The pattern of polymorphism in Arabidopsis thaliana

We resequenced 876 short fragments in a sample of 96 individuals of Arabidopsis thaliana that included stock center accessions as well as a hierarchical sample from natural populations. Although A. thaliana is a selfing weed, the pattern of polymorphism in general agrees with what is expected for a widely distributed, sexually reproducing species. Linkage disequilibrium decays rapidly, within 50 kb. Variation is shared worldwide, although population structure and isolation by distance are evident. The data fail to fit standard neutral models in several ways. There is a genome-wide excess of rare alleles, at least partially due to selection. There is too much variation between genomic regions in the level of polymorphism. The local level of polymorphism is negatively correlated with gene density and positively correlated with segmental duplications. Because the data do not fit theoretical null distributions, attempts to infer natural selection from polymorphism data will require genome-wide surveys of polymorphism in order to identify anomalous regions. Despite this, our data support the utility of A. thaliana as a model for evolutionary functional genomics.     

Evidence for a large-scale population structure of Arabidopsis thaliana from genome-wide single nucleotide polymorphism markers

Population-based methods for the genetic mapping of adaptive traits and the analysis of natural selection require that the population structure and demographic history of a species are taken into account. We characterized geographic patterns of genetic variation in the model plant Arabidopsis thaliana by genotyping 115 genome-wide single nucleotide polymorphism (SNP) markers in 351 accessions from the whole species range using a matrix-assisted laser desorption/ionization time-of-flight assay, and by sequencing of nine unlinked short genomic regions in a subset of 64 accessions. The observed frequency distribution of SNPs is not consistent with a constant-size neutral model of sequence polymorphism due to an excess of rare polymorphisms. There is evidence for a significant population structure as indicated by differences in genetic diversity between geographic regions. Accessions from Central Asia have a low level of polymorphism and an increased level of genome-wide linkage disequilibrium (LD) relative to accessions from the Iberian Peninsula and Central Europe. Cluster analysis with the structure program grouped Eurasian accessions into K=6 clusters. Accessions from the Iberian Peninsula and from Central Asia constitute distinct populations, whereas Central and Eastern European accessions represent admixed populations in which genomes were reshuffled by historical recombination events. These patterns likely result from a rapid postglacial recolonization of Eurasia from glacial refugial populations. Our analyses suggest that mapping populations for association or LD mapping should be chosen from regional rather than a species-wide sample or identified genetically as sets of individuals with similar average genetic distances. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Adaptive variation in beach mice produced by two interacting pigmentation genes

Little is known about the genetic basis of ecologically important morphological variation such as the diverse color patterns of mammals. Here we identify genetic changes contributing to an adaptive difference in color pattern between two subspecies of oldfield mice (Peromyscus polionotus). One mainland subspecies has a cryptic dark brown dorsal coat, while a younger beach-dwelling subspecies has a lighter coat produced by natural selection for camouflage on pale coastal sand dunes. Using genome-wide linkage mapping, we identified three chromosomal regions (two of major and one of minor effect) associated with differences in pigmentation traits. Two candidate genes, the melanocortin-1 receptor (Mc1r) and its antagonist, the Agouti signaling protein (Agouti), map to independent regions that together are responsible for most of the difference in pigmentation between subspecies. A derived mutation in the coding region of Mc1r, rather than change in its expression level, contributes to light pigmentation. Conversely, beach mice have a derived increase in Agouti mRNA expression but no changes in protein sequence. These two genes also interact epistatically: the phenotypic effects of Mc1r are visible only in genetic backgrounds containing the derived Agouti allele. These results demonstrate that cryptic coloration can be based largely on a few interacting genes of major effect.     

A Nonparametric Test Reveals Selection for Rapid Flowering in the Arabidopsis Genome

The detection of footprints of natural selection in genetic polymorphism data is fundamental to understanding the genetic basis of adaptation, and has important implications for human health. The standard approach has been to reject neutrality in favor of selection if the pattern of variation at a candidate locus was significantly different from the predictions of the standard neutral model. The problem is that the standard neutral model assumes more than just neutrality, and it is almost always possible to explain the data using an alternative neutral model with more complex demography. Today's wealth of genomic polymorphism data, however, makes it possible to dispense with models altogether by simply comparing the pattern observed at a candidate locus to the genomic pattern, and rejecting neutrality if the pattern is extreme. Here, we utilize this approach on a truly genomic scale, comparing a candidate locus to thousands of alleles throughout the Arabidopsis thaliana genome. We demonstrate that selection has acted to increase the frequency of early-flowering alleles at the vernalization requirement locus FRIGIDA. Selection seems to have occurred during the last several thousand years, possibly in response to the spread of agriculture. We introduce a novel test statistic based on haplotype sharing that embraces the problem of population structure, and so should be widely applicable.     

Genome-wide association mapping in Arabidopsis identifies previously known flowering time and pathogen resistance genes

There is currently tremendous interest in the possibility of using genome-wide association mapping to identify genes responsible for natural variation, particularly for human disease susceptibility. The model plant Arabidopsis thaliana is in many ways an ideal candidate for such studies, because it is a highly selfing hermaphrodite. As a result, the species largely exists as a collection of naturally occurring inbred lines, or accessions, which can be genotyped once and phenotyped repeatedly. Furthermore, linkage disequilibrium in such a species will be much more extensive than in a comparable outcrossing species. We tested the feasibility of genome-wide association mapping in A. thaliana by searching for associations with flowering time and pathogen resistance in a sample of 95 accessions for which genome-wide polymorphism data were available. In spite of an extremely high rate of false positives due to population structure, we were able to identify known major genes for all phenotypes tested, thus demonstrating the potential of genome-wide association mapping in A. thaliana and other species with similar patterns of variation. The rate of false positives differed strongly between traits, with more clinal traits showing the highest rate. However, the false positive rates were always substantial regardless of the trait, highlighting the necessity of an appropriate genomic control in association studies.     

Whole-Genome Analysis of Histone H3 Lysine 27 Trimethylation in Arabidopsis

Trimethylation of histone H3 lysine 27 (H3K27me3) plays critical roles in regulating animal development, and in several cases, H3K27me3 is also required for the proper expression of developmentally important genes in plants. However, the extent to which H3K27me3 regulates plant genes on a genome-wide scale remains unknown. In addition, it is not clear whether the establishment and spreading of H3K27me3 occur through the same mechanisms in plants and animals. We identified regions containing H3K27me3 in the genome of the flowering plant Arabidopsis thaliana using a high-density whole-genome tiling microarray. The results suggest that H3K27me3 is a major silencing mechanism in plants that regulates an unexpectedly large number of genes in Arabidopsis (~4,400), and that the maintenance of H3K27me3 is largely independent of other epigenetic pathways, such as DNA methylation or RNA interference. Unlike in animals, where H3K27m3 occupies large genomic regions, in Arabidopsis, we found that H3K27m3 domains were largely restricted to the transcribed regions of single genes. Furthermore, unlike in animals systems, H3K27m3 domains were not preferentially associated with low–nucleosome density regions. The results suggest that different mechanisms may underlie the establishment and spreading of H3K27me3 in plants and animals.     

Mapping Meiotic Single-Strand DNA Reveals a New Landscape of DNA Double-Strand Breaks in Saccharomyces cerevisiae

DNA double-strand breaks (DSBs), which are formed by the Spo11 protein, initiate meiotic recombination. Previous DSB-mapping studies have used rad50S or sae2Δ mutants, which are defective in break processing, to accumulate Spo11-linked DSBs, and report large (≥ 50 kb) “DSB-hot” regions that are separated by “DSB-cold” domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2Δ mutants. We therefore developed a novel method to map genome-wide, single-strand DNA (ssDNA)–associated DSBs that accumulate in processing-capable, repair-defective dmc1Δ and dmc1Δ rad51Δ mutants. DSBs were observed at known hot spots, but also in most previously identified “DSB-cold” regions, including near centromeres and telomeres. Although approximately 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1Δ shows that most of these regions have substantial DSB activity. Southern blot assays of DSBs in selected regions in dmc1Δ, rad50S, and wild-type cells confirm these findings. Thus, DSBs are distributed much more uniformly than was previously believed. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as a critical strand-exchange activity genome-wide, and confirm previous conclusions that Spo11-induced lesions initiate all meiotic recombination.     

Molecular-level variation affects population growth in a butterfly metapopulation

The dynamics of natural populations are thought to be dominated by demographic and environmental processes with little influence of intraspecific genetic variation and natural selection, apart from inbreeding depression possibly reducing population growth in small populations. Here we analyse hundreds of well-characterised local populations in a large metapopulation of the Glanville fritillary butterfly (Melitaea cinxia), which persists in a balance between stochastic local extinctions and recolonisations in a network of 4,000 discrete habitat patches. We show that the allelic composition of the glycolytic enzyme phosphoglucose isomerase (Pgi) has a significant effect on the growth of local populations, consistent with previously reported effects of allelic variation on flight metabolic performance and fecundity in the Glanville fritillary and Colias butterflies. The strength and the sign of the molecular effect on population growth are sensitive to the ecological context (the area and spatial connectivity of the habitat patches), which affects genotype-specific gene flow and the influence of migration on the dynamics of local populations. The biological significance of the results for Pgi is underscored by lack of any association between population growth and allelic variation at six other loci typed in the same material. In demonstrating, to our knowledge for the first time, that molecular variation in a candidate gene affects population growth, this study challenges the perception that differential performance of individual genotypes, leading to differential fitness, is irrelevant to population dynamics. These results also demonstrate that the spatial configuration of habitat and spatial dynamics of populations contribute to maintenance of Pgi polymorphism in this species.     

Accelerated Evolution of the ASPM Gene Controlling Brain Size Begins Prior to Human Brain Expansion

Primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by global reduction in cerebral cortical volume. The microcephalic brain has a volume comparable to that of early hominids, raising the possibility that some MCPH genes may have been evolutionary targets in the expansion of the cerebral cortex in mammals and especially primates. Mutations in ASPM, which encodes the human homologue of a fly protein essential for spindle function, are the most common known cause of MCPH. Here we have isolated large genomic clones containing the complete ASPM gene, including promoter regions and introns, from chimpanzee, gorilla, orangutan, and rhesus macaque by transformation-associated recombination cloning in yeast. We have sequenced these clones and show that whereas much of the sequence of ASPM is substantially conserved among primates, specific segments are subject to high Ka/Ks ratios (nonsynonymous/synonymous DNA changes) consistent with strong positive selection for evolutionary change. The ASPM gene sequence shows accelerated evolution in the African hominoid clade, and this precedes hominid brain expansion by several million years. Gorilla and human lineages show particularly accelerated evolution in the IQ domain of ASPM. Moreover, ASPM regions under positive selection in primates are also the most highly diverged regions between primates and nonprimate mammals. We report the first direct application of TAR cloning technology to the study of human evolution. Our data suggest that evolutionary selection of specific segments of the ASPM sequence strongly relates to differences in cerebral cortical size.     

Linkage of Osteoporosis to Chromosome 20p12 and Association to BMP2

Osteoporotic fractures are a major cause of morbidity and mortality in ageing populations. Osteoporosis, defined as low bone mineral density (BMD) and associated fractures, have significant genetic components that are largely unknown. Linkage analysis in a large number of extended osteoporosis families in Iceland, using a phenotype that combines osteoporotic fractures and BMD measurements, showed linkage to Chromosome 20p12.3 (multipoint allele-sharing LOD, 5.10; p value, 6.3 × 10⁻⁷), results that are statistically significant after adjusting for the number of phenotypes tested and the genome-wide search. A follow-up association analysis using closely spaced polymorphic markers was performed. Three variants in the bone morphogenetic protein 2 (BMP2) gene, a missense polymorphism and two anonymous single nucleotide polymorphism haplotypes, were determined to be associated with osteoporosis in the Icelandic patients. The association is seen with many definitions of an osteoporotic phenotype, including osteoporotic fractures as well as low BMD, both before and after menopause. A replication study with a Danish cohort of postmenopausal women was conducted to confirm the contribution of the three identified variants. In conclusion, we find that a region on the short arm of Chromosome 20 contains a gene or genes that appear to be a major risk factor for osteoporosis and osteoporotic fractures, and our evidence supports the view that BMP2 is at least one of these genes.     

Local Patterns of Nucleotide Polymorphism Are Highly Variable in the Selfing Species Arabidopsis thaliana

Neighboring genes predictably share similar evolutionary histories to an extent delineated by recombination. This correlation should extend across multiple linked genes in a selfing species such as Arabidopsis thaliana due to its low effective recombination rate. To test this prediction, we performed a molecular population genetics analysis of nucleotide polymorphism and divergence in chromosomal regions surrounding four low-diversity loci. Three of these loci, At1g67140, At3g03700, and TERMINAL FLOWER1 (TFL1), have been previously implicated as targets of selection and we would predict stronger correlations in polymorphism between neighboring loci due to genetic hitchhiking around these loci. The remaining locus, At1g04300, was identified in a study of linkage disequilibrium surrounding the CRYPTOCHROME2 (CRY2) locus. Although we found broad valleys of reduced nucleotide variation around two of our focal genes, At1g67140 and At3g03700, all chromosomal regions exhibited extreme variation in the patterns of polymorphism and evolution between neighboring loci. Although three of our four regions contained potential targets of selection, application of the composite-likelihood-ratio test of selection in conjunction with a goodness-of-fit test supports the selection hypothesis only for the region containing At3g03700. The degree of discordance in evolutionary histories between linked loci within each region generally correlated with estimates of recombination and linkage disequilibrium for that region, with the exception of the region containing At1g04300. We discuss the implications of these data for future population genetics analyses and genomics studies in A. thaliana. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genomic localization of endogenous mobile CACTA family transposons in natural variants of<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The differentiation between gene-rich and transposon-rich (gene-poor) regions is a common feature of plant genomes. This may be due to preferential integration of transposons into gene-poor regions or may be due to purifying selection against transposon insertion into gene-rich regions. We examined the distribution of a low-copy-number mobile subfamily of Arabidopsis CACTA transposons in the genomes of 19 natural variants (ecotypes) of A. thaliana, and compared that to the pattern of integrations induced in the laboratory by mutation of the DDM1 ( Decrease in DNA Methylation) gene. Sequences similar to mobile CACTA1 copies were distributed among the ecotypes and showed high degrees of polymorphism in genomic localization. Despite the high level of polymorphism, the copy number was low in all the ecotypes examined, and the elements were localized preferentially in pericentromeric and transposon-rich regions. This contrasts with the pattern of transposition induced by the ddm1 mutation, in which the range of integration sites is less biased and the copy number frequently increases. Based on these observations, we discuss the possible contribution of natural selection and chromatin structure to the distribution of transposons.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by M.-A. Grandbastien     

Natural Selection and Recombination Rate Variation Shape Nucleotide Polymorphism Across the Genomes of Three Related Populus Species

A central aim of evolutionary genomics is to identify the relative roles that various evolutionary forces have played in generating and shaping genetic variation within and among species. Here we use whole-genome resequencing data to characterize and compare genome-wide patterns of nucleotide polymorphism, site frequency spectrum, and population-scaled recombination rates in three species of Populus: Populus tremula, P. tremuloides, and P. trichocarpa. We find that P. tremuloides has the highest level of genome-wide variation, skewed allele frequencies, and population-scaled recombination rates, whereas P. trichocarpa harbors the lowest. Our findings highlight multiple lines of evidence suggesting that natural selection, due to both purifying and positive selection, has widely shaped patterns of nucleotide polymorphism at linked neutral sites in all three species. Differences in effective population sizes and rates of recombination largely explain the disparate magnitudes and signatures of linked selection that we observe among species. The present work provides the first phylogenetic comparative study on a genome-wide scale in forest trees. This information will also improve our ability to understand how various evolutionary forces have interacted to influence genome evolution among related species.     

A Scale-Corrected Comparison of Linkage Disequilibrium Levels between Genic and Non-Genic Regions

The understanding of non-random association between loci, termed linkage disequilibrium (LD), plays a central role in genomic research. Since causal mutations are generally not included in genomic marker data, LD between those and available markers is essential for capturing the effects of causal loci on localizing genes responsible for traits. Thus, the interpretation of association studies requires a detailed knowledge of LD patterns. It is well known that most LD measures depend on minor allele frequencies (MAF) of the considered loci and the magnitude of LD is influenced by the physical distances between loci. In the present study, a procedure to compare the LD structure between genomic regions comprising several markers each is suggested. The approach accounts for different scaling factors, namely the distribution of MAF, the distribution of pair-wise differences in MAF, and the physical extent of compared regions, reflected by the distribution of pair-wise physical distances. In the first step, genomic regions are matched based on similarity in these scaling factors. In the second step, chromosome- and genome-wide significance tests for differences in medians of LD measures in each pair are performed. The proposed framework was applied to test the hypothesis that the average LD is different in genic and non-genic regions. This was tested with a genome-wide approach with data sets for humans (Homo sapiens), a highly selected chicken line (Gallus gallus domesticus) and the model plant Arabidopsis thaliana. In all three data sets we found a significantly higher level of LD in genic regions compared to non-genic regions. About 31% more LD was detected genome-wide in genic compared to non-genic regions in Arabidopsis thaliana, followed by 13.6% in human and 6% chicken. Chromosome-wide comparison discovered significant differences on all 5 chromosomes in Arabidopsis thaliana and on one third of the human and of the chicken chromosomes.     

The Case for Selection at CCR5-Δ32

The C-C chemokine receptor 5, 32 base-pair deletion (CCR5-Δ32) allele confers strong resistance to infection by the AIDS virus HIV. Previous studies have suggested that CCR5-Δ32 arose within the past 1,000 y and rose to its present high frequency (5%–14%) in Europe as a result of strong positive selection, perhaps by such selective agents as the bubonic plague or smallpox during the Middle Ages. This hypothesis was based on several lines of evidence, including the absence of the allele outside of Europe and long-range linkage disequilibrium at the locus. We reevaluated this evidence with the benefit of much denser genetic maps and extensive control data. We find that the pattern of genetic variation at CCR5-Δ32 does not stand out as exceptional relative to other loci across the genome. Moreover using newer genetic maps, we estimated that the CCR5-Δ32 allele is likely to have arisen more than 5,000 y ago. While such results can not rule out the possibility that some selection may have occurred at C-C chemokine receptor 5 (CCR5), they imply that the pattern of genetic variation seen atCCR5-Δ32 is consistent with neutral evolution. More broadly, the results have general implications for the design of future studies to detect the signs of positive selection in the human genome.     

Functional Divergence Caused by Ancient Positive Selection of a Drosophila Hybrid Incompatibility Locus

Interspecific hybrid lethality and sterility are a consequence of divergent evolution between species and serve to maintain the discrete identities of species. The evolution of hybrid incompatibilities has been described in widely accepted models by Dobzhansky and Muller where lineage-specific functional divergence is the essential characteristic of hybrid incompatibility genes. Experimentally tractable models are required to identify and test candidate hybrid incompatibility genes. Several Drosophila melanogaster genes involved in hybrid incompatibility have been identified but none has yet been shown to have functionally diverged in accordance with the Dobzhansky-Muller model. By introducing transgenic copies of the X-linked Hybrid male rescue (Hmr) gene into D. melanogaster from its sibling species D. simulans and D. mauritiana, we demonstrate that Hmr has functionally diverged to cause F1 hybrid incompatibility between these species. Consistent with the Dobzhansky-Muller model, we find that Hmr has diverged extensively in the D. melanogaster lineage, but we also find extensive divergence in the sibling-species lineage. Together, these findings implicate over 13% of the amino acids encoded by Hmr as candidates for causing hybrid incompatibility. The exceptional level of divergence at Hmr cannot be explained by neutral processes because we use phylogenetic methods and population genetic analyses to show that the elevated amino-acid divergence in both lineages is due to positive selection in the distant past—at least one million generations ago. Our findings suggest that multiple substitutions driven by natural selection may be a general phenomenon required to generate hybrid incompatibility alleles.     

The genetics of speciation by reinforcement

Reinforcement occurs when natural selection strengthens behavioral discrimination to prevent costly interspecies matings, such as when matings produce sterile hybrids. This evolutionary process can complete speciation, thereby providing a direct link between Darwin's theory of natural selection and the origin of new species. Here, by examining a case of speciation by reinforcement in Drosophila, we present the first high-resolution genetic study of variation within species for female mating discrimination that is enhanced by natural selection. We show that reinforced mating discrimination is inherited as a dominant trait, exhibits variability within species, and may be influenced by a known set of candidate genes involved in olfaction. Our results show that the genetics of reinforced mating discrimination is different from the genetics of mating discrimination between species, suggesting that overall mating discrimination might be a composite phenomenon, which in Drosophila could involve both auditory and olfactory cues. Examining the genetics of reinforcement provides a unique opportunity for both understanding the origin of new species in the face of gene flow and identifying the genetic basis of adaptive female species preferences, two major gaps in our understanding of speciation.     

Genetic elucidation of human hyperosmia to isovaleric acid

The genetic basis of odorant-specific variations in human olfactory thresholds, and in particular of enhanced odorant sensitivity (hyperosmia), remains largely unknown. Olfactory receptor (OR) segregating pseudogenes, displaying both functional and nonfunctional alleles in humans, are excellent candidates to underlie these differences in olfactory sensitivity. To explore this hypothesis, we examined the association between olfactory detection threshold phenotypes of four odorants and segregating pseudogene genotypes of 43 ORs genome-wide. A strong association signal was observed between the single nucleotide polymorphism variants in OR11H7P and sensitivity to the odorant isovaleric acid. This association was largely due to the low frequency of homozygous pseudogenized genotype in individuals with specific hyperosmia to this odorant, implying a possible functional role of OR11H7P in isovaleric acid detection. This predicted receptor–ligand functional relationship was further verified using the Xenopus oocyte expression system, whereby the intact allele of OR11H7P exhibited a response to isovaleric acid. Notably, we also uncovered another mechanism affecting general olfactory acuity that manifested as a significant inter-odorant threshold concordance, resulting in an overrepresentation of individuals who were hyperosmic to several odorants. An involvement of polymorphisms in other downstream transduction genes is one possible explanation for this observation. Thus, human hyperosmia to isovaleric acid is a complex trait, contributed to by both receptor and other mechanisms in the olfactory signaling pathway.     

Evolutionary Processes Acting on Candidate cis-Regulatory Regions in Humans Inferred from Patterns of Polymorphism and Divergence

Analysis of polymorphism and divergence in the non-coding portion of the human genome yields crucial information about factors driving the evolution of gene regulation. Candidate cis-regulatory regions spanning more than 15,000 genes in 15 African Americans and 20 European Americans were re-sequenced and aligned to the chimpanzee genome in order to identify potentially functional polymorphism and to characterize and quantify departures from neutral evolution. Distortions of the site frequency spectra suggest a general pattern of selective constraint on conserved non-coding sites in the flanking regions of genes (CNCs). Moreover, there is an excess of fixed differences that cannot be explained by a Gamma model of deleterious fitness effects, suggesting the presence of positive selection on CNCs. Extensions of the McDonald-Kreitman test identified candidate cis-regulatory regions with high probabilities of positive and negative selection near many known human genes, the biological characteristics of which exhibit genome-wide trends that differ from patterns observed in protein-coding regions. Notably, there is a higher probability of positive selection in candidate cis-regulatory regions near genes expressed in the fetal brain, suggesting that a larger portion of adaptive regulatory changes has occurred in genes expressed during brain development. Overall we find that natural selection has played an important role in the evolution of candidate cis-regulatory regions throughout hominid evolution.