De novo sequence assembly and characterisation of a partial transcriptome for an evolutionarily distinct reptile, the tuatara (Sphenodon punctatus)

BACKGROUND: The tuatara (Sphenodon punctatus) is a species of extraordinary zoological interest, being the only surviving member of an entire order of reptiles which diverged early in amniote evolution. In addition to their unique phylogenetic placement, many aspects of tuatara biology, including temperature-dependent sex determination, cold adaptation and extreme longevity have the potential to inform studies of genome evolution and development. Despite increasing interest in the tuatara genome, genomic resources for the species are still very limited. We aimed to address this by assembling a transcriptome for tuatara from an early-stage embryo, which will provide a resource for genome annotation, molecular marker development and studies of development and adaptation in tuatara. RESULTS: We obtained 30 million paired-end 50 bp reads from an Illumina Genome Analyzer and assembled them with Velvet and Oases using a range of kmers. After removing redundancy and filtering out low quality transcripts, our transcriptome dataset contained 32911 transcripts, with an N50 of 675 and a mean length of 451 bp. Almost 50% (15965) of these transcripts could be annotated by comparison with the NCBI non-redundant (NR) protein database or the chicken, green anole and zebrafish UniGene sets. A scan of candidate genes and repetitive elements revealed genes involved in immune function, sex differentiation and temperature-sensitivity, as well as over 200 microsatellite markers. CONCLUSIONS: This dataset represents a major increase in genomic resources for the tuatara, increasing the number of annotated gene sequences from just 60 to almost 16,000. This will facilitate future research in sex determination, genome evolution, local adaptation and population genetics of tuatara, as well as inform studies on amniote evolution

Transcript and protein profiling identify candidate gene sets of potential adaptive significance in New Zealand Pachycladon

<p>Abstract</p> <p>Background</p> <p>Transcript profiling of closely related species provides a means for identifying genes potentially important in species diversification. However, the predictive value of transcript profiling for inferring downstream-physiological processes has been unclear. In the present study we use shotgun proteomics to validate inferences from microarray studies regarding physiological differences in three <it>Pachycladon </it>species. We compare transcript and protein profiling and evaluate their predictive value for inferring glucosinolate chemotypes characteristic of these species.</p> <p>Results</p> <p>Evidence from heterologous microarrays and shotgun proteomics revealed differential expression of genes involved in glucosinolate hydrolysis (myrosinase-associated proteins) and biosynthesis (methylthioalkylmalate isomerase and dehydrogenase), the interconversion of carbon dioxide and bicarbonate (carbonic anhydrases), water use efficiency (ascorbate peroxidase, 2 cys peroxiredoxin, 20 kDa chloroplastic chaperonin, mitochondrial succinyl CoA ligase) and others (glutathione-S-transferase, serine racemase, vegetative storage proteins, genes related to translation and photosynthesis). Differences in glucosinolate hydrolysis products were directly confirmed. Overall, prediction of protein abundances from transcript profiles was stronger than prediction of transcript abundance from protein profiles. Protein profiles also proved to be more accurate predictors of glucosinolate profiles than transcript profiles. The similarity of species profiles for both transcripts and proteins reflected previously inferred phylogenetic relationships while glucosinolate chemotypes did not.</p> <p>Conclusions</p> <p>We have used transcript and protein profiling to predict physiological processes that evolved differently during diversification of three <it>Pachycladon </it>species. This approach has also identified candidate genes potentially important in adaptation, which are now the focus of ongoing study. Our results indicate that protein profiling provides a valuable tool for validating transcript profiles in studies of adaptive divergence.</p

A Delphi study to determine the European core curriculum for Master programmes in genetic counselling.

Genetic counsellors have been working in some European countries for at least 30 years. Although there are great disparities between the numbers, education, practice and acceptance of these professionals across Europe, it is evident that genetic counsellors and genetic nurses in Europe are working autonomously within teams to deliver patient care. The aim of this study was to use the Delphi research method to develop a core curriculum to guide the educational preparation of these professionals in Europe. The Delphi method enables the researcher to utilise the views and opinions of a group of recognised experts in the field of study; this study consisted of four phases. Phases 1 and 4 consisted of expert workshops, whereas data were collected in phases 2 and 3 (n=35) via online surveys. All participants in the study were considered experts in the field of genetic counselling. The topics considered essential for genetic counsellor training have been organised under the following headings: (1) counselling; (2) psychological issues; (3) medical genetics; (4) human genetics; (5) ethics, law and sociology; (6) professional practice; and (7) education and research. Each topic includes the knowledge, skills and attitudes required to enable genetic counsellors to develop competence. In addition, it was considered by the experts that clinical practice should comprise 50% of the educational programme. The core Master programme curriculum will enable current courses to be assessed and inform the design of future educational programmes for European genetic counsellors

Reverse Genetics in Ecological Research

By precisely manipulating the expression of individual genetic elements thought to be important for ecological performance, reverse genetics has the potential to revolutionize plant ecology. However, untested concerns about possible side-effects of the transformation technique, caused by Agrobacterium infection and tissue culture, on plant performance have stymied research by requiring onerous sample sizes. We compare 5 independently transformed Nicotiana attenuata lines harboring empty vector control (EVC) T-DNA lacking silencing information with isogenic wild types (WT), and measured a battery of ecologically relevant traits, known to be important in plant-herbivore interactions: phytohormones, secondary metabolites, growth and fitness parameters under stringent competitive conditions, and transcriptional regulation with microarrays. As a positive control, we included a line silenced in trypsin proteinase inhibitor gene (TPI) expression, a potent anti-herbivore defense known to exact fitness costs in its expression, in the analysis. The experiment was conducted twice, with 10 and 20 biological replicates per genotype. For all parameters, we detected no difference between any EVC and WT lines, but could readily detect a fitness benefit of silencing TPI production. A statistical power analyses revealed that the minimum sample sizes required for detecting significant fitness differences between EVC and WT was 2–3 orders of magnitude larger than the 10 replicates required to detect a fitness effect of TPI silencing. We conclude that possible side-effects of transformation are far too low to obfuscate the study of ecologically relevant phenotypes

Reporting Helicopter Emergency Medical Services in Major Incidents: A Delphi Study

Peer reviewe

Different Transcript Patterns in Response to Specialist and Generalist Herbivores in the Wild Arabidopsis Relative Boechera divaricarpa

BACKGROUND: Plants defend themselves against herbivorous insects, utilizing both constitutive and inducible defenses. Induced defenses are controlled by several phytohormone-mediated signaling pathways. Here, we analyze transcriptional changes in the North American Arabidopsis relative Boechera divaricarpa in response to larval herbivory by the crucifer specialist lepidopteran Plutella xylostella (diamondback moth) and by the generalist lepidopteran Trichoplusia ni (cabbage semilooper), and compare them to wounding and exogenous phytohormone application. METHODOLOGY/PRINCIPAL FINDINGS: We use a custom macroarray constructed from B. divaricarpa herbivory-regulated cDNAs identified by suppression subtractive hybridization and from known stress-responsive A. thaliana genes for transcript profiling after insect herbivory, wounding and in response to jasmonate, salicylate and ethylene. In addition, we introduce path analysis as a novel approach to analyze transcript profiles. Path analyses reveal that transcriptional responses to the crucifer specialist P. xylostella are primarily determined by direct effects of the ethylene and salicylate pathways, whereas responses to the generalist T. ni are influenced by the ethylene and jasmonate pathways. Wound-induced transcriptional changes are influenced by all three pathways, with jasmonate having the strongest effect. CONCLUSIONS/SIGNIFICANCE: Our results show that insect herbivory is distinct from simple mechanical plant damage, and that different lepidopteran herbivores elicit different transcriptional responses

Brevicoryne brassicae aphids interfere with transcriptome responses of Arabidopsis thaliana to feeding by Plutella xylostella caterpillars in a density‑dependent manner

Plants are commonly attacked by multiple herbivorous species. Yet, little is known about transcriptional patterns underlying plant responses to multiple insect attackers feeding simultaneously. Here, we assessed= transcriptomic responses of Arabidopsis thaliana plants to simultaneous feeding by Plutella xylostella caterpillars and Brevicoryne brassicae aphids in comparison to plants infested by P. xylostella caterpillars alone, using microarray analysis. We particularly investigated how aphid feeding interferes with the transcriptomic response to P. xylostella caterpillars and whether this interference is dependent on aphid density and time since aphid attack. Various JA-responsive genes were up-regulated in response to feeding by P. xylostella caterpillars. The additional presence of aphids, both at low and high densities, clearly affected the transcriptional plant response to caterpillars. Interestingly, some important modulators of plant defense signalling, including WRKY transcription factor genes and ABA-dependent genes, were differentially induced in response to simultaneous aphid feeding at low or high density compared with responses to P. xylostella caterpillars feeding alone. Furthermore, aphids affected the P. xylostella-induced transcriptomic response in a density dependent manner, which caused an acceleration in plant response against dual insect attack at high aphid density compared to dual insect attack at low aphid density. In conclusion, our study provides evidence that aphids influence the caterpillar-induced transcriptional response of A. thaliana in a density-dependent manner. It highlights the importance of addressing insect density to understand how plant responses to single attackers interfere with responses to other attackers and thus underlines the importance of the dynamics of transcriptional plant responses to multiple herbivory