Elasmobranch qPCR reference genes: a case study of hypoxia preconditioned epaulette sharks

<p>Abstract</p> <p>Background</p> <p>Elasmobranch fishes are an ancient group of vertebrates which have high potential as model species for research into evolutionary physiology and genomics. However, no comparative studies have established suitable reference genes for quantitative PCR (qPCR) in elasmobranchs for any physiological conditions. Oxygen availability has been a major force shaping the physiological evolution of vertebrates, especially fishes. Here we examined the suitability of 9 reference candidates from various functional categories after a single hypoxic insult or after hypoxia preconditioning in epaulette shark (<it>Hemiscyllium ocellatum</it>).</p> <p>Results</p> <p>Epaulette sharks were caught and exposed to hypoxia. Tissues were collected from 10 controls, 10 individuals with single hypoxic insult and 10 individuals with hypoxia preconditioning (8 hypoxic insults, 12 hours apart). We produced sequence information for reference gene candidates and monitored mRNA expression levels in four tissues: cerebellum, heart, gill and eye. The stability of the genes was examined with analysis of variance, geNorm and NormFinder. The best ranking genes in our study were <it>eukaryotic translation elongation factor 1 beta </it>(<it>eef1b</it>), <it>ubiquitin </it>(<it>ubq</it>) and <it>polymerase (RNA) II (DNA directed) polypeptide F </it>(<it>polr2f</it>). The performance of the <it>ribosomal protein L6 </it>(<it>rpl6</it>) was tissue-dependent. Notably, in one tissue the analysis of variance indicated statistically significant differences between treatments for genes that were ranked as the most stable candidates by reference gene software.</p> <p>Conclusions</p> <p>Our results indicate that <it>eef1b </it>and <it>ubq </it>are generally the most suitable reference genes for the conditions and tissues in the present epaulette shark studies. These genes could also be potential reference gene candidates for other physiological studies examining stress in elasmobranchs. The results emphasise the importance of inter-group variation in reference gene evaluation.</p

Synthesis of global satellite observations of magmatic and volcanic deformation: implications for volcano monitoring & the lateral extent of magmatic domains

Global Synthetic Aperture Radar (SAR) measurements made over the past decades provide insights into the lateral extent of magmatic domains, and capture volcanic process on scales useful for volcano monitoring. Satellite-based SAR imagery has great potential for monitoring topographic change, the distribution of eruptive products and surface displacements (InSAR) at subaerial volcanoes. However, there are challenges in applying it routinely, as would be required for the reliable operational assessment of hazard. The deformation detectable depends upon satellite repeat time and swath widths, relative to the spatial and temporal scales of volcanological processes. We describe the characteristics of InSAR-measured volcano deformation over the past two decades, highlighting both the technique’s capabilities and its limitations as a monitoring tool. To achieve this, we draw on two global datasets of volcano deformation: the Smithsonian Institution Volcanoes of the World database and the Centre for the Observation and Modelling of Earthquakes, Volcanoes and Tectonics volcano deformation catalogue, as well as compiling some measurement characteristics and interpretations from the primary literature. We find that a higher proportion of InSAR observations capture non-eruptive and non-magmatic processes than those from ground-based instrument networks, and that both transient ( 5 years) deformation episodes are under-represented. However, satellite radar is already used to assess the development of extended periods of unrest and long-lasting eruptions, and improved spatial resolution and coverage have resulted in the detection of previously unrecognised deformation at both ends of the spatial scale (~ 10 to > 1000 km²). ‘Baseline’ records of past InSAR measurements, including ‘null’ results, are fundamental for any future interpretation of interferograms in terms of hazard‚ both by providing information about past deformation at an individual volcano, and for assessing the characteristics of deformation that are likely to be detectable (and undetectable) using InSAR. More than half of all InSAR deformation signals attributed to magmatic processes have sources in the shallow crust (< 5 km depth). While the depth distribution of InSAR-derived deformation sources is affected by measurement limitations, their lateral distribution provides information about the extent of active magmatic domains. Deformation is common (24% of all potentially magmatic events) at loci ≥5 km away from the nearest active volcanic vent. This demonstrates that laterally extensive active magmatic domains are not exceptional, but can comprise the shallowest part of trans-crustal magmatic systems in a range of volcanic settings

Genetic dissection of photoperiod response based on GWAS of pre-anthesis phase duration in spring barley

Heading time is a complex trait, and natural variation in photoperiod responses is a major factor controlling time to heading, adaptation and grain yield. In barley, previous heading time studies have been mainly conducted under field conditions to measure total days to heading. We followed a novel approach and studied the natural variation of time to heading in a world-wide spring barley collection (218 accessions), comprising of 95 photoperiod-sensitive (Ppd-H1) and 123 accessions with reduced photoperiod sensitivity (ppd-H1) to long-day (LD) through dissecting pre-anthesis development into four major stages and sub-phases. The study was conducted under greenhouse (GH) conditions (LD; 16/8 h; ∼20/∼16°C day/night). Genotyping was performed using a genome-wide high density 9K single nucleotide polymorphisms (SNPs) chip which assayed 7842 SNPs. We used the barley physical map to identify candidate genes underlying genome-wide association scans (GWAS). GWAS for pre-anthesis stages/sub-phases in each photoperiod group provided great power for partitioning genetic effects on floral initiation and heading time. In addition to major genes known to regulate heading time under field conditions, several novel QTL with medium to high effects, including new QTL having major effects on developmental stages/sub-phases were found to be associated in this study. For example, highly associated SNPs tagged the physical regions around HvCO1 (barley CONSTANS1) and BFL (BARLEY FLORICAULA/LEAFY) genes. Based upon our GWAS analysis, we propose a new genetic network model for each photoperiod group, which includes several newly identified genes, such as several HvCO-like genes, belonging to different heading time pathways in barley

Discovery of Rare Variants via Sequencing: Implications for the Design of Complex Trait Association Studies

There is strong evidence that rare variants are involved in complex disease etiology. The first step in implicating rare variants in disease etiology is their identification through sequencing in both randomly ascertained samples (e.g., the 1,000 Genomes Project) and samples ascertained according to disease status. We investigated to what extent rare variants will be observed across the genome and in candidate genes in randomly ascertained samples, the magnitude of variant enrichment in diseased individuals, and biases that can occur due to how variants are discovered. Although sequencing cases can enrich for casual variants, when a gene or genes are not involved in disease etiology, limiting variant discovery to cases can lead to association studies with dramatically inflated false positive rates

Accounting for Population Stratification in Practice: A Comparison of the Main Strategies Dedicated to Genome-Wide Association Studies

Genome-Wide Association Studies are powerful tools to detect genetic variants associated with diseases. Their results have, however, been questioned, in part because of the bias induced by population stratification. This is a consequence of systematic differences in allele frequencies due to the difference in sample ancestries that can lead to both false positive or false negative findings. Many strategies are available to account for stratification but their performances differ, for instance according to the type of population structure, the disease susceptibility locus minor allele frequency, the degree of sampling imbalanced, or the sample size. We focus on the type of population structure and propose a comparison of the most commonly used methods to deal with stratification that are the Genomic Control, Principal Component based methods such as implemented in Eigenstrat, adjusted Regressions and Meta-Analyses strategies. Our assessment of the methods is based on a large simulation study, involving several scenarios corresponding to many types of population structures. We focused on both false positive rate and power to determine which methods perform the best. Our analysis showed that if there is no population structure, none of the tests led to a bias nor decreased the power except for the Meta-Analyses. When the population is stratified, adjusted Logistic Regressions and Eigenstrat are the best solutions to account for stratification even though only the Logistic Regressions are able to constantly maintain correct false positive rates. This study provides more details about these methods. Their advantages and limitations in different stratification scenarios are highlighted in order to propose practical guidelines to account for population stratification in Genome-Wide Association Studies

The Genomic Signature of Crop-Wild Introgression in Maize

The evolutionary significance of hybridization and subsequent introgression has long been appreciated, but evaluation of the genome-wide effects of these phenomena has only recently become possible. Crop-wild study systems represent ideal opportunities to examine evolution through hybridization. For example, maize and the conspecific wild teosinte Zea mays ssp. mexicana, (hereafter, mexicana) are known to hybridize in the fields of highland Mexico. Despite widespread evidence of gene flow, maize and mexicana maintain distinct morphologies and have done so in sympatry for thousands of years. Neither the genomic extent nor the evolutionary importance of introgression between these taxa is understood. In this study we assessed patterns of genome-wide introgression based on 39,029 single nucleotide polymorphisms genotyped in 189 individuals from nine sympatric maize-mexicana populations and reference allopatric populations. While portions of the maize and mexicana genomes were particularly resistant to introgression (notably near known cross-incompatibility and domestication loci), we detected widespread evidence for introgression in both directions of gene flow. Through further characterization of these regions and preliminary growth chamber experiments, we found evidence suggestive of the incorporation of adaptive mexicana alleles into maize during its expansion to the highlands of central Mexico. In contrast, very little evidence was found for adaptive introgression from maize to mexicana. The methods we have applied here can be replicated widely, and such analyses have the potential to greatly informing our understanding of evolution through introgressive hybridization. Crop species, due to their exceptional genomic resources and frequent histories of spread into sympatry with relatives, should be particularly influential in these studies

Post-depositional fracturing and subsidence of pumice flow deposits: Lascar Volcano, Chile

Unconsolidated pyroclastic flow deposits of the 1993 eruption of Lascar Volcano, Chile, have, with time, become increasingly dissected by a network of deeply penetrating fractures. The fracture network comprises orthogonal sets of decimeter-wide linear voids that form a pseudo-polygonal grid visible on the deposit surface. In this work, we combine shallow surface geophysical imaging tools with remote sensing observations and direct field measurements of the deposit to investigate these fractures and their underlying causal mechanisms. Based on ground penetrating radar images, the fractures are observed to have propagated to depths of up to 10 m. In addition, orbiting radar interferometry shows that deposit subsidence of up to 1 cm/year occurred between 1993 and 1996 with continued subsidence occurring at a slower rate thereafter. In situ measurements show that 1 m below the surface, the 1993 deposits remain 5°C to 15°C hotter, 18 years after emplacement, than adjacent deposits. Based on the observed subsidence as well as estimated cooling rates, the fractures are inferred to be the combined result of deaeration, thermal contraction, and sedimentary compaction in the months to years following deposition. Significant environmental factors, including regional earthquakes in 1995 and 2007, accelerated settling at punctuated moments in time. The spatially variable fracture pattern relates to surface slope and lithofacies variations as well as substrate lithology. Similar fractures have been reported in other ignimbrites but are generally exposed only in cross section and are often attributed to formation by external forces. Here we suggest that such interpretations should be invoked with caution, and deformation including post-emplacement subsidence and fracturing of loosely packed ash-rich deposits in the months to years postemplacement is a process inherent in the settling of pyroclastic material

Clonal diversity and conservation genetics of the medicinal plant Carapichea ipecacuanha (Rubiaceae)

The roots of the understorey shrub Carapichea ipecacuanha (ipecac) have medicinal properties, and the uprooting of wild plants has supplied most of the world demand for this species. Although under severe population decline, C. ipecacuanha lacks legal protection. In the wild, the aerial stems of ipecac clump together to form clusters with well-defined borders. Cluster size may range from several to hundreds of aerial stems. To investigate the extent of clonality among aerial stems in ipecac clusters, we sampled 50 wild clusters (a total of 291 aerial stems) and screened them with 89 inter-simple sequence repeat (ISSR) markers. The 291 aerial stems were grouped into 42 putative clones. The clonal groups generally consisted of aerial stems from the same cluster, and there was little or no genetic differentiation among aerial stems at the cluster level. These findings suggest that strategies designed to conserve ipecac in situ should not rely upon census data, which are based on the number of aerial stems per cluster and the number of clusters per population, because such data greatly underestimate the species effective population size and genetic diversity. Our results also indicate that this species needs protection at a federal level

Improving the use of research evidence in guideline development: 10. Integrating values and consumer involvement

BACKGROUND: The World Health Organization (WHO), like many other organisations around the world, has recognised the need to use more rigorous processes to ensure that health care recommendations are informed by the best available research evidence. This is the 10(th )of a series of 16 reviews that have been prepared as background for advice from the WHO Advisory Committee on Health Research to WHO on how to achieve this. OBJECTIVES: We reviewed the literature on integrating values and consumers in guideline development. METHODS: We searched PubMed and three databases of methodological studies for existing systematic reviews and relevant methodological research. We reviewed the titles of all citations and retrieved abstracts and full text articles if the citations appeared relevant to the topic. We checked the reference lists of articles relevant to the questions and used snowballing as a technique to obtain additional information. We did not conduct a full systematic review ourselves. Our conclusions based on the available evidence, consideration of what WHO and other organisations are doing and logical arguments. KEY QUESTIONS AND ANSWERS: We did not find a systematic review of methods for integrating values in guidelines, but we found several systematic reviews that dealt with related topics. Whose values should WHO use when making recommendations? • Values, the relative importance or worth of a state or consequences of a decision (outcomes relating to benefits, harms, burden and costs), play a role in every recommendation. Ethical considerations, concepts that determine what is right, also play a role. • The values used in making recommendations should reflect those of the people affected. Judgements should be explicit and should be informed by input from those affected (including citizens, patients, clinicians and policy makers). • When differences in values may lead to different decisions or there is uncertainty about values, this should also be explicit. If differences in values are likely to affect a decision, such that people in different setting would likely make different choices about interventions or actions based on differences in their values, global recommendations should be explicit in terms of which values were applied and allow for adaptation after incorporating local values. How should WHO ensure that appropriate values are integrated in recommendations? • All WHO guideline groups should uniformly apply explicit, transparent and clearly described methods for integrating values. • WHO should consider involving relevant stakeholders if this is feasible and efficient. • WHO should develop a checklist for guidelines panels to help them to ensure that ethical considerations relevant to recommendations are addressed explicitly and transparently. How should users and consumers be involved in generating recommendations? • Including consumers in groups that are making global recommendations presents major challenges with respect to the impossibility of including a representative spectrum of consumers from a variety of cultures and settings. Nonetheless, consideration should be given to including consumers in groups who are able to challenge assumptions that are made about the values used for making recommendations, rather than represent the values of consumers around the world. • WHO should establish a network to facilitate involvement of users. • Draft recommendations should be reviewed by consumers, who should be asked explicitly to consider the values that were used. How should values be presented in recommendations? • Recommendations should include a description of how decisions were made about the relative importance of the consequences (benefits, harms and costs) of a decision. • Values that influence recommendations should be reported along with the research evidence underlying recommendations. • When differences in values would lead to different decisions or there is important uncertainty about values that are critical to a decision, this should be flagged and reflected in the strength of the recommendation. • Adaptable guideline templates that allow for integration of different values should be developed and used when differences in values are likely to be critical to a decision

A unified framework for multi-locus association analysis of both common and rare variants

<p>Abstract</p> <p>Background</p> <p>Common, complex diseases are hypothesized to result from a combination of common and rare genetic variants. We developed a unified framework for the joint association testing of both types of variants. Within the framework, we developed a union-intersection test suitable for genome-wide analysis of single nucleotide polymorphisms (SNPs), candidate gene data, as well as medical sequencing data. The union-intersection test is a composite test of association of genotype frequencies and differential correlation among markers.</p> <p>Results</p> <p>We demonstrated by computer simulation that the false positive error rate was controlled at the expected level. We also demonstrated scenarios in which the multi-locus test was more powerful than traditional single marker analysis. To illustrate use of the union-intersection test with real data, we analyzed a publically available data set of 319,813 autosomal SNPs genotyped for 938 cases of Parkinson disease and 863 neurologically normal controls for which no genome-wide significant results were found by traditional single marker analysis. We also analyzed an independent follow-up sample of 183 cases and 248 controls for replication.</p> <p>Conclusions</p> <p>We identified a single risk haplotype with a directionally consistent effect in both samples in the gene <it>GAK</it>, which is involved in clathrin-mediated membrane trafficking. We also found suggestive evidence that directionally inconsistent marginal effects from single marker analysis appeared to result from risk being driven by different haplotypes in the two samples for the genes <it>SYN3 </it>and <it>NGLY1</it>, which are involved in neurotransmitter release and proteasomal degradation, respectively. These results illustrate the utility of our unified framework for genome-wide association analysis of common, complex diseases.</p