Comparative architectures of mammalian and chicken genomes reveal highly variable rates of genomic rearrangements across different lineages

Molecular evolution studies are usually based on the analysis of individual genes and thus reflect only small-range variations in genomic sequences. A complementary approach is to study the evolutionary history of rearrangements in entire genomes based on the analysis of gene orders. The progress in whole genome sequencing provides an unprecedented level of detailed sequence data to infer genome rearrangements through comparative approaches. The comparative analysis of recently sequenced rodent genomes with the human genome revealed evidence for a larger number of rearrangements than previously thought and led to the reconstruction of the putative genomic architecture of the murid rodent ancestor, while the architecture of the ancestral mammalian genome and the rate of rearrangements in the human lineage remained unknown. Sequencing the chicken genome provides an opportunity to reconstruct the architecture of the ancestral mammalian genome by using chicken as an outgroup. Our analysis reveals a very low rate of rearrangements and, in particular, interchromosomal rearrangements in chicken, in the early mammalian ancestor, or in both. The suggested number of interchromosomal rearrangements between the mammalian ancestor and chicken, during an estimated 500 million years of evolution, only slightly exceeds the number of interchromosomal rearrangements that happened in the mouse lineage, over the course of about 87 million years.     

Chicken genome: new tools and concepts.

Last spring, the Second International Chicken Genome Workshop was held at the Stowers Institute for Medical Research, less than 2 months after the first draft of the chicken genome was publicly released in March, 2004. This major event was highly anticipated by the chicken community, because of the invaluable resources that would be newly provided. In addition, from an evolutionary standpoint, birds are the species most closely related to mammals, whose genome has been sequenced. The meeting gathered both agricultural and academic chicken communities and provided the opportunity to discuss the status of the chicken genome sequencing, the preliminary analysis of the chicken genome sequences freshly available, and the impact on avian genetic tools.     

Comparative genome sequencing of Drosophila pseudoobscura: chromosomal, gene, and cis-element evolution

We have sequenced the genome of a second Drosophila species, Drosophila pseudoobscura, and compared this to the genome sequence of Drosophila melanogaster, a primary model organism. Throughout evolution the vast majority of Drosophila genes have remained on the same chromosome arm, but within each arm gene order has been extensively reshuffled, leading to a minimum of 921 syntenic blocks shared between the species. A repetitive sequence is found in the D. pseudoobscura genome at many junctions between adjacent syntenic blocks. Analysis of this novel repetitive element family suggests that recombination between offset elements may have given rise to many paracentric inversions, thereby contributing to the shuffling of gene order in the D. pseudoobscura lineage. Based on sequence similarity and synteny, 10,516 putative orthologs have been identified as a core gene set conserved over 25–55 million years (Myr) since the pseudoobscura/melanogaster divergence. Genes expressed in the testes had higher amino acid sequence divergence than the genome-wide average, consistent with the rapid evolution of sex-specific proteins. Cis-regulatory sequences are more conserved than random and nearby sequences between the species—but the difference is slight, suggesting that the evolution of cis-regulatory elements is flexible. Overall, a pattern of repeat-mediated chromosomal rearrangement, and high coadaptation of both male genes and cis-regulatory sequences emerges as important themes of genome divergence between these species of Drosophila.     

Genome-wide characterization of centromeric satellites from multiple mammalian genomes

Despite its importance in cell biology and evolution, the centromere has remained the final frontier in genome assembly and annotation due to its complex repeat structure. However, isolation and characterization of the centromeric repeats from newly sequenced species are necessary for a complete understanding of genome evolution and function. In recent years, various genomes have been sequenced, but the characterization of the corresponding centromeric DNA has lagged behind. Here, we present a computational method (RepeatNet) to systematically identify higher-order repeat structures from unassembled whole-genome shotgun sequence and test whether these sequence elements correspond to functional centromeric sequences. We analyzed genome datasets from six species of mammals representing the diversity of the mammalian lineage, namely, horse, dog, elephant, armadillo, opossum, and platypus. We define candidate monomer satellite repeats and demonstrate centromeric localization for five of the six genomes. Our analysis revealed the greatest diversity of centromeric sequences in horse and dog in contrast to elephant and armadillo, which showed high-centromeric sequence homogeneity. We could not isolate centromeric sequences within the platypus genome, suggesting that centromeres in platypus are not enriched in satellite DNA. Our method can be applied to the characterization of thousands of other vertebrate genomes anticipated for sequencing in the near future, providing an important tool for annotation of centromeres.     

Lessons from the human genome: transitions between euchromatin and heterochromatin.

The publication of the human genome draft sequence provides, for the first time, a global view of the structural properties of the human genome. Initial sequence analysis, in combination with previous published reports, reveals that more than half of the transition regions between euchromatin and centromeric heterochromatin contain duplicated segments. The individual duplications originate from diverse euchromatic regions of the human genome, often containing intron-exon structure of known genes. Multiple duplicons are concatenated together to form larger blocks of wall-to-wall duplications. For a single chromosome, these paralogous segments can span >1 Mb of sequence and define a buffer zone between unique sequence and tandemly repeated satellite sequences. Unusual pericentromeric interspersed repeat elements have been identified at the junctions of many of these duplications. Phylogenetic and comparative studies of pericentromeric sequences suggest that this peculiar genome organization has emerged within the last 30 million years of human evolution and is a source of considerable genomic variation between closely related primate species. Interestingly, not all human pericentromeric regions show this proclivity to duplicate and transpose genomic sequence, suggesting at least two different models for the organization of these regions.     

Repetitive extragenic palindromic sequences in the Pseudomonas syringae pv. tomato DC3000 genome: extragenic signals for genome reannotation

Repetitive extragenic palindromic (REPs) sequences were first described in enterobacteriacea and later in Pseudomonas putida. We have detected a new variant (51 base pairs) of REP sequences that appears to be disseminated in more than 300 copies in the Pseudomonas syringae DC3000 genome. The finding of REP sequences in P. syringae confirms the broad presence of this type of repetitive sequence in bacteria. We analyzed the distribution of REP sequences and the structure of the clusters, and we show that palindromy is conserved. REP sequences appear to be allocated to the extragenic space, with a special preference for the intergenic spaces limited by convergent genes, while their presence is scarce between divergent genes. Using REP sequences as markers of extragenicity we re-annotated a set of genes of the P. syringae DC3000 genome demonstrating that REP sequences can be used for refinement of annotation of a genome. The similarity detected between virulence genes from evolutionarily distant pathogenic bacteria suggests the acquisition of clusters of virulence genes by horizontal gene transfer. We did not detect the presence of P. syringae REP elements in the principal pathogenicity gene clusters. This absence suggests that genome fragments lacking REP sequences could point to regions recently acquired from other organisms, and REP sequences might be new tracers for gaining insight into key aspects of bacterial genome evolution, especially when studying pathogenicity acquisition. In addition, as the P. syringae REP sequence is species-specific with respect to the sequenced genomes, it is an exceptional candidate for use as a fingerprint in precise genotyping and epidemiological studies.     

Cytogenetic repartition of chicken CR1 sequences evidenced by PRINS in Galliformes and some other birds

Chicken repeat 1 (CR1) belongs to the non-long repeat class of retrotransposons. Nearly 100000 repeats interspersed in the chicken genome are subdivided into at least six distinct subfamilies, each 300 bp long and all sharing substantial sequence similarity. CR1-like elements were found in genomes from invertebrates to mammals, suggesting their importance for genome structure and/or function. Moreover, numerous data support the hypothesis of their implication in regulation of gene expression. So, the chromosomal distribution of these CR1 sequences in vertebrates is of great interest to improve our knowledge about the genome structure, function and evolution. A comparison of the cytogenetic distribution of CR1 sequences was performed by PRINS using consensus chicken primers on the chromosomes of chicken and species of several bird orders: Galliformes, Anseriformes, Passeriformes and Falconiformes. The study revealed that CR1 repeats are spread over nearly all chicken chromosomes with a higher density on the macrochromosomes and in particular with hot spots on subtelomeric regions of chromosome 1, 2, 3q, 4q, 5q. Their distribution on the macrochromosomes forms a kind of banding pattern, which was not systematically matched with R- or G-banding. This banding pattern appears to be conserved on the chromosomes of the Galliformes studied, irrespective of their karyotypes, rearranged or not. CR1 primers also show similar signals on the chromosomes of birds phylogenetically more distant (Anseriformes, Passeriformes and Falconiformes). This fact confirms the importance of these sequences at the large scale of bird evolution and in the chromosomal structure. The location of CR1 sequences, and in particular of the hot spots, mainly within the richest CG areas are in conformity with the data on an epigenetic role of these highly conserved sequences.     

The genome of the leaf-cutting ant Acromyrmex echinatior suggests key adaptations to advanced social life and fungus farming

We present a high-quality (>100× depth) Illumina genome sequence of the leaf-cutting ant Acromyrmex echinatior, a model species for symbiosis and reproductive conflict studies. We compare this genome with three previously sequenced genomes of ants from different subfamilies and focus our analyses on aspects of the genome likely to be associated with known evolutionary changes. The first is the specialized fungal diet of A. echinatior, where we find gene loss in the ant's arginine synthesis pathway, loss of detoxification genes, and expansion of a group of peptidase proteins. One of these is a unique ant-derived contribution to the fecal fluid, which otherwise consists of "garden manuring" fungal enzymes that are unaffected by ant digestion. The second is multiple mating of queens and ejaculate competition, which may be associated with a greatly expanded nardilysin-like peptidase gene family. The third is sex determination, where we could identify only a single homolog of the feminizer gene. As other ants and the honeybee have duplications of this gene, we hypothesize that this may partly explain the frequent production of diploid male larvae in A. echinatior. The fourth is the evolution of eusociality, where we find a highly conserved ant-specific profile of neuropeptide genes that may be related to caste determination. These first analyses of the A. echinatior genome indicate that considerable genetic changes are likely to have accompanied the transition from hunter-gathering to agricultural food production 50 million years ago, and the transition from single to multiple queen mating 10 million years ago.     

Single nucleotide polymorphism mapping using genome-wide unique sequences

As more and more genomic DNAs are sequenced to characterize human genetic variations, the demand for a very fast and accurate method to genomically position these DNA sequences is high. We have developed a new mapping method that does not require sequence alignment. In this method, we first identified DNA fragments of 15 bp in length that are unique in the human genome and then used them to position single nucleotide polymorphism (SNP) sequences. By use of four desktop personal computers with AMD K7 (1 GHz) processors, our new method mapped more than 1.6 million SNP sequences in 20 hr and achieved a very good agreement with mapping results from alignment-based methods.     

Complete genome sequence and comparative genomics of Shigella flexneri serotype 2a strain 2457T

We determined the complete genome sequence of Shigella flexneri serotype 2a strain 2457T (4,599,354 bp). Shigella species cause >1 million deaths per year from dysentery and diarrhea and have a lifestyle that is markedly different from those of closely related bacteria, including Escherichia coli. The genome exhibits the backbone and island mosaic structure of E. coli pathogens, albeit with much less horizontally transferred DNA and lacking 357 genes present in E. coli. The strain is distinctive in its large complement of insertion sequences, with several genomic rearrangements mediated by insertion sequences, 12 cryptic prophages, 372 pseudogenes, and 195 S. flexneri-specific genes. The 2457T genome was also compared with that of a recently sequenced S. flexneri 2a strain, 301. Our data are consistent with Shigella being phylogenetically indistinguishable from E. coli. The S. flexneri-specific regions contain many genes that could encode proteins with roles in virulence. Analysis of these will reveal the genetic basis for aspects of this pathogenic organism's distinctive lifestyle that have yet to be explained.     

A microRNA catalog of the developing chicken embryo identified by a deep sequencing approach

MicroRNA (miRNA) and other types of small regulatory RNAs play a crucial role in the regulation of gene expression in eukaryotes. Several distinct classes of small regulatory RNAs have been discovered in recent years. To extend the repertoire of small regulatory RNAs characterized in chickens we used a deep sequencing approach developed by Solexa (now Illumina Inc.). We sequenced three small RNA libraries prepared from different developmental stages of the chicken embryo (days five, seven, and nine) to produce over 9.5 million short sequence reads. We developed a bioinformatics pipeline to distinguish authentic mature miRNA sequences from other classes of small RNAs and short RNA fragments represented in the sequencing data. Using this approach we detected almost all of the previously known chicken miRNAs and their respective miRNA* sequences. In addition we discovered 449 new chicken miRNAs including 88 miRNA candidates. Of these, 430 miRNAs appear to be specific to the avian lineage. Another six new miRNAs had evidence of evolutionary conservation in at least one vertebrate species outside of the bird lineage. The remaining 13 putative miRNAs appear to represent chicken orthologs of known vertebrate miRNAs. We discovered 39 additional putative miRNA candidates originating from miRNA generating intronic sequences known as mirtrons.     

Molecular characterization of full-length MLV-related endogenous retrovirus ChiRV1 from the chicken, Gallus gallus

We report the first full-length sequence of an endogenous retrovirus from the genome of domestic chicken, that is not related to the Avian leukemia viruses (ALV). This retrovirus, designated ChiRV1, clusters with Murine leukemia virus (MLV)-related retroviruses and hence is the first complete gammaretrovirus from the genome of a bird. Nevertheless it is not related to exogenous MLV-related retroviruses infecting chicken. The provirus is 9133 bp long and contains 90%-identical LTRs as well as reading frames for the gag, pol and env genes, interrupted by in-frame stop codons. Expression analysis showed that ChiRV1 is a transcribed provirus. Screening of the chicken genome database revealed 100 ChiRV1-related sequences that are grouped into three classes based upon LTR alignment and subsequent phylogenetic analysis.     

A comparative genomic analysis of diverse clonal types of enterotoxigenic Escherichia coli reveals pathovar-specific conservation

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal illness in children less than 5 years of age in low- and middle-income nations, whereas it is an emerging enteric pathogen in industrialized nations. Despite being an important cause of diarrhea, little is known about the genomic composition of ETEC. To address this, we sequenced the genomes of five ETEC isolates obtained from children in Guinea-Bissau with diarrhea. These five isolates represent distinct and globally dominant ETEC clonal groups. Comparative genomic analyses utilizing a gene-independent whole-genome alignment method demonstrated that sequenced ETEC strains share approximately 2.7 million bases of genomic sequence. Phylogenetic analysis of this "core genome" confirmed the diverse history of the ETEC pathovar and provides a finer resolution of the E. coli relationships than multilocus sequence typing. No identified genomic regions were conserved exclusively in all ETEC genomes; however, we identified more genomic content conserved among ETEC genomes than among non-ETEC E. coli genomes, suggesting that ETEC isolates share a genomic core. Comparisons of known virulence and of surface-exposed and colonization factor genes across all sequenced ETEC genomes not only identified variability but also indicated that some antigens are restricted to the ETEC pathovar. Overall, the generation of these five genome sequences, in addition to the two previously generated ETEC genomes, highlights the genomic diversity of ETEC. These studies increase our understanding of ETEC evolution, as well as provide insight into virulence factors and conserved proteins, which may be targets for vaccine development.     

Intraspecies sequence comparisons for annotating genomes

Analysis of sequence variation among members of a single species offers a potential approach to identify functional DNA elements responsible for biological features unique to that species. Due to its high rate of allelic polymorphism and ease of genetic manipulability, we chose the sea squirt, Ciona intestinalis, to explore intraspecies sequence comparisons for genome annotation. A large number of C. intestinalis specimens were collected from four continents, and a set of genomic intervals were amplified, resequenced, and analyzed to determine the mutation rates at each nucleotide in the sequence. We found that regions with low mutation rates efficiently demarcated functionally constrained sequences: these include a set of noncoding elements, which we showed in C. intestinalis transgenic assays to act as tissue-specific enhancers, as well as the location of coding sequences. This illustrates that comparisons of multiple members of a species can be used for genome annotation, suggesting a path for the annotation of the sequenced genomes of organisms occupying uncharacterized phylogenetic branches of the animal kingdom. It also raises the possibility that the resequencing of a large number of Homo sapiens individuals might be used to annotate the human genome and identify sequences defining traits unique to our species.     

Enrichment of segmental duplications in regions of breaks of synteny between the human and mouse genomes suggest their involvement in evolutionary rearrangements

The sequence of the mouse genome allows one to compare the conservation of synteny between the human and mouse genome and exploration of regions that might have been involved in major rearrangements during the evolution of these two species (evolutionary genome rearrangements). Recent segmental duplications (or duplicons) are paralogous DNA sequences with high sequence identity that account for about 3.5-5% of the human genome and have emerged during the past approximately 35 million years of evolution. These regions are susceptible to illegitimate recombination leading to rearrangements that result in genomic disorders or genomic mutations. A catalogue of several hundred segmental duplications potentially leading to genomic rearrangements has been reported. The authors and others have observed that some chromosome regions involved in genomic disorders are shuffled in orientation and order in the mouse genome and that regions flanked by segmental duplications are often polymorphic. We have compared the human and mouse genome sequences and demonstrate here that recent segmental duplications correlate with breaks of synteny between these two species. We also observed that nine primary regions involved in human genomic disorders show changes in the order or the orientation of mouse/human synteny segments, were often flanked by segmental duplications in the human sequence. We found that 53% of all evolutionary rearrangement breakpoints associate with segmental duplications, as compared with 18% expected in a random location of breaks along the chromosome (P<0.0001). Our data suggest that segmental duplications have participated in the recent evolution of the human genome, as driving forces for evolutionary rearrangements, chromosome structure polymorphisms and genomic disorders.     

Mosaic organization of orthologous sequences in grass genomes

Although comparative genetic mapping studies show extensive genome conservation among grasses, recent data provide many exceptions to gene collinearity at the DNA sequence level. Rice, sorghum, and maize are closely related grass species, once sharing a common ancestor. Because they diverged at different times during evolution, they provide an excellent model to investigate sequence divergence. We isolated, sequenced, and compared orthologous regions from two rice subspecies, sorghum, and maize to investigate the nature of their sequence differences. This study represents the most extensive sequence comparison among grasses, including the largest contiguous genomic sequences from sorghum (425 kb) and maize (435 kb) to date. Our results reveal a mosaic organization of the orthologous regions, with conserved sequences interspersed with nonconserved sequences. Gene amplification, gene movement, and retrotransposition account for the majority of the nonconserved sequences. Our analysis also shows that gene amplification is frequently linked with gene movement. Analyzing an additional 2.9 Mb of genomic sequence from rice not only corroborates our observations, but also suggests that a significant portion of grass genomes may consist of paralogous sequences derived from gene amplification. We propose that sequence divergence started from hotspots along chromosomes and expanded by accumulating small-scale genomic changes during evolution.     

Centromere satellites from Arabidopsis populations: maintenance of conserved and variable domains

The rapid evolution of centromere sequences between species has led to a debate over whether centromere activity is sequence-dependent. The Arabidopsis thaliana centromere regions contain approximately 20,000 copies of a 178-bp satellite repeat. Here, we analyzed satellites from 41 Arabidopsis ecotypes, providing the first broad population survey of satellite variation within a species. We found highly conserved segments and consistent sequence lengths in the Arabidopsis satellites and in the published collection of human alpha-satellites, supporting models for a functional role. Despite this conservation, polymorphisms are significantly enriched at some sites, yielding variation that could restrict binding proteins to a subset of repeat monomers. Some satellite regions vary considerably; at certain bases, consensus sequences derived from each ecotype diverge significantly from the Arabidopsis consensus, indicating substitutions sweep through a genome in less than 5 million years. Such rapid changes generate more variation within the set of Arabidopsis satellites than in genes from the chromosome arms or from the recombinationally suppressed centromere regions. These studies highlight a balance between the mechanisms that maintain particular satellite domains and the forces that disperse sequence changes throughout the satellite repeats in the genome.     

rpoB gene sequencing for identification of Corynebacterium species

The genus Corynebacterium is a heterogeneous group of species comprising human and animal pathogens and environmental bacteria. It is defined on the basis of several phenotypic characters and the results of DNA-DNA relatedness and, more recently, 16S rRNA gene sequencing. However, the 16S rRNA gene is not polymorphic enough to ensure reliable phylogenetic studies and needs to be completely sequenced for accurate identification. The almost complete rpoB sequences of 56 Corynebacterium species were determined by both PCR and genome walking methods. In all cases the percent similarities between different species were lower than those observed by 16S rRNA gene sequencing, even for those species with degrees of high similarity. Several clusters supported by high bootstrap values were identified. In order to propose a method for strain identification which does not require sequencing of the complete rpoB sequence (approximately 3,500 bp), we identified an area with a high degree of polymorphism, bordered by conserved sequences that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (434 to 452 bp) allows accurate species identification and may be used in the future for routine sequence-based identification of Corynebacterium species.