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In Drosophila melanogaster and other insects, the seminal fluid proteins (SFPs) and male sex pheromones that enter the female with sperm during mating are essential for fertility and induce profound post-mating effects on female physiology. The SFPs in D. melanogaster and other taxa include several members of the large gene family known as odorant binding proteins (Obps). Work in Drosophila has shown that some Obp genes are highly expressed in the antennae and can mediate behavioral responses to odorants, potentially by binding and carrying these molecules to odorant receptors. These observations have led to the hypothesis that the seminal Obps might act as molecular carriers for pheromones or other compounds important for male fertility, though functional evidence in any species is lacking. Here, we used functional genetics to test the role of the seven seminal Obps in D. melanogaster fertility and the post-mating response (PMR). We found that Obp56g is required for male fertility and the induction of the PMR, whereas the other six genes are dispensable. We found males lacking Obp56g fail to form a mating plug in the mated female’s reproductive tract, leading to ejaculate loss and reduced sperm storage, likely due to its expression in the male ejaculatory bulb. We also examined the evolutionary history of these seminal Obp genes, as several studies have documented rapid evolution and turnover of SFP genes across taxa. We found extensive lability in gene copy number and evidence of positive selection acting on two genes, Obp22a and Obp51a. Comparative RNAseq data from the male reproductive tract of multiple Drosophila species revealed that Obp56g shows high male reproductive tract expression in a subset of taxa, though conserved head expression across the phylogeny. Together, these functional and expression data suggest that Obp56g may have been co-opted for a reproductive function over evolutionary time.

Noncommunicable diseases (NCDs) are on the rise worldwide. Obesity, cardiovascular disease, and type 2 diabetes are among a long list of "lifestyle" diseases that were rare throughout human history but are now common. The evolutionary mismatch hypothesis posits that humans evolved in environments that radically differ from those we currently experience; consequently, traits that were once advantageous may now be "mismatched" and disease causing. At the genetic level, this hypothesis predicts that loci with a history of selection will exhibit "genotype by environment" (GxE) interactions, with different health effects in "ancestral" versus "modern" environments. To identify such loci, we advocate for combining genomic tools in partnership with subsistence-level groups experiencing rapid lifestyle change. In these populations, comparisons of individuals falling on opposite extremes of the "matched" to "mismatched" spectrum are uniquely possible. More broadly, the work we propose will inform our understanding of environmental and genetic risk factors for NCDs across diverse ancestries and cultures.

Recent experiments have produced several Anopheles gambiae homing gene drives that disrupt female fertility genes, thereby eventually inducing population collapse. Such drives may be highly effective tools to combat malaria. One such homing drive, based on the zpg promoter driving CRISPR/Cas9, was able to eliminate a cage population of mosquitoes. A second version, purportedly improved upon the first by incorporating an X-shredder element (which biases inheritance towards male offspring), was similarly successful. Here, we analyze experimental data from each of these gene drives to extract their characteristics and performance parameters and compare these to previous interpretations of their experimental performance. We assess each suppression drive within an individual-based simulation framework that models mosquito population dynamics in continuous space. We find that the combined homing/X-shredder drive is actually less effective at population suppression within the context of our mosquito population model. In particular, the combined drive often fails to completely suppress the population, instead resulting in an unstable equilibrium between drive and wild-type alleles. By contrast, otherwise similar drives based on the nos promoter may prove to be more promising candidates for future development than originally thought.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 provides a highly efficient and flexible genome editing technology with numerous potential applications ranging from gene therapy to population control. Some proposed applications involve the integration of CRISPR/Cas9 endonucleases into an organism’s genome, which raises questions about potentially harmful effects to the transgenic individuals. One example for which this is particularly relevant are CRISPR-based gene drives conceived for the genetic alteration of entire populations. The performance of such drives can strongly depend on fitness costs experienced by drive carriers, yet relatively little is known about the magnitude and causes of these costs. Here, we assess the fitness effects of genomic CRISPR/Cas9 expression in Drosophila melanogaster cage populations by tracking allele frequencies of four different transgenic constructs that allow us to disentangle ‘direct’ fitness costs due to the integration, expression, and target-site activity of Cas9, from fitness costs due to potential off-target cleavage. Using a maximum likelihood framework, we find that a model with no direct fitness costs but moderate costs due to off-target effects fits our cage data best. Consistent with this, we do not observe fitness costs for a construct with Cas9HF1, a high-fidelity version of Cas9. We further demonstrate that using Cas9HF1 instead of standard Cas9 in a homing drive achieves similar drive conversion efficiency. These results suggest that gene drives should be designed with high-fidelity endonucleases and may have implications for other applications that involve genomic integration of CRISPR endonucleases.

AbstractGene drives are engineered alleles that can bias inheritance in their favor, allowing them to spread throughout a population. They could potentially be used to modify or suppress pest populations, such as mosquitoes that spread diseases. CRISPR/Cas9 homing drives, which copy themselves by homology-directed repair in drive/wild-type heterozygotes, are a powerful form of gene drive, but they are vulnerable to resistance alleles that preserve the function of their target gene. Such resistance alleles can prevent successful population suppression. Here, we constructed a homing suppression drive in Drosophila melanogaster

Actomyosin contractility is a major engine of preimplantation morphogenesis, which starts at the 8-cell stage during mouse embryonic development. Contractility becomes first visible with the appearance of periodic cortical waves of contraction (PeCoWaCo), which travel around blastomeres in an oscillatory fashion. How contractility of the mouse embryo becomes active remains unknown. We have taken advantage of PeCoWaCo to study the awakening of contractility during preimplantation development. We find that PeCoWaCo become detectable in most embryos only after the second cleavage and gradually increase their oscillation frequency with each successive cleavage. To test the influence of cell size reduction during cleavage divisions, we use cell fusion and fragmentation to manipulate cell size across a 20- to 60-μm range. We find that the stepwise reduction in cell size caused by cleavage divisions does not explain the presence of PeCoWaCo or their accelerating rhythm. Instead, we discover that blastomeres gradually decrease their surface tensions until the 8-cell stage and that artificially softening cells enhances PeCoWaCo prematurely. We further identify the programmed down-regulation of the formin Fmnl3 as a required event to soften the cortex and expose PeCoWaCo. Therefore, during cleavage stages, cortical softening, mediated by Fmnl3 down-regulation, awakens zygotic contractility before preimplantation morphogenesis.

AbstractLittle is known about the genetic architecture of antifungal immunity in natural populations. Using two population genetic approaches, quantitative trait locus (QTL) mapping and evolve and resequence (E&R), we explored D. melanogasterBeauveria bassianaB. bassianaB. bassiana

AbstractSimple sequence tandem repeats are among the most rapidly evolving compartments of the genome. Some repeat expansions are associated with mammalian disease or meiotic segregation distortion, yet the rates of copy number change across generations are not well known. Here, we use 14 distinct sublineages of the C57BL/6 and C57BL/10 inbred mouse strains, which have been evolving independently over about 300 generations, to estimate the rates of copy number changes in genome-wide tandem repeats. Rates of change varied across repeats and across lines. Notably, CAG, whose expansions in coding regions are associated with many neurological and genetic disorders, was highly stable in copy number, likely indicating stabilizing selection. Rates of change were positively correlated with copy number, but the direction and magnitude of changes varied across lines. Some mouse lines experienced consistent losses or gains across most simple repeats, but this did not correlate with copy number changes in complex repeats. Rates of copy number change were similar between simple repeats and the more abundant complex repeats after normalization by copy number. Finally, the Y-specific centromeric repeat had a fourfold higher rate of change than the homologous centromeric repeat on other chromosomes. Structural differences in satellite complexity, or restriction to the Y chromosome and elevated mutation rates of the male germline, may explain the higher rate of change. Overall, our work underscores the mutational fluidity of long tandem arrays of repeats, and the correlations and constraints between genome-wide tandem repeats, which suggest that turnover is not a completely neutral process.

CRISPR-based homing gene drives have sparked both enthusiasm and deep concerns due to their potential for genetically altering entire species. This raises the question about our ability to prevent the unintended spread of such drives from the laboratory into a natural population. Here, we experimentally demonstrate the suitability of synthetic target site drives as well as split drives as flexible safeguarding strategies for gene drive experiments by showing that their performance closely resembles that of standard homing drives in Drosophila melanogaster. Using our split drive system, we further find that maternal deposition of both Cas9 and gRNA is required to form resistance alleles in the early embryo and that maternally-deposited Cas9 alone can power germline drive conversion in individuals that lack a genomic source of Cas9.

Selfish genetic elements (historically also referred to as selfish genes, ultra-selfish genes, selfish DNA, parasitic DNA, genomic outlaws) are genetic segments that can enhance their own transmission at the expense of other genes in the genome, even if this has no or a negative effect on organismal fitness. [1-6] Genomes have traditionally been viewed as cohesive units, with genes acting together to improve the fitness of the organism. However, when genes have some control over their own transmission, the rules can change, and so just like all social groups, genomes are vulnerable to selfish behaviour by their parts. Early observations of selfish genetic elements were made almost a century ago, but the topic did not get widespread attention until several decades later. Inspired by the gene-centred views of evolution popularized by George Williams[7] and Richard Dawkins,[8] two papers were published back-to-back in Nature in 1980-by Leslie Orgel and Francis Crick[9] and Ford Doolittle and Carmen Sapienza[10] respectively-introducing the concept of selfish genetic elements (at the time called "selfish DNA") to the wider scientific community. Both papers emphasized that genes can spread in a population regardless of their effect on organismal fitness as long as they have a transmission advantage. Selfish genetic elements have now been described in most groups of organisms, and they demonstrate a remarkable diversity in the ways by which they promote their own transmission.[11] Though long dismissed as genetic curiosities, with little relevance for evolution, they are now recognized to affect a wide swath of biological processes, ranging from genome size and architecture to speciation.[12].

Geographically limited dispersal can shape genetic population structure and result in a correlation between genetic and geographic distance, commonly called isolation-by-distance. Despite the prevalence of isolation-by-distance in nature, to date few studies have empirically demonstrated the processes that generate this pattern, largely because few populations have direct measures of individual dispersal and pedigree information. Intensive, long-term demographic studies and exhaustive genomic surveys in the Florida Scrub-Jay (Aphelocoma coerulescens) provide an excellent opportunity to investigate the influence of dispersal on genetic structure. Here, we used a panel of genome-wide SNPs and extensive pedigree information to explore the role of limited dispersal in shaping patterns of isolation-by-distance in both sexes, and at an exceedingly fine spatial scale (within ~10 km). Isolation-by-distance patterns were stronger in male-male and male-female comparisons than in female-female comparisons, consistent with observed differences in dispersal propensity between the sexes. Using the pedigree, we demonstrated how various genealogical relationships contribute to fine-scale isolation-by-distance. Simulations using field-observed distributions of male and female natal dispersal distances showed good agreement with the distribution of geographic distances between breeding individuals of different pedigree relationship classes. Furthermore, we built coalescent simulations parameterized by the observed dispersal curve, population density, and immigration rate, and showed how incorporating these extensions to Malécot's theory of isolation-by-distance allows us to accurately reconstruct observed sex-specific isolation-by-distance patterns in autosomal and Z-linked SNPs. Therefore, patterns of fine-scale isolation-by-distance in the Florida Scrub-Jay can be well understood as a result of limited dispersal over contemporary timescales.

A functioning gene drive system could fundamentally change our strategies for the control of vector-borne diseases by facilitating rapid dissemination of transgenes that prevent pathogen transmission or reduce vector capacity. CRISPR/Cas9 gene drive promises such a mechanism, which works by converting cells that are heterozygous for the drive construct into homozygotes, thereby enabling super-Mendelian inheritance. Although CRISPR gene drive activity has already been demonstrated, a key obstacle for current systems is their propensity to generate resistance alleles, which cannot be converted to drive alleles. In this study, we developed two CRISPR gene drive constructs based on the nanos and vasa promoters that allowed us to illuminate the different mechanisms by which resistance alleles are formed in the model organism Drosophila melanogaster. We observed resistance allele formation at high rates both prior to fertilization in the germline and post-fertilization in the embryo due to maternally deposited Cas9. Assessment of drive activity in genetically diverse backgrounds further revealed substantial differences in conversion efficiency and resistance rates. Our results demonstrate that the evolution of resistance will likely impose a severe limitation to the effectiveness of current CRISPR gene drive approaches, especially when applied to diverse natural populations.

Host genotype can influence the composition of the commensal bacterial community in some organisms. Composition, however, is only one parameter describing a microbial community. Here, we test whether a second parameter-abundance of bacteria-is a heritable trait by quantifying the presence of four commensal bacterial strains within 36 gnotobiotic inbred lines of Drosophila melanogaster. We find that D. melanogaster genotype exerts a significant effect on microbial levels within the fly. When introduced as monocultures into axenic flies, three of the four bacterial strains were reliably detected within the fly. The amounts of these different strains are strongly correlated, suggesting that the host regulates commensal bacteria through general, not bacteria-specific, means. While the correlation does not appear to be driven by simple variation in overall gut dimensions, a genetic association study suggests that variation in commensal bacterial load may largely be attributed to physical aspects of host cell growth and development.

Gene expression divergence between closely related species could be attributed to both cis- and trans- DNA sequence changes during evolution, but it is unclear how the evolutionary dynamics of epigenetic marks are regulated. In eutherian mammals, biparental DNA methylation marks are erased and reset during gametogenesis, resulting in paternal or maternal imprints, which lead to genomic imprinting. Whether DNA methylation reprogramming exists in insects is not known. Wasps of the genus Nasonia are non-social parasitoids that are emerging as a model for studies of epigenetic processes in insects. In this study, we quantified allele-specific expression and methylation genome-wide in Nasonia vitripennis and Nasonia giraulti and their reciprocal F1 hybrids. No parent-of-origin effect in allelic expression was found for >8,000 covered genes, suggesting a lack of genomic imprinting in adult Nasonia. As we expected, both significant cis- and trans- effects are responsible for the expression divergence between N. vitripennis and N. giraulti. Surprisingly, all 178 differentially methylated genes are also differentially methylated between the two alleles in F1 hybrid offspring, recapitulating the parental methylation status with nearly 100% fidelity, indicating the presence of strong cis-elements driving the target of gene body methylation. In addition, we discovered that total and allele-specific expression are positively correlated with allele-specific methylation in a subset of the differentially methylated genes. The 100% cis-regulation in F1 hybrids suggests the methylation machinery is conserved and DNA methylation is targeted by cis features in Nasonia. The lack of genomic imprinting and parent-of-origin differentially methylated regions in Nasonia, together with the stable inheritance of methylation status between generations, suggests either a cis-regulatory motif for methylation at the DNA level or highly stable inheritance of an epigenetic signal in Nasonia.

Abstract Progress in systems biology depends on accurate descriptions of biological networks. Connections in a regulatory network are identified as correlations of gene expression across a set of environmental or genetic perturbations. To use this information to predict system behavior, we must test how the nature of perturbations affects topologies of networks they reveal. To probe this question, we focused on metabolism of Drosophila melanogaster. Our source of perturbations is a set of crosses among 92 wild‐derived lines from five populations, replicated in a manner permitting separate assessment of the effects of genetic variation and environmental fluctuation. We directly assayed activities of enzymes and levels of metabolites. Using a multivariate Bayesian model, we estimated covariance among metabolic parameters and built fine‐grained probabilistic models of network topology. The environmental and genetic co‐regulation networks are substantially the same among five populations. However, genetic and environmental perturbations reveal qualitative differences in metabolic regulation, suggesting that environmental shifts, such as diet modifications, produce different systemic effects than genetic changes, even if the primary targets are the same.

Although host-parasitoid interactions are becoming well characterized at the organismal and cellular levels, much remains to be understood of the molecular bases for the host immune response and the parasitoids' ability to defeat this immune response. Leptopilina boulardi and L. heterotoma, two closely related, highly infectious natural parasitoids of Drosophila melanogaster, appear to use very different infection strategies at the cellular level. Here, we further characterize cellular level differences in the infection characteristics of these two wasp species using newly derived, virulent inbred strains, and then use whole genome microarrays to compare the transcriptional response of Drosophila to each. While flies attacked by the melanogaster group specialist L. boulardi (strain Lb17) up-regulate numerous genes encoding proteolytic enzymes, components of the Toll and JAK/STAT pathways, and the melanization cascade as part of a combined cellular and humoral innate immune response, flies attacked by the generalist L. heterotoma (strain Lh14) do not appear to initiate an immune transcriptional response at the time points post-infection we assayed, perhaps due to the rapid venom-mediated lysis of host hemocytes (blood cells). Thus, the specialist parasitoid appears to invoke a full-blown immune response in the host, but suppresses and/or evades downstream components of this response. Given that activation of the host immune response likely depletes the energetic resources of the host, the specialist's infection strategy seems relatively disadvantageous. However, we uncover the mechanism for one potentially important fitness tradeoff of the generalist's highly immune suppressive infection strategy.

Endoplasmic reticulum (ER) stress occurs when misfolded proteins accumulate in the ER. The cellular response to ER stress involves complex transcriptional and translational changes, important to the survival of the cell. ER stress is a primary cause and a modifier of many human diseases. A first step to understanding how the ER stress response impacts human disease is to determine how the transcriptional response to ER stress varies among individuals. The genetic diversity of the eight mouse Collaborative Cross (CC) founder strains allowed us to determine how genetic variation impacts the ER stress transcriptional response. We used tunicamycin, a drug commonly used to induce ER stress, to elicit an ER stress response in mouse embryonic fibroblasts (MEFs) derived from the CC founder strains and measured their transcriptional responses. We identified hundreds of genes that differed in response to ER stress across these genetically diverse strains. Strikingly, inflammatory response genes differed most between strains; major canonical ER stress response genes showed relatively invariant responses across strains. To uncover the genetic architecture underlying these strain differences in ER stress response, we measured the transcriptional response to ER stress in MEFs derived from a subset of F1 crosses between the CC founder strains. We found a unique layer of regulatory variation that is only detectable under ER stress conditions. Over 80% of the regulatory variation under ER stress derives from cis-regulatory differences. This is the first study to characterize the genetic variation in ER stress transcriptional response in the laboratory mouse. Our findings indicate that the ER stress transcriptional response is highly variable among strains and arises from genetic variation in individual downstream response genes, rather than major signaling transcription factors. These results have important implications for understanding how genetic variation impacts the ER stress response, an important component of many human diseases.

Whole genome analysis in large samples from a single population is needed to provide adequate power to assess relative strengths of natural selection across different functional components of the genome. In this study, we analyzed next-generation sequencing data from 962 European Americans, and found that as expected approximately 60% of the top 1% of positive selection signals lie in intergenic regions, 33% in intronic regions, and slightly over 1% in coding regions. Several detailed functional annotation categories in intergenic regions showed statistically significant enrichment in positively selected loci when compared to the null distribution of the genomic span of ENCODE categories. There was a significant enrichment of purifying selection signals detected in enhancers, transcription factor binding sites, microRNAs and target sites, but not on lincRNA or piRNAs, suggesting different evolutionary constraints for these domains. Loci in "repressed or low activity regions" and loci near or overlapping the transcription start site were the most significantly over-represented annotations among the top 1% of signals for positive selection.

Endoplasmic reticulum (ER) stress occurs when misfolded proteins accumulate in the lumen of the ER. A cell responds to ER stress with the unfolded protein response (UPR), a complex program of transcriptional and translational changes aimed at clearing misfolded proteins. Secretory tissues and cells are particularly well adapted to respond to ER stress because their function requires high protein production and secretory load. The insect male accessory gland (AG) is a secretory tissue involved in male fertility. The AG secretes many seminal fluid proteins (SFPs) essential for male reproduction. Among adult Drosophila tissues, we find that genes upregulated by ER stress are most highly expressed in the AG, suggesting that the AG is already undergoing high levels of ER stress due to its normal secretory functions. We hypothesized that induction of excessive ER stress in the AG above basal levels, would perturb normal function and provide a genetic tool for studying AG and SFP biology. To test this, we genetically induced excessive ER stress in the AG by conditional 1) expression of a misfolded protein or 2) knockdown of the UPR regulatory protein, BiP. Both genetic manipulations induced excessive ER stress in the AG, as indicated by the increase in Xbp1 splicing, a marker of ER stress. Both models resulted in a large decrease in or loss of SFP production and male infertility. Sperm production, motility, and transfer appeared unaffected. The induction of strong ER stress in the insect male AG may provide a simple way for studying or manipulating male fertility, as it eliminates AG function while preserving sperm production.

X inactivation--the transcriptional silencing of one X chromosome copy per female somatic cell--is universal among therian mammals, yet the choice of which X to silence exhibits considerable variation among species. X inactivation strategies can range from strict paternally inherited X inactivation (PXI), which renders females haploid for all maternally inherited alleles, to unbiased random X inactivation (RXI), which equalizes expression of maternally and paternally inherited alleles in each female tissue. However, the underlying evolutionary processes that might account for this observed diversity of X inactivation strategies remain unclear. We present a theoretical population genetic analysis of X inactivation evolution and specifically consider how conditions of dominance, linkage, recombination, and sex-differential selection each influence evolutionary trajectories of X inactivation. The results indicate that a single, critical interaction between allelic dominance and sex-differential selection can select for a broad and continuous range of X inactivation strategies, including unequal rates of inactivation between maternally and paternally inherited X chromosomes. RXI is favored over complete PXI as long as alleles deleterious to female fitness are sufficiently recessive, and the criteria for RXI evolution is considerably more restrictive when fitness variation is sexually antagonistic (i.e., alleles deleterious to females are beneficial to males) relative to variation that is deleterious to both sexes. Evolutionary transitions from PXI to RXI also generally increase mean relative female fitness at the expense of decreased male fitness. These results provide a theoretical framework for predicting and interpreting the evolution of chromosome-wide expression of X-linked genes and lead to several useful predictions that could motivate future studies of allele-specific gene expression variation.

The innate immune system in insects consists of a conserved core signaling network and rapidly diversifying effector and recognition components, often containing a high proportion of taxonomically-restricted genes. In the absence of functional annotation, genes encoding immune system proteins can thus be difficult to identify, as homology-based approaches generally cannot detect lineage-specific genes. Here, we use RNA-seq to compare the uninfected and infection-induced transcriptome in the parasitoid wasp Nasonia vitripennis to identify genes regulated by infection. We identify 183 genes significantly up-regulated by infection and 61 genes significantly down-regulated by infection. We also produce a new homology-based immune catalog in N. vitripennis, and show that most infection-induced genes cannot be assigned an immune function from homology alone, suggesting the potential for substantial novel immune components in less well-studied systems. Finally, we show that a high proportion of these novel induced genes are taxonomically restricted, highlighting the rapid evolution of immune gene content. The combination of functional annotation using RNA-seq and homology-based annotation provides a robust method to characterize the innate immune response across a wide variety of insects, and reveals significant novel features of the Nasonia immune response.

The parasitoid wasp Nasonia vitripennis is an emerging genetic model for functional analysis of DNA methylation. Here, we characterize genome-wide methylation at a base-pair resolution, and compare these results to gene expression across five developmental stages and to methylation patterns reported in other insects. An accurate assessment of DNA methylation across the genome is accomplished using bisulfite sequencing of adult females from a highly inbred line. One-third of genes show extensive methylation over the gene body, yet methylated DNA is not found in non-coding regions and rarely in transposons. Methylated genes occur in small clusters across the genome. Methylation demarcates exon-intron boundaries, with elevated levels over exons, primarily in the 5' regions of genes. It is also elevated near the sites of translational initiation and termination, with reduced levels in 5' and 3' UTRs. Methylated genes have higher median expression levels and lower expression variation across development stages than non-methylated genes. There is no difference in frequency of differential splicing between methylated and non-methylated genes, and as yet no established role for methylation in regulating alternative splicing in Nasonia. Phylogenetic comparisons indicate that many genes maintain methylation status across long evolutionary time scales. Nasonia methylated genes are more likely to be conserved in insects, but even those that are not conserved show broader expression across development than comparable non-methylated genes. Finally, examination of duplicated genes shows that those paralogs that have lost methylation in the Nasonia lineage following gene duplication evolve more rapidly, show decreased median expression levels, and increased specialization in expression across development. Methylation of Nasonia genes signals constitutive transcription across developmental stages, whereas non-methylated genes show more dynamic developmental expression patterns. We speculate that loss of methylation may result in increased developmental specialization in evolution and acquisition of methylation may lead to broader constitutive expression.

Characterizing and understanding the complex spectrum of lipids in higher organisms lags far behind our analysis of genome and transcriptome sequences. Here we generate and evaluate comprehensive lipid profiles (>200 lipids) of 92 inbred lines from five different Drosophila melanogaster populations. We find that the majority of lipid species are highly heritable, and even lipids with odd-chain fatty acids, which cannot be generated by the fly itself, also have high heritabilities. Abundance of the endosymbiont Wolbachia, a potential provider of odd-chained lipids, was positively correlated with this group of lipids. Additionally, we show that despite years of laboratory rearing on the same medium, the lipid profiles of the five geographic populations are sufficiently distinct for population discrimination. Our data predicts a strikingly different membrane fluidity for flies from the Netherlands, which is supported by their increased ethanol tolerance. We find that 18% of lipids show strong concentration differences between males and females. Through an analysis of the correlation structure of the lipid classes, we find modules of co-regulated lipids and begin to associate these with metabolic constraints. Our data provide a foundation for developing associations between variation in lipid composition with variation in other metabolic attributes, with genome-wide variation, and with metrics of health and overall reproductive fitness.

Various methods have been developed for identifying gene-gene interactions in genome-wide association studies (GWAS). However, most methods focus on individual markers as the testing unit, and the large number of such tests drastically erodes statistical power. In this study, we propose novel interaction tests of quantitative traits that are gene-based and that confer advantage in both statistical power and biological interpretation. The framework of gene-based gene-gene interaction (GGG) tests combine marker-based interaction tests between all pairs of markers in two genes to produce a gene-level test for interaction between the two. The tests are based on an analytical formula we derive for the correlation between marker-based interaction tests due to linkage disequilibrium. We propose four GGG tests that extend the following P value combining methods: minimum P value, extended Simes procedure, truncated tail strength, and truncated P value product. Extensive simulations point to correct type I error rates of all tests and show that the two truncated tests are more powerful than the other tests in cases of markers involved in the underlying interaction not being directly genotyped and in cases of multiple underlying interactions. We applied our tests to pairs of genes that exhibit a protein-protein interaction to test for gene-level interactions underlying lipid levels using genotype data from the Atherosclerosis Risk in Communities study. We identified five novel interactions that are not evident from marker-based interaction testing and successfully replicated one of these interactions, between SMAD3 and NEDD9, in an independent sample from the Multi-Ethnic Study of Atherosclerosis. We conclude that our GGG tests show improved power to identify gene-level interactions in existing, as well as emerging, association studies.

DNA sequences on X chromosomes often have a faster rate of evolution when compared to similar loci on the autosomes, and well articulated models provide reasons why the X-linked mode of inheritance may be responsible for the faster evolution of X-linked genes. We analyzed microarray and RNA-seq data collected from females and males of six Drosophila species and found that the expression levels of X-linked genes also diverge faster than autosomal gene expression, similar to the "faster-X" effect often observed in DNA sequence evolution. Faster-X evolution of gene expression was recently described in mammals, but it was limited to the evolutionary lineages shortly following the creation of the therian X chromosome. In contrast, we detect a faster-X effect along both deep lineages and those on the tips of the Drosophila phylogeny. In Drosophila males, the dosage compensation complex (DCC) binds the X chromosome, creating a unique chromatin environment that promotes the hyper-expression of X-linked genes. We find that DCC binding, chromatin environment, and breadth of expression are all predictive of the rate of gene expression evolution. In addition, estimates of the intraspecific genetic polymorphism underlying gene expression variation suggest that X-linked expression levels are not under relaxed selective constraints. We therefore hypothesize that the faster-X evolution of gene expression is the result of the adaptive fixation of beneficial mutations at X-linked loci that change expression level in cis. This adaptive faster-X evolution of gene expression is limited to genes that are narrowly expressed in a single tissue, suggesting that relaxed pleiotropic constraints permit a faster response to selection. Finally, we present a conceptional framework to explain faster-X expression evolution, and we use this framework to examine differences in the faster-X effect between Drosophila and mammals.

Total cholesterol, low-density lipoprotein cholesterol, triglyceride, and high-density lipoprotein cholesterol (HDL-C) levels are among the most important risk factors for coronary artery disease. We tested for gene-gene interactions affecting the level of these four lipids based on prior knowledge of established genome-wide association study (GWAS) hits, protein-protein interactions, and pathway information. Using genotype data from 9,713 European Americans from the Atherosclerosis Risk in Communities (ARIC) study, we identified an interaction between HMGCR and a locus near LIPC in their effect on HDL-C levels (Bonferroni corrected P(c) = 0.002). Using an adaptive locus-based validation procedure, we successfully validated this gene-gene interaction in the European American cohorts from the Framingham Heart Study (P(c) = 0.002) and the Multi-Ethnic Study of Atherosclerosis (MESA; P(c) = 0.006). The interaction between these two loci is also significant in the African American sample from ARIC (P(c) = 0.004) and in the Hispanic American sample from MESA (P(c) = 0.04). Both HMGCR and LIPC are involved in the metabolism of lipids, and genome-wide association studies have previously identified LIPC as associated with levels of HDL-C. However, the effect on HDL-C of the novel gene-gene interaction reported here is twice as pronounced as that predicted by the sum of the marginal effects of the two loci. In conclusion, based on a knowledge-driven analysis of epistasis, together with a new locus-based validation method, we successfully identified and validated an interaction affecting a complex trait in multi-ethnic populations.

Duplications play a significant role in both extremes of the phenotypic spectrum of newly arising mutations: they can have severe deleterious effects (e.g. duplications underlie a variety of diseases) but can also be highly advantageous. The phenotypic potential of newly arisen duplications has stimulated wide interest in both the mutational and selective processes shaping these variants in the genome. Here we take advantage of the Drosophila simulans-Drosophila melanogaster genetic system to further our understanding of both processes. Regarding mutational processes, the study of two closely related species allows investigation of the potential existence of shared duplication hotspots, and the similarities and differences between the two genomes can be used to dissect its underlying causes. Regarding selection, the difference in the effective population size between the two species can be leveraged to ask questions about the strength of selection acting on different classes of duplications. In this study, we conducted a survey of duplication polymorphisms in 14 different lines of D. simulans using tiling microarrays and combined it with an analogous survey for the D. melanogaster genome. By integrating the two datasets, we identified duplication hotspots conserved between the two species. However, unlike the duplication hotspots identified in mammalian genomes, Drosophila duplication hotspots are not associated with sequences of high sequence identity capable of mediating non-allelic homologous recombination. Instead, Drosophila duplication hotspots are associated with late-replicating regions of the genome, suggesting a link between DNA replication and duplication rates. We also found evidence supporting a higher effectiveness of selection on duplications in D. simulans than in D. melanogaster. This is also true for duplications segregating at high frequency, where we find evidence in D. simulans that a sizeable fraction of these mutations is being driven to fixation by positive selection.

A remarkable characteristic of the human major histocompatibility complex (MHC) is its extreme genetic diversity, which is maintained by balancing selection. In fact, the MHC complex remains one of the best-known examples of natural selection in humans, with well-established genetic signatures and biological mechanisms for the action of selection. Here, we present genetic and functional evidence that another gene with a fundamental role in MHC class I presentation, endoplasmic reticulum aminopeptidase 2 (ERAP2), has also evolved under balancing selection and contains a variant that affects antigen presentation. Specifically, genetic analyses of six human populations revealed strong and consistent signatures of balancing selection affecting ERAP2. This selection maintains two highly differentiated haplotypes (Haplotype A and Haplotype B), with frequencies 0.44 and 0.56, respectively. We found that ERAP2 expressed from Haplotype B undergoes differential splicing and encodes a truncated protein, leading to nonsense-mediated decay of the mRNA. To investigate the consequences of ERAP2 deficiency on MHC presentation, we correlated surface MHC class I expression with ERAP2 genotypes in primary lymphocytes. Haplotype B homozygotes had lower levels of MHC class I expressed on the surface of B cells, suggesting that naturally occurring ERAP2 deficiency affects MHC presentation and immune response. Interestingly, an ERAP2 paralog, endoplasmic reticulum aminopeptidase 1 (ERAP1), also shows genetic signatures of balancing selection. Together, our findings link the genetic signatures of selection with an effect on splicing and a cellular phenotype. Although the precise selective pressure that maintains polymorphism is unknown, the demonstrated differences between the ERAP2 splice forms provide important insights into the potential mechanism for the action of selection.

It is now well established that natural populations of Drosophila melanogaster harbor substantial genetic variation associated with physiological measures of immune function. In no case, however, have intermediate measures of immune function, such as transcriptional activity of immune-related genes, been tested as mediators of phenotypic variation in immunity. In this study, we measured bacterial load sustained after infection of D. melanogaster with Serratia marcescens, Providencia rettgeri, Enterococcus faecalis, and Lactococcus lactis in a panel of 94 third-chromosome substitution lines. We also measured transcriptional levels of 329 immune-related genes eight hours after infection with E. faecalis and S. marcescens in lines from the phenotypic tails of the test panel. We genotyped the substitution lines at 137 polymorphic markers distributed across 25 genes in order to test for statistical associations among genotype, bacterial load, and transcriptional dynamics. We find that genetic polymorphisms in the pathogen recognition genes (and particularly in PGRP-LC, GNBP1, and GNBP2) are most significantly associated with variation in bacterial load. We also find that overall transcriptional induction of effector proteins is a significant predictor of bacterial load after infection with E. faecalis, and that a marker upstream of the recognition gene PGRP-SD is statistically associated with variation in both bacterial load and transcriptional induction of effector proteins. These results show that polymorphism in genes near the top of the immune system signaling cascade can have a disproportionate effect on organismal phenotype due to the amplification of minor effects through the cascade.

Analysis of polymorphism and divergence in the non-coding portion of the human genome yields crucial information about factors driving the evolution of gene regulation. Candidate cis-regulatory regions spanning more than 15,000 genes in 15 African Americans and 20 European Americans were re-sequenced and aligned to the chimpanzee genome in order to identify potentially functional polymorphism and to characterize and quantify departures from neutral evolution. Distortions of the site frequency spectra suggest a general pattern of selective constraint on conserved non-coding sites in the flanking regions of genes (CNCs). Moreover, there is an excess of fixed differences that cannot be explained by a Gamma model of deleterious fitness effects, suggesting the presence of positive selection on CNCs. Extensions of the McDonald-Kreitman test identified candidate cis-regulatory regions with high probabilities of positive and negative selection near many known human genes, the biological characteristics of which exhibit genome-wide trends that differ from patterns observed in protein-coding regions. Notably, there is a higher probability of positive selection in candidate cis-regulatory regions near genes expressed in the fetal brain, suggesting that a larger portion of adaptive regulatory changes has occurred in genes expressed during brain development. Overall we find that natural selection has played an important role in the evolution of candidate cis-regulatory regions throughout hominid evolution.

Quantifying the distribution of fitness effects among newly arising mutations in the human genome is key to resolving important debates in medical and evolutionary genetics. Here, we present a method for inferring this distribution using Single Nucleotide Polymorphism (SNP) data from a population with non-stationary demographic history (such as that of modern humans). Application of our method to 47,576 coding SNPs found by direct resequencing of 11,404 protein coding-genes in 35 individuals (20 European Americans and 15 African Americans) allows us to assess the relative contribution of demographic and selective effects to patterning amino acid variation in the human genome. We find evidence of an ancient population expansion in the sample with African ancestry and a relatively recent bottleneck in the sample with European ancestry. After accounting for these demographic effects, we find strong evidence for great variability in the selective effects of new amino acid replacing mutations. In both populations, the patterns of variation are consistent with a leptokurtic distribution of selection coefficients (e.g., gamma or log-normal) peaked near neutrality. Specifically, we predict 27-29% of amino acid changing (nonsynonymous) mutations are neutral or nearly neutral (|s|1%). Our results are consistent with 10-20% of amino acid differences between humans and chimpanzees having been fixed by positive selection with the remainder of differences being neutral or nearly neutral. Our analysis also predicts that many of the alleles identified via whole-genome association mapping may be selectively neutral or (formerly) positively selected, implying that deleterious genetic variation affecting disease phenotype may be missed by this widely used approach for mapping genes underlying complex traits.

Imprinted genes display differential allelic expression in a manner that depends on the sex of the transmitting parent. The degree of imprinting is often tissue-specific and/or developmental stage-specific, and may be altered in some diseases including cancer. Here we applied Illumina/Solexa sequencing of the transcriptomes of reciprocal F1 mouse neonatal brains and identified 26 genes with parent-of-origin dependent differential allelic expression. Allele-specific Pyrosequencing verified 17 of them, including three novel imprinted genes. The known and novel imprinted genes all are found in proximity to previously reported differentially methylated regions (DMRs). Ten genes known to be imprinted in placenta had sufficient expression levels to attain a read depth that provided statistical power to detect imprinting, and yet all were consistent with non-imprinting in our transcript count data for neonatal brain. Three closely linked and reciprocally imprinted gene pairs were also discovered, and their pattern of expression suggests transcriptional interference. Despite the coverage of more than 5000 genes, this scan only identified three novel imprinted refseq genes in neonatal brain, suggesting that this tissue is nearly exhaustively characterized. This approach has the potential to yield an complete catalog of imprinted genes after application to multiple tissues and developmental stages, shedding light on the mechanism, bioinformatic prediction, and evolution of imprinted genes and diseases associated with genomic imprinting.

Tuberculosis (TB) is the leading cause of death worldwide due to an infectious agent. Data have accumulated over decades suggesting that variability in human susceptibility to TB disease has a genetic component. Toll-like receptors (TLRs) play a critical role in initiating the innate immune response to many pathogens in mouse models, but little is known about their role in human infections. Human TLRs have been reported to recognize mycobacterial antigens and initiate an immune response. We tested the hypothesis that amino acid-altering polymorphisms in five TLRs were associated with susceptibility to TB disease using a population-based case-control study with 1,312 adult TB patients and controls. Full-coding region sequencing of the five TLR genes in all 1,312 subjects yielded a data set in excess of 16 Mb. Rare nonsynonymous polymorphisms in TLR6-TLR1-TLR10 were significantly overrepresented among African-American TB cases compared with ethnically-matched control subjects. Common nonsynonymous polymorphisms in TLR6-TLR1-TLR10 also were significantly associated with TB disease in certain ethnic groups. Among African Americans, homozygotes for the common-variant haplotype TLR1-248S, TLR1-602I, and TLR6-249S had a significantly increased TB disease risk. A transmission/disequilibrium test on an independent sample found that the TLR1-248S variant was preferentially transmitted to diseased children, thereby confirming disease association. These results are consistent with recent reports implicating TLR1 variants, including TLR1-602, in significantly altered innate immune responses. Also consistent with disease association, rare TLR6 variants were defective in their ability to mediate NF-kappaB signal transduction in transfected human cells. Taken together, the data suggest that variant TLRs contribute to human susceptibility to TB disease. Extensive full-exon resequencing was critical for revealing new information about the role of TLRs in human-pathogen interactions and the genetic basis of innate immune function.

Identifying genomic locations that have experienced selective sweeps is an important first step toward understanding the molecular basis of adaptive evolution. Using statistical methods that account for the confounding effects of population demography, recombination rate variation, and single-nucleotide polymorphism ascertainment, while also providing fine-scale estimates of the position of the selected site, we analyzed a genomic dataset of 1.2 million human single-nucleotide polymorphisms genotyped in African-American, European-American, and Chinese samples. We identify 101 regions of the human genome with very strong evidence (p < 10(-5)) of a recent selective sweep and where our estimate of the position of the selective sweep falls within 100 kb of a known gene. Within these regions, genes of biological interest include genes in pigmentation pathways, components of the dystrophin protein complex, clusters of olfactory receptors, genes involved in nervous system development and function, immune system genes, and heat shock genes. We also observe consistent evidence of selective sweeps in centromeric regions. In general, we find that recent adaptation is strikingly pervasive in the human genome, with as much as 10% of the genome affected by linkage to a selective sweep.

The exploration of quantitative variation in human populations has become one of the major priorities for medical genetics. The successful identification of variants that contribute to complex traits is highly dependent on reliable assays and genetic maps. We have performed a genome-wide quantitative trait analysis of 630 genes in 60 unrelated Utah residents with ancestry from Northern and Western Europe using the publicly available phase I data of the International HapMap project. The genes are located in regions of the human genome with elevated functional annotation and disease interest including the ENCODE regions spanning 1% of the genome, Chromosome 21 and Chromosome 20q12-13.2. We apply three different methods of multiple test correction, including Bonferroni, false discovery rate, and permutations. For the 374 expressed genes, we find many regions with statistically significant association of single nucleotide polymorphisms (SNPs) with expression variation in lymphoblastoid cell lines after correcting for multiple tests. Based on our analyses, the signal proximal (cis-) to the genes of interest is more abundant and more stable than distal and trans across statistical methodologies. Our results suggest that regulatory polymorphism is widespread in the human genome and show that the 5-kb (phase I) HapMap has sufficient density to enable linkage disequilibrium mapping in humans. Such studies will significantly enhance our ability to annotate the non-coding part of the genome and interpret functional variation. In addition, we demonstrate that the HapMap cell lines themselves may serve as a useful resource for quantitative measurements at the cellular level.

Since the divergence of humans and chimpanzees about 5 million years ago, these species have undergone a remarkable evolution with drastic divergence in anatomy and cognitive abilities. At the molecular level, despite the small overall magnitude of DNA sequence divergence, we might expect such evolutionary changes to leave a noticeable signature throughout the genome. We here compare 13,731 annotated genes from humans to their chimpanzee orthologs to identify genes that show evidence of positive selection. Many of the genes that present a signature of positive selection tend to be involved in sensory perception or immune defenses. However, the group of genes that show the strongest evidence for positive selection also includes a surprising number of genes involved in tumor suppression and apoptosis, and of genes involved in spermatogenesis. We hypothesize that positive selection in some of these genes may be driven by genomic conflict due to apoptosis during spermatogenesis. Genes with maximal expression in the brain show little or no evidence for positive selection, while genes with maximal expression in the testis tend to be enriched with positively selected genes. Genes on the X chromosome also tend to show an elevated tendency for positive selection. We also present polymorphism data from 20 Caucasian Americans and 19 African Americans for the 50 annotated genes showing the strongest evidence for positive selection. The polymorphism analysis further supports the presence of positive selection in these genes by showing an excess of high-frequency derived nonsynonymous mutations.

Objectives To investigate causality of the association of serum vitamin D with the risk and severity of COVID-19 infection.Design Two-sample Mendelian randomisation study.Setting Summary data from genome-wide analyses in the population-based UK Biobank and SUNLIGHT Consortium, applied to meta-analysed results of genome-wide analyses in the COVID-19 Host Genetics Initiative.Participants 17 965 COVID-19 cases including 11 085 laboratory or physician-confirmed cases, 7885 hospitalised cases and 4336 severe respiratory cases, and 1 370 547 controls, primarily of European ancestry.Exposures Genetically predicted variation in serum vitamin D status, instrumented by genome-wide significant single nucleotide polymorphisms (SNPs) associated with serum vitamin D or risk of vitamin D deficiency/insufficiency.Main outcome measures Susceptibility to and severity of COVID-19 infection, including severe respiratory infection and hospitalisation.Results Mendelian randomisation analysis, sufficiently powered to detect effects comparable to those seen in observational studies, provided little to no evidence for an effect of genetically predicted serum vitamin D on susceptibility to or severity of COVID-19 infection. Using SNPs in loci related to vitamin D metabolism as genetic instruments for serum vitamin D concentrations, the OR per SD higher serum vitamin D was 1.04 (95% CI 0.92 to 1.18) for any COVID-19 infection versus population controls, 1.05 (0.84 to 1.31) for hospitalised COVID-19 versus population controls, 0.96 (0.64 to 1.43) for severe respiratory COVID-19 versus population controls, 1.15 (0.99 to 1.35) for COVID-19 positive versus COVID-19 negative and 1.44 (0.75 to 2.78) for hospitalised COVID-19 versus non-hospitalised COVID-19. Results were similar in analyses using SNPs with genome-wide significant associations with serum vitamin D (ie, including SNPs in loci with no known relationship to vitamin D metabolism) and in analyses using SNPs with genome-wide significant associations with risk of vitamin D deficiency or insufficiency.Conclusions These findings suggest that genetically predicted differences in long-term vitamin D nutritional status do not causally affect susceptibility to and severity of COVID-19 infection, and that associations observed in previous studies may have been driven by confounding. These results do not exclude the possibility of low-magnitude causal effects or causal effects of acute responses to therapeutic doses of vitamin D.