The African malaria mosquito, Anopheles gambiae sensu stricto (A. gambiae), provides a unique opportunity to study the evolution of reproductive isolation because it is divided into two sympatric, partially isolated subtaxa known as M form and S form. With the annotated genome of this species now available, high-throughput techniques can be applied to locate and characterize the genomic regions contributing to reproductive isolation. In order to quantify patterns of differentiation within A. gambiae, we hybridized population samples of genomic DNA from each form to Affymetrix GeneChip microarrays. We found that three regions, together encompassing less than 2.8 Mb, are the only locations where the M and S forms are significantly differentiated. Two of these regions are adjacent to centromeres, on Chromosomes 2L and X, and contain 50 and 12 predicted genes, respectively. Sequenced loci in these regions contain fixed differences between forms and no shared polymorphisms, while no fixed differences were found at nearby control loci. The third region, on Chromosome 2R, contains only five predicted genes; fixed differences in this region were also verified by direct sequencing. These “speciation islands” remain differentiated despite considerable gene flow, and are therefore expected to contain the genes responsible for reproductive isolation. Much effort has recently been applied to locating the genes and genetic changes responsible for reproductive isolation between species. Though much can be inferred about speciation by studying taxa that have diverged for millions of years, studying differentiation between taxa that are in the early stages of isolation will lead to a clearer view of the number and size of regions involved in the genetics of speciation. Despite appreciable levels of gene flow between the M and S forms of A. gambiae, we were able to isolate three small regions of differentiation where genes responsible for ecological and behavioral isolation are likely to be located. We expect reproductive isolation to be due to changes at a small number of loci, as these regions together contain only 67 predicted genes. Concentrating future mapping experiments on these regions should reveal the genes responsible for reproductive isolation between forms.

A powerful way to map functional genomic variation and reveal the genetic basis of local adaptation is to associate allele frequency across the genome with environmental conditions. Serpentine soils, characterized by high heavy-metal content and low calcium-to-magnesium ratios, are a classic context for studying adaptation of plants to local soil conditions. To investigate whether Arabidopsis lyrata is locally adapted to serpentine soil, and to map the polymorphisms responsible for such adaptation, we pooled DNA from individuals from serpentine and nonserpentine soils and sequenced each 'gene pool' with the Illumina Genome Analyzer. The polymorphisms that are most strongly associated with soil type are enriched at heavy-metal detoxification and calcium and magnesium transport loci, providing numerous candidate mutations for serpentine adaptation. Sequencing of three candidate loci in the European subspecies of A. lyrata indicates parallel differentiation of the same polymorphism at one locus, confirming ecological adaptation, and different polymorphisms at two other loci, which may indicate convergent evolution.

There is abundant variation in gene expression between individuals, populations, and species. The evolution of gene regulation and expression within and between species is thought to frequently contribute to adaptation. Yet considerable evidence suggests that the primary evolutionary force acting on variation in gene expression is stabilizing selection. We review here the results of recent studies characterizing the evolution of gene expression occurring in cis (via linked polymorphisms) or in trans (through diffusible products of other genes) and their contribution to adaptation and response to the environment. We review the evidence for buffering of variation in gene expression at the level of both transcription and translation, and the possible mechanisms for this buffering. Lastly, we summarize unresolved questions about the evolution of gene regulation.

Senescence, the decline in survivorship and fertility with increasing age, is a near-universal property of organisms. Senescence and limited lifespan are thought to arise because weak natural selection late in life allows the accumulation of mutations with deleterious late-age effects that are either neutral (the mutation accumulation hypothesis) or beneficial (the antagonistic pleiotropy hypothesis) early in life. Analyses of Drosophila spontaneous mutations, patterns of segregating variation and covariation, and lines selected for late-age fertility have implicated both classes of mutation in the evolution of aging, but neither their relative contributions nor the properties of individual loci that cause aging in nature are known. To begin to dissect the multiple genetic causes of quantitative variation in lifespan, we have conducted a genome-wide screen for quantitative trait loci (QTLs) affecting lifespan that segregate among a panel of recombinant inbred lines using a dense molecular marker map. Five autosomal QTLs were mapped by composite interval mapping and by sequential multiple marker analysis. The QTLs had large sex-specific effects on lifespan and agespecific effects on survivorship and mortality and mapped to the same regions as candidate genes with fertility, cellular aging, stress resistance and male-specific effects. Late ageof-onset QTL effects are consistent with the mutation accumulation hypothesis for the evolution of senescence, and sex-specific QTL effects suggest a novel mechanism for maintaining genetic variation for lifespan.

Sequence divergence scaled by variation within species has been used to infer the action of selection upon individual genes. Applying this approach to expression, we compared whole-genome whole-body RNA levels in 10 heterozygous Drosophila simulans genotypes and a pooled sample of 10 D. melanogaster lines using Affymetrix Genechip. For 972 genes expressed in D. melanogaster, the transcript level was below detection threshold in D. simulans, which may be explained either by sequence divergence between the primers on the chip and the mRNA transcripts or by down-regulation of these genes. Out of 6,707 genes that were expressed in both species, transcript level was significantly different between species for 534 genes (at P < 0.001). Genes whose expression is under stabilizing selection should exhibit reduced genetic variation within species and reduced divergence between species. Expression of genes under directional selection in D. simulans should be highly divergent from D. melanogaster, while showing low genetic variation in D. simulans. Finally, the genes with large variation within species but modest divergence between species are candidates for balancing selection. Rapidly diverging, low-polymorphism genes included those involved in reproduction (e.g., Mst 3Ba, 98Cb; Acps 26Aa, 63F; and sperm-specific dynein). Genes with high variation in transcript abundance within species included metallothionein and hairless, both hypothesized to be segregating in nature because of gene-by-environment interactions. Further, we compared expression divergence and DNA substitution rate in 195 genes. Synonymous substitution rate and expression divergences were uncorrelated, whereas there was a significant positive correlation between nonsynonymous substitution rate and expression divergence. We hypothesize that as a substantial fraction of nonsynonymous divergence has been shown to be adaptive, much of the observed expression divergence is likewise adaptive.

Assessment of the degree to which gene expression is additive and heritable has important implications for understanding the maintenance of variation, adaptation, phenotypic divergence, and the mapping of genotype onto phenotype. We used whole-genome transcript profiling using Agilent long-oligonucleotide microarrays representing 12,017 genes to demonstrate that gene transcription is pervasively nonadditive in Drosophila melanogaster. Comparison of adults of two isogenic lines and their reciprocal F 1 hybrids revealed 5820 genes as significantly different between at least two of the four genotypes in either males or females or across both sexes. Strikingly, while 25% of all genes differ between the two parents, 33% differ between both F 1 's and the parents, averaged across sexes. However, only 5% of genes show overdominance, suggesting that heterosis for expression is rare.

BackgroundRNA-seq is revolutionizing the way we study transcriptomes. mRNA can be surveyed without prior knowledge of gene transcripts. Alternative splicing of transcript isoforms and the identification of previously unknown exons are being reported. Initial reports of differences in exon usage, and splicing between samples as well as quantitative differences among samples are beginning to surface. Biological variation has been reported to be larger than technical variation. In addition, technical variation has been reported to be in line with expectations due to random sampling. However, strategies for dealing with technical variation will differ depending on the magnitude. The size of technical variance, and the role of sampling are examined in this manuscript.ResultsIn this study three independent Solexa/Illumina experiments containing technical replicates are analyzed. When coverage is low, large disagreements between technical replicates are apparent. Exon detection between technical replicates is highly variable when the coverage is less than 5 reads per nucleotide and estimates of gene expression are more likely to disagree when coverage is low. Although large disagreements in the estimates of expression are observed at all levels of coverage.ConclusionsTechnical variability is too high to ignore. Technical variability results in inconsistent detection of exons at low levels of coverage. Further, the estimate of the relative abundance of a transcript can substantially disagree, even when coverage levels are high. This may be due to the low sampling fraction and if so, it will persist as an issue needing to be addressed in experimental design even as the next wave of technology produces larger numbers of reads. We provide practical recommendations for dealing with the technical variability, without dramatic cost increases.

Natural diversity in aging and other life history patterns is a hallmark of organismal variation. Related species, populations, and individuals within populations show genetically based variation in life span and other aspects of age-related performance. Population differences are especially informative because these differences can be large relative to within-population variation and because they occur in organisms with otherwise similar genomes. We used experimental evolution to produce populations divergent for life span and late-age fertility and then used deep genome sequencing to detect sequence variants with nucleotide-level resolution. Several genes and genome regions showed strong signatures of selection, and the same regions were implicated in independent comparisons, suggesting that the same alleles were selected in replicate lines. Genes related to oogenesis, immunity, and protein degradation were implicated as important modifiers of late-life performance. Expression profiling and functional annotation narrowed the list of strong candidate genes to 38, most of which are novel candidates for regulating aging. Life span and early-age fecundity were negatively correlated among populations; therefore the alleles we identified also are candidate regulators of a major life-history trade-off. More generally, we argue that hitchhiking mapping can be a powerful tool for uncovering the molecular bases of quantitative genetic variation.