Gene expression networks in the Drosophila Genetic Reference Panel.

A major challenge in modern biology is to understand how naturally occurring variation in DNA sequences affects complex organismal traits through networks of intermediate molecular phenotypes. This question is best addressed in a genetic mapping population in which all molecular polymorphisms are known and for which molecular endophenotypes and complex traits are assessed on the same genotypes. Here, we performed deep RNA sequencing of 200 Drosophila Genetic Reference Panel inbred lines with complete genome sequences and for which phenotypes of many quantitative traits have been evaluated. We mapped expression quantitative trait loci for annotated genes, novel transcribed regions, transposable elements, and microbial species. We identified host variants that affect expression of transposable elements, independent of their copy number, as well as microbiome composition. We constructed sex-specific expression quantitative trait locus regulatory networks. These networks are enriched for novel transcribed regions and target genes in heterochromatin and euchromatic regions of reduced recombination, as well as genes regulating transposable element expression. This study provides new insights regarding the role of natural genetic variation in regulating gene expression and generates testable hypotheses for future functional analyses.

Natural genetic variation in a dopamine receptor is associated with variation in female fertility in Drosophila melanogaster.

Fertility is a major component of fitness but its genetic architecture remains poorly understood. Using a full diallel cross of 50 Drosophila Genetic Reference Panel inbred lines with whole genome sequences, we found substantial genetic variation in fertility largely attributable to females. We mapped genes associated with variation in female fertility by genome-wide association analysis of common variants in the fly genome. Validation of candidate genes by RNAi knockdown confirmed the role of the dopamine 2-like receptor (Dop2R) in promoting egg laying. We replicated the Dop2R effect in an independently collected productivity dataset and showed that the effect of the Dop2R variant was mediated in part by regulatory gene expression variation. This study demonstrates the strong potential of genome-wide association analysis in this diverse panel of inbred strains and subsequent functional analyses for understanding the genetic architecture of fitness traits.

The Drosophila melanogaster Genetic Reference Panel.

A major challenge of biology is understanding the relationship between molecular genetic variation and variation in quantitative traits, including fitness. This relationship determines our ability to predict phenotypes from genotypes and to understand how evolutionary forces shape variation within and between species. Previous efforts to dissect the genotype-phenotype map were based on incomplete genotypic information. Here, we describe the Drosophila melanogaster Genetic Reference Panel (DGRP), a community resource for analysis of population genomics and quantitative traits. The DGRP consists of fully sequenced inbred lines derived from a natural population. Population genomic analyses reveal reduced polymorphism in centromeric autosomal regions and the X chromosome, evidence for positive and negative selection, and rapid evolution of the X chromosome. Many variants in novel genes, most at low frequency, are associated with quantitative traits and explain a large fraction of the phenotypic variance. The DGRP facilitates genotype-phenotype mapping using the power of Drosophila genetics.

Genetic Architecture of Abdominal Pigmentation in Drosophila melanogaster.

Pigmentation varies within and between species and is often adaptive. The amount of pigmentation on the abdomen of Drosophila melanogaster is a relatively simple morphological trait, which serves as a model for mapping the genetic basis of variation in complex phenotypes. Here, we assessed natural variation in female abdominal pigmentation in 175 sequenced inbred lines of the Drosophila melanogaster Genetic Reference Panel, derived from the Raleigh, NC population. We quantified the proportion of melanization on the two most posterior abdominal segments, tergites 5 and 6 (T5, T6). We found significant genetic variation in the proportion of melanization and high broad-sense heritabilities for each tergite. Genome-wide association studies identified over 150 DNA variants associated with the proportion of melanization on T5 (84), T6 (34), and the difference between T5 and T6 (35). Several of the top variants associated with variation in pigmentation are in tan, ebony, and bric-a-brac1, genes known to affect D. melanogaster abdominal pigmentation. Mutational analyses and targeted RNAi-knockdown showed that 17 out of 28 (61%) novel candidate genes implicated by the genome-wide association study affected abdominal pigmentation. Several of these genes are involved in developmental and regulatory pathways, chitin production, cuticle structure, and vesicle formation and transport. These findings show that genetic variation may affect multiple steps in pathways involved in tergite development and melanization. Variation in these novel candidates may serve as targets for adaptive evolution and sexual selection in D. melanogaster.

Genetic basis of transcriptome diversity in Drosophila melanogaster.

Understanding how DNA sequence variation is translated into variation for complex phenotypes has remained elusive but is essential for predicting adaptive evolution, for selecting agriculturally important animals and crops, and for personalized medicine. Gene expression may provide a link between variation in DNA sequence and organismal phenotypes, and its abundance can be measured efficiently and accurately. Here we quantified genome-wide variation in gene expression in the sequenced inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP), increasing the annotated Drosophila transcriptome by 11%, including thousands of novel transcribed regions (NTRs). We found that 42% of the Drosophila transcriptome is genetically variable in males and females, including the NTRs, and is organized into modules of genetically correlated transcripts. We found that NTRs often were negatively correlated with the expression of protein-coding genes, which we exploited to annotate NTRs functionally. We identified regulatory variants for the mean and variance of gene expression, which have largely independent genetic control. Expression quantitative trait loci (eQTLs) for the mean, but not for the variance, of gene expression were concentrated near genes. Notably, the variance eQTLs often interacted epistatically with local variants in these genes to regulate gene expression. This comprehensive characterization of population-scale diversity of transcriptomes and its genetic basis in the DGRP is critically important for a systems understanding of quantitative trait variation.

Natural variation in genome architecture among 205 Drosophila melanogaster Genetic Reference Panel lines.

The Drosophila melanogaster Genetic Reference Panel (DGRP) is a community resource of 205 sequenced inbred lines, derived to improve our understanding of the effects of naturally occurring genetic variation on molecular and organismal phenotypes. We used an integrated genotyping strategy to identify 4,853,802 single nucleotide polymorphisms (SNPs) and 1,296,080 non-SNP variants. Our molecular population genomic analyses show higher deletion than insertion mutation rates and stronger purifying selection on deletions. Weaker selection on insertions than deletions is consistent with our observed distribution of genome size determined by flow cytometry, which is skewed toward larger genomes. Insertion/deletion and single nucleotide polymorphisms are positively correlated with each other and with local recombination, suggesting that their nonrandom distributions are due to hitchhiking and background selection. Our cytogenetic analysis identified 16 polymorphic inversions in the DGRP. Common inverted and standard karyotypes are genetically divergent and account for most of the variation in relatedness among the DGRP lines. Intriguingly, variation in genome size and many quantitative traits are significantly associated with inversions. Approximately 50% of the DGRP lines are infected with Wolbachia, and four lines have germline insertions of Wolbachia sequences, but effects of Wolbachia infection on quantitative traits are rarely significant. The DGRP complements ongoing efforts to functionally annotate the Drosophila genome. Indeed, 15% of all D. melanogaster genes segregate for potentially damaged proteins in the DGRP, and genome-wide analyses of quantitative traits identify novel candidate genes. The DGRP lines, sequence data, genotypes, quality scores, phenotypes, and analysis and visualization tools are publicly available.

Dopa decarboxylase (Ddc) affects variation in Drosophila longevity.

Mutational analyses in model organisms have shown that genes affecting metabolism and stress resistance regulate life span, but the genes responsible for variation in longevity in natural populations are largely unidentified. Previously, we mapped quantitative trait loci (QTLs) affecting variation in longevity between two Drosophila melanogaster strains. Here, we show that the longevity QTL in the 36E;38B cytogenetic interval on chromosome 2 contains multiple closely linked QTLs, including the Dopa decarboxylase (Ddc) locus. Complementation tests to mutations show that Ddc is a positional candidate gene for life span in these strains. Linkage disequilibrium (LD) mapping in a sample of 173 alleles from a single population shows that three common molecular polymorphisms in Ddc account for 15.5% of the genetic contribution to variance in life span from chromosome 2. The polymorphisms are in strong LD, and the effects of the haplotypes on longevity suggest that the polymorphisms are maintained by balancing selection. DDC catalyzes the final step in the synthesis of the neurotransmitters, dopamine and serotonin. Thus, these data implicate variation in the synthesis of bioamines as a factor contributing to natural variation in individual life span.

Genetic variation in senescence marker protein-30 is associated with natural variation in cold tolerance in Drosophila.

A comprehensive understanding of the genetic basis of phenotypic adaptation in nature requires the identification of the functional allelic variation underlying adaptive phenotypes. The manner in which organisms respond to temperature extremes is an adaptation in many species. In the current study, we investigate the role of molecular variation in senescence marker protein-30 (Smp-30) on natural phenotypic variation in cold tolerance in Drosophila melanogaster. Smp-30 encodes a product that is thought to be involved in the regulation of Ca2+ ion homeostasis and has been shown previously to be differentially expressed in response to cold stress. Thus, we sought to assess whether molecular variation in Smp-30 was associated with natural phenotypic variation in cold tolerance in a panel of naturally derived inbred lines from a population in Raleigh, North Carolina. We identified four non-coding polymorphisms that were strongly associated with natural phenotypic variation in cold tolerance. Interestingly, two polymorphisms that were in close proximity to one another (2 bp apart) exhibited opposite phenotypic effects. Consistent with the maintenance of a pair of antagonistically acting polymorphisms, tests of molecular evolution identified a significant excess of maintained variation in this region, suggesting balancing selection is acting to maintain this variation. These results suggest that multiple mutations in non-coding regions can have significant effects on phenotypic variation in adaptive traits within natural populations, and that balancing selection can maintain polymorphisms with opposite effects on phenotypic variation.

Genotype by environment interaction for gene expression in Drosophila melanogaster.

The genetics of phenotypic responses to changing environments remains elusive. Using whole-genome quantitative gene expression as a model, here we study how the genetic architecture of regulatory variation in gene expression changed in a population of fully sequenced inbred Drosophila melanogaster strains when flies developed in different environments (25 °C and 18 °C). We find a substantial fraction of the transcriptome exhibited genotype by environment interaction, implicating environmentally plastic genetic architecture of gene expression. Genetic variance in expression increases at 18 °C relative to 25 °C for most genes that have a change in genetic variance. Although the majority of expression quantitative trait loci (eQTLs) for the gene expression traits in the two environments are shared and have similar effects, analysis of the environment-specific eQTLs reveals enrichment of binding sites for two transcription factors. Finally, although genotype by environment interaction in gene expression could potentially disrupt genetic networks, the co-expression networks are highly conserved across environments. Genes with higher network connectivity are under stronger stabilizing selection, suggesting that stabilizing selection on expression plays an important role in promoting network robustness.

Phenotypic variation and natural selection at catsup, a pleiotropic quantitative trait gene in Drosophila.

Quantitative traits are shaped by networks of pleiotropic genes . To understand the mechanisms that maintain genetic variation for quantitative traits in natural populations and to predict responses to artificial and natural selection, we must evaluate pleiotropic effects of underlying quantitative trait genes and define functional allelic variation at the level of quantitative trait nucleotides (QTNs). Catecholamines up (Catsup), which encodes a negative regulator of tyrosine hydroxylase , the rate-limiting step in the synthesis of the neurotransmitter dopamine, is a pleiotropic quantitative trait gene in Drosophila melanogaster. We used association mapping to determine whether the same or different QTNs at Catsup are associated with naturally occurring variation in multiple quantitative traits. We sequenced 169 Catsup alleles from a single population and detected 33 polymorphisms with little linkage disequilibrium (LD). Different molecular polymorphisms in Catsup are independently associated with variation in longevity, locomotor behavior, and sensory bristle number. Most of these polymorphisms are potentially functional variants in protein coding regions, have large effects, and are not common. Thus, Catsup is a pleiotropic quantitative trait gene, but individual QTNs do not have pleiotropic effects. Molecular population genetic analyses of Catsup sequences are consistent with balancing selection maintaining multiple functional polymorphisms.

Association of polymorphisms in odorant-binding protein genes with variation in olfactory response to benzaldehyde in Drosophila.

Adaptive evolution of animals depends on behaviors that are essential for their survival and reproduction. The olfactory system of Drosophila melanogaster has emerged as one of the best characterized olfactory systems, which in addition to a family of odorant receptors, contains an approximately equal number of odorant-binding proteins (OBPs), encoded by a multigene family of 51 genes. Despite their abundant expression, little is known about their role in chemosensation, largely due to the lack of available mutations in these genes. We capitalized on naturally occurring mutations (polymorphisms) to gain insights into their functions. We analyzed the sequences of 13 Obp genes in two chromosomal clusters in a population of wild-derived inbred lines, and asked whether polymorphisms in these genes are associated with variation in olfactory responsiveness. Four polymorphisms in 3 Obp genes exceeded the statistical permutation threshold for association with responsiveness to benzaldehyde, suggesting redundancy and/or combinatorial recognition by these OBPs of this odorant. Model predictions of alternative pre-mRNA secondary structures associated with polymorphic sites suggest that alterations in Obp mRNA structure could contribute to phenotypic variation in olfactory behavior.

Quantitative analysis of bristle number in Drosophila mutants identifies genes involved in neural development.

BACKGROUND: The identification of the function of all genes that contribute to specific biological processes and complex traits is one of the major challenges in the postgenomic era. One approach is to employ forward genetic screens in genetically tractable model organisms. In Drosophila melanogaster, P element-mediated insertional mutagenesis is a versatile tool for the dissection of molecular pathways, and there is an ongoing effort to tag every gene with a P element insertion. However, the vast majority of P element insertion lines are viable and fertile as homozygotes and do not exhibit obvious phenotypic defects, perhaps because of the tendency for P elements to insert 5' of transcription units. Quantitative genetic analysis of subtle effects of P element mutations that have been induced in an isogenic background may be a highly efficient method for functional genome annotation. RESULTS: Here, we have tested the efficacy of this strategy by assessing the extent to which screening for quantitative effects of P elements on sensory bristle number can identify genes affecting neural development. We find that such quantitative screens uncover an unusually large number of genes that are known to function in neural development, as well as genes with yet uncharacterized effects on neural development, and novel loci. CONCLUSIONS: Our findings establish the use of quantitative trait analysis for functional genome annotation through forward genetics. Similar analyses of quantitative effects of P element insertions will facilitate our understanding of the genes affecting many other complex traits in Drosophila.

Candidate genes affecting Drosophila life span identified by integrating microarray gene expression analysis and QTL mapping.

The current increase in life expectancy observed in industrialized societies underscores the need to achieve a better understanding of the aging process that could help the development of effective strategies to achieve healthy aging. This will require not only identifying genes involved in the aging process, but also understanding how their effects are modulated by environmental factors, such as dietary intake and life style. Although the human genome has been sequenced, it may be impractical to study humans or other long-lived organisms to gain a mechanistic understanding about the aging process. Thus, short-lived animal models are essential to identifying the mechanisms and genes that affect the rate and quality of aging as a first step towards identifying genetic variants in humans. In this study, we investigated gene expression changes between two strains of Drosophila (Oregon and 2b) for which quantitative trait loci (QTLs) affecting life span were identified previously. We collected males and females from both strains at young and old ages, and assessed whole genome variation in transcript abundance using Affymetrix GeneChips. We observed 8217 probe sets with detectable transcripts. A total of 2371 probe sets, representing 2220 genes, exhibited significant changes in transcript abundance with age; and 839 probe sets were differentially expressed between Oregon and 2b. We focused on the 359 probe sets (representing 354 genes) that exhibited significant changes in gene expression both with age and between strains. We used these genes to integrate the analysis of microarray gene expression data, bioinformatics, and the results of genetic mapping studies reported previously, to identify 49 candidate genes and four pathways that could potentially be responsible for regulating life span and involved in the process of aging in Drosophila and humans.

Polygenic mutation in Drosophila melanogaster: Mapping spontaneous mutations affecting sensory bristle number.

Our ability to predict long-term responses to artificial and natural selection, and understand the mechanisms by which naturally occurring variation for quantitative traits is maintained, depends on detailed knowledge of the properties of spontaneous polygenic mutations, including the quantitative trait loci (QTL) at which mutations occur, mutation rates, and mutational effects. These parameters can be estimated by mapping QTL that cause divergence between mutation-accumulation lines that have been established from an inbred base population and selected for high and low trait values. Here, we have utilized quantitative complementation to deficiencies to map QTL at which spontaneous mutations affecting Drosophila abdominal and sternopleural bristle number have occurred in 11 replicate lines during 206 generations of divergent selection. Estimates of the numbers of mutations were consistent with diploid per-character mutation rates for bristle traits of 0.03. The ratio of the per-character mutation rate to total mutation rate (0.023) implies that >2% of the genome could affect just one bristle trait and that there must be extensive pleiotropy for quantitative phenotypes. The estimated mutational effects were not, however, additive and exhibited dependency on genetic background consistent with diminishing epistasis. However, these inferences must be tempered by the potential for epistatic interactions between spontaneous mutations and QTL affecting bristle number on the deficiency-bearing chromosomes, which could lead to overestimates in numbers of QTL and inaccurate inference of gene action.

Spontaneous mutations and the origin and maintenance of quantitative genetic variation.

Mutation and natural selection shape the genetic variation in natural populations. Here, we directly estimated the spontaneous mutation rate by sequencing new Drosophila mutation accumulation lines maintained with minimal natural selection. We inferred strong stabilizing natural selection on quantitative traits because genetic variation among wild-derived inbred lines was much lower than predicted from a neutral model and the mutational effects were much larger than allelic effects of standing polymorphisms. Stabilizing selection could act directly on the traits, or indirectly from pleiotropic effects on fitness. However, our data are not consistent with simple models of mutation-stabilizing selection balance; therefore, further empirical work is needed to assess the balance of evolutionary forces responsible for quantitative genetic variation.

hairy: A quantitative trait locus for drosophila sensory bristle number.

Advances in medicine, agriculture, and an understanding of evolution depend on resolving the genetic architecture of quantitative traits, which is challenging since variation for complex traits is caused by multiple interacting quantitative trait loci (QTL) with small and conditional effects. Here, we show that the key developmental gene, hairy (h), is a QTL for Drosophila sternopleural bristle number, a model quantitative trait. Near-isoallelic lines (NIL) for the h gene region exhibited significant variation in bristle number and failed to complement a hairy mutation. Sequencing 10 h alleles from a single population revealed 330 polymorphic sites in approximately 10 kb. Genotypes for 25 of these and 14 additional sites in the flanking regions were determined for the 57 NIL and associated with variation in bristle number in four genetic backgrounds. A highly significant association was found for a complicated insertion/deletion polymorphism upstream of the transcription start site. This polymorphism, present in 17.5% of the h alleles, was associated with an increase of 0.5 bristle and accounted for 31% of the genetic variance in bristle number in the NIL.

Transcription profiling in Drosophila eyes that overexpress the human glaucoma-associated trabecular meshwork-inducible glucocorticoid response protein/myocilin (TIGR/MYOC).

The availability of the human genome sequence together with sequenced genomes of several model organisms provides an unprecedented opportunity to utilize comparative genomic approaches for the discovery of genes that contribute to human disease. We have used transgenic flies to establish an experimental paradigm for the discovery of genes that might be involved in the development of glaucoma, a prevalent disease affecting a large segment of the population. Inherited mutations in the trabecular meshwork-inducible glucocorticoid response protein/myocilin (TIGR/MYOC) are associated with juvenile glaucoma and some cases of adult primary open angle glaucoma. The interrelationships between TIGR/MYOC and the development of glaucoma, however, are not understood. We show that overexpression of human TIGR/MYOC in the eyes of Drosophila melanogaster results in distortion of ommatidia accompanied by fluid discharge. High-density oligonucleotide microarrays identified altered expression of 50 transcripts in response to TIGR/MYOC overexpression, including homologs of aquaporin-4 and cytochrome-P450, previously associated with glaucoma, and several proteins of unknown function. We found that expression of Swiss Cheese, a neurodegenerative protein, increased 34-fold and that its human ortholog, neuropathy target esterase, is also upregulated in response to adenovirus-mediated overexpression of TIGR/MYOC in perfused postmortem human eyes. Our observations establish the Drosophila eye as an advantageous system for the discovery of genes that are associated with glaucoma.

Impaired olfactory behavior in mice deficient in the alpha subunit of G(o).

The ability to respond to chemical signals is essential for the survival and reproduction of most organisms. Olfactory signaling involves odorant receptor-mediated activation of G(olf), a homologue of G(s), on the dendrites of olfactory neurons. Olfactory receptor cells, however, also express Galpha(i2) and Galpha(o) on their axons, with all neurons expressing G(o) and a subset G(i2). Despite their abundance, possible contributions of G(o) and G(i2) to chemoreception remain unexplored. We investigated whether homologous recombinant mice deficient in the alpha subunit of G(o) are able to respond to odorants, whether possible olfactory impairments are dependent on genetic background, and whether formation of glomeruli in their olfactory bulbs is compromised. In an olfactory habituation/dishabituation test, G(o)-/- mice were unresponsive when exposed to odorants. Analysis of variance shows that performance of G(o)+/- mice crossed into the CD-1 background is also diminished in this test compared to their G(o)+/+ counterparts. Following food deprivation, G(o)-/- mice in the 129 Sv-ter/C57BL/6 genetic background were unable to locate a buried food pellet until they were approximately 10 weeks of age after which they performed as well as their litter mate controls. However, CD-1 G(o)-/- mice could locate a buried food pellet even when tested immediately after weaning. Despite their compromised olfactory responsiveness, histological examination did not reveal gross alterations in the olfactory bulbs of G(o)-/- mice. Thus, Galpha(o) is necessary for the expression of olfactory behavior under normal conditions and dependent on genetic background, but is not essential for the formation and maintenance of glomeruli.

Genomic response to selection for postponed senescence in Drosophila.

Limited lifespan and senescence are quantitative traits, controlled by many interacting genes with individually small and environmentally plastic effects, complicating genetic analysis. We performed genome wide analysis of gene expression for two Drosophila melanogaster lines selected for postponed senescence and one control, unselected line to identify candidate genes affecting lifespan as well as variation in lifespan. We obtained gene expression profiles for young flies of all lines, all lines at the time only 10% of the control lines survived, and the time at which 10% of the selected lines survived. Transcriptional responses to aging involved 19% of the genome. The transcriptional signature of aging involved the down-regulation of genes affecting proteolysis, metabolism, oxidative phosphorylation, and mitochrondrial function; and the up-regulation of genes affecting protein synthesis, immunity, defense responses, and the detoxification of xenobiotic substances. The transcriptional signature of postponed senescence involved the up-regulation of proteases and phosphatases and genes affecting detoxification of xenobiotics; and the down-regulation of genes affecting immunity, defense responses, metabolism and muscle function. Functional tests of 17 mutations confirmed 12 novel genes affecting Drosophila lifespan. Identification of genes affecting longevity by analysis of gene expression changes in lines selected for postponed senescence thus complements alternative genetic approaches.