Biology subjects

Macdonald, S. J.

Publications and source records attributed to Macdonald, S. J..

12 recordsLinked to original sources

bioRxiv2026

Pooled progeny sequencing effectively estimates polyandry in species with large families

Polyandry, multiple mating by females, both benefits females by increases genetic variation within families and provides an opportunity for sexual selection through sperm competition and cryptic female choice. Estimating polyandry in species with large clutches is challenging because genotyping all progeny individually can be impractical. We present a likelihood-based framework for estimating the number of sires and their relative contributions from the maternal genotype and pooled sequencing of entire progeny pools. The method evaluates the probability of observed pooled offspring read counts under alternative paternal genotype mixtures, accounting for Mendelian segregation and sampling variation from finite sequencing depth. We implement this framework for both biallelic SNPs and short-read mini-haplotype markers constructed from closely linked SNPs, which provide multi-allelic information comparable to traditional microsatellite markers. Simulations show that this approach accurately recovers sire number and sire contributions across a range of conditions. We then applied the method to 275 wild-caught Drosophila melanogaster females and their offspring from a Kansas population using 3,053 biallelic SNPs and 239 four-allele mini-haplotype markers. Estimates from the two marker sets were highly concordant, and combined analyses indicate that 57% of families were sired by a single male. These results demonstrate that pooled progeny sequencing provides an efficient genomic approach for estimating polyandry in species with large families.

genomics↗

bioRxiv2026

A genomewide association study for bristle number variation in Drosophila melanogaster

Decades of research has uncovered a wealth of mechanistic information about the development of sensory bristles in Drosophila melanogaster. By studying large-effect, often loss-of-function mutations, many genes have been associated with bristle development, morphology, patterning, and number. Equally, the number of bristles present in certain areas of the fly cuticle is a classic quantitative trait, the genetic basis of which has been studied using a range of tools, from artificial selection to QTL (Quantitative Trait Locus) mapping. Such studies have often implicated well-understood bristle development genes as contributing to natural variation in bristle number. Here we contribute to the study of bristle number genetic variation in flies by executing a GWAS (genomewide association study). We generated whole genome sequences for 897 phenotyped male D. melanogaster individuals derived from a wild-derived, but lab-adapted outbred population, revealing - following quality control and filtering - over 780,000 variants with frequencies greater than 5%. Using these data we estimated the SNP (Single Nucleotide Polymorphism) heritability for ABN (abdominal bristle number) and SBN (sternopleural bristle number) as 0.28 and 0.35, respectively. These values indicate that our set of genotyped variants collectively explain a substantial fraction of the variance in phenotype in the mapping panel. Subsequently, genome scans revealed 1085 (ABN) and 211 (SBN) genomewide significant sites, and - due to extensive LD (Linkage Disequilibrium) in our panel - nearly all these sites are clustered into three locations; We find a GWAS hit for ABN in the middle of chromosome 3L, and hits for SBN at the tip of the X chromosome (where several prior mapping studies have resolved QTL for bristle number), and on 2L. Surveying existing studies that identified genes that control bristle number/development, we highlight several candidates that may segregate for causative, functional variants.

genetics↗

bioRxiv2025

The Illusion of Polygenicity in Poolseq studies: Insufficient Power Can Mask Simple Genetic Architectures

Pool-seq (pooled sequencing) combines DNA from multiple individuals prior to sequencing, enabling population-level allele frequency estimation without individual genotyping. When employed in Genome Wide Association Studies (GWAS) pool-seq faces a fundamental power limitation in that errors on allele frequency estimates are proportional to sequence coverage. Although this power limitation is widely appreciated, pool-seq GWAS lacking unambiguous hits are often interpreted as showing a highly polygenic genetic architecture. We illustrate the limitation of inferring architecture from Manhattan plots using empirical data from a Drosophila zinc resistance mapping study. Despite achieving an average of >700x sequencing coverage in case and control pools, a directly ascertained SNP-based GWAS failed to reveal clear evidence for major-effect loci. A unique feature of the dataset is that an advanced intercross multiparent population, with known founders, was employed as the base population for the GWAS. We leverage this unique population structure to carry out a second GWAS using imputed haplotype frequency estimates, which in contrast revealed localized regions of major effect. A third reanalysis of the same data using imputed SNP genotypes derived from the founder haplotype frequency estimates uncovered a similar major gene architecture. The key difference between approaches lies in statistical power: directly ascertained SNP counts have errors proportional to sequencing coverage whereas known founder imputation-based approaches can be considerably more accurate. This work highlights that insufficiently powered GWAS studies can mask simple genetic architectures and create the illusion of polygenicity through statistical noise alone.

genetics↗

bioRxiv2025

High-throughput genetic mapping discovers novel zinc toxicity response loci in Drosophila melanogaster

Heavy metals are a widespread environmental contaminant, and even low levels of some metals can disrupt cellular processes and result in DNA damage. However, the consequences of metal exposure are variable among individuals, with susceptibility to metal toxicity representing a complex trait influenced by genetic and non-genetic factors. To uncover toxicity response genes, and better understand responses to metal toxicity, we sought to dissect resistance to zinc, a metal required for normal cellular function, which can be toxic at high doses. To facilitate efficient, powerful discovery of Quantitative Trait Loci (QTL) we employed extreme, or X-QTL mapping, leveraging a multiparental, recombinant Drosophila melanogaster population. Our approach involved bulk selection of zinc-resistant individuals, sequencing several replicate pools of selected and control animals, and identified QTL as genomic positions showing consistent allele frequency shifts between treatments. We successfully identified seven regions segregating for resistance/susceptibility alleles, and implicated several strong candidate genes. Phenotypic characterization of populations derived from selected or control animals revealed that our selection procedure resulted in greater egg-to-adult emergence, and a reduced developmental delay on zinc media. We subsequently measured emergence and development time for a series of midgut-specific RNAi gene knockdowns and matched genetic controls raised in both zinc-supplemented and normal media. This identified ten genes with significant genotype-by-treatment effects, including pHCl-2, which encodes a zinc sensor protein. Our work highlights recognized and novel contributors to zinc toxicity resistance in flies, and provides a pathway to a broader understanding of the biological impact of metal toxicity. ARTICLE SUMMARYStarting with an outbred Drosophila melanogaster population we repeatedly selected for groups of individuals showing high resistance to toxic levels of zinc during development. Pooled sequencing of these groups, along with matched groups of control individuals, enabled the identification of seven genomic regions - or QTL - contributing to zinc toxicity resistance. Midgut-specific RNAi of genes implicated by these QTL yielded ten genes impacting developmental traits in zinc-supplemented media, including MTF-1 (a metal response transcription factor) and pHCl-2 (a zinc sensor protein).

genetics↗

bioRxiv2024

Dynamic Changes in Gene Expression Through Aging in Drosophila melanogaster Heads

Work in many systems has shown large-scale changes in gene expression during aging. However, many studies employ just two, arbitrarily-chosen timepoints at which to measure expression, and can only observe an increase or a decrease in expression between "young" and "old" animals, failing to capture any dynamic, non-linear changes that occur throughout the aging process. We used RNA sequencing to measure expression in male head tissue at 15 timepoints through the lifespan of an inbred Drosophila melanogaster strain. We detected >6,000 significant, age-related genes, nearly all of which have been seen in previous fly aging expression studies, and which include several known to harbor lifespan-altering mutations. We grouped our gene set into 28 clusters via their temporal expression change, observing a diversity of trajectories; some clusters show a linear change over time, while others show more complex, non-linear patterns. Notably, re-analysis of our dataset comparing the earliest and latest timepoints - mimicking a two-timepoint design - revealed fewer differentially-expressed genes (around 4,500). Additionally, those genes exhibiting complex expression trajectories in our multi-timepoint analysis were most impacted in this re-analysis; Their identification, and the inferred change in gene expression with age, was often dependent on the timepoints chosen. Informed by our trajectory-based clusters, we executed a series of gene enrichment analyses, identifying enriched functions/pathways in all clusters, including the commonly seen increase in stress- and immune-related gene expression with age. Finally, we developed a pair of accessible shiny apps to enable exploration of our differential expression and gene enrichment results.

genomics↗

bioRxiv2023

Gene expression variation underlying tissue-specific response to copper stress in Drosophila melanogaster

Copper is one of a handful of biologically necessary heavy metals that is also a common environmental pollutant. Under normal conditions, copper ions are required for many key physiological processes. However, in excess, copper quickly results in cell and tissue damage that can range in severity from temporary injury to permanent neurological damage. Because of its biological relevance, and because many conserved copper-responsive genes also respond to other non-essential heavy metal pollutants, copper resistance in Drosophila melanogaster is a useful model system with which to investigate the genetic control of the response to heavy metal stress. Because heavy metal toxicity has the potential to differently impact specific tissues, we genetically characterized the control of the gene expression response to copper stress in a tissue- specific manner in this study. We assessed the copper stress response in head and gut tissue of 96 inbred strains from the Drosophila Synthetic Population Resource (DSPR) using a combination of differential expression analysis and expression quantitative trait locus (eQTL) mapping. Differential expression analysis revealed clear patterns of tissue-specific expression, primarily driven by a more pronounced gene expression response in gut tissue. eQTL mapping of gene expression under control and copper conditions as well as for the change in gene expression following copper exposure (copper response eQTL) revealed hundreds of genes with tissue- specific local cis-eQTL and many distant trans-eQTL. eQTL associated with MtnA, Mdr49, Mdr50, and Sod3 exhibited genotype by environment effects on gene expression under copper stress, illuminating several tissue- and treatment-specific patterns of gene expression control. Together, our data build a nuanced description of the roles and interactions between allelic and expression variation in copper-responsive genes, provide valuable insight into the genomic architecture of susceptibility to metal toxicity, and highlight many candidate genes for future functional characterization.

genetics↗

bioRxiv2022

Discovery of malathion resistance QTL in Drosophila melanogaster using a bulked phenotyping approach

Drosophila melanogaster has proven an effective system with which to understand the evolutionary genetics and molecular mechanisms of insecticide resistance. Insecticide use has left signatures of selection in the fly genome, and both functional and quantitative genetics studies in the system have identified genes and variants associated with resistance. Here, we use D. melanogaster and leverage a bulk phenotyping and pooled sequencing "extreme QTL" approach to genetically dissect variation in resistance to malathion, an organophosphate insecticide. We resolve two QTL (Quantitative Trait Loci), one of which implicates allelic variation at the cytochrome P450 gene Cyp6g1, a strong candidate based on previous work. The second shows no overlap with hits from a previous genomewide association study (GWAS) for malathion resistance, recapitulating other studies showing that different strategies for complex trait dissection in flies can yield apparently different architectures. Notably, we see no genetic signal at the Ace gene. Ace encodes the target of organophosphate insecticide inhibition, and GWAS have identified strong Ace-linked associations with resistance. The absence of QTL implicating Ace here is most likely because our mapping population does not segregate for several of the known functional polymorphisms impacting resistance at Ace, perhaps because our population is derived from flies collected prior to the widespread use of organophosphate insecticides. Our fundamental approach can be an efficient, powerful strategy to dissect genetic variation in resistance traits. Nonetheless, studies seeking to interrogate contemporary insecticide resistance variation may benefit from deriving mapping populations from more recently collected strains.

genetics↗

bioRxiv2021

Powerful, efficient QTL mapping in Drosophila melanogaster using bulked phenotyping and pooled sequencing

Despite the value of Recombinant Inbred Lines (RILs) for the dissection of complex traits, large panels can be difficult to maintain, distribute, and phenotype. An attractive alternative to RILs for many traits leverages selecting phenotypically-extreme individuals from a segregating population, and subjecting pools of selected and control individuals to sequencing. Under a bulked or extreme segregant analysis paradigm, genomic regions contributing to trait variation are revealed as frequency differences between pools. Here we describe such an extreme quantitative trait locus, or X-QTL mapping strategy that builds on an existing multiparental population, the DSPR (Drosophila Synthetic Population Resource), and involves phenotyping and genotyping a population derived by mixing hundreds of DSPR RILs. Simulations demonstrate that challenging, yet experimentally tractable X-QTL designs (>=4 replicates, >=5000 individuals/replicate, and a selection intensity of 5-10%) yield at least the same power as traditional RIL-based QTL mapping, and can localize variants with sub-centimorgan resolution. We empirically demonstrate the effectiveness of the approach using a 4-fold replicated X-QTL experiment that identifies 7 QTL for caffeine resistance. Two mapped X-QTL factors replicate loci previously identified in RILs, 6/7 are associated with excellent candidate genes, and RNAi knock-downs support the involvement of 4 genes in the genetic control of trait variation. For many traits of interest to drosophilists a bulked phenotyping/genotyping X-QTL design has considerable advantages.

genetics↗

bioRxiv2020

Characterizing the genetic basis of copper toxicity in Drosophila reveals a complex pattern of allelic, regulatory, and behavioral variation

A range of heavy metals are required for normal cell function and homeostasis. Equally, the anthropogenic release of heavy metals into soil and water sources presents a pervasive health threat. Copper is one such metal; it functions as a critical enzymatic cofactor, but at high concentrations is toxic, and can lead to the production of reactive oxygen species. Using a combination of quantitative trait locus (QTL) mapping and RNA sequencing in the Drosophila Synthetic Population Resource (DSPR), we demonstrate that resistance to the toxic effects of ingested copper in D. melanogaster is genetically complex, and influenced by allelic and expression variation at multiple loci. Additionally, we find that copper resistance is impacted by variation in behavioral avoidance of copper and may be subject to life-stage specific regulation. Multiple genes with known copper-specific functions, as well as genes that are involved in the regulation of other heavy metals were identified as potential candidates to contribute to variation in adult copper resistance. We demonstrate that nine of 16 candidates tested by RNAi knockdown influence adult copper resistance, a number of which may have pleiotropic effects since they have previously been shown to impact the response to other metals. Our work provides new understanding of the genetic complexity of copper resistance, highlighting the diverse mechanisms through which copper pollution can negatively impact organisms. Additionally, we further support the similarities between copper metabolism and that of other essential and nonessential heavy metals.

genetics↗

bioRxiv2018

Structural variants exhibit allelic heterogeneity and shape variation in complex traits

Despite extensive effort to reveal the genetic basis of complex phenotypic variation, studies typically explain only a fraction of trait heritability. It has been hypothesized that individually rare hidden structural variants (SVs) could account for a significant fraction of variation in complex traits. To investigate this hypothesis, we assembled 14 Drosophila melanogaster genomes and systematically identified more than 20,000 euchromatic SVs, of which [~]40% are invisible to high specificity short read genotyping approaches. SVs are common in Drosophila genes, with almost one third of diploid individuals harboring an SV in genes larger than 5kb, and nearly a quarter harboring multiple SVs in genes larger than 10kb. We show that SV alleles are rarer than amino acid polymorphisms, implying that they are more strongly deleterious. A number of functionally important genes harbor previously hidden structural variants that likely affect complex phenotypes (e.g., Cyp6g1, Drsl5, Cyp28d1&2, InR, and Gss1&2).Furthermore, SVs are overrepresented in quantitative trait locus candidate genes from eight Drosophila Synthetic Population Resource (DSPR) mapping experiments. We conclude that SVs are pervasive in genomes, are frequently present as heterogeneous allelic series, and can act as rare alleles of large effect.

genomics↗

bioRxiv2018

Dissection of complex, fitness-related traits in multiple Drosophila mapping populations offers insight into the genetic control of stress resistance

We leverage two complementary Drosophila melanogaster mapping panels to genetically dissect starvation resistance, an important fitness trait. Using >1600 genotypes of the multiparental Drosophila Synthetic Population Resource (DSPR) we map numerous starvation stress QTL that collectively explain a substantial fraction of trait heritability. QTL effects further allowed us to estimate DSPR founder phenotypes, predictions that were correlated with the actual founder phenotypes. Starvation resistance has been linked to triglyceride level, and while we observe a modest phenotypic correlation between the traits in the DSPR, overlap among the QTL identified for each trait is low. Since we show that DSPR strains with extreme starvation phenotypes also differ in desiccation resistance and activity level, our data imply that multiple physiological mechanisms contribute to starvation variability. We also exploit the Drosophila Genetic Reference Panel (DGRP), identifying a number of sequence variants associated with starvation resistance. Consistent with prior work these sites rarely fall within QTL intervals mapped in the DSPR. Two other groups previously measured starvation resistance in the DGRP, offering a unique opportunity to directly compare mapping results across labs. We found strong phenotypic correlations among studies, but extremely low overlap in the sets of genomewide significant sites. Despite this, our analyses revealed that the most highly-associated variants from each study typically showed the same additive effect sign in independent studies, in contrast to otherwise equivalent sets of random variants. This consistency provides evidence for reproducible trait-associated sites in a widely-used mapping panel, and highlights the polygenic nature of starvation resistance.

genetics↗

bioRxiv2017

Naturally-segregating Variation at Ugt86Dd Contributes to Nicotine Resistance in Drosophila melanogaster

Identifying the causative sequence polymorphisms underlying complex trait variation is a key goal of evolutionary and biomedical genetics. By knowing the precise molecular events that confer phenotypic changes we can gain insight into the pathways underlying complex traits and the evolutionary forces acting on variation. Genetic analysis of complex traits in model systems regularly starts by constructing QTL maps, but generally fails to identify causative nucleotide-level polymorphisms. Previously we mapped a series of QTL contributing to resistance to nicotine in a Drosophila melanogaster multiparental mapping resource, and here use a battery of functional tests to resolve QTL to the molecular level. One large-effect QTL resided over a cluster of UDP-glucuronosyltransferases, and quantitative complementation tests using deficiencies eliminating subsets of these detoxification genes revealed allelic variation impacting resistance. RNAseq showed that Ugt86Dd had significantly higher expression in genotypes that are more resistant to nicotine, and anterior midgut-specific RNAi of this gene reduced resistance. We discovered a naturally-segregating 22-bp frameshift deletion in Ugt86Dd, and overexpression of the insertion-containing allele in a range of tissues enhanced resistance. Accounting for the InDel event during mapping largely eliminates the QTL, implying the InDel explains the bulk of the effect associated with the mapped locus. Finally, we edited a relatively resistant genetic background to generate lesions in Ugt86Dd that recapitulate the naturally-occurring putative loss-of-function allele, and succeeded in radically reducing resistance. The putatively causative coding InDel in Ugt86Dd can be a launchpad for future mechanistic exploration of xenobiotic detoxification.\n\nARTICLE SUMMARYResolving the mutations that contribute to among-individual trait variation is a major goal of biomedical and evolutionary genetics. In general however, genetic mapping experiments do not allow immediate resolution of the underlying causative variants. Previous mapping work revealed several loci contributing to nicotine resistance in Drosophila melanogaster. We employed a battery of functional tests to demonstrate that the detoxification gene Ugt86Dd has a major phenotypic effect, and that a segregating frameshift mutation is likely causative. Editing the gene to introduce a premature stop codon led to a significant reduction in resistance, validating its role in xenobiotic detoxification.

genetics↗