The relative impact of parental and current environment on plant transcriptomes depends on type of stress and genotype.

Through developmental plasticity, an individual organism integrates influences from its immediate environment with those due to the environment of its parents. While both effects on phenotypes are well documented, their relative impact has been little studied in natural systems, especially at the level of gene expression. We examined this issue in four genotypes of the annual plant Persicaria maculosa by varying two key resources-light and soil moisture-in both generations. Transcriptomic analyses showed that the relative effects of parent and offspring environment on gene expression (i.e. the number of differentially expressed transcripts, DETs) varied both for the two types of resource stress and among genotypes. For light, immediate environment induced more DETs than parental environment for all genotypes, although the precise proportion of parental versus immediate DETs varied among genotypes. By contrast, the relative effect of soil moisture varied dramatically among genotypes, from 8-fold more DETs due to parental than immediate conditions to 10-fold fewer. These findings provide evidence at the transcriptomic level that the relative impacts of parental and immediate environment on the developing organism may depend on the environmental factor and vary strongly among genotypes, providing potential for the interplay of these developmental influences to evolve.

Genomics analysis of Drosophila sechellia response to Morinda citrifolia fruit diet.

Drosophila sechellia is an island endemic host specialist that has evolved to consume the toxic fruit of Morinda citrifolia, also known as noni fruit. Recent studies by our group and others have examined genome-wide gene expression responses of fruit flies to individual highly abundant compounds found in noni responsible for the fruit's unique chemistry and toxicity. In order to relate these reductionist experiments to the gene expression responses to feeding on noni fruit itself, we fed rotten noni fruit to adult female D. sechellia and performed RNA-sequencing. Combining the reductionist and more wholistic approaches, we have identified candidate genes that may contribute to each individual compound and those that play a more general role in response to the fruit as a whole. Using the compound specific and general responses, we used transcription factor prediction analyses to identify the regulatory networks and specific regulators involved in the responses to each compound and the fruit itself. The identified genes and regulators represent the possible genetic mechanisms and biochemical pathways that contribute to toxin resistance and noni specialization in D. sechellia.

Genomics analysis of hexanoic acid exposure in Drosophila species.

Drosophila sechellia is a dietary specialist endemic to the Seychelles islands that has evolved to consume the fruit of Morinda citrifolia. When ripe, the fruit of M. citrifolia contains octanoic acid and hexanoic acid, two medium-chain fatty acid volatiles that deter and are toxic to generalist insects. Drosophila sechellia has evolved resistance to these volatiles allowing it to feed almost exclusively on this host plant. The genetic basis of octanoic acid resistance has been the focus of multiple recent studies, but the mechanisms that govern hexanoic acid resistance in D. sechellia remain unknown. To understand how D. sechellia has evolved to specialize on M. citrifolia fruit and avoid the toxic effects of hexanoic acid, we exposed adult D. sechellia, D. melanogaster and D. simulans to hexanoic acid and performed RNA sequencing comparing their transcriptional responses to identify D. sechellia specific responses. Our analysis identified many more genes responding transcriptionally to hexanoic acid in the susceptible generalist species than in the specialist D. sechellia. Interrogation of the sets of differentially expressed genes showed that generalists regulated the expression of many genes involved in metabolism and detoxification whereas the specialist primarily downregulated genes involved in the innate immunity. Using these data, we have identified interesting candidate genes that may be critically important in aspects of adaptation to their food source that contains high concentrations of HA. Understanding how gene expression evolves during dietary specialization is crucial for our understanding of how ecological communities are built and how evolution shapes trophic interactions.

Comparative transcriptome analyses of the Drosophila pupal eye.

Tissue function is dependent on correct cellular organization and behavior. As a result, the identification and study of genes that contribute to tissue morphogenesis is of paramount importance to the fields of cell and developmental biology. Many of the genes required for tissue patterning and organization are highly conserved between phyla. This has led to the emergence of several model organisms and developmental systems that are used to study tissue morphogenesis. One such model is the Drosophila melanogaster pupal eye that has a highly stereotyped arrangement of cells. In addition, the pupal eye is postmitotic that allows for the study of tissue morphogenesis independent from any effects of proliferation. While the changes in cell morphology and organization that occur throughout pupal eye development are well documented, less is known about the corresponding transcriptional changes that choreograph these processes. To identify these transcriptional changes, we dissected wild-type Canton S pupal eyes and performed RNA-sequencing. Our analyses identified differential expression of many loci that are documented regulators of pupal eye morphogenesis and contribute to multiple biological processes including signaling, axon projection, adhesion, and cell survival. We also identified differential expression of genes not previously implicated in pupal eye morphogenesis such as components of the Toll pathway, several non-classical cadherins, and components of the muscle sarcomere, which could suggest these loci function as novel patterning factors.

Genome-wide effect of tetracycline, doxycycline and 4-epidoxycycline on gene expression in Saccharomyces cerevisiae.

Tetracycline (Tet) and derivative chemicals (e.g., doxycycline or Dox) have gained widespread recognition for their antibiotic properties since their introduction in the late 1970s, but recent work with these chemicals in the lab has shifted to include multiple techniques in all genetic model systems for the precise control of gene expression. The most widely used Tet-modulated methodology is the Tet-On/Tet-Off gene expression system. Tet is generally considered to have effects specific to bacteria; therefore, it should have few off-target effects when used in eukaryotic systems, and a previous study in the yeast Saccharomyces cerevisiae found that Dox had no effect on genome-wide gene expression as measured by microarray. In contrast, another study found that the use of Dox in common cell lines and several model organisms led to mitonuclear protein imbalance, suggesting an inhibitory role of Dox in eukaryotic mitochondria. Recently, a new Dox derivative, 4-epidoxycycline (4-ED) was developed that was shown to have less off-target consequences on mitochondrial health. To determine the best tetracycline family chemical to use for gene expression control in S. cerevisiae, we performed RNA sequencing (RNA-seq) on yeast grown on standard medium compared with growth on media supplemented with Tet, Dox or 4-ED. We found each caused dozens of genes to change expression, with Dox eliciting the greatest expression responses, suggesting that the specific tetracycline used in experiments should be tailored to the specific gene(s) of interest when using the Tet-On/Tet-Off system to reduce the consequences of confounding off-target responses.

Mask, a component of the Hippo pathway, is required for Drosophila eye morphogenesis.

Hippo signaling is an important regulator of tissue size, but it also has a lesser-known role in tissue morphogenesis. Here we use the Drosophila pupal eye to explore the role of the Hippo effector Yki and its cofactor Mask in morphogenesis. We found that Mask is required for the correct distribution and accumulation of adherens junctions and appropriate organization of the cytoskeleton. Accordingly, disrupting mask expression led to severe mis-patterning and similar defects were observed when yki was reduced or in response to ectopic wts. Further, the patterning defects generated by reducing mask expression were modified by Hippo pathway activity. RNA-sequencing revealed a requirement for Mask for appropriate expression of numerous genes during eye morphogenesis. These included genes implicated in cell adhesion and cytoskeletal organization, a comprehensive set of genes that promote cell survival, and numerous signal transduction genes. To validate our transcriptome analyses, we then considered two loci that were modified by Mask activity: FER and Vinc, which have established roles in regulating adhesion. Modulating the expression of either locus modified mask mis-patterning and adhesion phenotypes. Further, expression of FER and Vinc was modified by Yki. It is well-established that the Hippo pathway is responsive to changes in cell adhesion and the cytoskeleton, but our data indicate that Hippo signaling also regulates these structures.

Genomics Analysis of L-DOPA Exposure in Drosophila sechellia.

Drosophila sechellia is a dietary specialist fruit fly that evolved from a generalist ancestor to specialize on the toxic fruit of Morinda citrifolia This species pair has been the subject of numerous studies where the goal has largely been to determine the genetic basis of adaptations associated with host specialization. Because one of the most striking features of M. citrifolia fruit is the production of toxic volatile compounds that kill insects, most genomic studies in D. sechellia to date have focused on gene expression responses to the toxic compounds in its food. In this study, we aim to identify new genes important for host specialization by profiling gene expression response to 3,4-dihydroxyphenylalanine (L-DOPA). Recent work found it to be highly abundant in M. citrifolia, critical for reproductive success of D. sechellia, and supplementation of diet with the downstream pathway product dopamine can influence toxin resistance phenotypes in related species. Here we used a combination of functional genetics and genomics techniques to identify new genes that are important for D. sechellia ecological adaptation to this new niche. We show that L-DOPA exposure can affect toxin resistance phenotypes, identify genes with plastic responses to L-DOPA exposure, and functionally test an identified candidate gene. We found that knock-down of Esterase 6 (Est6) in a heterologous species alters toxin resistance suggesting Est6 may play an important role in D. sechellia host specialization.

Investigating the role of Osiris genes in Drosophila sechellia larval resistance to a host plant toxin.

The underlying genetic basis of adaptive phenotypic changes is generally poorly understood, yet a growing number of case studies are beginning to shed light on important questions about the molecular nature and pleiotropy of such changes. We use Drosophila sechellia, a dietary specialist fruit fly that evolved to specialize on a single toxic host plant, Morinda citrifolia, as a model for adaptive phenotypic change and seek to determine the genetic basis of traits associated with host specialization in this species. The fruit of M. citrifolia is toxic to other drosophilids, primarily due to high levels of the defense chemical octanoic acid (OA), yet D. sechellia has evolved resistance to OA. Our prior work identified three Osiris family genes that reside in a fine-mapped QTL for OA resistance: Osiris 6 (Osi6), Osi7, and Osi8, which can alter OA resistance in adult D. melanogaster when knocked down with RNA interference suggesting they may contribute to OA resistance in D. sechellia. Genetic mapping identified overlapping genomic regions involved in larval and adult OA resistance in D. sechellia, yet it remains unknown whether Osiris genes contribute to resistance in both life stages. Furthermore, because multiple genomic regions contribute to OA resistance, we aim to identify other gene(s) involved in this adaptation. Here, we identify candidate larval OA resistance genes using RNA sequencing to measure genome-wide differential gene expression in D. sechellia larvae after exposure to OA and functionally test identified genes for a role in OA resistance. We then test the Osiris genes previously shown to alter adult OA resistance for effects on OA resistance in larvae. We found that Osi8 knockdown decreased OA resistance in D. melanogaster larvae. These data suggest that evolved changes in Osi8 could impact OA resistance in multiple life stages while Osi6 and Osi7 may only impact adult resistance to OA.

Transcriptomic Analysis of Octanoic Acid Response in Drosophila sechellia Using RNA-Sequencing.

The dietary specialist fruit fly Drosophila sechellia has evolved to specialize on the toxic fruit of its host plant Morinda citrifolia Toxicity of Morinda fruit is primarily due to high levels of octanoic acid (OA). Using RNA interference (RNAi), prior work found that knockdown of Osiris family genes Osiris 6 (Osi6), Osi7, and Osi8 led to increased susceptibility to OA in adult D. melanogaster flies, likely representing genes underlying a Quantitative Trait Locus (QTL) for OA resistance in D. sechellia While genes in this major effect locus are beginning to be revealed, prior work has shown at least five regions of the genome contribute to OA resistance. Here, we identify new candidate OA resistance genes by performing differential gene expression analysis using RNA-sequencing (RNA-seq) on control and OA-exposed D. sechellia flies. We found 104 significantly differentially expressed genes with annotated orthologs in D. melanogaster, including six Osiris gene family members, consistent with previous functional studies and gene expression analyses. Gene ontology (GO) term enrichment showed significant enrichment for cuticle development in upregulated genes and significant enrichment of immune and defense responses in downregulated genes, suggesting important aspects of the physiology of D. sechellia that may play a role in OA resistance. In addition, we identified five candidate OA resistance genes that potentially underlie QTL peaks outside of the major effect region, representing promising new candidate genes for future functional studies.

Evolutionary Dynamics of Regulatory Changes Underlying Gene Expression Divergence among Saccharomyces Species.

Heritable changes in gene expression are important contributors to phenotypic differences within and between species and are caused by mutations in cis-regulatory elements and trans-regulatory factors. Although previous work has suggested that cis-regulatory differences preferentially accumulate with time, technical restrictions to closely related species and limited comparisons have made this observation difficult to test. To address this problem, we used allele-specific RNA-seq data from Saccharomyces species and hybrids to expand both the evolutionary timescale and number of species in which the evolution of regulatory divergence has been investigated. We find that as sequence divergence increases, cis-regulatory differences do indeed become the dominant type of regulatory difference between species, ultimately becoming a better predictor of expression divergence than trans-regulatory divergence. When both cis- and trans-regulatory differences accumulate for the same gene, they more often have effects in opposite directions than in the same direction, indicating widespread compensatory changes underlying the evolution of gene expression. The frequency of compensatory changes within and between species and the magnitude of effect for the underlying cis- and trans-regulatory differences suggests that compensatory changes accumulate primarily due to selection against divergence in gene expression as a result of weak stabilizing selection on gene expression levels. These results show that cis-regulatory differences and compensatory changes in regulation play increasingly important roles in the evolution of gene expression as time increases.

Molecular Mechanisms and Evolutionary Processes Contributing to Accelerated Divergence of Gene Expression on the Drosophila X Chromosome.

In species with a heterogametic sex, population genetics theory predicts that DNA sequences on the X chromosome can evolve faster than comparable sequences on autosomes. Both neutral and nonneutral evolutionary processes can generate this pattern. Complex traits like gene expression are not predicted to have accelerated evolution by these theories, yet a "faster-X" pattern of gene expression divergence has recently been reported for both Drosophila and mammals. Here, we test the hypothesis that accelerated adaptive evolution of cis-regulatory sequences on the X chromosome is responsible for this pattern by comparing the relative contributions of cis- and trans-regulatory changes to patterns of faster-X expression divergence observed between strains and species of Drosophila with a range of divergence times. We find support for this hypothesis, especially among male-biased genes, when comparing different species. However, we also find evidence that trans-regulatory differences contribute to a faster-X pattern of expression divergence both within and between species. This contribution is surprising because trans-acting regulators of X-linked genes are generally assumed to be randomly distributed throughout the genome. We found, however, that X-linked transcription factors appear to preferentially regulate expression of X-linked genes, providing a potential mechanistic explanation for this result. The contribution of trans-regulatory variation to faster-X expression divergence was larger within than between species, suggesting that it is more likely to result from neutral processes than positive selection. These data show how accelerated evolution of both coding and noncoding sequences on the X chromosome can lead to accelerated expression divergence on the X chromosome relative to autosomes.

Evolution of splicing regulatory networks in Drosophila.

The proteome expanding effects of alternative pre-mRNA splicing have had a profound impact on eukaryotic evolution. The events that create this diversity can be placed into four major classes: exon skipping, intron retention, alternative 5' splice sites, and alternative 3' splice sites. Although the regulatory mechanisms and evolutionary pressures among alternative splicing classes clearly differ, how these differences affect the evolution of splicing regulation remains poorly characterized. We used RNA-seq to investigate splicing differences in D. simulans, D. sechellia, and three strains of D. melanogaster. Regulation of exon skipping and tandem alternative 3' splice sites (NAGNAGs) were more divergent than other splicing classes. Splicing regulation was most divergent in frame-preserving events and events in noncoding regions. We further determined the contributions of cis- and trans-acting changes in splicing regulatory networks by comparing allele-specific splicing in F1 interspecific hybrids, because differences in allele-specific splicing reflect changes in cis-regulatory element activity. We find that species-specific differences in intron retention and alternative splice site usage are primarily attributable to changes in cis-regulatory elements (median ∼80% cis), whereas species-specific exon skipping differences are driven by both cis- and trans-regulatory divergence (median ∼50% cis). These results help define the mechanisms and constraints that influence splicing regulatory evolution and show that networks regulating the four major classes of alternative splicing diverge through different genetic mechanisms. We propose a model in which differences in regulatory network architecture among classes of alternative splicing affect the evolution of splicing regulation.

Tempo and mode of regulatory evolution in Drosophila.

Genetic changes affecting gene expression contribute to phenotypic divergence; thus, understanding how regulatory networks controlling gene expression change over time is critical for understanding evolution. Prior studies of expression differences within and between species have identified properties of regulatory divergence, but technical and biological differences among these studies make it difficult to assess the generality of these properties or to understand how regulatory changes accumulate with divergence time. Here, we address these issues by comparing gene expression among strains and species of Drosophila with a range of divergence times and use F1 hybrids to examine inheritance patterns and disentangle cis- and trans-regulatory changes. We find that the fixation of compensatory changes has caused the regulation of gene expression to diverge more rapidly than gene expression itself. Specifically, we observed that the proportion of genes with evidence of cis-regulatory divergence has increased more rapidly with divergence time than the proportion of genes with evidence of expression differences. Surprisingly, the amount of expression divergence explained by cis-regulatory changes did not increase steadily with divergence time, as was previously proposed. Rather, one species (Drosophila sechellia) showed an excess of cis-regulatory divergence that we argue most likely resulted from positive selection in this lineage. Taken together, this work reveals not only the rate at which gene expression evolves, but also the molecular and evolutionary mechanisms responsible for this evolution.

The roles of cis- and trans-regulation in the evolution of regulatory incompatibilities and sexually dimorphic gene expression.

Evolutionary changes in gene expression underlie many aspects of phenotypic diversity within and among species. Understanding the genetic basis for evolved changes in gene expression is therefore an important component of a comprehensive understanding of the genetic basis of phenotypic evolution. Using interspecific introgression hybrids, we examined the genetic basis for divergence in genome-wide patterns of gene expression between Drosophila simulans and Drosophila mauritiana. We find that cis-regulatory and trans-regulatory divergences differ significantly in patterns of genetic architecture and evolution. The effects of cis-regulatory divergence are approximately additive in heterozygotes, quantitatively different between males and females, and well predicted by expression differences between the two parental species. In contrast, the effects of trans-regulatory divergence are associated with largely dominant introgressed alleles, have similar effects in the two sexes, and generate expression levels in hybrids outside the range of expression in both parental species. Although the effects of introgressed trans-regulatory alleles are similar in males and females, expression levels of the genes they regulate are sexually dimorphic between the parental D. simulans and D. mauritiana strains, suggesting that pure-species genotypes carry unlinked modifier alleles that increase sexual dimorphism in expression. Our results suggest that independent effects of cis-regulatory substitutions in males and females may favor their role in the evolution of sexually dimorphic phenotypes, and that trans-regulatory divergence is an important source of regulatory incompatibilities.

Sex-specific effects of cis-regulatory variants in Drosophila melanogaster.

Sexual dimorphism at the level of gene expression is common and well documented, but much less is known about how different cis-regulatory alleles interact with the different trans-regulatory environments present in males and females. Here we show that sex-specific effects of cis-regulatory variants are common in Drosophila.

Sources of bias in measures of allele-specific expression derived from RNA-sequence data aligned to a single reference genome.

BACKGROUND: RNA-seq can be used to measure allele-specific expression (ASE) by assigning sequence reads to individual alleles; however, relative ASE is systematically biased when sequence reads are aligned to a single reference genome. Aligning sequence reads to both parental genomes can eliminate this bias, but this approach is not always practical, especially for non-model organisms. To improve accuracy of ASE measured using a single reference genome, we identified properties of differentiating sites responsible for biased measures of relative ASE. RESULTS: We found that clusters of differentiating sites prevented sequence reads from an alternate allele from aligning to the reference genome, causing a bias in relative ASE favoring the reference allele. This bias increased with greater sequence divergence between alleles. Increasing the number of mismatches allowed when aligning sequence reads to the reference genome and restricting analysis to genomic regions with fewer differentiating sites than the number of mismatches allowed almost completely eliminated this systematic bias. Accuracy of allelic abundance was increased further by excluding differentiating sites within sequence reads that could not be aligned uniquely within the genome (imperfect mappability) and reads that overlapped one or more insertions or deletions (indels) between alleles. CONCLUSIONS: After aligning sequence reads to a single reference genome, excluding differentiating sites with at least as many neighboring differentiating sites as the number of mismatches allowed, imperfect mappability, and/or an indel(s) nearby resulted in measures of allelic abundance comparable to those derived from aligning sequence reads to both parental genomes.

Long-term nitrogen amendment alters the diversity and assemblage of soil bacterial communities in tallgrass prairie.

Anthropogenic changes are altering the environmental conditions and the biota of ecosystems worldwide. In many temperate grasslands, such as North American tallgrass prairie, these changes include alteration in historically important disturbance regimes (e.g., frequency of fires) and enhanced availability of potentially limiting nutrients, particularly nitrogen. Such anthropogenically-driven changes in the environment are known to elicit substantial changes in plant and consumer communities aboveground, but much less is known about their effects on soil microbial communities. Due to the high diversity of soil microbes and methodological challenges associated with assessing microbial community composition, relatively few studies have addressed specific taxonomic changes underlying microbial community-level responses to different fire regimes or nutrient amendments in tallgrass prairie. We used deep sequencing of the V3 region of the 16S rRNA gene to explore the effects of contrasting fire regimes and nutrient enrichment on soil bacterial communities in a long-term (20 yrs) experiment in native tallgrass prairie in the eastern Central Plains. We focused on responses to nutrient amendments coupled with two extreme fire regimes (annual prescribed spring burning and complete fire exclusion). The dominant bacterial phyla identified were Proteobacteria, Verrucomicrobia, Bacteriodetes, Acidobacteria, Firmicutes, and Actinobacteria and made up 80% of all taxa quantified. Chronic nitrogen enrichment significantly impacted bacterial community diversity and community structure varied according to nitrogen treatment, but not phosphorus enrichment or fire regime. We also found significant responses of individual bacterial groups including Nitrospira and Gammaproteobacteria to long-term nitrogen enrichment. Our results show that soil nitrogen enrichment can significantly alter bacterial community diversity, structure, and individual taxa abundance, which have important implications for both managed and natural grassland ecosystems.

Genomic imprinting absent in Drosophila melanogaster adult females.

Genomic imprinting occurs when expression of an allele differs based on the sex of the parent that transmitted the allele. In D. melanogaster, imprinting can occur, but its impact on allelic expression genome-wide is unclear. Here, we search for imprinted genes in D. melanogaster using RNA-seq to compare allele-specific expression between pools of 7- to 10-day-old adult female progeny from reciprocal crosses. We identified 119 genes with allelic expression consistent with imprinting, and these genes showed significant clustering within the genome. Surprisingly, additional analysis of several of these genes showed that either genomic heterogeneity or high levels of intrinsic noise caused imprinting-like allelic expression. Consequently, our data provide no convincing evidence of imprinting for D. melanogaster genes in their native genomic context. Elucidating sources of false-positive signals for imprinting in allele-specific RNA-seq data, as done here, is critical given the growing popularity of this method for identifying imprinted genes.

Regulatory divergence in Drosophila revealed by mRNA-seq.

The regulation of gene expression is critical for organismal function and is an important source of phenotypic diversity between species. Understanding the genetic and molecular mechanisms responsible for regulatory divergence is therefore expected to provide insight into evolutionary change. Using deep sequencing, we quantified total and allele-specific mRNA expression levels genome-wide in two closely related Drosophila species (D. melanogaster and D. sechellia) and their F(1) hybrids. We show that 78% of expressed genes have divergent expression between species, and that cis- and trans-regulatory divergence affects 51% and 66% of expressed genes, respectively, with 35% of genes showing evidence of both. This is a relatively larger contribution of trans-regulatory divergence than was expected based on prior studies, and may result from the unique demographic history of D. sechellia. Genes with antagonistic cis- and trans-regulatory changes were more likely to be misexpressed in hybrids, consistent with the idea that such regulatory changes contribute to hybrid incompatibilities. In addition, cis-regulatory differences contributed more to divergent expression of genes that showed additive rather than nonadditive inheritance. A correlation between sequence similarity and the conservation of cis-regulatory activity was also observed that appears to be a general feature of regulatory evolution. Finally, we examined regulatory divergence that may have contributed to the evolution of a specific trait--divergent feeding behavior in D. sechellia. Overall, this study illustrates the power of mRNA sequencing for investigating regulatory evolution, provides novel insight into the evolution of gene expression in Drosophila, and reveals general trends that are likely to extend to other species.