Genomic Variation and Its Impact on Gene Expression in Drosophila melanogaster

Understanding the relationship between genetic and phenotypic variation is one of the great outstanding challenges in biology. To meet this challenge, comprehensive genomic variation maps of human as well as of model organism populations are required. Here, we present a nucleotide resolution catalog of single-nucleotide, multi-nucleotide, and structural variants in 39 Drosophila melanogaster Genetic Reference Panel inbred lines. Using an integrative, local assembly-based approach for variant discovery, we identify more than 3.6 million distinct variants, among which were more than 800,000 unique insertions, deletions (indels), and complex variants (1 to 6,000 bp). While the SNP density is higher near other variants, we find that variants themselves are not mutagenic, nor are regions with high variant density particularly mutation-prone. Rather, our data suggest that the elevated SNP density around variants is mainly due to population-level processes. We also provide insights into the regulatory architecture of gene expression variation in adult flies by mapping cis-expression quantitative trait loci (cis-eQTLs) for more than 2,000 genes. Indels comprise around 10% of all cis-eQTLs and show larger effects than SNP cis-eQTLs. In addition, we identified two-fold more gene associations in males as compared to females and found that most cis-eQTLs are sex-specific, revealing a partial decoupling of the genomic architecture between the sexes as well as the importance of genetic factors in mediating sex-biased gene expression. Finally, we performed RNA-seq-based allelic expression imbalance analyses in the offspring of crosses between sequenced lines, which revealed that the majority of strong cis-eQTLs can be validated in heterozygous individuals.     

Sex-Specific Weight Loss Mediates Sexual Size Dimorphism in Drosophila melanogaster

The selective pressures leading to the evolution of Sexual Size Dimorphism (SSD) have been well studied in many organisms, yet, the underlying developmental mechanisms are poorly understood. By generating a complete growth profile by sex in Drosophila melanogaster, we describe the sex-specific pattern of growth responsible for SSD. Growth rate and critical size for pupariation significantly contributed to adult SSD, whereas duration of growth did not. Surprisingly, SSD at peak larval mass was twice that of the uneclosed adult SSD with weight loss between peak larval mass and pupariation playing an important role in generating the final SSD. Our finding that weight loss is an important regulator of SSD adds additional complexity to our understanding of how body size is regulated in different sexes. Collectively, these data allow for the elucidation of the molecular-genetic mechanisms that generate SSD, an important component of understanding how SSD evolves.     

Age,but Not Experience,Affects Courtship Gene Expression in Male Drosophila melanogaster

Mutation screens in model organisms have helped identify the foundation of many fundamental organismal phenotypes. An emerging question in evolutionary and behavioral biology is the extent to which these “developmental” genes contribute to the subtle individual variation that characterizes natural populations. A related question is whether individual differences arise from static differences in gene expression that arose during previous life stages, or whether they are due to dynamic regulation of expression during the life stage under investigation. Here, we address these questions using genes that have been discovered to control the development of normal courtship behavior in male Drosophila melanogaster. We examined whether these genes have static or dynamic expression in the heads of adult male flies of different ages and with different levels of social experience. We found that 16 genes of the 25 genes examined were statically expressed, and 9 genes were dynamically expressed with changes related to adult age. No genes exhibited rapid dynamic expression changes due to social experience or age*experience interaction. We therefore conclude that a majority of fly “courtship” genes are statically expressed, while a minority are regulated in adults with respect to age, but not with respect to relevant social experience. These results are consistent with those from a recent microarray analysis that found none of the canonical courtship genes changed expression in male flies after brief exposure to females.     

A Genome-Wide Gene Function Prediction Resource for Drosophila melanogaster

Predicting gene functions by integrating large-scale biological data remains a challenge for systems biology. Here we present a resource for Drosophila melanogaster gene function predictions. We trained function-specific classifiers to optimize the influence of different biological datasets for each functional category. Our model predicted GO terms and KEGG pathway memberships for Drosophila melanogaster genes with high accuracy, as affirmed by cross-validation, supporting literature evidence, and large-scale RNAi screens. The resulting resource of prioritized associations between Drosophila genes and their potential functions offers a guide for experimental investigations.     

Effect of Spaceflight on the Circadian Rhythm,Lifespan and Gene Expression of Drosophila melanogaster

Space travelers are reported to experience circadian rhythm disruption during spaceflight. However, how the space environment affects circadian rhythm is yet to be determined. The major focus of this study was to investigate the effect of spaceflight on the Drosophila circadian clock at both the behavioral and molecular level. We used China’s Shenzhou-9 spaceship to carry Drosophila. After 13 days of spaceflight, behavior tests showed that the flies maintained normal locomotor activity rhythm and sleep pattern. The expression level and rhythm of major clock genes were also unaffected. However, expression profiling showed differentially regulated output genes of the circadian clock system between space flown and control flies, suggesting that spaceflight affected the circadian output pathway. We also investigated other physiological effects of spaceflight such as lipid metabolism and lifespan, and searched genes significantly affected by spaceflight using microarray analysis. These results provide new information on the effects of spaceflight on circadian rhythm, lipid metabolism and lifespan. Furthermore, we showed that studying the effect of spaceflight on gene expression using samples collected at different Zeitgeber time could obtain different results, suggesting the importance of appropriate sampling procedures in studies on the effects of spaceflight.     

Parallel Geographic Variation in Drosophila melanogaster

Drosophila melanogaster, an ancestrally African species, has recently spread throughout the world, associated with human activity. The species has served as the focus of many studies investigating local adaptation relating to latitudinal variation in non-African populations, especially those from the United States and Australia. These studies have documented the existence of shared, genetically determined phenotypic clines for several life history and morphological traits. However, there are no studies designed to formally address the degree of shared latitudinal differentiation at the genomic level. Here we present our comparative analysis of such differentiation. Not surprisingly, we find evidence of substantial, shared selection responses on the two continents, probably resulting from selection on standing ancestral variation. The polymorphic inversion In(3R)P has an important effect on this pattern, but considerable parallelism is also observed across the genome in regions not associated with inversion polymorphism. Interestingly, parallel latitudinal differentiation is observed even for variants that are not particularly strongly differentiated, which suggests that very large numbers of polymorphisms are targets of spatially varying selection in this species.     

Population Genomics of Inversion Polymorphisms in Drosophila melanogaster

Chromosomal inversions have been an enduring interest of population geneticists since their discovery in Drosophila melanogaster. Numerous lines of evidence suggest powerful selective pressures govern the distributions of polymorphic inversions, and these observations have spurred the development of many explanatory models. However, due to a paucity of nucleotide data, little progress has been made towards investigating selective hypotheses or towards inferring the genealogical histories of inversions, which can inform models of inversion evolution and suggest selective mechanisms. Here, we utilize population genomic data to address persisting gaps in our knowledge of D. melanogaster''s inversions. We develop a method, termed Reference-Assisted Reassembly, to assemble unbiased, highly accurate sequences near inversion breakpoints, which we use to estimate the age and the geographic origins of polymorphic inversions. We find that inversions are young, and most are African in origin, which is consistent with the demography of the species. The data suggest that inversions interact with polymorphism not only in breakpoint regions but also chromosome-wide. Inversions remain differentiated at low levels from standard haplotypes even in regions that are distant from breakpoints. Although genetic exchange appears fairly extensive, we identify numerous regions that are qualitatively consistent with selective hypotheses. Finally, we show that In(1)Be, which we estimate to be ∼60 years old (95% CI 5.9 to 372.8 years), has likely achieved high frequency via sex-ratio segregation distortion in males. With deeper sampling, it will be possible to build on our inferences of inversion histories to rigorously test selective models—particularly those that postulate that inversions achieve a selective advantage through the maintenance of co-adapted allele complexes.     

Anent the Genomics of Spermatogenesis in Drosophila melanogaster

An appreciable fraction of the Drosophila melanogaster genome is dedicated to male fertility. One approach to characterizing this subset of the genome is through the study of male-sterile mutations. We studied the relation between vital and male-fertility genes in three large autosomal regions that were saturated for lethal and male-sterile mutations. The majority of male-sterile mutations affect genes that are exclusively expressed in males. These genes are required only for male fertility, and several mutant alleles of each such gene were encountered. A few male-sterile mutations were alleles of vital genes that are expressed in both males and females. About one-fifth of the genes in Drosophila melanogaster show male-specific expression in adults. Although some earlier studies found a paucity of genes on the X chromosome showing male-biased expression, we did not find any significant differences between the X chromosome and the autosomes either in the relative frequencies of mutations to male sterility or in the frequencies of genes with male-specific expression in adults. Our results suggest that as much as 25% of the Drosophila genome may be dedicated to male fertility.     

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.     

Variation in Male Mate Choice in Drosophila melanogaster

Male mate choice has been reported in the fruit fly, Drosophila melanogaster, even though males of this species were previously thought to maximise their fitness by mating with all available females. To understand the evolution of male mate choice it is important to understand variation in male mating preferences. Two studies, using different stock populations and different methods, have reported contrasting patterns of variation in male mate choice in D. melanogaster. Two possible explanations are that there are evolved differences in each stock population or that the methods used to measure choice could have biased the results. We investigated these hypotheses here by repeating the methods used in one study in which variable male mate choice was found, using the stock population from the other study in which choice was not variable. The results showed a significant resource-independent male preference for less fecund, smaller females, which contrasts with previous observations of male mate choice. This indicates that different selection pressures between populations have resulted in evolved differences in the expression of male mate choice. It also reveals phenotypic plasticity in male mate choice in response to cues encountered in each choice environment. The results highlight the importance of variation in male mate choice, and of identifying mechanisms in order to understand the evolution of mate choice under varying ecological conditions.     

Quantitative Genetics of Food Intake in Drosophila melanogaster

Food intake is an essential animal activity, regulated by neural circuits that motivate food localization, evaluate nutritional content and acceptance or rejection responses through the gustatory system, and regulate neuroendocrine feedback loops that maintain energy homeostasis. Excess food consumption in people is associated with obesity and metabolic and cardiovascular disorders. However, little is known about the genetic basis of natural variation in food consumption. To gain insights in evolutionarily conserved genetic principles that regulate food intake, we took advantage of a model system, Drosophila melanogaster, in which food intake, environmental conditions and genetic background can be controlled precisely. We quantified variation in food intake among 182 inbred, sequenced lines of the Drosophila melanogaster Genetic Reference Panel (DGRP). We found significant genetic variation in the mean and within-line environmental variance of food consumption and observed sexual dimorphism and genetic variation in sexual dimorphism for both food intake traits (mean and variance). We performed genome wide association (GWA) analyses for mean food intake and environmental variance of food intake (using the coefficient of environmental variation, CV_E, as the metric for environmental variance) and identified molecular polymorphisms associated with both traits. Validation experiments using RNAi-knockdown confirmed 24 of 31 (77%) candidate genes affecting food intake and/or variance of food intake, and a test cross between selected DGRP lines confirmed a SNP affecting mean food intake identified in the GWA analysis. The majority of the validated candidate genes were novel with respect to feeding behavior, and many had mammalian orthologs implicated in metabolic diseases.     

Genome-Wide Fine-Scale Recombination Rate Variation in Drosophila melanogaster

Estimating fine-scale recombination maps of Drosophila from population genomic data is a challenging problem, in particular because of the high background recombination rate. In this paper, a new computational method is developed to address this challenge. Through an extensive simulation study, it is demonstrated that the method allows more accurate inference, and exhibits greater robustness to the effects of natural selection and noise, compared to a well-used previous method developed for studying fine-scale recombination rate variation in the human genome. As an application, a genome-wide analysis of genetic variation data is performed for two Drosophila melanogaster populations, one from North America (Raleigh, USA) and the other from Africa (Gikongoro, Rwanda). It is shown that fine-scale recombination rate variation is widespread throughout the D. melanogaster genome, across all chromosomes and in both populations. At the fine-scale, a conservative, systematic search for evidence of recombination hotspots suggests the existence of a handful of putative hotspots each with at least a tenfold increase in intensity over the background rate. A wavelet analysis is carried out to compare the estimated recombination maps in the two populations and to quantify the extent to which recombination rates are conserved. In general, similarity is observed at very broad scales, but substantial differences are seen at fine scales. The average recombination rate of the X chromosome appears to be higher than that of the autosomes in both populations, and this pattern is much more pronounced in the African population than the North American population. The correlation between various genomic features—including recombination rates, diversity, divergence, GC content, gene content, and sequence quality—is examined using the wavelet analysis, and it is shown that the most notable difference between D. melanogaster and humans is in the correlation between recombination and diversity.