Patterns of pan-genome occupancy and gene coexpression under water-deficit in Brachypodium distachyon.

Natural populations are characterized by abundant genetic diversity driven by a range of different types of mutation. The tractability of sequencing complete genomes has allowed new insights into the variable composition of genomes, summarized as a species pan-genome. These analyses demonstrate that many genes are absent from the first reference genomes, whose analysis dominated the initial years of the genomic era. Our field now turns towards understanding the functional consequence of these highly variable genomes. Here, we analysed weighted gene coexpression networks from leaf transcriptome data for drought response in the purple false brome Brachypodium distachyon and the differential expression of genes putatively involved in adaptation to this stressor. We specifically asked whether genes with variable "occupancy" in the pan-genome - genes which are either present in all studied genotypes or missing in some genotypes - show different distributions among coexpression modules. Coexpression analysis united genes expressed in drought-stressed plants into nine modules covering 72 hub genes (87 hub isoforms), and genes expressed under controlled water conditions into 13 modules, covering 190 hub genes (251 hub isoforms). We find that low occupancy pan-genes are under-represented among several modules, while other modules are over-enriched for low-occupancy pan-genes. We also provide new insight into the regulation of drought response in B. distachyon, specifically identifying one module with an apparent role in primary metabolism that is strongly responsive to drought. Our work shows the power of integrating pan-genomic analysis with transcriptomic data using factorial experiments to understand the functional genomics of environmental response.

Pangenome Analysis of Plant Transcripts and Coding Sequences.

The pangenome of a species is the sum of the genomes of its individuals. As coding sequences often represent only a small fraction of each genome, analyzing the pangene set can be a cost-effective strategy for plants with large genomes or highly heterozygous species. Here, we describe a step-by-step protocol to analyze plant pangene sets with the software GET_HOMOLOGUES-EST . After a short introduction, where the main concepts are illustrated, the remaining sections cover the installation and typical operations required to analyze and annotate pantranscriptomes and gene sets of plants. The recipes include instructions on how to call core and accessory genes, how to compute a presence-absence pangenome matrix, and how to identify and analyze private genes, present only in some genotypes. Downstream phylogenetic analyses are also discussed.

Comparative Genomics, Evolution, and Drought-Induced Expression of Dehydrin Genes in Model Brachypodium Grasses.

Dehydration proteins (dehydrins, DHNs) confer tolerance to water-stress deficit in plants. We performed a comparative genomics and evolutionary study of DHN genes in four model Brachypodium grass species. Due to limited knowledge on dehydrin expression under water deprivation stress in Brachypodium, we also performed a drought-induced gene expression analysis in 32 ecotypes of the genus' flagship species B. distachyon showing different hydric requirements. Genomic sequence analysis detected 10 types of dehydrin genes (Bdhn) across the Brachypodium species. Domain and conserved motif contents of peptides encoded by Bdhn genes revealed eight protein architectures. Bdhn genes were spread across several chromosomes. Selection analysis indicated that all the Bdhn genes were constrained by purifying selection. Three upstream cis-regulatory motifs (BES1, MYB124, ZAT) were detected in several Bdhn genes. Gene expression analysis demonstrated that only four Bdhn1-Bdhn2, Bdhn3, and Bdhn7 genes, orthologs of wheat, barley, rice, sorghum, and maize genes, were expressed in mature leaves of B. distachyon and that all of them were more highly expressed in plants under drought conditions. Brachypodium dehydrin expression was significantly correlated with drought-response phenotypic traits (plant biomass, leaf carbon and proline contents and water use efficiency increases, and leaf water and nitrogen content decreases) being more pronounced in drought-tolerant ecotypes. Our results indicate that dehydrin type and regulation could be a key factor determining the acquisition of water-stress tolerance in grasses.

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.

Reconstructing the origins and the biogeography of species' genomes in the highly reticulate allopolyploid-rich model grass genus Brachypodium using minimum evolution, coalescence and maximum likelihood approaches.

The identification of homeologous genomes and the biogeographical analyses of highly reticulate allopolyploid-rich groups face the challenge of incorrectly inferring the genomic origins and the biogeographical patterns of the polyploids from unreliable strictly bifurcating trees. Here we reconstruct a plausible evolutionary scenario of the diverging and merging genomes inherited by the diploid and allopolyploid species and cytotypes of the model grass genus Brachypodium. We have identified the ancestral Brachypodium genomes and inferred the paleogeographical ranges for potential hybridization events that originated its allopolyploid taxa. We also constructed a comprehensive phylogeny of Brachypodium from five nuclear and plastid genes using Species Tree Minimum Evolution allele grafting and Species Network analysis. The divergence ages of the lineages were estimated from a consensus maximum clade credibility tree using fossil calibrations, whereas ages of origin of the diploid and allopolyploid species were inferred from coalescence Bayesian methods. The biogeographical events of the genomes were reconstructed using a stratified Dispersal-Extinction-Colonization model with three temporal windows. Our combined Minimum Evolution-coalescence-Bayesian approach allowed us to infer the origins and the identities of the homeologous genomes of the Brachypodium allopolyploids, matching the expected ploidy levels of the hybrids. To date, the current extant progenitor genomes (species) are only known for B. hybridum. Putative ancestral homeologous genome have been inherited by B. mexicanum, ancestral and recent genomes by B. boissieri, and only recently evolved genomes by B. retusum and the core perennial clade allopolyploids (B. phoenicoides, B. pinnatum 4x, B. rupestre 4x). We dissected the complex spatio-temporal evolution of ancestral and recent genomes and have detected successive splitting, dispersal and merging events for dysploid homeologous genomes in diverse geographical scenarios that have led to the current extant taxa. Our data support Mid-Miocene splits of the Holarctic ancestral genomes that preceded the Late Miocene origins of Brachypodium ancestors of the modern diploid species. Successive divergences of the annual B. stacei and B. distachyon diploid genomes were implied to have occurred in the Mediterranean region during the Late Miocene-Pliocene. By contrast, a profusion of splits, range expansions and different genome mergings were inferred for the perennial diploid genomes in the Mediterranean and Eurasian regions, with sporadic colonizations and further mergings in other continents during the Quaternary. A reliable biogeographical scenario was obtained for the Brachypodium genomes and allopolyploids where homeologous genomes split from their respective diploid counterpart lineages in the same ancestral areas, showing similar or distinct dispersals. By contrast, the allopolyploid taxa remained in the same ancestral ranges after hybridization and genome doubling events. Our approach should have utility in deciphering the genomic composition and the historical biogeography of other allopolyploid-rich organismal groups, which are predominant in eukaryotes.

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.

Highly expressed captured genes and cross-kingdom domains present in Helitrons create novel diversity in Pleurotus ostreatus and other fungi.

BACKGROUND: Helitrons are class-II eukaryotic transposons that transpose via a rolling circle mechanism. Due to their ability to capture and mobilize gene fragments, they play an important role in the evolution of their host genomes. We have used a bioinformatics approach for the identification of helitrons in two Pleurotus ostreatus genomes using de novo detection and homology-based searching. We have analyzed the presence of helitron-captured genes as well as the expansion of helitron-specific helicases in fungi and performed a phylogenetic analysis of their conserved domains with other representative eukaryotic species. RESULTS: Our results show the presence of two helitron families in P. ostreatus that disrupt gene colinearity and cause a lack of synteny between their genomes. Both putative autonomous and non-autonomous helitrons were transcriptionally active, and some of them carried highly expressed captured genes of unknown origin and function. In addition, both families contained eukaryotic, bacterial and viral domains within the helitron's boundaries. A phylogenetic reconstruction of RepHel helicases using the Helitron-like and PIF1-like helicase conserved domains revealed a polyphyletic origin for eukaryotic helitrons. CONCLUSION: P. ostreatus helitrons display features similar to other eukaryotic helitrons and do not tend to capture host genes or gene fragments. The occurrence of genes probably captured from other hosts inside the helitrons boundaries pose the hypothesis that an ancient horizontal transfer mechanism could have taken place. The viral domains found in some of these genes and the polyphyletic origin of RepHel helicases in the eukaryotic kingdom suggests that virus could have played a role in a putative lateral transfer of helitrons within the eukaryotic kingdom. The high similarity of some helitrons, along with the transcriptional activity of its RepHel helicases indicates that these elements are still active in the genome of P. ostreatus.

Estudio comparativo de la punción arterial ecoguiada frente a la técnica clásica / Comparative study between ultrasound-guided arterial puncture vs. the traditional technique

OBJETIVO: analizar los beneficios de la punción arterial ecoguiada (PAE) frente a la técnica de punción clásica (TPC) en cuanto al porcentaje de éxito en el primer intento de punción, siendo objetivos secundarios el tiempo empleado y el grado de dolor autorreferido. MATERIAL Y MÉTODO: estudio experimental controlado y aleatorizado de comparación entre la TPC y la PAE en pacientes mayores de catorce años que precisaron de extracción de sangre arterial. Muestreo probabilístico de 208 pacientes. Las variables analizadas fueron: el éxito a la primera punción, el tiempo empleado en realizar la técnica y el dolor autorreferido por el paciente postpunción medido a través de la escala visual numérica. RESULTADOS: se obtuvieron dos grupos de pacientes: 105 so-metidos a PAE y 103 a TPC, sin diferencias significativas en la edad y el sexo. El éxito en el primer intento de punción fue de 87,6% con la PAE frente al 58,3% con la TCP (p< 0,000); el tiempo empleado fue menor de 4 minutos en el 97,1% de PAE vs 75,7% de TPC (p< 0,001) y el dolor autorreferido fue valorado con una media de 3,1±2,2 después de la PAE vs 4,7±2,6 tras la TPC (p< 0,001). CONCLUSIONES: la PAE reduce el número de punciones para la obtención de la muestra arterial y disminuye el dolor autorreferido. La reducción de los tiempos diagnósticos, junto con la seguridad de obtención de sangre arterial, contribuye a una atención de Enfermería de mayor calidad

OBJECTIVE: to analyze the benefits of Ultrasound-Guided Arterial Puncture (UGAP) vs. Traditional Puncture Technique (TPT), in terms of success rate at the first puncture attempt, with time required and self-reported pain level as secondary objectives. MATERIALS AND METHOD: experimental study, controlled and randomized, between UGAP and TPT in patients over 14-year-old which required arterial blood extraction. Probability sample of 208 patients. Those variables analyzed were: success at first puncture, time required to conduct the technique, and post-puncture pain, self-reported by patients through the Visual Numeric Scale. RESULTS: two groups of patients were recruited: 105 undergoing UGAP and 103 undergoing TPT. There were no significant differences in age and gender. Success at the first puncture attempt was 87.6% with UGAP vs 58.3% with TPT (p< 0.000); the time required was under 4 minutes for 97.1% of UGAP vs 75.7% for TPT (p< 0.001), and self-reported pain was assessed with a mean of 3.1±2.2 after UGAP vs 4.7±2.6 after TPT (p< 0.001). CONCLUSIONS: UGAP reduces the number of punctures for acquiring the arterial sample, and reduces self-reported pain. A reduction in diagnostic times, as well as the safety in acquiring arterial blood, will contribute to a higher quality in Nursing care