Drosophila Genetic Reference Panel Research Articles

Identification of genetic modifiers of lifespan on a high sugar diet in the Drosophila Genetic Reference Panel

Genome-wide association studies (GWAS) have become beneficial in identifying genetic variants underlying susceptibility to various complex diseases and conditions, including obesity. Utilizing the Drosophila Genetic Reference Panel (DGRP), we performed a GWAS of lifespan of 193 genetically distinct lines on a high sugar diet (HSD). The DGRP analysis pipeline determined the most significant lifespan associated polymorphisms were within loci of genes involved in: neural processes, behavior, development, and apoptosis, among other functions. Next, based on the relevance to obesity pathology, and the availability of transgenic RNAi lines targeting the genes we identified, whole-body in vivo knockdown of several candidate genes was performed. We utilized the GAL4-UAS binary expression system to independently validate the impacts of these loci on Drosophila lifespan during HSD. These loci were largely confirmed to affect lifespan in that HSD setting, as well as a normal diet setting. However, we also detected unexpected dietary effects of the HSD, including inconsistent diet effects on lifespan relative to a normal diet and a strong downregulation of feeding quantity.

Genetic basis of variation in cocaine and methamphetamine consumption in outbred populations of Drosophila melanogaster

We used Drosophila melanogaster to map the genetic basis of naturally occurring variation in voluntary consumption of cocaine and methamphetamine. We derived an outbred advanced intercross population (AIP) from 37 sequenced inbred wild-derived lines of the Drosophila melanogaster Genetic Reference Panel (DGRP), which are maximally genetically divergent, have minimal residual heterozygosity, are not segregating for common inversions, and are not infected with Wolbachia pipientis We assessed consumption of sucrose, methamphetamine-supplemented sucrose, and cocaine-supplemented sucrose and found considerable phenotypic variation for consumption of both drugs, in both sexes. We performed whole-genome sequencing and extreme quantitative trait locus (QTL) mapping on the top 10% of consumers for each replicate, sex, and condition and an equal number of randomly selected flies. We evaluated changes in allele frequencies among high consumers and control flies and identified 3,033 variants significantly (P < 1.9 × 10-8) associated with increased consumption, located in or near 1,962 genes. Many of these genes are associated with nervous system development and function, and 77 belong to a known gene-gene interaction subnetwork. We assessed the effects of RNA interference (RNAi) on drug consumption for 22 candidate genes; 17 had a significant effect in at least one sex. We constructed allele-specific AIPs that were homozygous for alternative candidate alleles for 10 single-nucleotide polymorphisms (SNPs) and measured average consumption for each population; 9 SNPs had significant effects in at least one sex. The genetic basis of voluntary drug consumption in Drosophila is polygenic and implicates genes with human orthologs and associated variants with sex- and drug-specific effects.

Genome-wide analysis in Drosophila reveals diet-by-gene interactions and uncovers diet-responsive genes.

Genetic and environmental factors play a major role in metabolic health. However, they do not act in isolation, as a change in an environmental factor such as diet may exert different effects based on an individual’s genotype. Here, we sought to understand how such gene–diet interactions influenced nutrient storage and utilization, a major determinant of metabolic disease. We subjected 178 inbred strains from the Drosophila genetic reference panel (DGRP) to diets varying in sugar, fat, and protein. We assessed starvation resistance, a holistic phenotype of nutrient storage and utilization that can be robustly measured. Diet influenced the starvation resistance of most strains, but the effect varied markedly between strains such that some displayed better survival on a high carbohydrate diet (HCD) compared to a high-fat diet while others had opposing responses, illustrating a considerable gene × diet interaction. This demonstrates that genetics plays a major role in diet responses. Furthermore, heritability analysis revealed that the greatest genetic variability arose from diets either high in sugar or high in protein. To uncover the genetic variants that contribute to the heterogeneity in starvation resistance, we mapped 566 diet-responsive SNPs in 293 genes, 174 of which have human orthologs. Using whole-body knockdown, we identified two genes that were required for glucose tolerance, storage, and utilization. Strikingly, flies in which the expression of one of these genes, CG4607 a putative homolog of a mammalian glucose transporter, was reduced at the whole-body level, displayed lethality on a HCD. This study provides evidence that there is a strong interplay between diet and genetics in governing survival in response to starvation, a surrogate measure of nutrient storage efficiency and obesity. It is likely that a similar principle applies to higher organisms thus supporting the case for nutrigenomics as an important health strategy.

Characterizing dopaminergic neuron vulnerability using genome-wide analysis

Parkinson's disease (PD) is primarily characterized by the loss of dopaminergic (DA) neurons in the brain. However, little is known about why DA neurons are selectively vulnerable to PD. To identify genes that are associated with DA neuron loss, we screened through 201 wild-caught populations of Drosophila melanogaster as part of the Drosophila Genetic Reference Panel. Here, we identify the top-associated genes containing single-nucleotide polymorphisms that render DA neurons vulnerable. These genes were further analyzed by using mutant analysis and tissue-specific knockdown for functional validation. We found that this loss of DA neurons caused progressive locomotor dysfunction in mutants and gene knockdown analysis. The identification of genes associated with the progressive loss of DA neurons should help to uncover factors that render these neurons vulnerable in PD, and possibly develop strategies to make these neurons more resilient.

Genetic architecture of natural variations of cardiac performances in flies: Conserved features with humans

Deciphering the genetic architecture of cardiac disorders is of fundamental importance but their underlying complexity is a major hurdle. Drosophila has gained importance as a useful model to study heart development and function and allows the analysis of organismal traits in a physiologically relevant and accessible system. Our aim was to (i) identify in flies the variants associated to natural variations of cardiac performances among a natural population, (ii) decipher how these variants interact with each other and with the environment to impact cardiac traits, (iii) gain insights about the molecular and cellular processes affected, (iv) determine whether the genetic architecture of cardiac disorders is conserved with humans. We used the Drosophila Genetic Reference Panel, a community resource of sequenced inbred lines. Genome Wide Associations (GWA) for single markers and epistatic interactions were analyzed on cardiac traits related to rhythm and contractility. Genetic networks were unraveled and extensively validated in vivo. Non-coding variants were used to map potential regulatory non-coding regions and to predict Transcription Factors (TFs) binding sites. Cognate TFs were validated by heart specific knockdown. Natural variations of cardiac traits variance revealed unique features of the phenotypic plasticity of cardiac performance. Importantly, correlations between genes associated to GWAS for cardiac traits both in flies and humans supported conservation of the genetic architecture of cardiac disorders from arthropods to mammals. In addition, several conserved genes and pathways were validated in human iPSC-derived cardiomyocytes. We provide an in-depth analysis of the genetic architecture of natural variations of cardiac performance in flies. Our data may guide the analysis of cardiac disorders in humans.

Genetic basis of offspring number–body weight tradeoff in Drosophila melanogaster

Drosophila melanogaster egg production, a proxy for fecundity, is an extensively studied life-history trait with a strong genetic basis. As eggs develop into larvae and adults, space and resource constraints can put pressure on the developing offspring, leading to a decrease in viability, body size, and lifespan. Our goal was to map the genetic basis of offspring number and weight under the restriction of a standard laboratory vial. We screened 143 lines from the Drosophila Genetic Reference Panel for offspring numbers and weights to create an “offspring index” that captured the number vs weight tradeoff. We found 18 genes containing 30 variants associated with variation in the offspring index. Validation of hid, Sox21b, CG8312, and mub candidate genes using gene disruption mutants demonstrated a role in adult stage viability, while mutations in Ih and Rbp increased offspring number and increased weight, respectively. The polygenic basis of offspring number and weight, with many variants of small effect, as well as the involvement of genes with varied functional roles, support the notion of Fisher’s “infinitesimal model” for this life-history trait.

Laboratory diet influences cold tolerance in a genotype-dependent manner in Drosophila melanogaster

Cold stress can reduce insect fitness and is an important determinant of species distributions and responses to climate change. Cold tolerance is influenced by genotype and environmental conditions, with factors such as day length and temperature having a particularly strong influence. Recent studies also indicate that diet impacts cold tolerance, but it is unclear whether diet-mediated shifts in cold tolerance are consistent across distinct genotypes. The goal of this study was to determine the extent to which commonly used artificial diets influence cold tolerance in Drosophila melanogaster, and whether these effects are consistent across genetically distinct lines. Specifically, we tested the impact of different fly diets on 1) ability to survive cold stress, 2) critical thermal minimum (CTmin), and 3) the ability to maintain reproduction after cold stress. Experiments were conducted across six isogenic lines from the Drosophila Genetic Reference Panel, and these lines were reared on different fly diets. Cold shock survival, CTmin, and reproductive output pre- and post-cold exposure varied considerably across diet and genotype combinations, suggesting strong genotype by environment interactions shape nutritionally mediated changes in cold tolerance. For example, in some lines cold shock survival remained consistently high or low across diets, while in others cold shock survival ranged from 5% to 75% depending on diet. Ultimately, these results add to a growing literature that cold tolerance is shaped by complex interactions between genotype and environment and inform practical considerations when selecting a laboratory diet for thermal tolerance experiments in Drosophila.

Genetic basis of susceptibility to low-dose paraquat and variation between the sexes in Drosophila melanogaster.

Toxicant resistance is a complex trait, affected both by genetics and the environment. Like most complex traits, it can exhibit sexual dimorphism, yet sex is often overlooked as a factor in studies of toxicant resistance. Paraquat, one such toxicant, is a commonly used herbicide and is known to produce mitochondrial oxidative stress, decrease dopaminergic neurons and dopamine (DA) levels, and decrease motor ability. While the main effects of paraquat are well-characterized, less is known about the naturally occurring variation in paraquat susceptibility. The purpose of this study was to map the genes contributing to low-dose paraquat susceptibility in Drosophila melanogaster, and to determine if susceptibility differs between the sexes. One hundred of the Drosophila Genetic Reference Panel (DGRP) lines were scored for susceptibility via climbing ability and used in a genome-wide association study (GWAS). Variation in seventeen genes in females and thirty-five genes in males associated with paraquat susceptibility. Only two candidate genes overlapped between the sexes despite a significant positive correlation between male and female susceptibilities. Many associated polymorphisms had significant interactions with sex, with most having conditionally neutral effects. Conditional neutrality between the sexes probably stems from sex-biased expression which may result from partial resolution of sexual conflict. Candidate genes were verified with RNAi knockdowns, gene expression analyses, and DA quantification. Several of these genes are novel associations with paraquat susceptibility. This research highlights the importance of assessing both sexes when studying toxicant susceptibility.

GWAS reveal a role for the central nervous system in regulating weight and weight change in response to exercise

Body size and weight show considerable variation both within and between species. This variation is controlled in part by genetics, but also strongly influenced by environmental factors including diet and the level of activity experienced by the individual. Due to the increasing obesity epidemic in much of the world, there is considerable interest in the genetic factors that control body weight and how weight changes in response to exercise treatments. Here, we address this question in the Drosophila model system, utilizing 38 strains of the Drosophila Genetics Reference Panel. We use GWAS to identify the molecular pathways that control weight and weight changes in response to exercise. We find that there is a complex set of molecular pathways controlling weight, with many genes linked to the central nervous system (CNS). The CNS also plays a role in the weight change with exercise, in particular, signaling from the CNS. Additional analyses revealed that weight in Drosophila is driven by two factors, animal size, and body composition, as the amount of fat mass versus lean mass impacts the density. Thus, while the CNS appears to be important for weight and exercise-induced weight change, signaling pathways are particularly important for determining how exercise impacts weight.

Accurate, ultra-low coverage genome reconstruction and association studies in Hybrid Swarm mapping populations.

Genetic association studies seek to uncover the link between genotype and phenotype, and often utilize inbred reference panels as a replicable source of genetic variation. However, inbred reference panels can differ substantially from wild populations in their genotypic distribution, patterns of linkage-disequilibrium, and nucleotide diversity. As a result, associations discovered using inbred reference panels may not reflect the genetic basis of phenotypic variation in natural populations. To address this problem, we evaluated a mapping population design where dozens to hundreds of inbred lines are outbred for few generations, which we call the Hybrid Swarm. The Hybrid Swarm approach has likely remained underutilized relative to pre-sequenced inbred lines due to the costs of genome-wide genotyping. To reduce sequencing costs and make the Hybrid Swarm approach feasible, we developed a computational pipeline that reconstructs accurate whole genomes from ultra-low-coverage (0.05X) sequence data in Hybrid Swarm populations derived from ancestors with phased haplotypes. We evaluate reconstructions using genetic variation from the Drosophila Genetic Reference Panel as well as variation from neutral simulations. We compared the power and precision of Genome-Wide Association Studies using the Hybrid Swarm, inbred lines, recombinant inbred lines (RILs), and highly outbred populations across a range of allele frequencies, effect sizes, and genetic architectures. Our simulations show that these different mapping panels vary in their power and precision, largely depending on the architecture of the trait. The Hybrid Swam and RILs outperform inbred lines for quantitative traits, but not for monogenic ones. Taken together, our results demonstrate the feasibility of the Hybrid Swarm as a cost-effective method of fine-scale genetic mapping.

Prediction of complex phenotypes using the Drosophila melanogaster metabolome.

Understanding the genotype-phenotype map and how variation at different levels of biological organization is associated are central topics in modern biology. Fast developments in sequencing technologies and other molecular omic tools enable researchers to obtain detailed information on variation at DNA level and on intermediate endophenotypes, such as RNA, proteins and metabolites. This can facilitate our understanding of the link between genotypes and molecular and functional organismal phenotypes. Here, we use the Drosophila melanogaster Genetic Reference Panel and nuclear magnetic resonance (NMR) metabolomics to investigate the ability of the metabolome to predict organismal phenotypes. We performed NMR metabolomics on four replicate pools of male flies from each of 170 different isogenic lines. Our results show that metabolite profiles are variable among the investigated lines and that this variation is highly heritable. Second, we identify genes associated with metabolome variation. Third, using the metabolome gave better prediction accuracies than genomic information for four of five quantitative traits analyzed. Our comprehensive characterization of population-scale diversity of metabolomes and its genetic basis illustrates that metabolites have large potential as predictors of organismal phenotypes. This finding is of great importance, e.g., in human medicine, evolutionary biology and animal and plant breeding.

Natural genetic variation in Drosophila melanogaster reveals genes associated with Coxiella burnetii infection.

The gram-negative bacterium Coxiella burnetii is the causative agent of Query (Q) fever in humans and coxiellosis in livestock. Host genetics are associated with C. burnetii pathogenesis both in humans and animals; however, it remains unknown if specific genes are associated with severity of infection. We employed the Drosophila Genetics Reference Panel to perform a genome-wide association study to identify host genetic variants that affect host survival to C. burnetii infection. The genome-wide association study identified 64 unique variants (P < 10−5) associated with 25 candidate genes. We examined the role each candidate gene contributes to host survival during C. burnetii infection using flies carrying a null mutation or RNAi knockdown of each candidate. We validated 15 of the 25 candidate genes using at least one method. This is the first report establishing involvement of many of these genes or their homologs with C. burnetii susceptibility in any system. Among the validated genes, FER and tara play roles in the JAK/STAT, JNK, and decapentaplegic/TGF-β signaling pathways which are components of known innate immune responses to C. burnetii infection. CG42673 and DIP-ε play roles in bacterial infection and synaptic signaling but have no previous association with C. burnetii pathogenesis. Furthermore, since the mammalian ortholog of CG13404 (PLGRKT) is an important regulator of macrophage function, CG13404 could play a role in host susceptibility to C. burnetii through hemocyte regulation. These insights provide a foundation for further investigation regarding the genetics of C. burnetii susceptibility across a wide variety of hosts.

Testing Implications of the Omnigenic Model for the Genetic Analysis of Loci Identified through Genome-wide Association

Organismal phenotypes usually have a quantitative distribution, and their genetic architecture can be studied by genome-wide association (GWA) mapping approaches. In most of such studies, it has become clear that many genes of moderate or small effects contribute to the phenotype.1-4 Hence, the attention has turned toward the loci falling below the GWA cut-off, which may contribute to the phenotype through modifier interactions with a set of core genes, as proposed in the omnigenic model.5 One can thus predict that both moderate effect GWA-derived candidate genes and randomly chosen genes should have a similar likelihood to affect a given phenotype when they are analyzed via gene disruption assays. We have tested this hypothesis by using an automated phenotyping system for Drosophila pupal phenotypes.6,7 We first identified candidate genes for pupal length in a GWA based on the Drosophila Genetic Reference Panel (DGRP)8,9 and showed that most of these candidate genes are indeed involved in the phenotype. We then randomly chose genes below a GWA significance threshold and found that three-quarters of them had also an effect on the trait with comparable effect sizes as the GWA candidate genes. We further tested the effects of these knockout lines on an independent behavioral pupal trait (pupation site choice) and found that a similar fraction had a significant effect as well. Our data thus confirm the implication that a large number of genes can influence independent quantitative traits.

Beta-blockers reduce intestinal permeability and early mortality following traumatic brain injury in Drosophila.

Traumatic brain injury (TBI) frequently leads to non-neurological consequences such as intestinal permeability. The beta-blocker drug labetalol, which inhibits binding of catecholamine neurotransmitters to adrenergic receptors, reduces intestinal permeability in a rat TBI model. Using a Drosophila melanogaster TBI model, we previously found a strong positive correlation between intestinal permeability and mortality within 24 hours of TBI in a standard laboratory line (w1118) and across genetically diverse inbred lines from the Drosophila Genetic Reference Panel (DGRP). Here, we report that feeding injured w1118flies the beta-blockers labetalol and metoprolol reduced intestinal permeability and mortality. Additionally, metoprolol reduced intestinal permeability when 18 DGRP fly lines were analyzed in aggregate, but neither beta-blocker affected mortality. These data indicate that the mechanism underlying disruption of the intestinal barrier by adrenergic signaling following TBI is conserved between humans and flies and that mortality following TBI in flies is not strictly dependent on disruption of the intestinal barrier. Thus, the fly TBI model is useful for shedding light on the mechanism and consequences of adrenergic signaling between the brain and intestine following TBI in humans.

Leveraging Multiple Layers of Data To Predict Drosophila Complex Traits.

The ability to accurately predict complex trait phenotypes from genetic and genomic data are critical for the implementation of personalized medicine and precision agriculture; however, prediction accuracy for most complex traits is currently low. Here, we used data on whole genome sequences, deep RNA sequencing, and high quality phenotypes for three quantitative traits in the ∼200 inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) to compare the prediction accuracies of gene expression and genotypes for three complex traits. We found that expression levels (r = 0.28 and 0.38, for females and males, respectively) provided higher prediction accuracy than genotypes (r = 0.07 and 0.15, for females and males, respectively) for starvation resistance, similar prediction accuracy for chill coma recovery (null for both models and sexes), and lower prediction accuracy for startle response (r = 0.15 and 0.14 for female and male genotypes, respectively; and r = 0.12 and 0.11, for females and male transcripts, respectively). Models including both genotype and expression levels did not outperform the best single component model. However, accuracy increased considerably for all the three traits when we included gene ontology (GO) category as an additional layer of information for both genomic variants and transcripts. We found strongly predictive GO terms for each of the three traits, some of which had a clear plausible biological interpretation. For example, for starvation resistance in females, GO:0033500 (r = 0.39 for transcripts) and GO:0032870 (r = 0.40 for transcripts), have been implicated in carbohydrate homeostasis and cellular response to hormone stimulus (including the insulin receptor signaling pathway), respectively. In summary, this study shows that integrating different sources of information improved prediction accuracy and helped elucidate the genetic architecture of three Drosophila complex phenotypes.

Musashi expression in intestinal stem cells attenuates radiation-induced decline in intestinal permeability and survival in Drosophila

Exposure to genotoxic stress by environmental agents or treatments, such as radiation therapy, can diminish healthspan and accelerate aging. We have developed a Drosophila melanogaster model to study the molecular effects of radiation-induced damage and repair. Utilizing a quantitative intestinal permeability assay, we performed an unbiased GWAS screen (using 156 strains from the Drosophila Genetic Reference Panel) to search for natural genetic variants that regulate radiation-induced gut permeability in adult D. melanogaster. From this screen, we identified an RNA binding protein, Musashi (msi), as one of the possible genes associated with changes in intestinal permeability upon radiation. The overexpression of msi promoted intestinal stem cell proliferation, which increased survival after irradiation and rescued radiation-induced intestinal permeability. In summary, we have established D. melanogaster as an expedient model system to study the effects of radiation-induced damage to the intestine in adults and have identified msi as a potential therapeutic target.

Genetic architecture of natural variations of cardiac perfomances in flies

Drosophila, the simplest model system with a beating heart, has tremendous advantages for the identification and validation of genetic factors affecting heart development, function and aging. Our project aims to analyse the genetic architecture of the natural variation of heart function in Drosophila and to identify its main characteristics. In particular, we want to take advantage of this model system to disentangle the complex relationship between genetic and environmental factors influencing these complex traits. Cardiac performances were analysed in the Drosophila Genetic Reference Panel (DGRP), and Genome Wide Association Studies (GWAS) were conducted to identify variants associated to several cardiac traits (rate, rhythm, cardiac output, fractional shortening, etc). The compendium of variants and associated genes identified were studied using approaches based on the analysis of gene and protein interaction networks. Genetic and protein networks analyses pointed towards several signalling pathways involved, that were further validated in vivo by targeted gene knockdown. Phenotypic variance among genetically identical individuals was also analysed and associated variants were compared to those affecting variations of phenotypic mean, revealing that distinct though overlapping gene networks were affected. In addition, our results show that more than 90% of variants are located within non-coding regions, and we characterized and validated some aspects of the transcriptional regulatory network affected. We provide a comprehensive characterization of population scale diversity of cardiac traits and of their genetic basis. Our work may provide a framework to interpret similar data from highers eukaryotes, including humans.

Genetic Basis of Natural Variation in Spontaneous Grooming in Drosophila melanogaster.

Spontaneous grooming behavior is a component of insect fitness. We quantified spontaneous grooming behavior in 201 sequenced lines of the Drosophila melanogaster Genetic Reference Panel and observed significant genetic variation in spontaneous grooming, with broad-sense heritabilities of 0.25 and 0.24 in females and males, respectively. Although grooming behavior is highly correlated between males and females, we observed significant sex by genotype interactions, indicating that the genetic basis of spontaneous grooming is partially distinct in the two sexes. We performed genome-wide association analyses of grooming behavior, and mapped 107 molecular polymorphisms associated with spontaneous grooming behavior, of which 73 were in or near 70 genes and 34 were over 1 kilobase from the nearest gene. The candidate genes were associated with a wide variety of gene ontology terms, and several of the candidate genes were significantly enriched in a genetic interaction network. We performed functional assessments of 29 candidate genes using RNA interference, and found that 11 affected spontaneous grooming behavior. The genes associated with natural variation in Drosophila grooming are involved with glutamate metabolism (Gdh) and transport (Eaat); interact genetically with (CCKLR-17D1) or are in the same gene family as (PGRP-LA) genes previously implicated in grooming behavior; are involved in the development of the nervous system and other tissues; or regulate the Notch and Epidermal growth factor receptor signaling pathways. Several DGRP lines exhibited extreme grooming behavior. Excessive grooming behavior can serve as a model for repetitive behaviors diagnostic of several human neuropsychiatric diseases.

Genetic variation for resistance to the specific fly pathogen Entomophthora muscae

We found substantial variation in resistance to the fly-specific pathogen Entomophthora muscae 'Berkeley' (Entomophthoromycota), in 20 lines from the Drosophila melanogaster Genetic Reference Panel (DGRP). Resistance to E. muscae is positively (r = 0.55) correlated with resistance to the broad host range ascomycete entomopathogen Metarhizium anisopliae (Ma549), indicative of generalist (non-specific) defenses. Most of the lines showing above average resistance to Ma549 showed cross-resistance to E. muscae. However, lines that succumbed quickly to Ma549 exhibited the full range of resistance to E. muscae. This suggests fly populations differ in E. muscae-specific resistance mechanisms as well as generic defences effective against both Ma549 and E. muscae. We looked for trade-offs that could account for inter-line variation, but increases (decreases) in disease resistance to E. muscae are not consistently associated with increases (decreases) of resistance to oxidative stress, starvation stress and sleep indices. That these pathogens are dynamic agents of selection on hosts is reflected in this genetic variation for resistance in lines derived from wild populations.

Integrating GWAS and Transcriptomics to Identify the Molecular Underpinnings of Thermal Stress Responses in Drosophila melanogaster.

Thermal tolerance of an organism depends on both the ability to dynamically adjust to a thermal stress and preparatory developmental processes that enhance thermal resistance. However, the extent to which standing genetic variation in thermal tolerance alleles influence dynamic stress responses vs. preparatory processes is unknown. Here, using the model species Drosophila melanogaster, we used a combination of Genome Wide Association mapping (GWAS) and transcriptomic profiling to characterize whether genes associated with thermal tolerance are primarily involved in dynamic stress responses or preparatory processes that influence physiological condition at the time of thermal stress. To test our hypotheses, we measured the critical thermal minimum (CTmin) and critical thermal maximum (CTmax) of 100 lines of the Drosophila Genetic Reference Panel (DGRP) and used GWAS to identify loci that explain variation in thermal limits. We observed greater variation in lower thermal limits, with CTmin ranging from 1.81 to 8.60°C, while CTmax ranged from 38.74 to 40.64°C. We identified 151 and 99 distinct genes associated with CTmin and CTmax, respectively, and there was strong support that these genes are involved in both dynamic responses to thermal stress and preparatory processes that increase thermal resistance. Many of the genes identified by GWAS were involved in the direct transcriptional response to thermal stress (72/151 for cold; 59/99 for heat), and overall GWAS candidates were more likely to be differentially expressed than other genes. Further, several GWAS candidates were regulatory genes that may participate in the regulation of stress responses, and gene ontologies related to development and morphogenesis were enriched, suggesting many of these genes influence thermal tolerance through effects on development and physiological status. Overall, our results suggest that thermal tolerance alleles can influence both dynamic plastic responses to thermal stress and preparatory processes that improve thermal resistance. These results also have utility for directly comparing GWAS and transcriptomic approaches for identifying candidate genes associated with thermal tolerance.