Stuart J. Macdonald

Genome-Wide Transcript Profiles in Aging and Calorically Restricted Drosophila melanogaster

BACKGROUND We characterized RNA transcript levels for the whole Drosophila genome during normal aging. We compared age-dependent profiles from animals aged under full-nutrient conditions with profiles obtained from animals maintained on a low-calorie medium to determine if caloric restriction slows the aging process. Specific biological functions impacted by caloric restriction were identified using the Gene Ontology annotation. We used the global patterns of expression profiles to test if particular genomic regions contribute differentially to changes in transcript profiles with age and if global disregulation of gene expression occurs during aging. RESULTS Whole-genome transcript profiles contained a statistically powerful genetic signature of normal aging. Nearly 23% of the genome changed in transcript representation with age. Caloric restriction was accompanied by a slowing of the progression of normal, age-related changes in transcript levels. Many genes, including those associated with stress response and oogenesis, showed age-dependent transcript representation. Caloric restriction resulted in the downregulation of genes primarily involved in cell growth, metabolism, and reproduction. We found no evidence that age-dependent changes in transcription level were confined to genes localized to specific regions of the genome and found no support for widespread disregulation of gene expression with age. CONCLUSIONS Aging is characterized by highly dynamic changes in the expression of many genes, which provides a powerful molecular description of the normal aging process. Caloric restriction extends life span by slowing down the rate of normal aging. Transcription levels of genes from a wide variety of biological functions and processes are impacted by age and dietary conditions.

Genetic dissection of a model complex trait using the Drosophila Synthetic Population Resource

Genetic dissection of complex, polygenic trait variation is a key goal of medical and evolutionary genetics. Attempts to identify genetic variants underlying complex traits have been plagued by low mapping resolution in traditional linkage studies, and an inability to identify variants that cumulatively explain the bulk of standing genetic variation in genome-wide association studies (GWAS). Thus, much of the heritability remains unexplained for most complex traits. Here we describe a novel, freely available resource for the Drosophila community consisting of two sets of recombinant inbred lines (RILs), each derived from an advanced generation cross between a different set of eight highly inbred, completely resequenced founders. The Drosophila Synthetic Population Resource (DSPR) has been designed to combine the high mapping resolution offered by multiple generations of recombination, with the high statistical power afforded by a linkage-based design. Here, we detail the properties of the mapping panel of >1600 genotyped RILs, and provide an empirical demonstration of the utility of the approach by genetically dissecting alcohol dehydrogenase (ADH) enzyme activity. We confirm that a large fraction of the variation in this classic quantitative trait is due to allelic variation at the Adh locus, and additionally identify several previously unknown modest-effect trans-acting QTL (quantitative trait loci). Using a unique property of multiparental linkage mapping designs, for each QTL we highlight a relatively small set of candidate causative variants for follow-up work. The DSPR represents an important step toward the ultimate goal of a complete understanding of the genetics of complex traits in the Drosophila model system.

Properties and Power of the Drosophila Synthetic Population Resource for the Routine Dissection of Complex Traits

The Drosophila Synthetic Population Resource (DSPR) is a newly developed multifounder advanced intercross panel consisting of >1600 recombinant inbred lines (RILs) designed for the genetic dissection of complex traits. Here, we describe the inference of the underlying mosaic founder structure for the full set of RILs from a dense set of semicodominant restriction-site–associated DNA (RAD) markers and use simulations to explore how variation in marker density and sequencing coverage affects inference. For a given sequencing effort, marker density is more important than sequence coverage per marker in terms of the amount of genetic information we can infer. We also assessed the power of the DSPR by assigning genotypes at a hidden QTL to each RIL on the basis of the inferred founder state and simulating phenotypes for different experimental designs, different genetic architectures, different sample sizes, and QTL of varying effect sizes. We found the DSPR has both high power (e.g., 84% power to detect a 5% QTL) and high mapping resolution (e.g., ∼1.5 cM for a 5% QTL).

Joint Estimates of Quantitative Trait Locus Effect and Frequency Using Synthetic Recombinant Populations of Drosophila melanogaster

We develop and implement a strategy to map QTL in two synthetic populations of Drosophila melanogaster each initiated with eight inbred founder strains. These recombinant populations allow simultaneous estimates of QTL location, effect, and frequency. Five X-linked QTL influencing bristle number were resolved to intervals of ∼1.3 cM. We confirm previous observations of bristle number QTL distal to 4A at the tip of the chromosome and identify two novel QTL in 7F–8C, an interval that does not include any classic bristle number candidate genes. If QTL at the tip of the X are biallelic they appear to be intermediate in frequency, although there is evidence that these QTL may reside in multiallelic haplotypes. Conversely, the two QTL mapping to the middle of the X chromosome are likely rare: in each case the minor allele is observed in only 1 of the 16 founders. Assuming additivity and biallelism we estimate that identified QTL contribute 1.0 and 8.7%, respectively, to total phenotypic variation in male abdominal and sternopleural bristle number in nature. Models that seek to explain the maintenance of genetic variation make different predictions about the population frequency of QTL alleles. Thus, mapping QTL in eight-way recombinant populations can distinguish between these models.

Genome Sequence of Herpes Simplex Virus 1 Strain KOS

ABSTRACT Herpes simplex virus type 1 (HSV-1) strain KOS has been extensively used in many studies to examine HSV-1 replication, gene expression, and pathogenesis. Notably, strain KOS is known to be less pathogenic than the first sequenced genome of HSV-1, strain 17. To understand the genotypic differences between KOS and other phenotypically distinct strains of HSV-1, we sequenced the viral genome of strain KOS. When comparing strain KOS to strain 17, there are at least 1,024 small nucleotide polymorphisms (SNPs) and 172 insertions/deletions (indels). The polymorphisms observed in the KOS genome will likely provide insights into the genes, their protein products, and the cis elements that regulate the biology of this HSV-1 strain.

Abundance and Distribution of Transposable Elements in Two Drosophila QTL Mapping Resources

Here we present computational machinery to efficiently and accurately identify transposable element (TE) insertions in 146 next-generation sequenced inbred strains of Drosophila melanogaster. The panel of lines we use in our study is composed of strains from a pair of genetic mapping resources: the Drosophila Genetic Reference Panel (DGRP) and the Drosophila Synthetic Population Resource (DSPR). We identified 23,087 TE insertions in these lines, of which 83.3% are found in only one line. There are marked differences in the distribution of elements over the genome, with TEs found at higher densities on the X chromosome, and in regions of low recombination. We also identified many more TEs per base pair of intronic sequence and fewer TEs per base pair of exonic sequence than expected if TEs are located at random locations in the euchromatic genome. There was substantial variation in TE load across genes. For example, the paralogs derailed and derailed-2 show a significant difference in the number of TE insertions, potentially reflecting differences in the selection acting on these loci. When considering TE families, we find a very weak effect of gene family size on TE insertions per gene, indicating that as gene family size increases the number of TE insertions in a given gene within that family also increases. TEs are known to be associated with certain phenotypes, and our data will allow investigators using the DGRP and DSPR to assess the functional role of TE insertions in complex trait variation more generally. Notably, because most TEs are very rare and often private to a single line, causative TEs resulting in phenotypic differences among individuals may typically fail to replicate across mapping panels since individual elements are unlikely to segregate in both panels. Our data suggest that “burden tests” that test for the effect of TEs as a class may be more fruitful.

The Effect of Polymorphisms in the Enhancer of split Gene Complex on Bristle Number Variation in a Large Wild-Caught Cohort of Drosophila melanogaster

The Enhancer of split complex [E(spl)-C] in Drosophila encompasses a variety of functional elements controlling bristle patterning and on the basis of prior work is a strong candidate for harboring alleles having subtle effects on bristle number variation. Here we extend earlier studies identifying associations between complex phenotypes and polymorphisms segregating among inbred laboratory lines of Drosophila and test the influence of E(spl)-C on bristle number variation in a natural cohort. We describe results from an association mapping study using 203 polymorphisms spread throughout the E(spl)-C genotyped in 2000 wild-caught Drosophila melanogaster. Despite power to detect associations accounting for as little as 2% of segregating variation for bristle number, and saturating the region with single-nucleotide polymorphisms (SNPs), we identified no single SNP marker showing a significant (additive over loci) effect after correcting for multiple tests. Using a newly developed test we conservatively identify six regions of the E(spl)-C in which the insertion of transposable elements as a class contributes to variation in bristle number, apparently in a sex- or trait-limited fashion. Finally, we carry out all possible 20,503 two-way tests for epistasis and identify a slight excess of marginally significant interactions, although none survive multiple-testing correction. It may not be straightforward to extend the results of laboratory-based association studies to natural populations.

Genetic Dissection of the Drosophila melanogaster Female Head Transcriptome Reveals Widespread Allelic Heterogeneity

Modern genetic mapping is plagued by the “missing heritability” problem, which refers to the discordance between the estimated heritabilities of quantitative traits and the variance accounted for by mapped causative variants. One major potential explanation for the missing heritability is allelic heterogeneity, in which there are multiple causative variants at each causative gene with only a fraction having been identified. The majority of genome-wide association studies (GWAS) implicitly assume that a single SNP can explain all the variance for a causative locus. However, if allelic heterogeneity is prevalent, a substantial amount of genetic variance will remain unexplained. In this paper, we take a haplotype-based mapping approach and quantify the number of alleles segregating at each locus using a large set of 7922 eQTL contributing to regulatory variation in the Drosophila melanogaster female head. Not only does this study provide a comprehensive eQTL map for a major community genetic resource, the Drosophila Synthetic Population Resource, but it also provides a direct test of the allelic heterogeneity hypothesis. We find that 95% of cis-eQTLs and 78% of trans-eQTLs are due to multiple alleles, demonstrating that allelic heterogeneity is widespread in Drosophila eQTL. Allelic heterogeneity likely contributes significantly to the missing heritability problem common in GWAS studies.

A low-cost open-source SNP genotyping platform for association mapping applications

Association mapping aimed at identifying DNA polymorphisms that contribute to variation in complex traits entails genotyping a large number of single-nucleotide polymorphisms (SNPs) in a very large panel of individuals. Few technologies, however, provide inexpensive high-throughput genotyping. Here, we present an efficient approach developed specifically for genotyping large fixed panels of diploid individuals. The cost-effective, open-source nature of our methodology may make it particularly attractive to those working in nonmodel systems.

Dissecting complex traits using the Drosophila Synthetic Population Resource

For most complex traits we have a poor understanding of the positions, phenotypic effects, and population frequencies of the underlying genetic variants contributing to their variation. Recently, several groups have developed multi-parent advanced intercross mapping panels in different model organisms in an attempt to improve our ability to characterize causative genetic variants. These panels are powerful and are particularly well suited to the dissection of phenotypic variation generated by rare alleles and loci segregating multiple functional alleles. We describe studies using one such panel, the Drosophila Synthetic Population Resource (DSPR), and the implications for our understanding of the genetic basis of complex traits. In particular, we note that many loci of large effect appear to be multiallelic. If multiallelism is a general rule, analytical approaches designed to identify multiallelic variants should be a priority for both genome-wide association studies (GWASs) and multi-parental panels.