Characterization of human-specific DNA sequences obtained by genome subtraction

Recent studies on molecular evolution using nucleotide sequence data to clarify phylogenetic relationships among humans and the African great apes, have revealed that humans are more closely related to chimpanzees than to gorillas. However, the genetic basis of human uniqueness remains unclear. This is because phylogenetic studies have merely evaluated the degree of similarity by calculating the accumulation of nucleotide substitutions that have occurred in neutral DNA regions commonly present in all the species examined. In contrast, the genome subtraction method recently developed by us has revealed dissimilarity even among the genomes of the most closely related species. Here we describe the characteristics of the DNA sequences obtained by genome subtraction between humans and chimpanzees.     

Phylogenetic analysis of C, M, N, and U genomes and their relationships with Triticum and other related genomes as revealed by LMW-GS genes at Glu-3 loci

Phylogenetic relationships between the C, U, N, and M genomes of Aegilops species and the genomes of common wheat and other related species were investigated by using three types of low-molecular-weight glutenin subunit (LMW-GS) genes at Glu-3 loci. A total of 20 LMW-GS genes from Aegilops and Triticum species were isolated, including 11 LMW-m type and 9 LMW-i type genes. Particularly, four LMW-m type and three LMW-i type subunits encoded by the genes on the C, N, and U genomes possessed an extra cysteine residue at conserved positions, which could provide useful information for understanding phylogenetic relationships among Aegilops and Triticum genomes. Phylogenetic trees constructed by using either LMW-i or the combination of LMW-m and LMW-s, as well as analysis of all the three types of LMW-GS genes together, demonstrated that the C and U genomes were closely related to the A genome, whereas the N and M genomes were closely related to the D genome. Our results support previous findings that the A genome was derived from Triticum uratu, the B genome was from Aegilops speltoides, and the D genome was from Aegilops tauschii. In addition, phylogenetic relationships among different genomes analysed in this study support the concept that Aegilops is not monophyletic.     

Genetic Relationships Among Five Basic Genomes St,E,A,B and D in Triticeae Revealed by Genomic Southern and in situ Hybridization

The St and E are two important basic genomes in the perennial tribe Triticeae (Poaceae). They exist in many perennialspecies and are very closely related to the A, B and D genomes of bread wheat (Triticum aestivum L.). Genomic Southernhybridization and genomic in situ hybridization (GISH) were used to analyze the genomic relationships between the twogenomes (St and E) and the three basic genomes (A, B and D) of T. aestivum. The semi-quantitative analysis of the Southernhybridization suggested that both St and E genomes are most closely related to the D genome, then the A genome, andrelatively distant to the B genome. GISH analysis using St and E genomic DNA as probes further confirmed the conclusion.St and E are the two basic genomes of Thinopyrum ponticum (StStE~eE~bE~x) and Th. intermedium (StE~eE~b), two perennialspecies successfully used in wheat improvement. Therefore, this paper provides a possible answer as to why most of thespontaneous wheat-Thinopyrum translocations and substitutions usually happen in the D genome, some in the A genomeand rarely in the B genome. This would develop further use of alien species for wheat improvement, especially thosecontaining St or E in their genome components.     

Genetic Relationships Among Five Basic Genomes St, E, A, B and D in Triticeae Revealed by Genomic Southern and in situ Hybridization

The St and E are two important basic genomes in the perennial tribe Triticeae （Poaceae）. They exist in many perennial species and are very closely related to the A, B and D genomes of bread wheat （Triticum aestivum L.）. Genomic Southern hybridization and genomic in situ hybridization （GISH） were used to analyze the genomic relationships between the two genomes （St and E） and the three basic genomes （A, B and D） of T. aestivum. The semi-quantitative analysis of the Southern hybridization suggested that both St and E genomes are most closely related to the D genome, then the A genome, and relatively distant to the B genome. GISH analysis using St and E genomic DNA as probes further confirmed the conclusion. St and E are the two basic genomes of Thinopyrum ponticum （StStE^eE^bE^x） and Th. intermedium （StE^eE^b）, two perennial species successfully used in wheat improvement. Therefore, this paper provides a possible answer as to why most of the spontaneous wheat-Thinopyrum translocations and substitutions usually happen in the D genome, some in the A genome and rarely in the B genome. This would develop further use of alien species for wheat improvement, especially those containing St or E in their genome components.     

Incorporating Genomics into the Toolkit of Nematology

The study of nematode genomes over the last three decades has relied heavily on the model organism Caenorhabditis elegans, which remains the best-assembled and annotated metazoan genome. This is now changing as a rapidly expanding number of nematodes of medical and economic importance have been sequenced in recent years. The advent of sequencing technologies to achieve the equivalent of the $1000 human genome promises that every nematode genome of interest will eventually be sequenced at a reasonable cost. As the sequencing of species spanning the nematode phylum becomes a routine part of characterizing nematodes, the comparative approach and the increasing use of ecological context will help us to further understand the evolution and functional specializations of any given species by comparing its genome to that of other closely and more distantly related nematodes. We review the current state of nematode genomics and discuss some of the highlights that these genomes have revealed and the trend and benefits of ecological genomics, emphasizing the potential for new genomes and the exciting opportunities this provides for nematological studies.     

A versatile computational pipeline for bacterial genome annotation improvement and comparative analysis, with Brucella as a use case

We present a bacterial genome computational analysis pipeline, called GenVar. The pipeline, based on the program GeneWise, is designed to analyze an annotated genome and automatically identify missed gene calls and sequence variants such as genes with disrupted reading frames (split genes) and those with insertions and deletions (indels). For a given genome to be analyzed, GenVar relies on a database containing closely related genomes (such as other species or strains) as well as a few additional reference genomes. GenVar also helps identify gene disruptions probably caused by sequencing errors. We exemplify GenVar's capabilities by presenting results from the analysis of four Brucella genomes. Brucella is an important human pathogen and zoonotic agent. The analysis revealed hundreds of missed gene calls, new split genes and indels, several of which are species specific and hence provide valuable clues to the understanding of the genome basis of Brucella pathogenicity and host specificity.     

Systematic determination of the mosaic structure of bacterial genomes: species backbone versus strain-specific loops

Background  

Public databases now contain multitude of complete bacterial genomes, including several genomes of the same species. The available data offers new opportunities to address questions about bacterial genome evolution, a task that requires reliable fine comparison data of closely related genomes. Recent analyses have shown, using pairwise whole genome alignments, that it is possible to segment bacterial genomes into a common conserved backbone and strain-specific sequences called loops.     

Plant genome evolution: lessons from comparative genomics at the DNA level

Angiosperm genomes show tremendous variability in genome size and chromosome number. Nevertheless, comparative genetic mapping has revealed genome collinearity of closely related species. Sequence-based comparisons were used to assess the conservation of gene arrangements. Numerous small rearrangements, insertions/deletions, duplications, inversions and translocations have been detected. Importantly, comparative sequence analyses have unambiguously shown micro-collinearity of distantly related plant species. Duplications and subsequent gene loss have been identified as a particular important factor in the evolution of plant genomes.     

Genome analysis of Elytrigia pycnantha and Thinopyrum junceiforme and of their putative natural hybrid using the GISH technique.

Genomic in situ hybridization (GISH), using genomic DNA probes from Thinopyrum elongatum (Host) D.R. Dewey (E genome, 2n = 14), Th. bessarabicum (Savul. & Rayss) A. Love (J genome, 2n = 14), Pseudoroegneria stipifolia (Czern. ex Nevski) Love (S genome, 2n = 14), and Agropyron cristatum (L.) Gaertner (P genome, 2n = 14), was used to characterize the genome constitution of the polyploid species Elytrigia pycnantha (2n = 6x = 42) and Thinopyrum junceiforme (2n = 4x = 28) and of one hybrid population (2n = 5x = 35). GISH results indicated that E. pycnantha contains S, E, and P genomes; the first of these was closely related to the S genome of Ps. stipifolia, the second was closely related to to the E genome of Th. elongatum, and the third was specifically related to A. cristatum. The E and P genomes included 2 and 10 chromosomes, respectively, with S genome DNA sequences in the centromeric region. GISH analysis of Th. junceiforme showed the presence of two sets of the E genome, except for fewer than 10 chromosomes for which the telomeric regions were not identified. Based on these results, the genome formula SSPsPsEsEs is proposed for E. pycnantha and that of EEEE is proposed for Th. junceiforme. The genomic constitution of the pentaploid hybrid comprised one S genome (seven chromosomes), one P genome (seven chromosomes), and three E genomes (21 chromosomes). The E and P genomes both included mosaic chromosomes (chromosomes 1 and 5, respectively) with the centromere region closely related to S-genome DNA. On the basis of these data, the genome formula SPSESEE is suggested for this hybrid and it is also suggested that the two species E. pycnantha and Th. junceiforme are the parents of the pentaploid hybrid.     

A HERV-K provirus in chimpanzees, bonobos and gorillas, but not humans

Evidence from DNA sequencing studies strongly indicated that humans and chimpanzees are more closely related to each other than either is to gorillas [1-4]. However, precise details of the nature of the evolutionary separation of the lineage leading to humans from those leading to the African great apes have remained uncertain. The unique insertion sites of endogenous retroviruses, like those of other transposable genetic elements, should be useful for resolving phylogenetic relationships among closely related species. We identified a human endogenous retrovirus K (HERV-K) provirus that is present at the orthologous position in the gorilla and chimpanzee genomes, but not in the human genome. Humans contain an intact preintegration site at this locus. These observations provide very strong evidence that, for some fraction of the genome, chimpanzees, bonobos, and gorillas are more closely related to each other than they are to humans. They also show that HERV-K replicated as a virus and reinfected the germline of the common ancestor of the four modern species during the period of time when the lineages were separating and demonstrate the utility of using HERV-K to trace human evolution.     

Synteny: recent advances and future prospects

Their small sizes have meant that the Arabidopsis and rice genomes are the best-studied of all plant genomes. Although even closely related plant species can show large variations in genome size, extensive genome colinearity has been established at the genetic level and recently also at the gene level. This allows the transfer of information and resources assembled for rice and Arabidopsis to be used in the genome analysis of many other plants.     

A Novel Construction of Genome Space with Biological Geometry

A genome space is a moduli space of genomes. In this space, each point corresponds to a genome. The natural distance between two points in the genome space reflects the biological distance between these two genomes. Currently, there is no method to represent genomes by a point in a space without losing biological information. Here, we propose a new graphical representation for DNA sequences. The breakthrough of the subject is that we can construct the moment vectors from DNA sequences using this new graphical method and prove that the correspondence between moment vectors and DNA sequences is one-to-one. Using these moment vectors, we have constructed a novel genome space as a subspace in R^N. It allows us to show that the SARS-CoV is most closely related to a coronavirus from the palm civet not from a bird as initially suspected, and the newly discovered human coronavirus HCoV-HKU1 is more closely related to SARS than to any other known member of group 2 coronavirus. Furthermore, we reconstructed the phylogenetic tree for 34 lentiviruses (including human immunodeficiency virus) based on their whole genome sequences. Our genome space will provide a new powerful tool for analyzing the classification of genomes and their phylogenetic relationships.     

Heteroplasmy in the Mitochondrial Genomes of Human Lice and Ticks Revealed by High Throughput Sequencing

The typical mitochondrial (mt) genomes of bilateral animals consist of 37 genes on a single circular chromosome. The mt genomes of the human body louse, Pediculus humanus, and the human head louse, Pediculus capitis, however, are extensively fragmented and contain 20 minichromosomes, with one to three genes on each minichromosome. Heteroplasmy, i.e. nucleotide polymorphisms in the mt genome within individuals, has been shown to be significantly higher in the mt cox1 gene of human lice than in humans and other animals that have the typical mt genomes. To understand whether the extent of heteroplasmy in human lice is associated with mt genome fragmentation, we sequenced the entire coding regions of all of the mt minichromosomes of six human body lice and six human head lice from Ethiopia, China and France with an Illumina HiSeq platform. For comparison, we also sequenced the entire coding regions of the mt genomes of seven species of ticks, which have the typical mitochondrial genome organization of bilateral animals. We found that the level of heteroplasmy varies significantly both among the human lice and among the ticks. The human lice from Ethiopia have significantly higher level of heteroplasmy than those from China and France (P_t<0.05). The tick, Amblyomma cajennense, has significantly higher level of heteroplasmy than other ticks (P_t<0.05). Our results indicate that heteroplasmy level can be substantially variable within a species and among closely related species, and does not appear to be determined by single factors such as genome fragmentation.     

The origins of the genomes of Triticum biunciale,t. ovatum,t. neglectum,t. columnare,and t. rectum (poaceae) based on variation in repeated nucleotide sequences

The origins of the genomes of allotetraploid species Triticum biunciale, T. ovatum, T. neglectum, and T. columnare, and allohexaploid T. rectum were investigated by examining the presence of specific restriction fragments of repeated nucleotide sequences in DNAs of the polyploid species. The restriction fragments were detectable either in a single diploid Triticum species (unique characters) or a group of diploid species (unique shared characters). The analysis showed that Triticum biunciale and T. ovatum are closely related. In both species, one pair of genomes is closely related to the genome of T. umbellulatum and the other is a modified genome of T. comosum. The same genome formula, UUM°M°, is proposed for T. biunciale and T. ovatum. Potential reasons for the modification of the M° genome are discussed. Triticum neglectum and T. columnare are also closely related to each other and have the same genomes. They share the U genome with T. biunciale and T. ovatum, but their second pair of genomes is unrelated to the M° genome. No relationship was found of this genome to a genome of any extant diploid species of Triticum or any phylogenetic lineage leading to the extant diploid species. This unknown genome is designated X'.∗∗∗ The proposed genome formula for T. neglectum and T. columnare is UUX'X'∗∗∗. Hexaploid T. rectum originated from hybridization of one of the tetraploid species with the formula UUX'X', likely T. neglectum, with T. uniaristatum (genome N), and its genome formula is UUX'X'NN.     

Comparative genomics of closely related salmonellae

As the number of completed genome sequences increases, there is increasing emphasis on comparative genomic analysis of closely related organisms. Comparison of the similarities and differences between the five publicly available Salmonella genome sequences reveals extensive sequence conservation among the Salmonella serovars. However, horizontal gene transfer has provided each genome with between 10% and 12% of unique DNA. Genome comparisons of the closely related salmonellae emphasize the insights that can be gleaned from sequencing genomes of a single species.     

A novel repetitive DNA sequence in the genus Oryza

Summary Repetitive DNA sequences in the genus Oryza (rice) represent a large fraction of the nuclear DNA. The isolation and characterization of major repetitive DNA sequences will lead to a better understanding of rice genome organization and evolution. Here we report the characterization of a novel repetitive sequence, CC-1, from the CC genome. This repetitive sequence is present as long tandem arrays with a repeat unit 194 bp in length in the CC-diploid genome but 172 bp in length in the BBCC and CCDD tetraploid genomes. This repetitive sequence is also present, though at lower copy numbers, in the AA and BB genomes, but is absent in the EE and FF genomes. Hybridization experiments revealed considerable differences both in copy numbers and in restriction fragment patterns of CC-1 both between and within rice species. The results support the hypothesis that the CC genome is more closely related to the AA genome than to the BB genome, and most distantly related to the EE and FF genomes.     

The mosaic of ancestral karyotype blocks in the Sinapis alba L. genome

The organisation of the Sinapis alba genome, comprising 12 linkage groups (n = 12), was compared with the Brassicaceae ancestral karyotype (AK) genomic blocks previously described in other crucifer species. Most of the S. alba genome falls into conserved triplicated genomic blocks that closely match the AK-defined genomic blocks found in other crucifer species including the A, B, and C genomes of closely related Brassica species. In one instance, an S. alba linkage group (S05) was completely collinear with one AK chromosome (AK1), the first time this has been observed in a member of the Brassiceae tribe. However, as observed for other members of the Brassiceae tribe, ancestral genomic blocks were fragmented in the S. alba genome, supporting previously reported comparative chromosome painting describing rearrangements of the AK karyotype prior to the divergence of the Brassiceae from other crucifers. The presented data also refute previous phylogenetic reports that suggest S. alba was more closely related to Brassica nigra (B genome) than to B. rapa (A genome) and B. oleracea (C genome). A comparison of the S. alba and Arabidopsis thaliana genomes revealed many regions of conserved gene order, which will facilitate access to the rich genomic resources available in the model species A. thaliana for genetic research in the less well-resourced crop species S. alba.     

Genome-wide targeted search for human specific and polymorphic L1 integrations

Retroelements (REs) occupy up to 40% of the human genome. Newly integrated REs can change the pattern of expression of pre-existing host genes and therefore might play a significant role in evolution. In particular, human- and primate-specific REs could affect the divergence of the Hominoidea superfamily. A comparative genome-wide analysis of RE sites of integration, neighboring genes, and their regulatory interplay in human and ape genomes would be of help in understanding the impact of REs on evolution and genome regulation. We have developed a technique for the genome-wide comparison of the integrations of transposable elements in genomic DNAs of closely related species. The technique called targeted genome differences analysis (TGDA) is also useful for the detection of deletion/insertion polymorphisms of REs. The technique is based on an enhanced version of subtractive hybridization and does not require preliminary knowledge of the genome sequences under comparison. In this report, we describe its application to the detection and analysis of human specific L1 integrations and their polymorphisms. We obtained a library highly enriched in human-specific L1 insertions and identified 24 such new insertions. Many of these insertions are polymorphic in human populations. The total number of human-specific L1 inserts was estimated to be approximately 4000. The results suggest that TGDA is a universal method that can be successfully used for the detection of evolutionary and polymorphic markers in any closely related genomes.     

Clustering of main orthologs for multiple genomes

The identification of orthologous genes shared by multiple genomes is critical for both functional and evolutionary studies in comparative genomics. While it is usually done by sequence similarity search and reconciled tree construction in practice, recently a new combinatorial approach and high-throughput system MSOAR for ortholog identification between closely related genomes based on genome rearrangement and gene duplication has been proposed in Fu et al. MSOAR assumes that orthologous genes correspond to each other in the most parsimonious evolutionary scenario, minimizing the number of genome rearrangement and (postspeciation) gene duplication events. However, the parsimony approach used by MSOAR limits it to pairwise genome comparisons. In this paper, we extend MSOAR to multiple (closely related) genomes and propose an ortholog clustering method, called MultiMSOAR, to infer main orthologs in multiple genomes. As a preliminary experiment, we apply MultiMSOAR to rat, mouse, and human genomes, and validate our results using gene annotations and gene function classifications in the public databases. We further compare our results to the ortholog clusters predicted by MultiParanoid, which is an extension of the well-known program InParanoid for pairwise genome comparisons. The comparison reveals that MultiMSOAR gives more detailed and accurate orthology information, since it can effectively distinguish main orthologs from inparalogs.