Composite Effects of Polymorphisms near Multiple Regulatory Elements Create a Major-Effect QTL

Many agriculturally, evolutionarily, and medically important characters vary in a quantitative fashion. Unfortunately, the genes and sequence variants accounting for this variation remain largely unknown due to a variety of biological and technical challenges. Drosophila melanogaster contains high levels of sequence variation and low linkage disequilibrium, allowing us to dissect the effects of many causative variants within a single locus. Here, we take advantage of these features to identify and characterize the sequence polymorphisms that comprise major effect QTL alleles segregating at the bric-a-brac locus. We show that natural bric-a-brac alleles with large effects on cuticular pigmentation reflect a cumulative impact of polymorphisms that affect three functional regions: a promoter, a tissue-specific enhancer, and a Polycomb response element. Analysis of allele-specific expression at the bric-a-brac locus confirms that these polymorphisms modulate transcription at the cis-regulatory level. Our results establish that a single QTL can act through a confluence of multiple molecular mechanisms and that sequence variation in regions flanking experimentally validated functional elements can have significant quantitative effects on transcriptional activity and phenotype. These findings have important design and conceptual implications for basic and medical genomics

Widespread Selection Across Coding and Noncoding DNA in the Pea Aphid Genome

Genome-wide patterns of diversity and selection are critical measures for understanding how evolution has shaped the genome. Yet, these population genomic estimates are available for only a limited number of model organisms. Here we focus on the population genomics of the pea aphid (Acyrthosiphon pisum). The pea aphid is an emerging model system that exhibits a range of intriguing biological traits not present in classic model systems. We performed low-coverage genome resequencing of 21 clonal pea aphid lines collected from alfalfa host plants in North America to characterize genome-wide patterns of diversity and selection. We observed an excess of low-frequency polymorphisms throughout coding and noncoding DNA, which we suggest is the result of a founding event and subsequent population expansion in North America. Most gene regions showed lower levels of Tajima’s D than synonymous sites, suggesting that the majority of the genome is not evolving neutrally but rather exhibits significant constraint. Furthermore, we used the pea aphid’s unique manner of X-chromosome inheritance to assign genomic scaffolds to either autosomes or the X chromosome. Comparing autosomal vs. X-linked sequence variation, we discovered that autosomal genes show an excess of low frequency variants indicating that purifying selection acts more efficiently on the X chromosome. Overall, our results provide a critical first step in characterizing the genetic diversity and evolutionary pressures on an aphid genome

Abiotic and Biotic Stressors Causing Equivalent Mortality Induce Highly Variable Transcriptional Responses in the Soybean Aphid

Environmental stress affects basic organismal functioning and can cause physiological, developmental, and reproductive impairment. However, in many nonmodel organisms, the core molecular stress response remains poorly characterized and the extent to which stress-induced transcriptional changes differ across qualitatively different stress types is largely unexplored. The current study examines the molecular stress response of the soybean aphid (Aphis glycines) using RNA sequencing and compares transcriptional responses to multiple stressors (heat, starvation, and plant defenses) at a standardized stress level (27% adult mortality). Stress-induced transcriptional changes showed remarkable variation, with starvation, heat, and plant defensive stress altering the expression of 3985, 510, and 12 genes, respectively. Molecular responses showed little overlap across all three stressors. However, a common transcriptional stress response was identified under heat and starvation, involved with up-regulation of glycogen biosynthesis and molecular chaperones and down-regulation of bacterial endosymbiont cellular and insect cuticular components. Stressor-specific responses indicated heat affected expression of heat shock proteins and cuticular components, whereas starvation altered a diverse set of genes involved in primary metabolism, oxidative reductive processes, nucleosome and histone assembly, and the regulation of DNA repair and replication. Exposure to host plant defenses elicited the weakest response, of which half of the genes were of unknown function. This study highlights the need for standardizing stress levels when comparing across stress types and provides a basis for understanding the role of general vs. stressor specific molecular responses in aphids

Abiotic and Biotic Stressors Causing Equivalent Mortality Induce Highly Variable Transcriptional Responses in the Soybean Aphid

Environmental stress affects basic organismal functioning and can cause physiological, developmental, and reproductive impairment. However, in many nonmodel organisms, the core molecular stress response remains poorly characterized and the extent to which stress-induced transcriptional changes differ across qualitatively different stress types is largely unexplored. The current study examines the molecular stress response of the soybean aphid (Aphis glycines) using RNA sequencing and compares transcriptional responses to multiple stressors (heat, starvation, and plant defenses) at a standardized stress level (27% adult mortality). Stress-induced transcriptional changes showed remarkable variation, with starvation, heat, and plant defensive stress altering the expression of 3985, 510, and 12 genes, respectively. Molecular responses showed little overlap across all three stressors. However, a common transcriptional stress response was identified under heat and starvation, involved with up-regulation of glycogen biosynthesis and molecular chaperones and down-regulation of bacterial endosymbiont cellular and insect cuticular components. Stressor-specific responses indicated heat affected expression of heat shock proteins and cuticular components, whereas starvation altered a diverse set of genes involved in primary metabolism, oxidative reductive processes, nucleosome and histone assembly, and the regulation of DNA repair and replication. Exposure to host plant defenses elicited the weakest response, of which half of the genes were of unknown function. This study highlights the need for standardizing stress levels when comparing across stress types and provides a basis for understanding the role of general vs. stressor specific molecular responses in aphids

Allele-specific expression assays using Solexa

<p>Abstract</p> <p>Background</p> <p>Allele-specific expression (ASE) assays can be used to identify <it>cis</it>, <it>trans</it>, and <it>cis</it>-by-<it>trans </it>regulatory variation. Understanding the source of expression variation has important implications for disease susceptibility, phenotypic diversity, and adaptation. While ASE is commonly measured via relative fluorescence at a SNP, next generation sequencing provides an opportunity to measure ASE in an accurate and high-throughput manner using read counts.</p> <p>Results</p> <p>We introduce a Solexa-based method to perform large numbers of ASE assays using only a single lane of a Solexa flowcell. In brief, transcripts of interest, which contain a known SNP, are PCR enriched and barcoded to enable multiplexing. Then high-throughput sequencing is used to estimate allele-specific expression using sequencing counts. To validate this method, we measured the allelic bias in a dilution series and found high correlations between measured and expected values (r>0.9, p < 0.001). We applied this method to a set of 5 genes in a <it>Drosophila simulans </it>parental mix, F1 and introgression and found that for these genes the majority of expression divergence can be explained by <it>cis</it>-regulatory variation.</p> <p>Conclusion</p> <p>We present a new method with the capacity to measure ASE for large numbers of assays using as little as one lane of a Solexa flowcell. This will be a valuable technique for molecular and population genetic studies, as well as for verification of genome-wide data sets.</p

The Pea Aphid Uses a Version of the Terminal System during Oviparous, but not Viviparous, Development

Background: In most species of aphid, female nymphs develop into either sexual or asexual adults depending on the length of the photoperiod to which their mothers were exposed. The progeny of these sexual and asexual females, in turn, develop in dramatically different ways. The fertilized oocytes of sexual females begin embryogenesis after being deposited on leaves (oviparous development) while the oocytes of asexual females complete embryogenesis within the mother (viviparous development). Compared with oviparous development, viviparous development involves a smaller transient oocyte surrounded by fewer somatic epithelial cells and a smaller early embryo that comprises fewer cells. To investigate whether patterning mechanisms differ between the earliest stages of the oviparous and viviparous modes of pea aphid development, we examined the expression of pea aphid orthologs of genes known to specify embryonic termini in other insects. Results: Here we show that pea aphid oviparous ovaries express torso-like in somatic posterior follicle cells and activate ERK MAP kinase at the posterior of the oocyte. In addition to suggesting that some posterior features of the terminal system are evolutionarily conserved, our detection of activated ERK in the oocyte, rather than in the embryo, suggests that pea aphids may transduce the terminal signal using a mechanism distinct from the one used in Drosophila. In contrast with oviparous development, the pea aphid version of the terminal system does not appear to be used during viviparous development, since we did not detect expression of torso-like in the somatic epithelial cells that surround either the oocyte or the blastoderm embryo and we did not observe restricted activated ERK in the oocyte. Conclusions: We suggest that while oviparous oocytes and embryos may specify posterior fate through an aphid terminal system, viviparous oocytes and embryos employ a different mechanism, perhaps one that does not rely on an interaction between the oocyte and surrounding somatic cells. Together, these observations provide a striking example of a difference in the fundamental events of early development that is both environmentally induced and encoded by the same genome

The pea aphid uses a version of the terminal system during oviparous, but not viviparous, development

BACKGROUND: In most species of aphid, female nymphs develop into either sexual or asexual adults depending on the length of the photoperiod to which their mothers were exposed. The progeny of these sexual and asexual females, in turn, develop in dramatically different ways. The fertilized oocytes of sexual females begin embryogenesis after being deposited on leaves (oviparous development) while the oocytes of asexual females complete embryogenesis within the mother (viviparous development). Compared with oviparous development, viviparous development involves a smaller transient oocyte surrounded by fewer somatic epithelial cells and a smaller early embryo that comprises fewer cells. To investigate whether patterning mechanisms differ between the earliest stages of the oviparous and viviparous modes of pea aphid development, we examined the expression of pea aphid orthologs of genes known to specify embryonic termini in other insects. RESULTS: Here we show that pea aphid oviparous ovaries express torso-like in somatic posterior follicle cells and activate ERK MAP kinase at the posterior of the oocyte. In addition to suggesting that some posterior features of the terminal system are evolutionarily conserved, our detection of activated ERK in the oocyte, rather than in the embryo, suggests that pea aphids may transduce the terminal signal using a mechanism distinct from the one used in Drosophila. In contrast with oviparous development, the pea aphid version of the terminal system does not appear to be used during viviparous development, since we did not detect expression of torso-like in the somatic epithelial cells that surround either the oocyte or the blastoderm embryo and we did not observe restricted activated ERK in the oocyte. CONCLUSIONS: We suggest that while oviparous oocytes and embryos may specify posterior fate through an aphid terminal system, viviparous oocytes and embryos employ a different mechanism, perhaps one that does not rely on an interaction between the oocyte and surrounding somatic cells. Together, these observations provide a striking example of a difference in the fundamental events of early development that is both environmentally induced and encoded by the same genome

The Leeds Evaluation of Efficacy of Detoxification Study (LEEDS) prisons project: a randomised controlled trial comparing dihydrocodeine and buprenorphine for opiate detoxification

<p>Abstract</p> <p>Background</p> <p>Many opiate users entering British prisons require prescribed medication to help them achieve abstinence. This commonly takes the form of a detoxification regime. Previously, a range of detoxification agents have been prescribed without a clear evidence base to recommend a drug of choice. There are few trials and very few in the prison setting. This study compares dihydrocodeine with buprenorphine.</p> <p>Methods</p> <p>Open label, pragmatic, randomised controlled trial in a large remand prison in the North of England. Ninety adult male prisoners requesting an opiate detoxification were randomised to receive either daily sublingual buprenorphine or daily oral dihydrocodeine, given in the context of routine care. All participants gave written, informed consent. Reducing regimens were within a standard regimen of not more than 20 days and were at the discretion of the prescribing doctor. Primary outcome was abstinence from illicit opiates as indicated by a urine test at five days post detoxification. Secondary outcomes were collected during the detoxification period and then at one, three and six months post detoxification. Analysis was undertaken using relative risk tests for categorical data and unpaired t-tests for continuous data.</p> <p>Results</p> <p>64% of those approached took part in the study. 63 men (70%) gave a urine sample at five days post detoxification. At the completion of detoxification, by intention to treat analysis, a higher proportion of people allocated to buprenorphine provided a urine sample negative for opiates (abstinent) compared with those who received dihydrocodeine (57% vs 35%, RR 1.61 CI 1.02–2.56). At the 1, 3 and 6 month follow-up points, there were no significant differences for urine samples negative for opiates between the two groups. Follow up rates were low for those participants who had subsequently been released into the community.</p> <p>Conclusion</p> <p>These findings would suggest that dihydrocodeine should not be routinely used for detoxification from opiates in the prison setting. The high relapse rate amongst those achieving abstinence would suggest the need for an increased emphasis upon opiate maintenance programmes in the prison setting.</p> <p>Trial registration</p> <p>Current Controlled Trials ISRCTN07752728</p

Contrasting Patterns of Sequence Evolution at the Functionally Redundant bric à brac Paralogs in Drosophila melanogaster

Genes with overlapping expression and function may gradually diverge despite retaining some common functions. To test whether such genes show distinct patterns of molecular evolution within species, we examined sequence variation at the bric à brac (bab) locus of Drosophila melanogaster. This locus is composed of two anciently duplicated paralogs, bab1 and bab2, which are involved in patterning the adult abdomen, legs, and ovaries. We have sequenced the 148 kb genomic region spanning the bab1 and bab2 genes from 94 inbred lines of D. melanogaster sampled from a single location. Two non-coding regions, one in each paralog, appear to be under selection. The strongest evidence of directional selection is found in a region of bab2 that has no known functional role. The other region is located in the bab1 paralog and is known to contain a cis-regulatory element that controls sex-specific abdominal pigmentation. The coding region of bab1 appears to be under stronger functional constraint than the bab2 coding sequences. Thus, the two paralogs are evolving under different selective regimes in the same natural population, illuminating the different evolutionary trajectories of partially redundant duplicate genes

Circulating microRNAs in sera correlate with soluble biomarkers of immune activation but do not predict mortality in ART treated individuals with HIV-1 infection: A case control study

Introduction: The use of anti-retroviral therapy (ART) has dramatically reduced HIV-1 associated morbidity and mortality. However, HIV-1 infected individuals have increased rates of morbidity and mortality compared to the non-HIV-1 infected population and this appears to be related to end-organ diseases collectively referred to as Serious Non-AIDS Events (SNAEs). Circulating miRNAs are reported as promising biomarkers for a number of human disease conditions including those that constitute SNAEs. Our study sought to investigate the potential of selected miRNAs in predicting mortality in HIV-1 infected ART treated individuals. Materials and Methods: A set of miRNAs was chosen based on published associations with human disease conditions that constitute SNAEs. This case: control study compared 126 cases (individuals who died whilst on therapy), and 247 matched controls (individuals who remained alive). Cases and controls were ART treated participants of two pivotal HIV-1 trials. The relative abundance of each miRNA in serum was measured, by RTqPCR. Associations with mortality (all-cause, cardiovascular and malignancy) were assessed by logistic regression analysis. Correlations between miRNAs and CD4+ T cell count, hs-CRP, IL-6 and D-dimer were also assessed. Results: None of the selected miRNAs was associated with all-cause, cardiovascular or malignancy mortality. The levels of three miRNAs (miRs -21, -122 and -200a) correlated with IL-6 while miR-21 also correlated with D-dimer. Additionally, the abundance of miRs -31, -150 and -223, correlated with baseline CD4+ T cell count while the same three miRNAs plus miR- 145 correlated with nadir CD4+ T cell count. Discussion: No associations with mortality were found with any circulating miRNA studied. These results cast doubt onto the effectiveness of circulating miRNA as early predictors of mortality or the major underlying diseases that contribute to mortality in participants treated for HIV-1 infection