, rapid generation time, simple genetics, and transformability. Here, we provide a high-quality genome assembly and annotation for B. sylvaticum, an essential resource for a modern model system. In addition, we conducted transcriptomic studies under 4 abiotic stresses (water, heat, salt, and freezing). Our results indicate that crowns are more responsive to freezing than leaves which may help them overwinter. We observed extensive transcriptional responses with varying temporal dynamics to all abiotic stresses, including classic heat-responsive genes. These results can be used to form testable hypotheses about how perennial grasses respond to these stresses. Taken together, these results will allow B. sylvaticum to serve as a truly tractable perennial model system.

The effect of population density on outcomes of major trauma patients in Ireland.

BACKGROUND: Time-sensitive emergencies in areas of low population density have statistically poorer outcomes. This includes incidents of major trauma. This study assesses the effect that population density at a receiving hospital of a major trauma patient has on survival. METHODS: Patients meeting Trauma Audit Research Network criteria for major trauma from 2016 to 2020 in Ireland were included in this retrospective observational study. Incident data were retrieved from the Major Trauma Audit, while data on population density were calculated from Irish state sources. The primary outcome measure of survival to discharge was compared to population density using logistic regression, adjusted for demographic and incident variables. Records were divided into population density tertiles to assess for between-group differences in potential predictor variables. RESULTS: Population density at a receiving hospital had no impact on mortality in Irish major trauma patients from our logistic regression model (OR = 1.01, 95% CI 0.98-1.05, p = 0.53). Factors that did have an impact were age, Charlson Comorbidity Index, Injury Severity Score, and the presence of an Orthopaedic Surgery service at the receiving hospital (all p < 0.001). Age and Charlson Comorbidity Index differed slightly by population density tertile; both were higher in areas of high population density (all p < 0.001). CONCLUSIONS: Survival to discharge in Irish major trauma patients does not differ substantially based on population density. This is an important finding as Ireland moves to a new trauma system, with features based on population distribution. An Orthopaedic Surgery service is an important feature of a major trauma receiving hospital and its presence improves outcomes.

Object motion influences feedforward motor responses during mechanical stopping of virtual projectiles: a preliminary study.

An important window into sensorimotor function is how humans interact and stop moving projectiles, such as stopping a door from closing shut or catching a ball. Previous studies have suggested that humans time the initiation and modulate the amplitude of their muscle activity based on the momentum of the approaching object. However, real-world experiments are constrained by laws of mechanics, which cannot be manipulated experimentally to probe the mechanisms of sensorimotor control and learning. An augmented-reality variant of such tasks allows for experimental manipulation of the relationship between motion and force to obtain novel insights into how the nervous system prepares motor responses to interact with moving stimuli. Existing paradigms for studying interactions with moving projectiles use massless objects and are primarily focused on quantifying gaze and hand kinematics. Here, we developed a novel collision paradigm using a robotic manipulandum where participants mechanically stopped a virtual object moving in the horizontal plane. On each block of trials, we varied the virtual object's momentum by increasing either its velocity or mass. Participants stopped the object by applying a force impulse that matched the object momentum. We observed that hand force increased as a function of object momentum linked to changes in virtual mass or velocity, similar to results from studies involving catching free-falling objects. In addition, increasing object velocity resulted in later onset of hand force relative to the impending time-to-contact. These findings show that the present paradigm can be used to determine how humans process projectile motion for hand motor control.

Study of He*/Ne*+Ar, Kr, N2, H2, D2 Chemi-Ionization Reactions by Electron Velocity-Map Imaging.

The chemi-ionization of Ar, Kr, N2, H2, and D2 by Ne(3P2) and of Ar, Kr, and N2 by He(3S1) was studied by electron velocity map imaging (e-VMI) in a crossed molecular beam experiment. A curved magnetic hexapole was used to state-select the metastable species. Collision energies of 60 meV were obtained by individually controlling the beam velocities of both reactants. The chemi-ionization of atoms and molecules can proceed along different channels, among them Penning ionization and associative ionization. The evolution of the reaction is influenced by the internal redistribution of energy, which happens at the first reaction step that involves the emission of an electron. We designed and built an e-VMI spectrometer in order to investigate the electron kinetic energy distribution, which is related to the internal state distribution of the ionic reaction products. The analysis of the electron kinetic energy distributions allows an estimation of the ratio between the two-reaction channel Penning and associative ionization. In the molecular cases the vibrational or electronic excitation enhanced the conversion of internal energy into the translational energy of the forming ions, thus influencing the reaction outcome.

Identification of Candidate Susceptibility Genes to Puccinia graminis f. sp. tritici in Wheat.

Wheat stem rust disease caused by Puccinia graminis f. sp. tritici (Pgt) is a global threat to wheat production. Fast evolving populations of Pgt limit the efficacy of plant genetic resistance and constrain disease management strategies. Understanding molecular mechanisms that lead to rust infection and disease susceptibility could deliver novel strategies to deploy crop resistance through genetic loss of disease susceptibility. We used comparative transcriptome-based and orthology-guided approaches to characterize gene expression changes associated with Pgt infection in susceptible and resistant Triticum aestivum genotypes as well as the non-host Brachypodium distachyon. We targeted our analysis to genes with differential expression in T. aestivum and genes suppressed or not affected in B. distachyon and report several processes potentially linked to susceptibility to Pgt, such as cell death suppression and impairment of photosynthesis. We complemented our approach with a gene co-expression network analysis to identify wheat targets to deliver resistance to Pgt through removal or modification of putative susceptibility genes.

Gradual polyploid genome evolution revealed by pan-genomic analysis of Brachypodium hybridum and its diploid progenitors.

Our understanding of polyploid genome evolution is constrained because we cannot know the exact founders of a particular polyploid. To differentiate between founder effects and post polyploidization evolution, we use a pan-genomic approach to study the allotetraploid Brachypodium hybridum and its diploid progenitors. Comparative analysis suggests that most B. hybridum whole gene presence/absence variation is part of the standing variation in its diploid progenitors. Analysis of nuclear single nucleotide variants, plastomes and k-mers associated with retrotransposons reveals two independent origins for B. hybridum, ~1.4 and ~0.14 million years ago. Examination of gene expression in the younger B. hybridum lineage reveals no bias in overall subgenome expression. Our results are consistent with a gradual accumulation of genomic changes after polyploidization and a lack of subgenome expression dominance. Significantly, if we did not use a pan-genomic approach, we would grossly overestimate the number of genomic changes attributable to post polyploidization evolution.

The fate of 35S rRNA genes in the allotetraploid grass Brachypodium hybridum.

Nucleolar dominance (ND) consists of the reversible silencing of 35S/45S rDNA loci inherited from one of the ancestors of an allopolyploid. The molecular mechanisms by which one ancestral rDNA set is selected for silencing remain unclear. We applied a combination of molecular (Southern blot hybridization and reverse-transcription cleaved amplified polymorphic sequence analysis), genomic (analysis of variants) and cytogenetic (fluorescence in situ hybridization) approaches to study the structure, expression and epigenetic landscape of 35S rDNA in an allotetraploid grass that exhibits ND, Brachypodium hybridum (genome composition DDSS), and its putative progenitors, Brachypodium distachyon (DD) and Brachypodium stacei (SS). In progenitor genomes, B. stacei showed a higher intragenomic heterogeneity of rDNA compared with B. distachyon. In all studied accessions of B. hybridum, there was a reduction in the copy number of S homoeologues, which was accompanied by their inactive transcriptional status. The involvement of DNA methylation in CG and CHG contexts in the silencing of the S-genome rDNA loci was revealed. In the B. hybridum allotetraploid, ND is stabilized towards the D-genome units, irrespective of the polyphyletic origin of the species, and does not seem to be influenced by homoeologous 35S rDNA ratios and developmental stage.

Sub-Kelvin Stereodynamics of the Ne(

We present an experimental study of the low-energy stereodynamics of the Ne(^{3}P_{2})+N_{2} reaction. Supersonic expansions of the two reactants are superposed in a merged beam experiment, where individual velocity control of the two beams allows us to reach average relative velocities of zero, yielding minimum collision energies around 60 mK. We combine the merged beam technique with the orientation of the metastable neon atoms and measure the branching between two reaction channels, Penning ionization and associative ionization, as a function of neon orientation and collision energy, covering the range 0.06-700 K. We find that we lose the ability to orient Ne below ≈100 K due to dynamic reorientation. Associative ionization products Ne-N_{2}^{+} predissociate with a probability of 30%-60% and that associative ionization is entirely due to reactions of the Ω=2 state, where the singly occupied p orbital of the Ne^{*} is oriented along the interatomic axis.

PolyCRACKER, a robust method for the unsupervised partitioning of polyploid subgenomes by signatures of repetitive DNA evolution.

BACKGROUND: Our understanding of polyploid genomes is limited by our inability to definitively assign sequences to a specific subgenome without extensive prior knowledge like high resolution genetic maps or genome sequences of diploid progenitors. In theory, existing methods for assigning sequences to individual species from metagenome samples could be used to separate subgenomes in polyploid organisms, however, these methods rely on differences in coarse genome properties like GC content or sequences from related species. Thus, these approaches do not work for subgenomes where gross features are indistinguishable and related genomes are lacking. Here we describe a method that uses rapidly evolving repetitive DNA to circumvent these limitations. RESULTS: By using short, repetitive, DNA sequences as species-specific signals we separated closely related genomes from test datasets and subgenomes from two polyploid plants, tobacco and wheat, without any prior knowledge. CONCLUSION: This approach is ideal for separating the subgenomes of polyploid species with unsequenced or unknown progenitor genomes.

RDH1 suppresses adiposity by promoting brown adipose adaptation to fasting and re-feeding.

RDH1 is one of the several enzymes that catalyze the first of the two reactions to convert retinol into all-trans-retinoic acid (atRA). Here, we show that Rdh1-null mice fed a low-fat diet gain more weight as adiposity (17% males, 13% females) than wild-type mice by 20 weeks old, despite neither consuming more calories nor decreasing activity. Glucose intolerance and insulin resistance develop following increased adiposity. Despite the increase in white fat pads, epididymal white adipose does not express Rdh1, nor does muscle. Brown adipose tissue (BAT) and liver express Rdh1 at relatively high levels compared to other tissues. Rdh1 ablation lowered body temperatures during ambient conditions. Given the decreased body temperature, we focused on BAT. A lack of differences in BAT adipogenic gene expression between Rdh1-null mice and wild-type mice, including Pparg, Prdm16, Zfp516 and Zfp521, indicated that the phenotype was not driven by brown adipose hyperplasia. Rather, Rdh1 ablation eliminated the increase in BAT atRA that occurs after re-feeding. This disruption of atRA homeostasis increased fatty acid uptake, but attenuated lipolysis in primary brown adipocytes, resulting in increased lipid content and larger lipid droplets. Rdh1 ablation also decreased mitochondrial proteins, including CYCS and UCP1, the mitochondria oxygen consumption rate, and disrupted the mitochondria membrane potential, further reflecting impaired BAT function, resulting in both BAT and white adipose hypertrophy. RNAseq revealed dysregulation of 424 BAT genes in null mice, which segregated predominantly into differences after fasting vs after re-feeding. Exceptions were Rbp4 and Gbp2b, which increased during both dietary conditions. Rbp4 encodes the serum retinol-binding protein-an insulin desensitizer. Gbp2b encodes a GTPase. Because Gbp2b increased several hundred-fold, we overexpressed it in brown adipocytes. This caused a shift to larger lipid droplets, suggesting that GBP2b affects signaling downstream of the ß-adrenergic receptor during basal thermogenesis. Thus, Rdh1-generated atRA in BAT regulates multiple genes that promote BAT adaptation to whole-body energy status, such as fasting and re-feeding. These gene expression changes promote optimum mitochondria function and thermogenesis, limiting adiposity. Attenuation of adiposity and insulin resistance suggests that RDH1 mitigates metabolic syndrome.

Energy and orientation independence of the channel branching in Ne* + ND3 chemi-ionisation.

Collisions of excited neon atoms with ammonia molecules can lead to two reaction processes, dissociative ionisation and Penning ionisation. Both processes result in the ionisation of the ammonia molecule and redistribution of the electronic energy into the internal ammonia ion rovibrational modes. We performed energy dependent, crossed-beam stereodynamics studies of the branching ratio between the two ionisation processes. It was found that the branching ratio is totally and completely insensitive to both the neon orientation and the collision energy across the range we sampled, 370-520 cm-1. The total lack of stereodynamics can be explained by the structure of the ammonia and that its orientation, which we do not attempt to control, is the critical factor in the reaction outcome.

Global Diversity of the Brachypodium Species Complex as a Resource for Genome-Wide Association Studies Demonstrated for Agronomic Traits in Response to Climate.

The development of model systems requires a detailed assessment of standing genetic variation across natural populations. The Brachypodium species complex has been promoted as a plant model for grass genomics with translation to small grain and biomass crops. To capture the genetic diversity within this species complex, thousands of Brachypodium accessions from around the globe were collected and genotyped by sequencing. Overall, 1897 samples were classified into two diploid or allopolyploid species, and then further grouped into distinct inbred genotypes. A core set of diverse B. distachyon diploid lines was selected for whole genome sequencing and high resolution phenotyping. Genome-wide association studies across simulated seasonal environments was used to identify candidate genes and pathways tied to key life history and agronomic traits under current and future climatic conditions. A total of 8, 22, and 47 QTL were identified for flowering time, early vigor, and energy traits, respectively. The results highlight the genomic structure of the Brachypodium species complex, and the diploid lines provided a resource that allows complex trait dissection within this grass model species.

The genomic landscape of molecular responses to natural drought stress in Panicum hallii.

Environmental stress is a major driver of ecological community dynamics and agricultural productivity. This is especially true for soil water availability, because drought is the greatest abiotic inhibitor of worldwide crop yields. Here, we test the genetic basis of drought responses in the genetic model for C4 perennial grasses, Panicum hallii, through population genomics, field-scale gene-expression (eQTL) analysis, and comparison of two complete genomes. While gene expression networks are dominated by local cis-regulatory elements, we observe three genomic hotspots of unlinked trans-regulatory loci. These regulatory hubs are four times more drought responsive than the genome-wide average. Additionally, cis- and trans-regulatory networks are more likely to have opposing effects than expected under neutral evolution, supporting a strong influence of compensatory evolution and stabilizing selection. These results implicate trans-regulatory evolution as a driver of drought responses and demonstrate the potential for crop improvement in drought-prone regions through modification of gene regulatory networks.

Quantum-state-controlled channel branching in cold Ne(3P2)+Ar chemi-ionization.

A prerequisite to gain a complete understanding of the most basic aspects of chemical reactions is the ability to perform experiments with complete control over the reactant degrees of freedom. By controlling these, details of a reaction mechanism can be investigated and ultimately manipulated. Here, we present a study of chemi-ionization-a fundamental energy-transfer reaction-under completely controlled conditions. The collision energy of the reagents was tuned from 0.02 K to 1,000 K, with the orientation of the excited Ne atom relative to Ar fully specified by an external magnetic field. Chemi-ionization of Ne(3P2) and Ar in these conditions enables a detailed investigation of how the reaction proceeds, and provides us with a means to control the branching ratio between the two possible reaction outcomes. The merged-beam experimental technique used here allows access to a low-energy regime in which the atoms dynamically reorient into a favourable configuration for reaction, irrespective of their initial orientations.

Genome-wide scans of selection highlight the impact of biotic and abiotic constraints in natural populations of the model grass Brachypodium distachyon.

Grasses are essential plants for ecosystem functioning. Quantifying the selective pressures that act on natural variation in grass species is therefore essential regarding biodiversity maintenance. In this study, we investigate the selection pressures that act on two distinct populations of the grass model Brachypodium distachyon without prior knowledge about the traits under selection. We took advantage of whole-genome sequencing data produced for 44 natural accessions of B. distachyon and used complementary genome-wide selection scans (GWSS) methods to detect genomic regions under balancing and positive selection. We show that selection is shaping genetic diversity at multiple temporal and spatial scales in this species, and affects different genomic regions across the two populations. Gene ontology annotation of candidate genes reveals that pathogens may constitute important factors of positive and balancing selection in B. distachyon. We eventually cross-validated our results with quantitative trait locus data available for leaf-rust resistance in this species and demonstrate that, when paired with classical trait mapping, GWSS can help pinpointing candidate genes for further molecular validation. Thanks to a near base-perfect reference genome and the large collection of freely available natural accessions collected across its natural range, B. distachyon appears as a prime system for studies in ecology, population genomics and evolutionary biology.

Stereodynamics of Ne(3P2) reacting with Ar, Kr, Xe, and N2.

Stereodynamics experiments of Ne(3P2) reacting with Ar, Kr, Xe, and N2 leading to Penning and associative ionization have been performed in a crossed molecular beam apparatus. A curved magnetic hexapole was used to state-select and polarize Ne(3P2) atoms which were then oriented in a rotatable magnetic field and crossed with a beam of Ar, Kr, Xe, or N2. The ratio of associative to Penning ionization was recorded as a function of the magnetic field direction for collision energies between 320 cm-1 and 500 cm-1. Reactivities are obtained for individual states that differ only in Ω, the projection of the neon total angular momentum vector on the inter-particle axis. The results are rationalized on the basis of a model involving a long-range and a short-range reaction mechanism. Substantially lower probability for associative ionization was observed for N2, suggesting that predissociation plays a critical role in the overall reaction pathway.

Recent Activity in Expanding Populations and Purifying Selection Have Shaped Transposable Element Landscapes across Natural Accessions of the Mediterranean Grass Brachypodium distachyon.

Transposable element (TE) activity has emerged as a major cause of variation in genome size and structure among species. To what extent TEs contribute to genetic variation and divergence within species, however, is much less clear, mainly because population genomic data have so far only been available for the classical model organisms. In this study, we use the annual Mediterranean grass Brachypodium distachyon to investigate TE dynamics in natural populations. Using whole-genome sequencing data for 53 natural accessions, we identified more than 5,400 TE polymorphisms across the studied genomes. We found, first, that while population bottlenecks and expansions have shaped genetic diversity in B. distachyon, these events did not lead to lineage-specific activations of TE families, as observed in other species. Instead, the same families have been active across the species range and TE activity is homogeneous across populations, indicating the presence of conserved regulatory mechanisms. Second, almost half of the TE insertion polymorphisms are accession-specific, most likely because of recent activity in expanding populations and the action of purifying selection. And finally, although TE insertion polymorphisms are underrepresented in and around genes, more than 1,000 of them occur in genic regions and could thus contribute to functional divergence. Our study shows that while TEs in B. distachyon are "well-behaved" compared with TEs in other species with larger genomes, they are an abundant source of lineage-specific genetic variation and may play an important role in population divergence and adaptation.

Comparative plastome genomics and phylogenomics of Brachypodium: flowering time signatures, introgression and recombination in recently diverged ecotypes.

Few pan-genomic studies have been conducted in plants, and none of them have focused on the intraspecific diversity and evolution of their plastid genomes. We address this issue in Brachypodium distachyon and its close relatives B. stacei and B. hybridum, for which a large genomic data set has been compiled. We analyze inter- and intraspecific plastid comparative genomics and phylogenomic relationships within a family-wide framework. Major indel differences were detected between Brachypodium plastomes. Within B. distachyon, we detected two main lineages, a mostly Extremely Delayed Flowering (EDF+) clade and a mostly Spanish (S+) - Turkish (T+) clade, plus nine chloroplast capture and two plastid DNA (ptDNA) introgression and micro-recombination events. Early Oligocene (30.9 million yr ago (Ma)) and Late Miocene (10.1 Ma) divergence times were inferred for the respective stem and crown nodes of Brachypodium and a very recent Mid-Pleistocene (0.9 Ma) time for the B. distachyon split. Flowering time variation is a main factor driving rapid intraspecific divergence in B. distachyon, although it is counterbalanced by repeated introgression between previously isolated lineages. Swapping of plastomes between the three different genomic groups, EDF+, T+, S+, probably resulted from random backcrossing followed by stabilization through selection pressure.

Extensive gene content variation in the Brachypodium distachyon pan-genome correlates with population structure.

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.