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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.

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 30bp 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.

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.

The first steps of animal speciation are thought to be the development of sexual isolating mechanisms. In contrast to recent progress in understanding the genetic basis of postzygotic isolating mechanisms, little is known about the genetic architecture of sexual isolation. Here, we have subjected Drosophila melanogaster to 29 generations of replicated divergent artificial selection for mating speed. The phenotypic response to selection was highly asymmetrical in the direction of reduced mating speed, with estimates of realized heritability averaging 7%. The selection response was largely attributable to a reduction in female receptivity. We assessed the whole genome transcriptional response to selection for mating speed using Affymetrix GeneChips and a rigorous statistical analysis. Remarkably, >3,700 probe sets (21% of the array elements) exhibited a divergence in message levels between the Fast and Slow replicate lines. Genes with altered transcriptional abundance in response to selection fell into many different biological process and molecular function Gene Ontology categories, indicating substantial pleiotropy for this complex behavior. Future functional studies are necessary to test the extent to which transcript profiling of divergent selection lines accurately predicts genes that directly affect the selected trait.

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.

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 ~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.

The magnitude of segregating variation for bristle number in Drosophila melanogaster exceeds that predicted from models of mutation-selection balance. To evaluate the hypothesis that genotype-environment interaction (GEI) maintains variation for bristle number in nature, we quantified the extent of GEI for abdominal and sternopleural bristles among 98 recombinant inbred lines, derived from two homozygous laboratory strains, in three temperature environments. There was considerable GEl for both bristle traits, which was mainly attributable to changes in rank order of line means. We conducted a genome-wide screen for quantitative trait loci (QTLs) affecting bristle number in each sex and temperature environment, using a dense (3.2-cM) marker map of polymorphic insertion sites of roo transposable elements. Nine sternopleural and 11 abdominal bristle number QTLs were detected. Significant GEI was exhibited by 14 QTLs, but there was heterogeneity among QTLs in their sensitivity to thermal and sexual environments. To further evaluate the hypothesis that GEl maintains variation for bristle number, we require estimates of allelic effects across environments at genetic loci affecting the traits. This level of resolution may be achievable for Drosophila bristle number because candidate loci affecting bristle development often map to the same location as bristle number QTLs.

A restriction enzyme survey of a 57-kb region including the gene Delta uncovered 53 polymorphic molecular markers in a sample of 55 naturally occurring chromosomes. A permutation test, which assesses the significance of the molecular marker with the largest effect on bristle variation in four genetic backgrounds relative to permuted data-sets, found two sites that were independently associated with variation in bristle number. A common site in the second intron of Delta affected only sternopleural bristle number, and another common site in the fifth intron affected only abdominal bristle number in females. Under an additive genetic model, the polymorphism in the second intron may account for 12% of the total genetic variation in sternopleural bristle number due to third chromosomes, and the site in the fifth intron may account for 6% of the total variation in female abdominal bristle number due to the third chromosomes. These results suggest the following: (1) models that incorporate balancing selection are more consistent with observations than deleterious mutation-selection equilibrium models, (2) mapped quantitative trait loci of large effect may not represent a single variable site at a genetic locus, and (3) linkage disequilibrium can be used as a tool for understanding the molecular basis of quantitative variation.

Delta (Dl) and Hairless (H) are two chromosome 3 candidate neurogenic loci that might contribute to naturally occurring quantitative variation for sensory bristle number. To evaluate this hypothesis, we assessed quantitative genetic variation in abdominal and sternopleural bristle numbers among homozygous isogenic third chromosomes sampled from nature and substituted into the Samarkand (Sam) inbred chromosome 1 and 2 background; among homozygous lines in which the wild-derived Dl-H gene region was introgressed into the Sam chromosome 3 background; and among Dl-H region introgression lines as heterozygotes against the Sam wild-type strain and derivatives of Sam into which mutant Dl and H alleles had been introgressed. Variation among the Dl-H region introgression lines accounted for 36% (8.3%) of the total chromosome 3 among line variance in abdominal (sternopleural) bristle number and for 53% of the chromosome 3 sex X line variance in abdominal bristle number. Naturally occurring alleles in the Dl-H region failed to complement a Dl mutant allele for female abdominal bristle number and sternopleural bristle number in both sexes, and an H mutant allele for both bristle traits in males and females. These results are consistent with the hypothesis that naturally occurring alleles at Dl and H contribute to quantitative genetic variation in sensory bristle number.

Quantitative genetic variation in behavioral response to the odorant, benzaldehyde, was assessed among a sample of 43 X and 35 third chromosomes extracted from a natural population and substituted into a common inbred background. Significant genetic variation among chromosome lines was detected. Heritability estimates for olfactory response, however, were low, as is typical for traits under natural selection. Furthermore, the loci affecting naturally occurring variation in olfactory response to benzaldehyde were not the same in males and females, since the genetic correlation between the sexes was low and not significantly different from zero for the chromosome 3 lines. Competitive fitness, viability anti fertility of the chromosome 3 lines were estimated using the balancer equilibrium technique. Genetic correlations between fitness and odor-guided behavior were not significantly different from zero, suggesting the number of loci causing variation in olfactory response is small relative to the number of loci causing variation in fitness. Since different genes affect variation in olfactory response in males and females, genetic variation for olfactory response could be maintained by genotype X sex environment interaction. This unusual genetic architecture implies that divergent evolutionary trajectories for olfactory behavior may occur in males and females.

Single P-element mutagenesis was used to construct 1094 lines with P[lArB] inserts on all three major chromosomes in an isogenic background previously free of P elements. The effects of insertions on bristle number and on viability were assessed by comparison to 392 control lines. The variance and effects of P-element inserts on bristle number and viability were larger than those inferred from spontaneous mutations. The distributions of effects on bristle number were symmetrical and highly leptokurtic, such that a few inserts with large effects caused most of the increase in variance. The distribution of effects on viability were negatively skewed and platykurtic. On average, the effects of P-element insertions on bristle number were partly recessive and on viability were completely recessive. P-element inserts with large effects on bristle number tended to have reduced viability, but the correlation between the absolute value of the effects on bristle number and on viability was not strong. Fifty P-element inserts tagging quantitative trait loci (QTLs) with large effects on bristle number were mapped cytogenetically. Two P-element-induced scabrous alleles and five extramacrochaetae alleles were generated. Single P-element mutagenesis is a powerful method for identifying QTLs at the level of genetic locus.

Single P-element (P[lArB]) insertional mutagenesis of an isogenic strain was used to identify autosomal loci affecting odor-guided behavior of Drosophila melanogaster. The avoidance response to benzaldehyde of 379 homozygous P[lArB] element-containing insert lines was evaluated quantitatively. Fourteen smell impaired (smi) lines were identified in which P[lArB] element insertion caused different degrees of hyposmia in one or both sexes. The smi loci map to different cytological locations and probably are novel olfactory genes. Enhancer trap analysis of the smi lines indicates that expression of at least 10 smi genes is controlled by olfactory tissue-specific promoter/enhancer elements.

The polygenic mutation in Drosophila melanogaster is investigated by developing an autoregressive model which fits to the line of variance. The investigation reveals that the variance between the sternopleural bristle number and abdominal lines increases only after 100 generations, which are subjected to inbreeding. The autoregressive model also indicates the presence of the phenomena of strong natural selection.

An average divergence, following 125 generations of selection of 12.0 abdominal and 8.2 sternopleural bristles from the accumulation of new mutations influencing bristle number, resulted from replicated divergent artificial selection for abdominal and sternopleural responses from an inbred strain of Drosophila melanogaster. Extremely asymmetrical responses to selection were evident. Low abdominal and high sternopleural bristle numbers exhibited greater numbers.

The homozygous and heterozygous effects of P element insertions on the third chromosomal lines of Drosophila melanogaster were investigated. Ninety-four third chromosome lines which contained on average 3.1 stable P element inserts were created by P element mutagenesis. Average homozygous viability was reduced by about 38% while heterozygous viability was reduced by 17%. The mutational variance for bristle traits per homozygous and heterozygous inserts for both sexes showed similar 0.03 environmental variance.

A restriction enzyme survey of a 110-kb region including the achaete scute complex (ASC) examined 14 polymorphic molecular markers in a sample of 56 naturally occurring chromosomes. Large insertions as a class were associated with a reduction in both sternopleural and abdominal bristle number, supporting deleterious mutation-selection equilibrium models tot the maintenance of quantitative genetic variation. Two polymorphic sites were independently associated with variation in bristle number measured in two genetic backgrounds as assessed by a permutation test. A 6-bp deletion near sc [Alpha] is associated with sternopleural bristle number variation in both sexes and a 3.4-kb insertion between sc [Beta] and sc [Gamma] is associated with abdominal bristle number variation in females. Under an additive genetic model, the small deletion polymorphism near sc [Alpha] accounts for 25% of the total X chromosome genetic variation in sternopleural bristle number, and the 3.4 kb insertion accounts for 22% of the total X chromosome variation in female abdominal bristle number. The observation of common polymorphisms associated with variation in bristle number is more parsimoniously explained by models that incorporate balancing selection or assume variants affecting bristle number are neutral, than mutation-selection equilibrium models.

The nature of genetic variation for Drosophila longevity in a population of recombinant inbred lines was investigated by estimating quantitative genetic parameters and mapping quantitative trait loci (QTL) for adult life span in five environments: standard culture conditions, high and low temperature, and heatshock and starvation stress. There was highly significant genetic variation for life span within each sex and environment. In the analysis of variance of life span pooled over sexes and environments, however, the significant genetic variation appeared in the genotype x sex and genotype x environment interaction terms. The genetic correlation of longevity across the sexes and environments was not significantly different from zero in these lines. We estimated map positions and effects of QTL affecting life span by linkage to highly polymorphic roo transposable element markers, using a multiple-trait composite interval mapping procedure. A minimum of 17 QTL were detected; all were sex and/or environment-specific. Ten of the QTL had sexually antagonistic or antagonistic pleiotropic effects in different environments. These data provide support for the pleiotropy theory of senescence and the hypothesis that variation for longevity might be maintained by opposing selection pressures in males and females and variable environments. Further work is necessary to assess the generality of these results, using different strains, to determine heterozygous effects and to map the life span QTL to the level of genetic loci.