Julie M. Cridland

The Drosophila melanogaster Genetic Reference Panel

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.

Pollinator specialization and pollination syndromes of three related North American Silene

Community and biogeographic surveys often conclude that plant-pollinator interactions are highly generalized. Thus, a central implication of the pollination syndrome concept, that floral trait evolution occurs primarily via specialized interactions of plants with their pollinators, has been questioned. However, broad surveys may not distinguish whether flower visitors are actual pollen vectors and hence lack power to assess the relationship between syndrome traits and the pollinators responsible for their evolution. Here we address whether the floral traits of three closely related hermaphroditic Silene spp. native to eastern North America (S. caroliniana, S. virginica, and S. stellata) correspond to predicted specialized pollination based on floral differences among the three species and the congruence of these floral features with recognized pollination syndromes. A nocturnal/diurnal pollinator exclusion experiment demonstrated that all three Silene spp. have diurnal pollinators, and only S. stellata has nocturnal pollinators. Multiyear studies of visitation rates demonstrated that large bees, hummingbirds, and nocturnal moths were the most frequent pollinators of S. caroliniana, S. virginica, and S. stellata, respectively. Estimates of pollen grains deposited and removed per visit generally corroborated the visitation rate results for all three species. However, the relatively infrequent diurnal hawkmoth pollinators of S. caroliniana were equally effective and more efficient than the most frequent large bee visitors. Pollinator importance (visitation X deposition) of each of the animal visitors to each species was estimated and demonstrated that in most years large bees and nocturnal moths were the most important pollinators of S. caroliniana and S. stellata, respectively. By quantifying comprehensive aspects of the pollination process we determined that S. virginica and S. stellata were specialized on hummingbirds and nocturnal moths, respectively, and S. caroliniana was the least specialized with diurnal hawkmoth and large bee pollinators. Compared across the Silene species, divergent floral character states are consistent with increasing the attraction and/or pollen transfer efficiency of their respective major pollinators, which suggests that the pollinators are past and/or contemporary selective agents for floral trait evolution in these three Silene species. We conclude that the pollination syndrome concept allows us to effectively relate the functional significance of floral morphology to the major pollinators of these Silene species.

Genome of Drosophila suzukii, the Spotted Wing Drosophila

Drosophila suzukii Matsumura (spotted wing drosophila) has recently become a serious pest of a wide variety of fruit crops in the United States as well as in Europe, leading to substantial yearly crop losses. To enable basic and applied research of this important pest, we sequenced the D. suzukii genome to obtain a high-quality reference sequence. Here, we discuss the basic properties of the genome and transcriptome and describe patterns of genome evolution in D. suzukii and its close relatives. Our analyses and genome annotations are presented in a web portal, SpottedWingFlyBase, to facilitate public access.

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.

Landscape of Standing Variation for Tandem Duplications in Drosophila yakuba and Drosophila simulans

We have used whole genome paired-end Illumina sequence data to identify tandem duplications in 20 isofemale lines of Drosophila yakuba and 20 isofemale lines of D. simulans and performed genome wide validation with PacBio long molecule sequencing. We identify 1,415 tandem duplications that are segregating in D. yakuba as well as 975 duplications in D. simulans, indicating greater variation in D. yakuba. Additionally, we observe high rates of secondary deletions at duplicated sites, with 8% of duplicated sites in D. simulans and 17% of sites in D. yakuba modified with deletions. These secondary deletions are consistent with the action of the large loop mismatch repair system acting to remove polymorphic tandem duplication, resulting in rapid dynamics of gain and loss in duplicated alleles and a richer substrate of genetic novelty than has been previously reported. Most duplications are present in only single strains, suggesting that deleterious impacts are common. Drosophila simulans shows larger numbers of whole gene duplications in comparison to larger proportions of gene fragments in D. yakuba. Drosophila simulans displays an excess of high-frequency variants on the X chromosome, consistent with adaptive evolution through duplications on the D. simulans X or demographic forces driving duplicates to high frequency. We identify 78 chimeric genes in D. yakuba and 38 chimeric genes in D. simulans, as well as 143 cases of recruited noncoding sequence in D. yakuba and 96 in D. simulans, in agreement with rates of chimeric gene origination in D. melanogaster. Together, these results suggest that tandem duplications often result in complex variation beyond whole gene duplications that offers a rich substrate of standing variation that is likely to contribute both to detrimental phenotypes and disease, as well as to adaptive evolutionary change.

Validation of Rearrangement Break Points Identified by Paired-End Sequencing in Natural Populations of Drosophila melanogaster

Several recent studies have focused on the evolution of recently duplicated genes in Drosophila. Currently, however, little is known about the evolutionary forces acting upon duplications that are segregating in natural populations. We used a high-throughput, paired-end sequencing platform (Illumina) to identify structural variants in a population sample of African D. melanogaster. Polymerase chain reaction and sequencing confirmation of duplications detected by multiple, independent paired-ends showed that paired-end sequencing reliably uncovered the break points of structural rearrangements and allowed us to identify a number of tandem duplications segregating within a natural population. Our confirmation experiments show that rates of confirmation are very high, even at modest coverage. Our results also compare well with previous studies using microarrays (Emerson J, Cardoso-Moreira M, Borevitz JO, Long M. 2008. Natural selection shapes genome wide patterns of copy-number polymorphism in Drosophila melanogaster. Science. 320:1629–1631. and Dopman EB, Hartl DL. 2007. A portrait of copy-number polymorphism in Drosophila melanogaster. Proc Natl Acad Sci U S A. 104:19920–19925.), which both gives us confidence in the results of this study as well as confirms previous microarray results. We were also able to identify whole-gene duplications, such as a novel duplication of Or22a, an olfactory receptor, and identify copy-number differences in genes previously known to be under positive selection, like Cyp6g1, which confers resistance to dichlorodiphenyltrichloroethane. Several “hot spots” of duplications were detected in this study, which indicate that particular regions of the genome may be more prone to generating duplications. Finally, population frequency analysis of confirmed events also showed an excess of rare variants in our population, which indicates that duplications segregating in the population may be deleterious and ultimately destined to be lost from the population.

Gene Expression Variation in Drosophila melanogaster Due to Rare Transposable Element Insertion Alleles of Large Effect

Transposable elements are a common source of genetic variation that may play a substantial role in contributing to gene expression variation. However, the contribution of transposable elements to expression variation thus far consists of a handful of examples. We used previously published gene expression data from 37 inbred Drosophila melanogaster lines from the Drosophila Genetic Reference Panel to perform a genome-wide assessment of the effects of transposable elements on gene expression. We found thousands of transcripts with transposable element insertions in or near the transcript and that the presence of a transposable element in or near a transcript is significantly associated with reductions in expression. We estimate that within this example population, ∼2.2% of transcripts have a transposable element insertion, which significantly reduces expression in the line containing the transposable element. We also find that transcripts with insertions within 500 bp of the transcript show on average a 0.67 standard deviation decrease in expression level. These large decreases in expression level are most pronounced for transposable element insertions close to transcripts and the effect diminishes for more distant insertions. This work represents the first genome-wide analysis of gene expression variation due to transposable elements and suggests that transposable elements are an important class of mutation underlying expression variation in Drosophila and likely in other systems, given the ubiquity of these mobile elements in eukaryotic genomes.

Tandem Duplications and the Limits of Natural Selection in Drosophila yakuba and Drosophila simulans

Tandem duplications are an essential source of genetic novelty, and their variation in natural populations is expected to influence adaptive walks. Here, we describe evolutionary impacts of recently-derived, segregating tandem duplications in Drosophila yakuba and Drosophila simulans. We observe an excess of duplicated genes involved in defense against pathogens, insecticide resistance, chorion development, cuticular peptides, and lipases or endopeptidases associated with the accessory glands across both species. The observed agreement is greater than expectations on chance alone, suggesting large amounts of convergence across functional categories. We document evidence of widespread selection on the D. simulans X, suggesting adaptation through duplication is common on the X. Despite the evidence for positive selection, duplicates display an excess of low frequency variants consistent with largely detrimental impacts, limiting the variation that can effectively facilitate adaptation. Standing variation for tandem duplications spans less than 25% of the genome in D. yakuba and D. simulans, indicating that evolution will be strictly limited by mutation, even in organisms with large population sizes. Effective whole gene duplication rates are low at 1.17 × 10−9 per gene per generation in D. yakuba and 6.03 × 10−10 per gene per generation in D. simulans, suggesting long wait times for new mutations on the order of thousands of years for the establishment of sweeps. Hence, in cases where adaptation depends on individual tandem duplications, evolution will be severely limited by mutation. We observe low levels of parallel recruitment of the same duplicated gene in different species, suggesting that the span of standing variation will define evolutionary outcomes in spite of convergence across gene ontologies consistent with rapidly evolving phenotypes.

The Adaptive Significance of Natural Genetic Variation in the DNA Damage Response of Drosophila melanogaster.

Despite decades of work, our understanding of the distribution of fitness effects of segregating genetic variants in natural populations remains largely incomplete. One form of selection that can maintain genetic variation is spatially varying selection, such as that leading to latitudinal clines. While the introduction of population genomic approaches to understanding spatially varying selection has generated much excitement, little successful effort has been devoted to moving beyond genome scans for selection to experimental analysis of the relevant biology and the development of experimentally motivated hypotheses regarding the agents of selection; it remains an interesting question as to whether the vast majority of population genomic work will lead to satisfying biological insights. Here, motivated by population genomic results, we investigate how spatially varying selection in the genetic model system, Drosophila melanogaster, has led to genetic differences between populations in several components of the DNA damage response. UVB incidence, which is negatively correlated with latitude, is an important agent of DNA damage. We show that sensitivity of early embryos to UVB exposure is strongly correlated with latitude such that low latitude populations show much lower sensitivity to UVB. We then show that lines with lower embryo UVB sensitivity also exhibit increased capacity for repair of damaged sperm DNA by the oocyte. A comparison of the early embryo transcriptome in high and low latitude embryos provides evidence that one mechanism of adaptive DNA repair differences between populations is the greater abundance of DNA repair transcripts in the eggs of low latitude females. Finally, we use population genomic comparisons of high and low latitude samples to reveal evidence that multiple components of the DNA damage response and both coding and non-coding variation likely contribute to adaptive differences in DNA repair between populations.

The complex demographic history and evolutionary origin of the western honey bee, Apis mellifera

The western honey bee, Apis mellifera, provides critical pollination services to agricultural crops worldwide. However, despite substantial interest and prior investigation, the early evolution and subsequent diversification of this important pollinator remain uncertain. The primary hypotheses place the origin of A. mellifera in either Asia or Africa, with subsequent radiations proceeding from one of these regions. Here, we use two publicly available whole-genome data sets plus newly sequenced genomes and apply multiple population genetic analysis methods to investigate the patterns of ancestry and admixture in native honey bee populations from Europe, Africa, and the Middle East. The combination of these data sets is critical to the analyses, as each contributes samples from geographic locations lacking in the other, thereby producing the most complete set of honey bee populations available to date. We find evidence supporting an origin of A. mellifera in the Middle East or North Eastern Africa, with the A and Y lineages representing the earliest branching lineages. This finding has similarities with multiple contradictory hypotheses and represents a disentangling of genetic relationships, geographic proximity, and secondary contact to produce a more accurate picture of the origins of A. mellifera. We also investigate how previous studies came to their various conclusions based on incomplete sampling of populations, and illustrate the importance of complete sampling in understanding evolutionary processes. These results provide fundamental knowledge about genetic diversity within Old World honey bee populations and offer insight into the complex history of an important pollinator.