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Existing human genome assemblies have almost entirely excluded repetitive sequences within and near centromeres, limiting our understanding of their organization, evolution, and functions, which include facilitating proper chromosome segregation. Now, a complete, telomere-to-telomere human genome assembly (T2T-CHM13) has enabled us to comprehensively characterize pericentromeric and centromeric repeats, which constitute 6.2% of the genome (189.9 megabases). Detailed maps of these regions revealed multimegabase structural rearrangements, including in active centromeric repeat arrays. Analysis of centromere-associated sequences uncovered a strong relationship between the position of the centromere and the evolution of the surrounding DNA through layered repeat expansions. Furthermore, comparisons of chromosome X centromeres across a diverse panel of individuals illuminated high degrees of structural, epigenetic, and sequence variation in these complex and rapidly evolving regions.

Understanding the genomic and environmental basis of cold adaptation is key to understand how plants survive and adapt to different environmental conditions across their natural range. Univariate and multivariate genome-wide association (GWAS) and genotype-environment association (GEA) analyses were used to test associations among genome-wide SNPs obtained from whole-genome resequencing, measures of growth, phenology, emergence, cold hardiness, and range-wide environmental variation in coastal Douglas-fir (Pseudotsuga menziesii). Results suggest a complex genomic architecture of cold adaptation, in which traits are either highly polygenic or controlled by both large and small effect genes. Newly discovered associations for cold adaptation in Douglas-fir included 130 genes involved in many important biological functions such as primary and secondary metabolism, growth and reproductive development, transcription regulation, stress and signaling, and DNA processes. These genes were related to growth, phenology and cold hardiness and strongly depend on variation in environmental variables such degree days below 0c, precipitation, elevation and distance from the coast. This study is a step forward in our understanding of the complex interconnection between environment and genomics and their role in cold-associated trait variation in boreal tree species, providing a baseline for the species' predictions under climate change.

BackgroundBoth a source of diversity and the development of genomic tools, such as reference genomes and molecular markers, are equally important to enable faster progress in plant breeding. Pear (Pyrus spp.) lags far behind other fruit and nut crops in terms of employment of available genetic resources for new cultivar development. To address this gap, we designed a high-density, high-efficiency and robust single nucleotide polymorphism (SNP) array for pear, with the main objectives of conducting genetic diversity and genome-wide association studies.ResultsBy applying a two-step design process, which consisted of the construction of a first 'draft' array for the screening of a small subset of samples, we were able to identify the most robust and informative SNPs to include in the Applied Biosystems™ Axiom™ Pear 70 K Genotyping Array, currently the densest SNP array for pear. Preliminary evaluation of this 70 K array in 1416 diverse pear accessions from the USDA National Clonal Germplasm Repository (NCGR) in Corvallis, OR identified 66,616 SNPs (93% of all the tiled SNPs) as high quality and polymorphic (PolyHighResolution). We further used the Axiom Pear 70 K Genotyping Array to construct high-density linkage maps in a bi-parental population, and to make a direct comparison with available genotyping-by-sequencing (GBS) data, which suggested that the SNP array is a more robust method of screening for SNPs than restriction enzyme reduced representation sequence-based genotyping.ConclusionsThe Axiom Pear 70 K Genotyping Array, with its high efficiency in a widely diverse panel of Pyrus species and cultivars, represents a valuable resource for a multitude of molecular studies in pear. The characterization of the USDA-NCGR collection with this array will provide important information for pear geneticists and breeders, as well as for the optimization of conservation strategies for Pyrus.

Despite critical roles in chromosome segregation and disease, the repetitive structure and vast size of centromeres and their surrounding heterochromatic regions impede studies of genomic variation. Here we report the identification of large-scale haplotypes (cenhaps) in humans that span the centromere-proximal regions of all metacentric chromosomes, including the arrays of highly repeated α-satellites on which centromeres form. Cenhaps reveal deep diversity, including entire introgressed Neanderthal centromeres and equally ancient lineages among Africans. These centromere-spanning haplotypes contain variants, including large differences in α-satellite DNA content, which may influence the fidelity and bias of chromosome transmission. The discovery of cenhaps creates new opportunities to investigate their contribution to phenotypic variation, especially in meiosis and mitosis, as well as to more incisively model the unexpectedly rich evolution of these challenging genomic regions.

Over the last 20 years, global production of Persian walnut (Juglans regia L.) has grown enormously, likely reflecting increased consumption due to its numerous benefits to human health. However, advances in genome-wide association (GWA) studies and genomic selection (GS) for agronomically important traits in walnut remain limited due to the lack of powerful genomic tools. Here, we present the development and validation of a high-density 700K single nucleotide polymorphism (SNP) array in Persian walnut. Over 609K high-quality SNPs have been thoroughly selected from a set of 9.6 m genome-wide variants, previously identified from the high-depth re-sequencing of 27 founders of the Walnut Improvement Program (WIP) of University of California, Davis. To validate the effectiveness of the array, we genotyped a collection of 1284 walnut trees, including 1167 progeny of 48 WIP families and 26 walnut cultivars. More than half of the SNPs (55.7%) fell in the highest quality class of 'Poly High Resolution' (PHR) polymorphisms, which were used to assess the WIP pedigree integrity. We identified 151 new parent-offspring relationships, all confirmed with the Mendelian inheritance test. In addition, we explored the genetic variability among cultivars of different origin, revealing how the varieties from Europe and California were differentiated from Asian accessions. Both the reconstruction of the WIP pedigree and population structure analysis confirmed the effectiveness of the Applied Biosystems™ Axiom™ J. regia 700K SNP array, which initiates a novel genomic and advanced phase in walnut genetics and breeding.

Genomic analysis in Juglans (walnuts) is expected to transform the breeding and agricultural production of both nuts and lumber. To that end, we report here the determination of reference sequences for six additional relatives of Juglans regia: Juglans sigillata (also from section Dioscaryon), Juglans nigra, Juglans microcarpa, Juglans hindsii (from section Rhysocaryon), Juglans cathayensis (from section Cardiocaryon), and the closely related Pterocarya stenoptera While these are 'draft' genomes, ranging in size between 640Mbp and 990Mbp, their contiguities and accuracies can support powerful annotations of genomic variation that are often the foundation of new avenues of research and breeding. We annotated nucleotide divergence and synteny by creating complete pairwise alignments of each reference genome to the remaining six. In addition, we have re-sequenced a sample of accessions from four Juglans species (including regia). The variation discovered in these surveys comprises a critical resource for experimentation and breeding, as well as a solid complementary annotation. To demonstrate the potential of these resources the structural and sequence variation in and around the polyphenol oxidase loci, PPO1 and PPO2 were investigated. As reported for other seed crops variation in this gene is implicated in the domestication of walnuts. The apparently Juglandaceae specific PPO1 duplicate shows accelerated divergence and an excess of amino acid replacement on the lineage leading to accessions of the domesticated nut crop species, Juglans regia and sigillata.

The 22-gigabase genome of loblolly pine (Pinus taeda) is one of the largest ever sequenced. The draft assembly published in 2014 was built entirely from short Illumina reads, with lengths ranging from 100 to 250 base pairs (bp). The assembly was quite fragmented, containing over 11 million contigs whose weighted average (N50) size was 8206 bp. To improve this result, we generated approximately 12-fold coverage in long reads using the Single Molecule Real Time sequencing technology developed at Pacific Biosciences. We assembled the long and short reads together using the MaSuRCA mega-reads assembly algorithm, which produced a substantially better assembly, P. taeda version 2.0. The new assembly has an N50 contig size of 25 361, more than three times as large as achieved in the original assembly, and an N50 scaffold size of 107 821, 61% larger than the previous assembly.

A reference genome sequence for Pseudotsuga menziesii var. menziesii (Mirb.) Franco (Coastal Douglas-fir) is reported, thus providing a reference sequence for a third genus of the family Pinaceae. The contiguity and quality of the genome assembly far exceeds that of other conifer reference genome sequences (contig N50 = 44,136 bp and scaffold N50 = 340,704 bp). Incremental improvements in sequencing and assembly technologies are in part responsible for the higher quality reference genome, but it may also be due to a slightly lower exact repeat content in Douglas-fir vs. pine and spruce. Comparative genome annotation with angiosperm species reveals gene-family expansion and contraction in Douglas-fir and other conifers which may account for some of the major morphological and physiological differences between the two major plant groups. Notable differences in the size of the NDH-complex gene family and genes underlying the functional basis of shade tolerance/intolerance were observed. This reference genome sequence not only provides an important resource for Douglas-fir breeders and geneticists but also sheds additional light on the evolutionary processes that have led to the divergence of modern angiosperms from the more ancient gymnosperms.

We investigate the utility and scalability of new read cloud technologies to improve the draft genome assemblies of the colossal, and largely repetitive, genomes of conifers. Synthetic long read technologies have existed in various forms as a means of reducing complexity and resolving repeats since the outset of genome assembly. Recently, technologies that combine subhaploid pools of high molecular weight DNA with barcoding on a massive scale have brought new efficiencies to sample preparation and data generation. When combined with inexpensive light shotgun sequencing, the resulting data can be used to scaffold large genomes. The protocol is efficient enough to consider routinely for even the largest genomes. Conifers represent the largest reference genome projects executed to date. The largest of these is that of the conifer Pinus lambertiana (sugar pine), with a genome size of 31 billion bp. In this paper, we report on the molecular and computational protocols for scaffolding the P. lambertiana genome using the library technology from 10× Genomics. At 247,000 bp, the NG50 of the existing reference sequence is the highest scaffold contiguity among the currently published conifer assemblies; this new assembly's NG50 is 1.94 million bp, an eightfold increase.

The 22-gigabase genome of loblolly pine (Pinus taeda) is one of the largest ever sequenced. The draft assembly published in 2014 was built entirely from short Illumina reads, with lengths ranging from 100 to 250 base pairs (bp). The assembly was quite fragmented, containing over 11 million contigs whose weighted average (N50) size was 8206 bp. To improve this result, we generated approximately 12-fold coverage in long reads using the Single Molecule Real Time sequencing technology developed at Pacific Biosciences. We assembled the long and short reads together using the MaSuRCA mega-reads assembly algorithm, which produced a substantially better assembly, P. taeda version 2.0. The new assembly has an N50 contig size of 25 361, more than three times as large as achieved in the original assembly, and an N50 scaffold size of 107 821, 61% larger than the previous assembly.

Sugar pine (Pinus lambertiana Douglas) is within the subgenus Strobus with an estimated genome size of 31 Gbp. Transcriptomic resources are of particular interest in conifers due to the challenges presented in their megagenomes for gene identification. In this study, we present the first comprehensive survey of the P. lambertiana transcriptome through deep sequencing of a variety of tissue types to generate more than 2.5 billion short reads. Third generation, long reads generated through PacBio Iso-Seq have been included for the first time in conifers to combat the challenges associated with de novo transcriptome assembly. A technology comparison is provided here to contribute to the otherwise scarce comparisons of second and third generation transcriptome sequencing approaches in plant species. In addition, the transcriptome reference was essential for gene model identification and quality assessment in the parallel project responsible for sequencing and assembly of the entire genome. In this study, the transcriptomic data were also used to address questions surrounding lineage-specific Dicer-like proteins in conifers. These proteins play a role in the control of transposable element proliferation and the related genome expansion in conifers.

Until very recently, complete characterization of the megagenomes of conifers has remained elusive. The diploid genome of sugar pine (Pinus lambertiana Dougl.) has a highly repetitive, 31 billion bp genome. It is the largest genome sequenced and assembled to date, and the first from the subgenus Strobus, or white pines, a group that is notable for having the largest genomes among the pines. The genome represents a unique opportunity to investigate genome "obesity" in conifers and white pines. Comparative analysis of P. lambertiana and P. taeda L. reveals new insights on the conservation, age, and diversity of the highly abundant transposable elements, the primary factor determining genome size. Like most North American white pines, the principal pathogen of P. lambertiana is white pine blister rust (Cronartium ribicola J.C. Fischer ex Raben.). Identification of candidate genes for resistance to this pathogen is of great ecological importance. The genome sequence afforded us the opportunity to make substantial progress on locating the major dominant gene for simple resistance hypersensitive response, Cr1 We describe new markers and gene annotation that are both tightly linked to Cr1 in a mapping population, and associated with Cr1 in unrelated sugar pine individuals sampled throughout the species' range, creating a solid foundation for future mapping. This genomic variation and annotated candidate genes characterized in our study of the Cr1 region are resources for future marker-assisted breeding efforts as well as for investigations of fundamental mechanisms of invasive disease and evolutionary response.

Oak represents a valuable natural resource across Northern Hemisphere ecosystems, attracting a large research community studying its genetics, ecology, conservation, and management. Here we introduce a draft genome assembly of valley oak (Quercus lobata) using Illumina sequencing of adult leaf tissue of a tree found in an accessible, well-studied, natural southern California population. Our assembly includes a nuclear genome and a complete chloroplast genome, along with annotation of encoded genes. The assembly contains 94,394 scaffolds, totaling 1.17 Gb with 18,512 scaffolds of length 2 kb or longer, with a total length of 1.15 Gb, and a N50 scaffold size of 278,077 kb. The k-mer histograms indicate an diploid genome size of ∼720-730 Mb, which is smaller than the total length due to high heterozygosity, estimated at 1.25%. A comparison with a recently published European oak (Q. robur) nuclear sequence indicates 93% similarity. The Q. lobata chloroplast genome has 99% identity with another North American oak, Q. rubra Preliminary annotation yielded an estimate of 61,773 predicted protein-coding genes, of which 71% had similarity to known protein domains. We searched 956 Benchmarking Universal Single-Copy Orthologs, and found 863 complete orthologs, of which 450 were present in > 1 copy. We also examined an earlier version (v0.5) where duplicate haplotypes were removed to discover variants. These additional sources indicate that the predicted gene count in Version 1.0 is overestimated by 37-52%. Nonetheless, this first draft valley oak genome assembly represents a high-quality, well-annotated genome that provides a tool for forest restoration and management practices.

The 1966 GENETICS papers by John Hubby and Richard Lewontin were a landmark in the study of genome-wide levels of variability. They used the technique of gel electrophoresis of enzymes and proteins to study variation in natural populations of Drosophila pseudoobscura, at a set of loci that had been chosen purely for technical convenience, without prior knowledge of their levels of variability. Together with the independent study of human populations by Harry Harris, this seminal study provided the first relatively unbiased picture of the extent of genetic variability in protein sequences within populations, revealing that many genes had surprisingly high levels of diversity. These papers stimulated a large research program that found similarly high electrophoretic variability in many different species and led to statistical tools for interpreting the data in terms of population genetics processes such as genetic drift, balancing and purifying selection, and the effects of selection on linked variants. The current use of whole-genome sequences in studies of variation is the direct descendant of this pioneering work.

Hundreds of wild-derived Drosophila melanogaster genomes have been published, but rigorous comparisons across data sets are precluded by differences in alignment methodology. The most common approach to reference-based genome assembly is a single round of alignment followed by quality filtering and variant detection. We evaluated variations and extensions of this approach and settled on an assembly strategy that utilizes two alignment programs and incorporates both substitutions and short indels to construct an updated reference for a second round of mapping prior to final variant detection. Utilizing this approach, we reassembled published D. melanogaster population genomic data sets and added unpublished genomes from several sub-Saharan populations. Most notably, we present aligned data from phase 3 of the Drosophila Population Genomics Project (DPGP3), which provides 197 genomes from a single ancestral range population of D. melanogaster (from Zambia). The large sample size, high genetic diversity, and potentially simpler demographic history of the DPGP3 sample will make this a highly valuable resource for fundamental population genetic research. The complete set of assemblies described here, termed the Drosophila Genome Nexus, presently comprises 623 consistently aligned genomes and is publicly available in multiple formats with supporting documentation and bioinformatic tools. This resource will greatly facilitate population genomic analysis in this model species by reducing the methodological differences between data sets.

An estimated 80% of genomic DNA in eukaryotes is packaged as nucleosomes, which, together with the remaining interstitial linker regions, generate higher order chromatin structures [1]. Nucleosome sequences isolated from diverse organisms exhibit ∼10 bp periodic variations in AA, TT and GC dinucleotide frequencies. These sequence elements generate intrinsically curved DNA and help establish the histone-DNA interface. We investigated an important unanswered question concerning the interplay between chromatin organization and genome evolution: do the DNA sequence preferences inherent to the highly conserved histone core exert detectable natural selection on genomic divergence and polymorphism? To address this hypothesis, we isolated nucleosomal DNA sequences from Drosophila melanogaster embryos and examined the underlying genomic variation within and between species. We found that divergence along the D. melanogaster lineage is periodic across nucleosome regions with base changes following preferred nucleotides, providing new evidence for systematic evolutionary forces in the generation and maintenance of nucleosome-associated dinucleotide periodicities. Further, Single Nucleotide Polymorphism (SNP) frequency spectra show striking periodicities across nucleosomal regions, paralleling divergence patterns. Preferred alleles occur at higher frequencies in natural populations, consistent with a central role for natural selection. These patterns are stronger for nucleosomes in introns than in intergenic regions, suggesting selection is stronger in transcribed regions where nucleosomes undergo more displacement, remodeling and functional modification. In addition, we observe a large-scale (∼180 bp) periodic enrichment of AA/TT dinucleotides associated with nucleosome occupancy, while GC dinucleotide frequency peaks in linker regions. Divergence and polymorphism data also support a role for natural selection in the generation and maintenance of these super-nucleosomal patterns. Our results demonstrate that nucleosome-associated sequence periodicities are under selective pressure, implying that structural interactions between nucleosomes and DNA sequence shape sequence evolution, particularly in introns.

The long time scale of adaptive evolution makes it difficult to directly observe the spread of most beneficial mutations through natural populations. Therefore, inferring attributes of beneficial mutations by studying the genomic signals left by directional selection is an important component of population genetics research. One kind of signal is a trough in nearby neutral genetic variation due to selective fixation of initially rare alleles, a phenomenon known as "genetic hitchhiking." Accumulated evidence suggests that a considerable fraction of substitutions in the Drosophila genome results from positive selection, most of which are expected to have small selection coefficients and influence the population genetics of sites in the immediate vicinity. Using Drosophila melanogaster population genomic data, we found that the heterogeneity in synonymous polymorphism surrounding different categories of coding fixations is readily observable even within 25 bp of focal substitutions, which we interpret as the result of small-scale hitchhiking effects. The strength of natural selection on different sites appears to be quite heterogeneous. Particularly, neighboring fixations that changed amino acid polarities in a way that maintained the overall polarities of a protein were under stronger selection than other categories of fixations. Interestingly, we found that substitutions in slow-evolving genes are associated with stronger hitchhiking effects. This is consistent with the idea that adaptive evolution may involve few substitutions with large effects or many substitutions with small effects. Because our approach only weakly depends on the numbers of recent nonsynonymous substitutions, it can provide a complimentary view to the adaptive evolution inferred by other divergence-based evolutionary genetic methods.

Conifers are the predominant gymnosperm. The size and complexity of their genomes has presented formidable technical challenges for whole-genome shotgun sequencing and assembly. We employed novel strategies that allowed us to determine the loblolly pine (Pinus taeda) reference genome sequence, the largest genome assembled to date. Most of the sequence data were derived from whole-genome shotgun sequencing of a single megagametophyte, the haploid tissue of a single pine seed. Although that constrained the quantity of available DNA, the resulting haploid sequence data were well-suited for assembly. The haploid sequence was augmented with multiple linking long-fragment mate pair libraries from the parental diploid DNA. For the longest fragments, we used novel fosmid DiTag libraries. Sequences from the linking libraries that did not match the megagametophyte were identified and removed. Assembly of the sequence data were aided by condensing the enormous number of paired-end reads into a much smaller set of longer "super-reads," rendering subsequent assembly with an overlap-based assembly algorithm computationally feasible. To further improve the contiguity and biological utility of the genome sequence, additional scaffolding methods utilizing independent genome and transcriptome assemblies were implemented. The combination of these strategies resulted in a draft genome sequence of 20.15 billion bases, with an N50 scaffold size of 66.9 kbp.

BackgroundThe size and complexity of conifer genomes has, until now, prevented full genome sequencing and assembly. The large research community and economic importance of loblolly pine, Pinus taeda L., made it an early candidate for reference sequence determination.ResultsWe develop a novel strategy to sequence the genome of loblolly pine that combines unique aspects of pine reproductive biology and genome assembly methodology. We use a whole genome shotgun approach relying primarily on next generation sequence generated from a single haploid seed megagametophyte from a loblolly pine tree, 20-1010, that has been used in industrial forest tree breeding. The resulting sequence and assembly was used to generate a draft genome spanning 23.2 Gbp and containing 20.1 Gbp with an N50 scaffold size of 66.9 kbp, making it a significant improvement over available conifer genomes. The long scaffold lengths allow the annotation of 50,172 gene models with intron lengths averaging over 2.7 kbp and sometimes exceeding 100 kbp in length. Analysis of orthologous gene sets identifies gene families that may be unique to conifers. We further characterize and expand the existing repeat library based on the de novo analysis of the repetitive content, estimated to encompass 82% of the genome.ConclusionsIn addition to its value as a resource for researchers and breeders, the loblolly pine genome sequence and assembly reported here demonstrates a novel approach to sequencing the large and complex genomes of this important group of plants that can now be widely applied.

The largest genus in the conifer family Pinaceae is Pinus, with over 100 species. The size and complexity of their genomes (∼20-40 Gb, 2n = 24) have delayed the arrival of a well-annotated reference sequence. In this study, we present the annotation of the first whole-genome shotgun assembly of loblolly pine (Pinus taeda L.), which comprises 20.1 Gb of sequence. The MAKER-P annotation pipeline combined evidence-based alignments and ab initio predictions to generate 50,172 gene models, of which 15,653 are classified as high confidence. Clustering these gene models with 13 other plant species resulted in 20,646 gene families, of which 1554 are predicted to be unique to conifers. Among the conifer gene families, 159 are composed exclusively of loblolly pine members. The gene models for loblolly pine have the highest median and mean intron lengths of 24 fully sequenced plant genomes. Conifer genomes are full of repetitive DNA, with the most significant contributions from long-terminal-repeat retrotransposons. In depth analysis of the tandem and interspersed repetitive content yielded a combined estimate of 82%.

A primary goal of evolutionary genetics is to discover and explain the genetic basis of fitness-related traits and how this genetic basis evolves within natural populations. Unprecedented technological advances have fueled the discovery of genetic variants associated with ecologically relevant phenotypes in many different life forms, as well as the ability to scan genomes for deviations from selectively neutral models of evolution. Theoretically, the degree of overlap between lists of genomic regions identified using each approach is related to the genetic architecture of fitness-related traits and the strength and type of natural selection molding variation at these traits within natural populations. Here we address for the first time in a plant the degree of overlap between these lists, using patterns of nucleotide diversity and divergence for >7000 unique amplicons described from the extensive expressed sequence tag libraries generated for loblolly pine (Pinus taeda L.) in combination with the >1000 published genetic associations. We show that loci associated with phenotypic traits are distinct with regard to neutral expectations. Phenotypes measured at the whole plant level (e.g., disease resistance) exhibit an approximately twofold increase in the proportion of adaptive nonsynonymous substitutions over the genome-wide average. As expected for polygenic traits, these signals were apparent only when loci were considered at the level of functional sets. The ramifications of this result are discussed in light of the continued efforts to dissect the genetic basis of quantitative traits.

The relative importance of mutation, selection, and biased gene conversion to patterns of base composition variation in Drosophila melanogaster, and to a lesser extent, D. simulans, has been investigated for many years. However, genomic data from sufficiently large samples to thoroughly characterize patterns of base composition polymorphism within species have been lacking. Here, we report a genome-wide analysis of coding and noncoding polymorphism in a large sample of inbred D. melanogaster strains from Raleigh, North Carolina. Consistent with previous results, we observed that AT mutations fix more frequently than GC mutations in D. melanogaster. Contrary to predictions of previous models of codon usage in D. melanogaster, we found that synonymous sites segregating for derived AT polymorphisms were less skewed toward low frequencies compared with sites segregating a derived GC polymorphism. However, no such pattern was observed for comparable base composition polymorphisms in noncoding DNA. These results suggest that AT-ending codons could currently be favored by natural selection in the D. melanogaster lineage.

Drosophila melanogaster has played a pivotal role in the development of modern population genetics. However, many basic questions regarding the demographic and adaptive history of this species remain unresolved. We report the genome sequencing of 139 wild-derived strains of D. melanogaster, representing 22 population samples from the sub-Saharan ancestral range of this species, along with one European population. Most genomes were sequenced above 25X depth from haploid embryos. Results indicated a pervasive influence of non-African admixture in many African populations, motivating the development and application of a novel admixture detection method. Admixture proportions varied among populations, with greater admixture in urban locations. Admixture levels also varied across the genome, with localized peaks and valleys suggestive of a non-neutral introgression process. Genomes from the same location differed starkly in ancestry, suggesting that isolation mechanisms may exist within African populations. After removing putatively admixed genomic segments, the greatest genetic diversity was observed in southern Africa (e.g. Zambia), while diversity in other populations was largely consistent with a geographic expansion from this potentially ancestral region. The European population showed different levels of diversity reduction on each chromosome arm, and some African populations displayed chromosome arm-specific diversity reductions. Inversions in the European sample were associated with strong elevations in diversity across chromosome arms. Genomic scans were conducted to identify loci that may represent targets of positive selection within an African population, between African populations, and between European and African populations. A disproportionate number of candidate selective sweep regions were located near genes with varied roles in gene regulation. Outliers for Europe-Africa F(ST) were found to be enriched in genomic regions of locally elevated cosmopolitan admixture, possibly reflecting a role for some of these loci in driving the introgression of non-African alleles into African populations.

Heterozygosity is a major challenge to efficient, high-quality genomic assembly and to the full genomic survey of polymorphism and divergence. In Drosophila melanogaster lines derived from equatorial populations are particularly resistant to inbreeding, thus imposing a major barrier to the determination and analyses of genomic variation in natural populations of this model organism. Here we present a simple genome sequencing protocol based on the whole-genome amplification of the gynogenetically derived haploid genome of a progeny of females mated to males homozygous for the recessive male sterile mutation, ms(3)K81. A single "lane" of paired-end sequences (2 × 76 bp) provides a good syntenic assembly with >95% high-quality coverage (more than five reads). The amplification of the genomic DNA moderately inflates the variation in coverage across the euchromatic portion of the genome. It also increases the frequency of chimeric clones. But the low frequency and random genomic distribution of the chimeric clones limits their impact on the final assemblies. This method provides a solid path forward for population genomic sequencing and offers applications to many other systems in which small amounts of genomic DNA have unique experimental relevance.

Abstract Background In today's age of genomic discovery, no attempt has been made to comprehensively sequence a gymnosperm genome. The largest genus in the coniferous family Pinaceae is Pinus, whose 110-120 species have extremely large genomes (c. 20-40 Gb, 2N = 24). The size and complexity of these genomes have prompted much speculation as to the feasibility of completing a conifer genome sequence. Conifer genomes are reputed to be highly repetitive, but there is little information available on the nature and identity of repetitive units in gymnosperms. The pines have extensive genetic resources, with approximately 329000 ESTs from eleven species and genetic maps in eight species, including a dense genetic map of the twelve linkage groups in Pinus taeda. Results We present here the Sanger sequence and annotation of ten P. taeda BAC clones and Genome Analyzer II whole genome shotgun (WGS) sequences representing 7.5% of the genome. Computational annotation of ten BACs predicts three putative protein-coding genes and at least fifteen likely pseudogenes in nearly one megabase of sequence. We found three conifer-specific LTR retroelements in the BACs, and tentatively identified at least 15 others based on evidence from the distantly related angiosperms. Alignment of WGS sequences to the BACs indicates that 80% of BAC sequences have similar copies (≥ 75% nucleotide identity) elsewhere in the genome, but only 23% have identical copies (99% identity). The three most common repetitive elements in the genome were identified and, when combined, represent less than 5% of the genome. Conclusions This study indicates that the majority of repeats in the P. taeda genome are 'novel' and will therefore require additional BAC or genomic sequencing for accurate characterization. The pine genome contains a very large number of diverged and probably defunct repetitive elements. This study also provides new evidence that sequencing a pine genome using a WGS approach is a feasible goal.

The population genetic perspective is that the processes shaping genomic variation can be revealed only through simultaneous investigation of sequence polymorphism and divergence within and between closely related species. Here we present a population genetic analysis of Drosophila simulans based on whole-genome shotgun sequencing of multiple inbred lines and comparison of the resulting data to genome assemblies of the closely related species, D. melanogaster and D. yakuba. We discovered previously unknown, large-scale fluctuations of polymorphism and divergence along chromosome arms, and significantly less polymorphism and faster divergence on the X chromosome. We generated a comprehensive list of functional elements in the D. simulans genome influenced by adaptive evolution. Finally, we characterized genomic patterns of base composition for coding and noncoding sequence. These results suggest several new hypotheses regarding the genetic and biological mechanisms controlling polymorphism and divergence across the Drosophila genome, and provide a rich resource for the investigation of adaptive evolution and functional variation in D. simulans. © 2007 Begun et al.

While many functional elements of the meiotic process are well characterized in model organisms, the genetic basis of most of the natural phenotypic variation observed in meiotic pathways has not been determined. To begin to address this issue, we characterized patterns of polymorphism and divergence in the protein-coding regions of 33 genes across 31 lines of Drosophila melanogaster and 6 lines of Drosophila simulans. We sequenced genes known to be involved in chromosome segregation, recombination, DNA repair, and related heterochromatin binding. As expected, we found several of the genes to be highly conserved, consistent with purifying selection. However, a subset of genes showed patterns of polymorphism and divergence typical of other types of natural selection. Moreover, several intriguing differences between the two Drosophila lineages were evident: along the D. simulans lineage we consistently found evidence of adaptive protein evolution, whereas along the D. melanogaster lineage several loci exhibited patterns consistent with the maintenance of protein variation.

DNA sequence surveys in yeast and humans suggest that the forces shaping telomeric polymorphism and divergence are distinctly more dynamic than those in the euchromatic, gene-rich regions of the chromosomes. However, the generality of this pattern across outbreeding, multicellular eukaryotes has not been determined. To characterize the structure and evolution of Drosophila telomeres, we collected and analyzed molecular population genetics data from the X chromosome subtelomere in 58 lines of North American Drosophila melanogaster and 29 lines of African D. melanogaster. We found that Drosophila subtelomeres exhibit high levels of both structural and substitutional polymorphism relative to linked euchromatic regions. We also observed strikingly different patterns of variation in the North American and African samples. Moreover, our analyses of the polymorphism data identify a localized hotspot of recombination in the most-distal portion of the X subtelomere. While the levels of polymorphism decline sharply and in parallel with rates of crossing over per physical length over the distal first euchromatic megabase pairs of the X chromosome, our data suggest that they rise again sharply in the subtelomeric region ([approximately equal to] 80 kbp). These patterns of historical recombination and geographic differentiation indicate that, similar to yeast and humans, Drosophila subtelomeric DNA is evolving very differently from euchromatic DNA.

Author(s): Drosophila 12 Genomes Consortium; Project Leaders; Andrew G. Clark (corresponding author) [1]; Michael B. Eisen (corresponding author) [2, 3]; Douglas R. Smith (corresponding author) [4]; Casey M. Bergman (corresponding [...]

Disease resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) in the host species Lycopersicon esculentum, the cultivated tomato, and the closely related L. pimpinellifolium is triggered by the physical interaction between the protein products of the host resistance (R) gene Pto and the pathogen avirulence genes AvrPto and AvrPtoB. Sequence variation at the Pto locus was surveyed in natural populations of seven species of Lycopersicon to test hypotheses of host-parasite coevolution and functional adaptation of the Pto gene. Pto shows significantly higher nonsynonymous polymorphism than 14 other non-R-gene loci in the same samples of Lycopersicon species, while showing no difference in synonymous polymorphism, suggesting that the maintenance of amino acid polymorphism at this locus is mediated by pathogen selection. Also, a larger proportion of ancestral variation is maintained at Pto as compared to these non-R-gene loci. The frequency spectrum of amino acid polymorphisms known to negatively affect Pto function is skewed toward low frequency compared to amino acid polymorphisms that do not affect function or silent polymorphisms. Therefore, the evolution of Pto appears to be influenced by a mixture of both purifying and balancing selection.