The evolution of antifungal peptides in Drosophila

An essential component of the immune system of animals is the production of antimicrobial peptides (AMPs). In vertebrates and termites the protein sequence of some AMPs evolves rapidly under positive selection, suggesting that they may be coevolving with pathogens. However, antibacterial peptides in Drosophila tend to be highly conserved. We have inferred the selection pressures acting on Drosophila antifungal peptides (drosomycins) from both the divergence of drosomycin genes within and between five species of Drosophila and polymorphism data from Drosophila simulans and D. melanogaster. In common with Drosophila antibacterial peptides, there is no evidence of adaptive protein evolution in any of the drosomycin genes, suggesting that they do not coevolve with pathogens. It is possible that this reflects a lack of specific fungal and bacterial parasites in Drosophila populations. The polymorphism data from both species differed from neutrality at one locus, but this was not associated with changes in the protein sequence. The synonymous site diversity was greater in D. simulans than in D. melanogaster, but the diversity both upstream of the genes and at nonsynonymous sites was similar. This can be explained if both upstream and nonsynonymous mutations are slightly deleterious and are removed more effectively from D. simulans due to its larger effective population size.     

Genomewide comparative analysis of the highly abundant transposable element DINE-1 suggests a recent transpositional burst in Drosophila yakuba

DINE-1 (Drosophila interspersed element) is the most abundant repetitive sequence in the Drosophila genome derived from transposable elements. It comprises >1% of the Drosophila melanogaster genome (DMG) and is believed to be a relic from an ancient transpositional burst that occurred approximately 5-10 MYA. We performed a genomewide comparison of the abundance, sequence variation, and chromosomal distribution of DINE-1 in D. melanogaster and D. yakuba. Unlike the highly diverged copies in the DMG (pairwise distance approximately 15%), DINE-1's in the Drosophila yakuba genome (DYG) have diverged by only 3.4%. Moreover, the chromosomal distribution of DINE-1 in the two species is very different, with a significant number of euchromatic insertions found only in D. yakuba. We propose that these different patterns are caused by a second transpositional burst of DINE-1's in the D. yakuba genome approximately 1.5 MYA. On the basis of the sequence of these recently transposed copies, we conclude that DINE-1 is likely to be a family of nonautomomous DNA transposons. Analysis of the chromosomal distribution of two age groups of DINE-1's in D. yakuba indicates that (1) there is a negative correlation between recombination rates and the density of DINE-1's and (2) younger copies are more evenly distributed in the chromosome arms, while older copies are mostly located near the centromere regions. Our results fit the predictions of a selection-transposition balance model. Our data on whole-genome comparison of a highly abundant TE among Drosophila sibling species demonstrate the unexpectedly dynamic nature of TE activity in different host genomes.     

Population structure and genetic diversity in two species of Hawaiian picture-winged Drosophila

Over the last several decades many picture-winged Drosophila have become less common in both geographical distribution and local population size (pers. obs., Foote pers. comm., Montgomerey pers. comm.). Here we report on a study of two Hawaiian Drosophila species, D. engyochracea, and D. hawaiiensis, to determine the impact that changes in population sizes over the past thirty years have had on the genetic diversity of these species. D. engyochracea is known from only two locations on the Island of Hawai'i (Kipuka Ki and Kipuka Pua'ulu), while D. hawaiiensis is currently more wide spread across Hawai'i Island. We collected 65 D. hawaiiensis and 66 D. engyochracea from two forest patches (kipuka) isolated by a 400 year old volcanic ash deposit. DNA sequence data for 515 bases of the mitochondrial gene COII was analyzed for both species to estimate relative total genetic diversity as well as inter-kipuka gene flow. The more wide spread species, D. hawaiiensis, has more genetic diversity (23 vs. 11 unique haplotypes) than the rarer species, D. engyochracea. The distribution of haplotypes in the kipuka is consistent with more gene flow in D. engyochracea than in D. hawaiiensis. Phylogenetic analysis indicates a small number of individuals morphologically identified as one species but have DNA sequence diagnostic for the other species. These results are consistent with these individuals being descendant from hybrids between species.     

Rapid sequence turnover at an intergenic locus in Drosophila

Closely related species of Drosophila tend to have similar genome sizes. The strong imbalance in favor of small deletions relative to insertions implies that the unconstrained DNA in Drosophila is unlikely to be passively inherited from even closely related ancestors, and yet most DNA in Drosophila genomes is intergenic and potentially unconstrained. In an attempt to investigate the maintenance of this intergenic DNA, we studied the evolution of an intergenic locus on the fourth chromosome of the Drosophila melanogaster genome. This 1.2-kb locus is marked by two distinct, large insertion events: a nuclear transposition of a mitochondrial sequence and a transposition of a nonautonomous DNA transposon DNAREP1_DM. Because we could trace the evolutionary histories of these sequences, we were able to reconstruct the length evolution of this region in some detail. We sequenced this locus in all four species of the D. melanogaster species complex: D. melanogaster, D. simulans, D. sechellia, and D. mauritiana. Although this locus is similar in size in these four species, less than 10% of the sequence from the most recent common ancestor remains in D. melanogaster and all of its sister species. This region appears to have increased in size through several distinct insertions in the ancestor of the D. melanogaster species complex and has been shrinking since the split of these lineages. In addition, we found no evidence suggesting that the size of this locus has been maintained over evolutionary time; these results are consistent with the model of a dynamic equilibrium between persistent DNA loss through small deletions and more sporadic DNA gain through less frequent but longer insertions. The apparent stability of genome size in Drosophila may belie very rapid sequence turnover at intergenic loci.     

Large-scale trends in the evolution of gene structures within 11 animal genomes

We have used the annotations of six animal genomes (Homo sapiens, Mus musculus, Ciona intestinalis, Drosophila melanogaster, Anopheles gambiae, and Caenorhabditis elegans) together with the sequences of five unannotated Drosophila genomes to survey changes in protein sequence and gene structure over a variety of timescales—from the less than 5 million years since the divergence of D. simulans and D. melanogaster to the more than 500 million years that have elapsed since the Cambrian explosion. To do so, we have developed a new open-source software library called CGL (for “Comparative Genomics Library”). Our results demonstrate that change in intron–exon structure is gradual, clock-like, and largely independent of coding-sequence evolution. This means that genome annotations can be used in new ways to inform, corroborate, and test conclusions drawn from comparative genomics analyses that are based upon protein and nucleotide sequence similarities.     

Adaptive evolution of recently duplicated accessory gland protein genes in desert Drosophila

The relationship between animal mating system variation and patterns of protein polymorphism and divergence is poorly understood. Drosophila provides an excellent system for addressing this issue, as there is abundant interspecific mating system variation. For example, compared to D. melanogaster subgroup species, repleta group species have higher remating rates, delayed sexual maturity, and several other interesting differences. We previously showed that accessory gland protein genes (Acp's) of Drosophila mojavensis and D. arizonae evolve more rapidly than Acp's in the D. melanogaster subgroup and that adaptive Acp protein evolution is likely more common in D. mojavensis/D. arizonae than in D. melanogaster/D. simulans. These findings are consistent with the idea that greater postcopulatory selection results in more adaptive evolution of seminal fluid proteins in the repleta group flies. Here we report another interesting evolutionary difference between the repleta group and the D. melanogaster subgroup Acp's. Acp gene duplications are present in D. melanogaster, but their high sequence divergence indicates that the fixation rate of duplicated Acp's has been low in this lineage. Here we report that D. mojavensis and D. arizonae genomes contain several very young duplicated Acp's and that these Acp's have experienced very rapid, adaptive protein divergence. We propose that rapid remating of female desert Drosophila generates selection for continuous diversification of the male Acp complement to improve male fertilization potential. Thus, mating system variation may be associated with adaptive protein divergence as well as with duplication of Acp's in Drosophila.     

A Snapshot of Histone Modifications within Transposable Elements in Drosophila Wild Type Strains

Transposable elements (TEs) are a major source of genetic variability in genomes, creating genetic novelty and driving genome evolution. Analysis of sequenced genomes has revealed considerable diversity in TE families, copy number, and localization between different, closely related species. For instance, although the twin species Drosophila melanogaster and D. simulans share the same TE families, they display different amounts of TEs. Furthermore, previous analyses of wild type derived strains of D. simulans have revealed high polymorphism regarding TE copy number within this species. Several factors may influence the diversity and abundance of TEs in a genome, including molecular mechanisms such as epigenetic factors, which could be a source of variation in TE success. In this paper, we present the first analysis of the epigenetic status of four TE families (roo, tirant, 412 and F) in seven wild type strains of D. melanogaster and D. simulans. Our data shows intra- and inter-specific variations in the histone marks that adorn TE copies. Our results demonstrate that the chromatin state of common TEs varies among TE families, between closely related species and also between wild type strains.     

Ancestral polymorphism and recent invasion of transposable elements in Drosophila species

ABSTRACT: BACKGROUND: During the evolutionary history of transposable elements, some processes, such as ancestral polymorphisms and horizontal transfer of sequences between species, can produce incongruences in phylogenies. We investigated the evolutionary history of the transposable elements Bari and 412 in the sequenced genomes of the Drosophila melanogaster group and in the sibling species D. melanogaster and D. simulans using traditional phylogenetic and network approaches. RESULTS: The maximum likelihood (ML) phylogenetic analyses revealed incongruences and unresolved relationships for both the Bari and 412 elements. The DNA transposon Bari within the D. ananassae genome is more closely related to the element of the melanogaster complex than to the sequence in D. erecta, which is inconsistent with the species phylogeny. Divergence analysis and the comparison of the rate of synonymous substitutions per synonymous site of the Bari and host gene sequences explain the incongruence as an ancestral polymorphism inherited stochastically by the derived species. Unresolved relationships were observed in the ML phylogeny of both elements involving D. melanogaster, D. simulans and D. sechellia. A network approach was used to attempt to resolve these relationships. The resulting tree suggests recent transfers of both elements between D. melanogaster and D. simulans. The divergence values of the elements between these species support this conclusion. CONCLUSIONS: We showed that an ancestral polymorphism and recent invasion of genomes due to introgression or horizontal transfer between species occurred during the evolutionary history of the Bari and 412 elements in the melanogaster group. These invasions likely occurred in Africa during the Pleistocene, before the worldwide expansion of D. melanogaster and D. simulans.     

Different regulatory mechanisms underlie similar transposable element profiles in pufferfish and fruitflies

Comparative analysis of recently sequenced eukaryotic genomes has uncovered extensive variation in transposable element (TE) abundance, diversity, and distribution. The TE profile in the sequenced pufferfish genomes is more similar to that of Drosophila melanogaster than to human or mouse, in that pufferfish TEs exhibit low overall abundance, high family diversity, and localization in the heterochromatin. It has been suggested that selection against the deleterious effects of ectopic recombination between TEs has structured the TE profile in Drosophila and pufferfish but not in humans. We test this hypothesis by measuring the sample frequency of 48 euchromatic TE insertions in the genome of the green spotted pufferfish (Tetraodon nigroviridis). We estimate the strength of selection acting on recent insertions by analyzing the site frequency spectrum using a maximum-likelihood approach. We show that in contrast to Drosophila, euchromatic TE insertions in Tetraodon are selectively neutral and that the low copy number and compartmentalized distribution of TEs in the Tetraodon genome must be caused by regulation by means other than purifying selection acting on recent insertions. Inference of regulatory processes governing TE profiles should take into account factors such as effective population size, incidence of inbreeding/outcrossing, and other species-specific traits.     

De novo Assembly of highly diverse viral populations

ABSTRACT: BACKGROUND: Extensive genetic diversity in viral populations within infected hosts and the divergence of variants from existing reference genomes impede the analysis of deep viral sequencing data. A de novo population consensus assembly is valuable both as a single linear representation of the population and, as a backbone on which intra-host variants can be accurately mapped. The availability of consensus assemblies and robustly mapped variants are crucial to the genetic study of viral disease progression, transmission dynamics, and viral evolution. Existing de novo assembly techniques fail to robustly assemble ultra-deep sequence data from genetically heterogeneous populations such as viruses into full-length genomes due to the presence of extensive genetic variability, contaminants, and variable sequence coverage. RESULTS: We present VICUNA, a de novo assembly algorithm suitable for generating consensus assemblies from genetically heterogeneous populations. We demonstrate its effectiveness on Dengue, Human Immunodeficiency and West Nile viral populations, representing a range of intra-host diversity. Compared to state-of-the-art assemblers designed for haploid or diploid systems, VICUNA recovers full-length consensus and captures insertion/deletion polymorphisms in diverse samples. Final assemblies maintain a high base calling accuracy. VICUNA program is publicly available at: http://www.broadinstitute.org/scientific-community/science/projects/viral-genomics/viral-genomics-analysis-software CONCLUSIONS: We developed VICUNA, a publicly available software tool, that enables consensus assembly of ultra-deep sequence derived from diverse viral populations. While VICUNA was developed for the analysis of viral populations, its application to other heterogeneous sequence data sets such as metagenomic or tumor cell population samples may prove beneficial in these fields of research.     

Mitochondrial gene diversity associated with the atp9 stop codon in natural populations of wild carrot (Daucus carota ssp. carota)

Mitochondrial genomes extracted from the wild populations of Daucus carota have been used as a genetic resource by breeders of cultivated carrot, yet little is known concerning the extent of their diversity in nature. Of special interest is an SNP in the putative stop codon of the mitochondrial gene atp9 that has been associated previously with male-sterile and male-fertile phenotypic variants. In this study, either the sequence or PCR/RFLP genotypes were obtained from the mitochondrial genes atp1, atp9, and cox1 found in D. carota individuals collected from 24 populations in the eastern United States. More than half of the 128 individuals surveyed had a CAA or AAA, rather than TAA, genotype at the position usually thought to function as an atp9 stop codon in this species. We also found no evidence for mitochondrial RNA editing (Cytosine to Uridine) of the CAA stop codon in either floral or leaf tissue. Evidence for intragenic recombination, as opposed to the more common intergenic recombination in plant mitochondrial genomes, in our data set is presented. Indel and SNP variants elsewhere in atp9, and in the other 2 genes surveyed, were nonrandomly associated with the 3 atp9 stop codon variants, though further analysis suggested that multilocus genotypic diversity had been enhanced by recombination. Overall the mitochondrial genetic diversity was only modestly structured among populations with an F(ST) of 0.34.     

Whole-genome sequencing and analysis of the Malaysian cynomolgus macaque (Macaca fascicularis) genome

ABSTRACT: BACKGROUND: The genetic background of the cynomolgus macaque (Macaca fascicularis) is made complex by the high genetic diversity, population structure, and gene introgression from the closely related rhesus macaque (Macaca mulatta). Herein we report the whole-genome sequence of a Malaysian cynomolgus macaque male with more than 40-fold coverage, which was determined using a resequencing method based on the Indian rhesus macaque genome. RESULTS: We identified approximately 9.7 million single nucleotide variants (SNVs) between the Malaysian cynomolgus and the Indian rhesus macaque genomes. Compared with humans, a smaller nonsynonymous/synonymous SNV ratio in the cynomolgus macaque suggests more effective removal of slightly deleterious mutations. Comparison of two cynomolgus (Malaysian and Vietnamese) and two rhesus (Indian and Chinese) macaque genomes, including previously published macaque genomes, suggests that Indochinese cynomolgus macaques have been more affected by gene introgression from rhesus macaques. We further identified 60 nonsynonymous SNVs that completely differentiated the cynomolgus and rhesus macaque genomes, and that could be important candidate variants for determining species-specific responses to drugs and pathogens. The demographic inference using the genome sequence data revealed that Malaysian cynomolgus macaques have experienced at least three population bottlenecks. CONCLUSIONS: This list of whole-genome SNVs will be useful for many future applications, such as an array-based genotyping system for macaque individuals. High-quality whole-genome sequencing of the cynomolgus macaque genome may aid studies on finding genetic differences that are responsible for phenotypic diversity in macaques and may help control genetic backgrounds among individuals.     

Eucaryotic transposable genetic elements with inverted terminal repeats

DNA carrying inverted repeats was tested for transposition within the Drosophila genome. Five Bam HI segments containing related inverted repeats were isolated from D. melanogaster and analyzed by electron microscopy and restriction mapping. Southern blot experiments using single-copy flanking sequences as probes allowed the study of DNA arrangements at specific sites in the genomes of five closely related strains. We found that in some genomes the sequences with inverted repeats were present at a particular site, whereas in other genomes they were absent from this site. These results indicated that three of the sequences are transposable genetic elements. In one case we have purified the two corresponding DNA segments, with and without the sequence containing inverted repeats, thereby confirming the mobility of this sequence. These DNA elements were found to be distinct in two ways from copia and others previously described: first, they contain inverted terminal repeats, and second, they have a more heterogeneous construction.     

Ten years of bacterial genome sequencing: comparative-genomics-based discoveries

It has been more than 10 years since the first bacterial genome sequence was published. Hundreds of bacterial genome sequences are now available for comparative genomics, and searching a given protein against more than a thousand genomes will soon be possible. The subject of this review will address a relatively straightforward question: “What have we learned from this vast amount of new genomic data?” Perhaps one of the most important lessons has been that genetic diversity, at the level of large-scale variation amongst even genomes of the same species, is far greater than was thought. The classical textbook view of evolution relying on the relatively slow accumulation of mutational events at the level of individual bases scattered throughout the genome has changed. One of the most obvious conclusions from examining the sequences from several hundred bacterial genomes is the enormous amount of diversity—even in different genomes from the same bacterial species. This diversity is generated by a variety of mechanisms, including mobile genetic elements and bacteriophages. An examination of the 20 Escherichia coli genomes sequenced so far dramatically illustrates this, with the genome size ranging from 4.6 to 5.5 Mbp; much of the variation appears to be of phage origin. This review also addresses mobile genetic elements, including pathogenicity islands and the structure of transposable elements. There are at least 20 different methods available to compare bacterial genomes. Metagenomics offers the chance to study genomic sequences found in ecosystems, including genomes of species that are difficult to culture. It has become clear that a genome sequence represents more than just a collection of gene sequences for an organism and that information concerning the environment and growth conditions for the organism are important for interpretation of the genomic data. The newly proposed Minimal Information about a Genome Sequence standard has been developed to obtain this information.