Analysis of the Pythium ultimum transcriptome using Sanger and Pyrosequencing approaches

<p>Abstract</p> <p>Background</p> <p><it>Pythium </it>species are an agriculturally important genus of plant pathogens, yet are not understood well at the molecular, genetic, or genomic level. They are closely related to other oomycete plant pathogens such as <it>Phytophthora </it>species and are ubiquitous in their geographic distribution and host rage. To gain a better understanding of its gene complement, we generated Expressed Sequence Tags (ESTs) from the transcriptome of <it>Pythium ultimum </it>DAOM BR144 (= ATCC 200006 = CBS 805.95) using two high throughput sequencing methods, Sanger-based chain termination sequencing and pyrosequencing-based sequencing-by-synthesis.</p> <p>Results</p> <p>A single half-plate pyrosequencing (454 FLX) run on adapter-ligated cDNA from a normalized cDNA population generated 90,664 reads with an average read length of 190 nucleotides following cleaning and removal of sequences shorter than 100 base pairs. After clustering and assembly, a total of 35,507 unique sequences were generated. In parallel, 9,578 reads were generated from a library constructed from the same normalized cDNA population using dideoxy chain termination Sanger sequencing, which upon clustering and assembly generated 4,689 unique sequences. A hybrid assembly of both Sanger- and pyrosequencing-derived ESTs resulted in 34,495 unique sequences with 1,110 sequences (3.2%) that were solely derived from Sanger sequencing alone. A high degree of similarity was seen between <it>P. ultimum </it>sequences and other sequenced plant pathogenic oomycetes with 91% of the hybrid assembly derived sequences > 500 bp having similarity to sequences from plant pathogenic <it>Phytophthora </it>species. An analysis of Gene Ontology assignments revealed a similar representation of molecular function ontologies in the hybrid assembly in comparison to the predicted proteomes of three <it>Phytophthora </it>species, suggesting a broad representation of the <it>P. ultimum </it>transcriptome was present in the normalized cDNA population. <it>P. ultimum </it>sequences with similarity to oomycete RXLR and Crinkler effectors, Kazal-like and cystatin-like protease inhibitors, and elicitins were identified. Sequences with similarity to thiamine biosynthesis enzymes that are lacking in the genome sequences of three <it>Phytophthora </it>species and one downy mildew were identified and could serve as useful phylogenetic markers. Furthermore, we identified 179 candidate simple sequence repeats that can be used for genotyping strains of <it>P. ultimum</it>.</p> <p>Conclusion</p> <p>Through these two technologies, we were able to generate a robust set (~10 Mb) of transcribed sequences for <it>P. ultimum</it>. We were able to identify known sequences present in oomycetes as well as identify novel sequences. An ample number of candidate polymorphic markers were identified in the dataset providing resources for phylogenetic and diagnostic marker development for this species. On a technical level, in spite of the depth possible with 454 FLX platform, the Sanger and pyro-based sequencing methodologies were complementary as each method generated sequences unique to each platform.</p

Aquatic Bacterial Communities Associated With Land Use and Environmental Factors in Agricultural Landscapes Using a Metabarcoding Approach

This study applied a 16S rRNA gene metabarcoding approach to characterize bacterial community compositional and functional attributes for surface water samples collected within, primarily, agriculturally dominated watersheds in Ontario and Québec, Canada. Compositional heterogeneity was best explained by stream order, season, and watercourse discharge. Generally, community diversity was higher at agriculturally dominated lower order streams, compared to larger stream order systems such as small to large rivers. However, during times of lower relative water flow and cumulative 2-day rainfall, modestly higher relative diversity was found in the larger watercourses. Bacterial community assemblages were more sensitive to environmental/land use changes in the smaller watercourses, relative to small-to-large river systems, where the proximity of the sampled water column to bacteria reservoirs in the sediments and adjacent terrestrial environment was greater. Stream discharge was the environmental variable most significantly correlated (all positive) with bacterial functional groups, such as C/N cycling and plant pathogens. Comparison of the community structural similarity via network analyses helped to discriminate sources of bacteria in freshwater derived from, for example, wastewater treatment plant effluent and intensity and type of agricultural land uses (e.g., intensive swine production vs. dairy dominated cash/livestock cropping systems). When using metabarcoding approaches, bacterial community composition and coexisting pattern rather than individual taxonomic lineages, were better indicators of environmental/land use conditions (e.g., upstream land use) and bacterial sources in watershed settings. Overall, monitoring changes and differences in aquatic microbial communities at regional and local watershed scales has promise for enhancing environmental footprinting and for better understanding nutrient cycling and ecological function of aquatic systems impacted by a multitude of stressors and land uses

DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes

On passion and moral behavior in achievement settings: The mediating role of pride

The Dualistic Model of Passion (Vallerand et al., 2003) distinguishes two types of passion: harmonious passion (HP) and obsessive passion (OP) that predict adaptive and less adaptive outcomes, respectively. In the present research, we were interested in understanding the role of passion in the adoption of moral behavior in achievement settings. It was predicted that the two facets of pride (authentic and hubristic; Tracy & Robins, 2007) would mediate the passion-moral behavior relationship. Specifically, because people who are passionate about a given activity are highly involved in it, it was postulated that they should typically do well and thus experience high levels of pride when engaged in the activity. However, it was also hypothesized that while both types of passion should be conducive to authentic pride, only OP should lead to hubristic pride. Finally, in line with past research on pride (Carver, Sinclair, & Johnson, 2010; Tracy et al., 2009), only hubristic pride was expected to negatively predict moral behavior, while authentic pride was expected to positively predict moral behavior. Results of two studies conducted with paintball players (N=163, Study 1) and athletes (N=296, Study 2) supported the proposed model. Future research directions are discussed in light of the Dualistic Model of Passion

The Amsterdam Declaration on Fungal Nomenclature

The Amsterdam Declaration on Fungal Nomenclature was agreed at an international symposium convened in Amsterdam on 19–20 April 2011 under the auspices of the International Commission on the Taxonomy of Fungi (ICTF). The purpose of the symposium was to address the issue of whether or how the current system of naming pleomorphic fungi should be maintained or changed now that molecular data are routinely available. The issue is urgent as mycologists currently follow different practices, and no consensus was achieved by a Special Committee appointed in 2005 by the International Botanical Congress to advise on the problem. The Declaration recognizes the need for an orderly transitition to a single-name nomenclatural system for all fungi, and to provide mechanisms to protect names that otherwise then become endangered. That is, meaning that priority should be given to the first described name, except where that is a younger name in general use when the first author to select a name of a pleomorphic monophyletic genus is to be followed, and suggests controversial cases are referred to a body, such as the ICTF, which will report to the Committee for Fungi. If appropriate, the ICTF could be mandated to promote the implementation of the Declaration. In addition, but not forming part of the Declaration, are reports of discussions held during the symposium on the governance of the nomenclature of fungi, and the naming of fungi known only from an environmental nucleic acid sequence in particular. Possible amendments to the Draft BioCode (2011) to allow for the needs of mycologists are suggested for further consideration, and a possible example of how a fungus only known from the environment might be described is presented

Global burden of 288 causes of death and life expectancy decomposition in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BACKGROUND Regular, detailed reporting on population health by underlying cause of death is fundamental for public health decision making. Cause-specific estimates of mortality and the subsequent effects on life expectancy worldwide are valuable metrics to gauge progress in reducing mortality rates. These estimates are particularly important following large-scale mortality spikes, such as the COVID-19 pandemic. When systematically analysed, mortality rates and life expectancy allow comparisons of the consequences of causes of death globally and over time, providing a nuanced understanding of the effect of these causes on global populations. METHODS The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 cause-of-death analysis estimated mortality and years of life lost (YLLs) from 288 causes of death by age-sex-location-year in 204 countries and territories and 811 subnational locations for each year from 1990 until 2021. The analysis used 56 604 data sources, including data from vital registration and verbal autopsy as well as surveys, censuses, surveillance systems, and cancer registries, among others. As with previous GBD rounds, cause-specific death rates for most causes were estimated using the Cause of Death Ensemble model-a modelling tool developed for GBD to assess the out-of-sample predictive validity of different statistical models and covariate permutations and combine those results to produce cause-specific mortality estimates-with alternative strategies adapted to model causes with insufficient data, substantial changes in reporting over the study period, or unusual epidemiology. YLLs were computed as the product of the number of deaths for each cause-age-sex-location-year and the standard life expectancy at each age. As part of the modelling process, uncertainty intervals (UIs) were generated using the 2·5th and 97·5th percentiles from a 1000-draw distribution for each metric. We decomposed life expectancy by cause of death, location, and year to show cause-specific effects on life expectancy from 1990 to 2021. We also used the coefficient of variation and the fraction of population affected by 90% of deaths to highlight concentrations of mortality. Findings are reported in counts and age-standardised rates. Methodological improvements for cause-of-death estimates in GBD 2021 include the expansion of under-5-years age group to include four new age groups, enhanced methods to account for stochastic variation of sparse data, and the inclusion of COVID-19 and other pandemic-related mortality-which includes excess mortality associated with the pandemic, excluding COVID-19, lower respiratory infections, measles, malaria, and pertussis. For this analysis, 199 new country-years of vital registration cause-of-death data, 5 country-years of surveillance data, 21 country-years of verbal autopsy data, and 94 country-years of other data types were added to those used in previous GBD rounds. FINDINGS The leading causes of age-standardised deaths globally were the same in 2019 as they were in 1990; in descending order, these were, ischaemic heart disease, stroke, chronic obstructive pulmonary disease, and lower respiratory infections. In 2021, however, COVID-19 replaced stroke as the second-leading age-standardised cause of death, with 94·0 deaths (95% UI 89·2-100·0) per 100 000 population. The COVID-19 pandemic shifted the rankings of the leading five causes, lowering stroke to the third-leading and chronic obstructive pulmonary disease to the fourth-leading position. In 2021, the highest age-standardised death rates from COVID-19 occurred in sub-Saharan Africa (271·0 deaths [250·1-290·7] per 100 000 population) and Latin America and the Caribbean (195·4 deaths [182·1-211·4] per 100 000 population). The lowest age-standardised death rates from COVID-19 were in the high-income super-region (48·1 deaths [47·4-48·8] per 100 000 population) and southeast Asia, east Asia, and Oceania (23·2 deaths [16·3-37·2] per 100 000 population). Globally, life expectancy steadily improved between 1990 and 2019 for 18 of the 22 investigated causes. Decomposition of global and regional life expectancy showed the positive effect that reductions in deaths from enteric infections, lower respiratory infections, stroke, and neonatal deaths, among others have contributed to improved survival over the study period. However, a net reduction of 1·6 years occurred in global life expectancy between 2019 and 2021, primarily due to increased death rates from COVID-19 and other pandemic-related mortality. Life expectancy was highly variable between super-regions over the study period, with southeast Asia, east Asia, and Oceania gaining 8·3 years (6·7-9·9) overall, while having the smallest reduction in life expectancy due to COVID-19 (0·4 years). The largest reduction in life expectancy due to COVID-19 occurred in Latin America and the Caribbean (3·6 years). Additionally, 53 of the 288 causes of death were highly concentrated in locations with less than 50% of the global population as of 2021, and these causes of death became progressively more concentrated since 1990, when only 44 causes showed this pattern. The concentration phenomenon is discussed heuristically with respect to enteric and lower respiratory infections, malaria, HIV/AIDS, neonatal disorders, tuberculosis, and measles. INTERPRETATION Long-standing gains in life expectancy and reductions in many of the leading causes of death have been disrupted by the COVID-19 pandemic, the adverse effects of which were spread unevenly among populations. Despite the pandemic, there has been continued progress in combatting several notable causes of death, leading to improved global life expectancy over the study period. Each of the seven GBD super-regions showed an overall improvement from 1990 and 2021, obscuring the negative effect in the years of the pandemic. Additionally, our findings regarding regional variation in causes of death driving increases in life expectancy hold clear policy utility. Analyses of shifting mortality trends reveal that several causes, once widespread globally, are now increasingly concentrated geographically. These changes in mortality concentration, alongside further investigation of changing risks, interventions, and relevant policy, present an important opportunity to deepen our understanding of mortality-reduction strategies. Examining patterns in mortality concentration might reveal areas where successful public health interventions have been implemented. Translating these successes to locations where certain causes of death remain entrenched can inform policies that work to improve life expectancy for people everywhere. FUNDING Bill & Melinda Gates Foundation

Cluster oligonucleotide signatures for rapid identification by sequencing

Abstract Background Oligonucleotide signatures (signatures) have been widely used for studying microbial diversity and function in wet-lab settings, but using them for accurate in silico identification of organisms from high-throughput sequencing (HTS) data is only a proof of concept. Existing signature design programs for sequence signatures (signatures matching exactly one sequence) or clade signatures (signatures matching every sequence in a phylogenetic clade) are not able to identify all possible polymorphic sites for sequences with high similarity and perform poorly when handling large genome sequencing datasets. Results We introduce cluster signatures: subsequences that match perfectly and exclusively any group of sequences in a data set. Cluster signatures provide complete recall for primer/probe design and increased discrimination between sequences beyond that of clade signatures. Using cluster signatures for in silico identification of HTS targets achieves good precision/recall and running time performance. This method has been implemented into an open source tool, the Automated Oligonucleotide Design Pipeline (adop), included in supplementary material and available at: https://bitbucket.org/wenchen_aafc/aodp_v2.0_release. Conclusions Cluster signatures provide a rapid and universal analysis tool to identify all possible short diagnostic DNA markers and variants from any DNA sequencing dataset. They are particularly useful in discriminating genetic material from closely related organisms and in detecting deleterious mutations in highly or perfectly conserved genomic sites

Intraspecific rDNA Restriction Fragment Length Polymorphism in the Xiphinema americanum group

Summary -Xlphimma americanum sensu law is a plant parasitic nematode vector of nepoviruses, which is widely distributed in North America. Considerable variation in the capabiliry to vector nepoviruses and in many morphological characters is found between different populations. The concept of this species has been considerably restricted by sorne authors while others recognized the variabiliry but hesitated to split the species. In this study we separated populations of the X. americanum group using restriction fragment length differences in the 5.8s gene and the internaI transcribed spacers (lTS) of ribosomal DNA. Two plasmid clones from Xiphinema bricolensis (Xb) 18s and 26s ribosomal genes were isolated from a genomic library using a complete repeat of the ribosomaJ cistron of Caenorhabdilis elegans (Ce) as a probe. Conserved sequences between Xb and Ce were identified and two 21-mer oligonucleotides primers were designed to amplify the ITS region using the polymerase chain reaction (PCR). The 1.5 Kb amplified product from the ITS region of each of sixteen populations of the X. americanum group was analyzed for restriction length polymorphisms (RFLP&apos;s). The RFPL&apos;s were recorded, dissimilariry coefficients were calculated, and a cluster analysis was generated arranging the sixteen populations as a dendrogram with five clusters. Two populations of X. rivesi were well separated from other X. americanum populations. X. bricolensis and two populations from Washington State were grouped together, while X. pacificum and an undescribed population from California were in another cluster. Mixed populations of X. n&apos;vesi and X. americanum from Pennsylvania and West Virginia could not be resolved. The taxonomie separation of this complex of species is an important step towards determining the vectors of nepoviruses in North America. Résumé -Polymorphisme intraspécifique de fragments de restrù:tion de l&apos;ADN ribosomal dans le groupe Xiphinema americanum -Xiphimma amen&apos;canum sensu lato, parasite de plantes et vecteur de nepovirus est présent dans nombre de régions d&apos;Amérique du Nord. Il existe dans les différentes populations une variabilité importante chez de nombreux caractères morphologiques. La définition de cette espèce a été restreinte d&apos;une manière considérable par certains auteurs tandis que d&apos;autres, tout en reconnaissant la variabilité, ont hésité à diviser l&apos;espèce. Cette étude sépare cettaines populations du groupe X. americanum en utilisant la différence de longueur des fragments de restriction de séquences du gène 5.8s et des régions intergéniques transcrites de l&apos;ADN ribosomaJ. Deux plasmides contenant des fragments clones des gènes ribosomaux 18s et 26s de Xlphinema bricolensis ont été isolés à partir d&apos;une librairie génomique en utilisant comme sonde l&apos;opéron complet de l&apos;ADN ribosomal de Caenorhabdilis elegans (Ce). Les séquences homologues entre Xb et Ce ont été utilisées comme amorces d&apos;amplification des ITS utilisant la réaction de polymérase en chaîne. Le produit d&apos;amplification de chacune des seize populations du groupe X. americanum, un segment d&apos;environ 1.5 Kb, a été analysé pour la longueur des fragments de restriction (RFLP&apos;s). Les polymorphismes ont été notés, des coefficients de dissimilarité entre populations ont été calculés, et une analyse en grappes a permis de grouper les seize populations en cinq groupes. Deux populations de X. rivesi sont bien séparées des autres populations du groupe X. americanum. X. bricolensis et deux populations de l&apos;état de Washington sont regroupées, tandis que X. panfieum et une population de Californie non encore identifiée forment une autre branche. Des populations de Pennsylvanie et de Virginie de l&apos;Ouest, contenant un mélange de X. americanum et de X. n&apos;vesi, n&apos;ont pu être séparées. L&apos;étude taxinomique de ce complexe d&apos;espèces est un pas important vers la détermination des vecteurs de nepovirus d&apos;Amérique du Nord