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The origin of “orphan” genes, species-specific sequences that lack detectable homologues, has remained mysterious since the dawn of the genomic era. There are two dominant explanations for orphan genes: complete sequence divergence from ancestral genes, such that homologues are not readily detectable; and de novo emergence from ancestral non-genic sequences, such that homologues genuinely do not exist. The relative contribution of the two processes remains unknown. Here, we harness the special circumstance of conserved synteny to estimate the contribution of complete divergence to the pool of orphan genes. By separately comparing yeast, fly and human genes to related taxa using conservative criteria, we find that complete divergence accounts, on average, for at most a third of eukaryotic orphan and taxonomically restricted genes. We observe that complete divergence occurs at a stable rate within a phylum but at different rates between phyla, and is frequently associated with gene shortening akin to pseudogenization. Two cancer-related human genes, DEC1 and DIRC1, have likely originated via this route in a primate ancestor.

Circadian rhythms are biological rhythms with a period of approximately 24 h. While canonical circadian clock genes and their regulatory mechanisms appear highly conserved, the evolution of clock gene families is still unclear due to several rounds of whole genome duplication in vertebrates. The spotted gar (Lepisosteus oculatus), as a non-teleost ray-finned fish, represents a fish lineage that diverged before the teleost genome duplication (TGD), providing an outgroup for exploring the evolutionary mechanisms of circadian clocks after whole-genome duplication. In this study, we interrogated the spotted gar draft genome sequences and found that spotted gar contains 26 circadian clock genes from 11 families. Phylogenetic analysis showed that 9 of these 11 spotted gar circadian clock gene families have the same number of genes as humans, while the members of the nfil3 and cry families are different between spotted gar and humans. Using phylogenetic and syntenic analyses, we found that nfil3-1 is conserved in vertebrates, while nfil3-2 and nfil3-3 are maintained in spotted gar, teleost fish, amphibians, and reptiles, but not in mammals. Following the two-round vertebrate genome duplication (VGD), spotted gar retained cry1a, cry1b, and cry2, and cry3 is retained in spotted gar, teleost fish, turtles, and birds, but not in mammals. We hypothesize that duplication of core clock genes, such as (nfil3 and cry), likely facilitated diversification of circadian regulatory mechanisms in teleost fish. We also found that the transcription factor binding element (Ahr::Arnt) is retained only in one of the per1 or per2 duplicated paralogs derived from the TGD in the teleost fish, implicating possible subfuctionalization cases. Together, these findings help decipher the repertoires of the spotted gar&rsquo, s circadian system and shed light on how the vertebrate circadian clock systems have evolved.

Circadian rhythms are biological rhythms with a period of approximately 24 h. While canonical circadian clock genes and their regulatory mechanisms appear highly conserved, the evolution of clock gene families is still unclear due to several rounds of whole genome duplication in vertebrates. The spotted gar (Lepisosteus oculatus), as a non-teleost ray-finned fish, represents a fish lineage that diverged before the teleost genome duplication (TGD), providing an outgroup for exploring the evolutionary mechanisms of circadian clocks after whole-genome duplication. In this study, we interrogated the spotted gar draft genome sequences and found that spotted gar contains 26 circadian clock genes from 11 families. Phylogenetic analysis showed that 9 of these 11 spotted gar circadian clock gene families have the same number of genes as humans, while the members of the nfil3 and cry families are different between spotted gar and humans. Using phylogenetic and syntenic analyses, we found that nfil3-1 is conserved in vertebrates, while nfil3-2 and nfil3-3 are maintained in spotted gar, teleost fish, amphibians, and reptiles, but not in mammals. Following the two-round vertebrate genome duplication (VGD), spotted gar retained cry1a, cry1b, and cry2, and cry3 is retained in spotted gar, teleost fish, turtles, and birds, but not in mammals. We hypothesize that duplication of core clock genes, such as (nfil3 and cry), likely facilitated diversification of circadian regulatory mechanisms in teleost fish. We also found that the transcription factor binding element (Ahr::Arnt) is retained only in one of the per1 or per2 duplicated paralogs derived from the TGD in the teleost fish, implicating possible subfuctionalization cases. Together, these findings help decipher the repertoires of the spotted gar’s circadian system and shed light on how the vertebrate circadian clock systems have evolved.

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One difficulty when identifying alternative splicing (AS) events in plants is distinguishing functional AS from splicing noise. One way to add confidence to the validity of a splice isoform is to observe that it is conserved across evolutionarily related species. We use a high throughput method to identify junction-based conserved AS events from RNA-Seq data across nine plant species, including five grass monocots (maize, sorghum, rice, Brachpodium, and foxtail millet), plus two nongrass monocots (banana and African oil palm), the eudicot Arabidopsis, and the basal angiosperm Amborella. In total, 9804 AS events were found to be conserved between two or more species studied. In grasses containing large regions of conserved synteny, the frequency of conserved AS events is twice that observed for genes outside of conserved synteny blocks. In plant-specific RS and RS2Z subfamilies of the serine/arginine (SR) splice-factor proteins, we observe both conservation and divergence of AS events after the whole genome duplication in maize. In addition, plant-specific RS and RS2Z splice-factor subfamilies are highly connected with R2R3-MYB in STRING functional protein association networks built using genes exhibiting conserved AS. Furthermore, we discovered that functional protein association networks constructed around genes harboring conserved AS events are enriched for phosphatases, kinases, and ubiquitylation genes, which suggests that AS may participate in regulating signaling pathways. These data lay the foundation for identifying and studying conserved AS events in the monocots, particularly across grass species, and this conserved AS resource identifies an additional layer between genotype to phenotype that may impact future crop improvement efforts.

Zoo-FISH (fluorescence in situ hybridization) (Scherthan et al. 1994), also known as cross-species chromosome painting or comparative chromosome painting, essentially describes the use of whole chromosome- or chromosomal arm- or region-specific painting probes to delimit homologous segments (chromosome or chromosomal segments with evolutionarily conserved synteny) in other species by means of FISH. Like most conventional cytogenetic methodologies, the technique of chromosome painting was first introduced to human cytogenetics in 1988 (Pinkel et al. 1988). Wienberg and colleagues (1990) were among the pioneers who intro-duced chromosomal painting to the field of comparative cytogenetics of primates. They established the first genome-wide chromosome maps between human and a Japanese macaque. In the beginning, painting probes were derived from libraries of human chromosome-specific DNA clones. Limited by the availability of painting probes and technical difficulties in comparing distantly related species, early cross-species chromosome painting experiments primarily used apes and Old World monkeys (Jauch et al. 1992). Scherthan and colleagues (1994) were among the first to demonstrate the feasibility of comparing species as distantly related as those from different orders. Meanwhile, the invention of degenerate oligonucleotide-primed PCR (DOP-PCR, Telenius et al. 1992a), coupled with chromosomal sorting by flow cytometry, made it possible to generate painting probes for any given vertebrate species (Telenius et al 1992b; Rabbitts et al. 1995; Yang et al. 1995) and to carry out multidirectional cross-species chromosome painting. The whole set of human chromosome-specific painting probes derived from DOP-PCR were made commercially available around 1992, and the whole set of chromosome painting probes for mouse became available in late 1995. Human chromosome-specific

The genes involved in host defences are known to undergo rapid evolution. Therefore, it is often difficult to assign orthologs in multigene families among various vertebrate species. Chemokines are a large family of small cytokines that orchestrate cell migration in health and disease. Herein, we have surveyed the genomes of 18 representative vertebrate species for chemokine genes and identified a total of 553 genes. We have determined their orthologous relationships and classified them in accordance with the current systematic chemokine nomenclature system. Our study reveals an interesting evolutionary history that gave origin and diversification to the vertebrate chemokine superfamily.

The microbial genome database for comparative analysis (MBGD, available at http://mbgd.genome.ad.jp/) is a platform for microbial genome comparison based on orthology analysis. As its unique feature, MBGD allows users to conduct orthology analysis among any specified set of organisms; this flexibility allows MBGD to adapt to a variety of microbial genomic study. Reflecting the huge diversity of microbial world, the number of microbial genome projects now becomes several thousands. To efficiently explore the diversity of the entire microbial genomic data, MBGD now provides summary pages for pre-calculated ortholog tables among various taxonomic groups. For some closely related taxa, MBGD also provides the conserved synteny information (core genome alignment) pre-calculated using the CoreAligner program. In addition, efficient incremental updating procedure can create extended ortholog table by adding additional genomes to the default ortholog table generated from the representative set of genomes. Combining with the functionalities of the dynamic orthology calculation of any specified set of organisms, MBGD is an efficient and flexible tool for exploring the microbial genome diversity.

Chantier qualité GA; All salmonid species investigated to date have been characterized with a male heterogametic sex-determination system. However, as these species do not share any Y-chromosome conserved synteny, there remains a debate on whether they share a common master sex-determining gene. In this study, we investigated the extent of conservation and evolution of the rainbow trout (Oncorhynchus mykiss) master sex-determining gene, sdY (sexually dimorphic on the Y-chromosome), in 15 different species of salmonids. We found that the sdY sequence is highly conserved in all salmonids and that sdY is a male-specific Y-chromosome gene in the majority of these species. These findings demonstrate that most salmonids share a conserved sex-determining locus and also strongly suggest that sdY may be this conserved master sex-determining gene. However, in two whitefish species (subfamily Coregoninae), sdY was found both in males and females, suggesting that alternative sex-determination systems may have also evolved in this family. Based on the wide conservation of sdY as a male-specific Y-chromosome gene, efficient and easy molecular sexing techniques can now be developed that will be of great interest for studying these economically and environmentally important species.

Chantier qualité GA; DNA fingerprints and end sequences from bacterial artificial chromosomes (BACs) from two new libraries were generated to improve the first generation integrated physical and genetic map of the rainbow trout (Oncorhynchus mykiss) genome. The current version of the physical map is composed of 167,989 clones of which 158,670 are assembled into contigs and 9,319 are singletons. The number of contigs was reduced from 4,173 to 3,220. End sequencing of clones from the new libraries generated a total of 11,958 high quality sequence reads. The end sequences were used to develop 238 new microsatellites of which 42 were added to the genetic map. Conserved synteny between the rainbow trout genome and model fish genomes was analyzed using 188,443 BAC end sequence (BES) reads. The fractions of BES reads with significant BLASTN hits against the zebrafish, medaka, and stickleback genomes were 8.8%, 9.7%, and 10.5%, respectively, while the fractions of significant BLASTX hits against the zebrafish, medaka, and stickleback protein databases were 6.2%, 5.8%, and 5.5%, respectively. The overall number of unique regions of conserved synteny identified through grouping of the rainbow trout BES into fingerprinting contigs was 2,259, 2,229, and 2,203 for stickleback, medaka, and zebrafish, respectively. These numbers are approximately three to five times greater than those we have previously identified using BAC paired ends. Clustering of the conserved synteny analysis results by linkage groups as derived from the integrated physical and genetic map revealed that despite the low sequence homology, large blocks of macrosynteny are conserved between chromosome arms of rainbow trout and the model fish species.

Autosomal recessive nonsyndromic hearing impairment (ARNSHI) segregating in three unrelated, large consanguineous Pakistani families (PKDF528, PKDF859 and PKDF326) is linked to markers on chromosome 12q14.2-q15. This novel locus is designated DFNB74. Maximum two-point limit of detection (LOD) scores of 5.6, 5.7 and 2.6 were estimated for markers D12S313,D12S83 and D12S75 at theta = 0 for recessive deafness segregating in these three families. Haplotype analyses identified a critical linkage interval of 5.35 cM (5.36 Mb) defined by D12S329 at 74.58 cM and D12S313 at 79.93 cM. DFNB74 is the second ARNSHI locus mapped to chromosome 12, but the physical intervals do not overlap with one another. A locus contributing to the early onset, rapidly progressing hearing loss of A/J mice (ahl4, age-related hearing loss 4) was reported to map to chromosome 10 in a region of conserved synteny to DFNB74, suggesting that ahl4 and DFNB74 may be due to mutations of the same gene in these two species.

A large number of significant QTL for economically important traits including average daily gain have been located on SSC1q, which, as shown by chromosome painting, corresponds to four human chromosomes (HSA9, 14, 15 and 18). To provide a comprehensive comparative map for efficient selection of candidate genes, 81 and 34 genes localized on HSA9 and HSA14 respectively were mapped to SSC1q using a porcine 7000-rad radiation hybrid panel (IMpRH). This study, together with the cytogenetic map (http://www2.toulouse.inra.fr/lgc/pig/cyto/genmar/htm/1GM.HTM), demonstrates that SSC1q2.1-q2.13 corresponds to the region ranging from 44.6 to 63.2 Mb on HSA14q21.1-q23.1, the region from 86.5 to 86.8 Mb on HSA15q24-q25, the region from 0.9 to 27.2 Mb on HSA9p24.3-p21, the region from 35.1 to 38.0 Mb on HSA9p13, the region from 70.3 to 79.3 Mb on HSA9q13-q21 and the region from 96.4 to 140.0 Mb on HSA9q22.3-q34. The conserved synteny between HSA9 and SSC1q is interrupted by at least six sites, and the synteny between HSA14 and SSC1q is interrupted by at least one site.

The successful assignment of the existing genetic linkage groups (LGs) to individual chromosomes and the second-generation linkage map obtained by mapping a large number of bacterial artificial chromosome (BAC) contigs in the silkworm, Bombyx mori , together with public nucleotide sequence databases, offer a powerful tool for the study of synteny between karyotypes of B. mori and other lepidopteran species. Conserved synteny of genes between particular chromosomes can be identified by comparatively mapping orthologous genes of the corresponding linkage groups with the help of BAC-FISH (fluorescent in situ hybridization). This technique was established in B. mori for 2 differently labeled BAC probes simultaneously hybridized to pachytene bivalents. To achieve higher-throughput comparative mapping using BAC-FISH in Lepidoptera, we developed a protocol for five-color BAC-FISH, which allowed us to map simultaneously 6 different BAC probes to chromosome 15 in B. mori. We identified orthologs of 6 B. mori LG15 genes (RpP0, RpS8, eIF3, RpL7A, RpS23, and Hsc70) for the tobacco hornworm, Manduca sexta , and selected the ortholog-containing BAC clones from an M. sexta BAC library. All 6 M. sexta BAC clones hybridized to a single M. sexta bivalent in pachytene spermatocytes. Thus, we have confirmed the conserved synteny between the B. mori chromosome 15 and the corresponding M. sexta chromosome (hence provisionally termed chromosome 15).

Conservation of synteny of mammalian imprinted genes between chicken and human suggested that highly conserved gene clusters were selected long before these genes were recruited for genomic imprinting in mammals. Here we have applied in silico mapping of orthologous genes in pipid frog, zebrafish, spotted green and Japanese pufferfish to show considerable conservation of synteny in lower vertebrates. More than 400 million years ago in a common ancestor of teleost fish and tetrapods, ‘preimprinted’ chromosome regions homologous to human 6q25, 7q21, 7q32, 11p15, and 15q11→q12 already contained most present-day mammalian imprinted genes. Interestingly, some imprinted gene orthologues which are isolated from imprinted clusters in mouse and human could be linked to preimprinted regions in lower vertebrates, indicating that separation occurred during mammalian evolution. On the contrary, newly arisen genes by segmental duplication in the mammalian lineage, i.e. SNRPN and FRAT3, were transposed or translocated to imprinted clusters and recruited for parent-specific activity. By analysis of currently available sequences of non-mammalian vertebrates, the imprinted gene clusters homologous to human chromosomes 14q32 and 19q12 are only poorly conserved in chicken, frog, and fish and, therefore, may not have evolved from ancestral preimprinted gene arrays. Evidently, evolution of imprinted gene clusters is an ongoing and dynamic process in mammals. In general, imprinted gene orthologues do not show a higher degree of synteny conservation in vertebrates than non-imprinted genes interspersed with or adjacent to an imprinted cluster.

Summary Twenty-two and eight significant quantitative trait loci for economically important traits have been located on porcine chromosomes (SSC) 2q and SSC16 respectively, both of which have been shown to correspond to human chromosome 5 (HSA5) by chromosome painting. To provide a comprehensive comparative map for efficient selection of candidate genes, we assigned 117 genes from HSA5 using a porcine radiation hybrid (IMpRH) panel. Sixty-six genes were assigned to SSC2 and 48 to SSC16. One gene was suggested to link to SSC2 markers and another to SSC6. One gene did not link to any gene, expressed sequence tag or marker in the map, including those in the present investigation. This study demonstrated the following: (1) SSC2q21–q28 corresponds to the region ranging from 74.0 to 148.2 Mb on HSA5q13–q32 and the region from 176.0 to 179.3 Mb on HSA5q35; (2) SSC16 corresponds to the region from 1.4 to 68.7 Mb on HSA5p–q13 and to the region from 150.4 to 169.1 Mb on HSA5q32–q35 and (3) the conserved synteny between HSA5 and SSC2q21–q28 is interrupted by at least two sites and the synteny between HSA5 and SSC16 is also interrupted by at least two sites.

A 5-Mb YAC contig, partly supplemented with BAC contigs, was created from the distal Mhc class I region on mouse Chr 17. The gene order of Znf173-Tctex5-Mog-D17Tu42-D17Leh 89 is conserved between mouse and human but not the physical distance, supporting the independent expansion of Mhc class I genes in the so-called accordion model of Mhc evolution. The distal H2-M region includes the breakpoint of conserved synteny between mouse and human as well as the In(17)4 t-inversion. The H2-M region is rich in L1 repeats, implying that the insertion of L1 repeats may be associated with the evolutionary flexibility to break a chromosome.

Summary We present herein a bovine chromosome 24 (BTA24) radiation hybrid (RH) map using40 markers scored on a panel of 90 RHs. Of these markers, 29 loci were ordered with oddsof at least 1000:1 in a framework map. An average retention frequency of 17.4% wasobserved, with relatively higher frequencies near the centromere. The length of the com-prehensive map was 640 centiray 5000 (cR 5000 ) with an average marker interval ofapproximately 17.3 cR 5000 . The observed locus order is generally consistent with currentlypublished bovine linkage and physical maps. Nineteen markers were either Type I loci orclosely associated with expressed sequences and thus could be used to compare the BTA24RH map with human mapping information. All genes located on BTA24 were located onhuman chromosome 18, and previously reported regions of conserved synteny wereextended. The comparative data revealed the presence of at least six conserved regionsbetween these chromosomes.Keywords bovine chromosome 24, radiation hybrid map.

Enhancers can regulate the transcription of genes over long genomic distances. This is thought to lead to selection against genomic rearrangements within such regions that may disrupt this functional linkage. Here we test this concept experimentally using the human X chromosome. We describe a scoring method to identify evolutionary maintenance of linkage between conserved noncoding elements and neighbouring genes. Chromatin marks associated with enhancer function are strongly correlated with this linkage score. We test >1,000 putative enhancers by transgenesis assays in zebrafish to ascertain the identity of the target gene. The majority of active enhancers drive a transgenic expression in a pattern consistent with the known expression of a linked gene. These results show that evolutionary maintenance of linkage is a reliable predictor of an enhancer's function, and provide new information to discover the genetic basis of diseases caused by the mis-regulation of gene expression., Enhancers regulate the transcription of genes over long genomic distances. Here, the authors show that enhancer function is correlated with maintenance of linkage between non-coding elements and neighbouring genes in the human X chromosome and that enhancers in zebrafish drive expression in a pattern consistent with the expression of a linked gene.

PHOSPHO1 is a recently identified phosphatase expressed at high levels in the chicken growth plate and which may be involved in generating inorganic phosphate for skeletal matrix mineralization. Using a degenerate RT-PCR approach a fragment of human PHOSPHO1 was cloned. This enabled the identification of the human orthologue on HSA17q21, and the mouse orthologue on a region of MMU11 that exhibits conservation of synteny with HSA17q21. Chicken PHOSPHO1 was mapped by SSCP analysis to position 44 cM on GGA27, adjacent to the HOXB@ (44 cM) and COL1A1 (36 cM) loci. Comparison of genes on GGA27 with their orthologues on the preliminary draft of the human genome identifies regions of conserved synteny equivalent to 25 Mb on HSA17q21.2-23.3 and approximately 20 Mb on GGA27 in which the gene order appears to be conserved. Mapping of the PHOSPHO1 genes to regions of HSA17q21.3, MMU11 and GGA27 that exhibit conservation of synteny provides strong evidence that they are orthologous.

In 1997, neuronal ceroid lipofuscinosis (NCL) was identified for the first time in Merino sheep in Australia. A homozygosity mapping approach localized the disease gene in Merino sheep to the same region on chromosome 7 in which NCL was recently mapped in South Hampshire sheep. This region shows conserved synteny with the region on human chromosome 15 in which the human late infantile NCL variant CLN6 was mapped. NCL in Merino and South Hampshire sheep are therefore potential animal models for the human late infantile variant CLN6.

SummaryThe evolutionary origins of the microsporidia, a group of intracellular eukaryotic pathogens, have been unclear. Genome analysis of a sex locus and other gene clusters has now revealed conserved synteny with zygomycete fungi, indicating that microsporidia are true fungi descended from a zygomycete ancestor.

The annotation of known genes on linkage maps provides an informative framework for synteny mapping. In comparative gene mapping, conserved synteny is broadly defined as groups of two or more linked markers that are also linked in two or more species. Although many anonymous markers have been placed on the chicken genome map, locating known genes will augment the number of conserved syntenic groups and consolidate linkage groups. In this report, 21 additional genes have been assigned to linkage groups or chromosomes; five syntenic groups were identified. Ultimately, conserved syntenic groups may help to pinpoint important quantitative trait loci.

This project had as its goals the understanding of the ecophysiology of the genus Shewanella using various genomics approaches. As opposed to other programs involving Shewanella, this one branched out into the various areas in which Shewanella cells are active, and included both basic and applied studies. All of the work was, to some extent, related to the ability of the bacteria to accomplish electron exchange between the cell and solid state electron acceptors and/or electron donors, a process we call Extracellular Electron Transport, or EET. The major accomplishments related to several different areas: Basic Science Studies: 1. Genetics and genomics of nitrate reduction, resulting in elucidation of atypical nitrate reduction systems in Shewanella oneidensis (MR-1)[2]. 2. Influence of bacterial strain and growth conditions on iron reduction, showing that rates of reduction, extents of reduction, and the formation of secondary minerals were different for different strains of Shewanella [3,4,9]. 3. Comparative genomics as a tool for comparing metabolic capacities of different Shewanella strains, and for predicting growth and metabolism [6,10,15]. In these studies, collaboration with ORNL, PNNL, and 4. Basic studies of electron transport in strain MR-1, both to poised electrodes, and via conductive nanowires [12,13]. This included the firstmore » accurate measurements of electrical energy generation by a single cell during electrode growth [12], and the demonstration of electrical conductivity along the length of bacterial nanowires [13]. 5. Impact of surface charge and electron flow on cell movement, cell attachment, cell growth, and biofilm formation [7.18]. The demonstration that interaction with solid state electron acceptors resulted in increased motility [7] led to the description of a phenomenon called electrokinesis. The importance of this for biofilm formation and for electron flow was hypothesized by Nealson & Finkel [18], and is now under study in several laboratories. Applications: 1. Corrosion: Electron flow is often part of the corrosive process, and several studies were done in concert with this proposal with regard to the ability of EET-capable bacteria to enhance, inhibit, or detect corrosion. These included using EET-capable bacteria to detect corrosion in its earliest stages [5], to use corrosion-causing bacteria for the study of the microbe/mineral interface during corrosion [1], and to study the groups of microbes involved with corrosion of natural systems [19]. 2. Bioenergy and microbial fuel cells: The production of electricity by Shewanella was shown early in this program (several years ago) to be dependent on the genes for extracellular electron transport (EET), and applied work involved the testing of various strains and conditions for the optimization of current production by the shewanellae [11,14,16]. 3. Identification of shewanellae strains: Based on similarities seen in genomic comparisons, a rapid method was employed for distinguishing between shewanellae strains [17]. Interactions with other laboratories: This grant was an extension of a grant involving the so-called ?Shewanella Federation?, and as such, a number of our publications were joint with other members of this group. The groups included: 1. Pacific Northwest Laboratories ? 2. Oak Ridge National Labs 3. Michigan State University 4. University of Oklahoma 5. Naval Research Laboratory, Washington DC 6. Burnham Medical Research Institute, San Diego 7. J. Craig Venter Institute, San Diego Education: Graduate Students: Michael Waters, Ph.D. ? at NIST, Washington D.C. Lewis Hsu, Ph.D. ? at NRL, San Diego Howard Harris, Ph.D. ? Postdoc at University, France Everett Salas, Ph.D. ? Scientist at Chevron McLean, Jeffrey, Ph.D. ? Scientist at J. Craig Venter Institute McCrow, John, Ph.D. ? Scientist at J. Craig Venter Institute Postdocs: Mohamed El-Naggar ? Professor of Physics, USC Jinjun Kan ? Senior Researcher at Undergraduatges: During this year, we had three undergraduate researchers in the lab, each working on an aspect of this work, and each involved with an undergraduate honors research project. Publications produced as a result of this funding. 1. Waters, M.S., M.Y. El-Naggar, L. Hsu, C.A. Sturm, A. Luttge, F.E. Udwadia, D.G. Cvitkovitch, S.D. Goodman, and K.H. Nealson. 2009. Simultaneous interferometric measurement of corrosive or demineralizing bacteria and their mineral interfaces. Appl. Environ. Microbiol. 75:1445-1449. 2. Gao, H., Z.K. Yang, S. Barua, S. B. Reed, M.F. Romine, K.H. Nealson, J.K. Fredrickson, J.M. Tiedje, and J. Zhou. 2009. Reduction of nitrate in Shewanella oneidensis depends on atypical NAP and NRF systems with NapB as a preferred electron transport protein from CymA to NapA. The ISME J. 3:966-976. 3. Salas, E.C., W.M. Berelson, D.E. Hammond, A.R. Kampf and K. H. Nealson. 2009. The impact of bacterial strain on the products of dissimilatory iron reduction. Geochim. Cosmochim. Acta. (in press: doi: 10.1016/j.gca.2009.10.039) 4. Salas, E.C., W.M. Berelson, D.E. Hammond, A.R. Kampf, and K. H. Nealson. 2009. The influence of carbon source on the products of dissimilatory iron reduction. Geomicrobiol. J. 26:451-462 [7,18]. 5. Waters, M.S., E.C. Salas, S.D. Goodman, F.E. Udwadia, and K.H. Nealson. 2009. Early detection of oxidized surfaces using Shewanella oneidensis MR-1 as a tool. Biofouling. 25:163-172. 6. Konstantinidis, K., et al. 2009. Comparative systems biology across an evolutionary gradient within the Shewanella genus. Proc. Nat. Acad. Sci. USA 106: 15909-15914. 7. Harris, H.W., M.Y. El-Naggar, O. Bretschger, M.J. Ward, M. F. Romine, A.Y. Obraztsova, and K.H. Nealson. 2010. Electrokinesis is a microbial behavior that requires extracellular electron transport. Proc. Nat. Acad. Sci. 107:326-331 8. Nealson, K.H. 2010. Sediment reactions defy dogma. Nature 463:1033-1034. 9. Salas, E.C., W.M. Berelson, D.E. Hammond, A.R. Kampf, and K.H. Nealson. 2010. The impact of bacterial strain on the products of dissimilatory iron reduction. Geochim. Cosmochim. Acta. 74:574-583. 10. Karpinets, T.V., A.Y Obraztsova, Y. Wang, D.D. Schmoyer, G.H. Kora, B.H. Park, M.H. Serres, M.F. Ropmine, M.L. Land, T.B. Kothe, J.K. Fredrickson, K.H. Nealson, and E.C. Uberbacher 2010. Conserved synteny at the protein family level reveals genes underlying Shewanella species? cold tolerance and predicts their novel phenotypes. Funct. Integr. Genomics 10: 97 ? 110. (DOI 10.1007/s10143-009-0142-y) 11. Bretschger, O., A.C.M. Cheung, F. Mansfeld, and K.H. Nealson. 2010. Comparative microbial fuel cell evaluations of Shewanella spp. Electroanalysis 22: 883-894. 12. McLean, J.S., G. Wanger, Y.A. Gorby, M. Wainstein, J. McQuaid, Shun?ichi Ishii, O. Bretschger, H. Beyanal, K.H. Nealson. 2010. Quantification of electron transfer rates to a solid phase electron acceptor through the stages of biofilm formation from single cells to multicellular communities. Env. Sci. Technol. 44:2721-2717. 13. El-Naggar, M., G. Wanger, K.M. Leung, T.D. Yuzvinsky, G. Southam, J. Yang, W.M. Lau, K.H. Nealson, and Y.A. Gorby. 2010. Electrical Transport Along Bacterial Nanowires from Shewanella oneidensis MR-1 Proc. Nat. Acad. Sci. USA 107:18127-18131. 14. Biffinger, J.C., L.A. Fitzgerald, R. Ray, B.J. Little, S.E. Lizewski, E.R. Petersen, B.R. Ringeisen, W.C. Sanders, P.E. Sheehan, J.J. Pietron, J.W. Baldwin, L.J. Nadeau, G.R. Johnson, M. Ribbens, S.E. Finkel, K.H. Nealson. 2010. The utility of Shewanella japonica for microbial fuel cells. Bioresource Technol. 102:290-297. 15. Rodionov, D. , C. Yang, X. Li, I. Rodionova, Y. Wang, A.Y. Obraztsova, O. P. Zagnitko, R. Overbeek, M. F. Romine, S. Reed, J.K. Fredrickson, K.H. Nealson, A.L. Osterman. 2010. Genomic encyclopedia of sugar utilization pathways in the Shewanella genus. BMC Genomics 2010, 11:494 16. Kan, J., L. Hsu, A.C.M. Cheung, M. Pirbazari, and K.H. Nealson. 2011. Current production by bacterial communities in microbial fuel cells enriched from wastewater sludge with different electron donors. Env. Sci. Technol. 45: 1139-1146. 17. Kan, J. B. Flood, J.P. McCrow, J.S. Kim, L. Tan, and K.H. Nealson. 2011. A rapid fingerprinting approach to distinguish between closely related strains of Shewanella. J. Microbiol. Methods. 86: 62-68. 18. Nealson, K.H. and S.E. Finkel. 2011. Electron flow and biofilms. MRS Bull. 36:380-384. 19. Kan, J., P. Chellamuthu, A. Obraztsova, J.E. Moore, and K.H. Nealson. 2011. Diverse bacterial groups are associated with corrosive lesions at a Granite Mountain Record Vault (GMRV). J. Appl. Microbiol. 111: 329-337. Patents and Inventions: No patents or inventions were created during this funded work.« less

National audience; Insyght proposes a new way to explore the landscape of conserved and idiosyncratic genomic regions across multiple genomes and their rearrangements throughout evolution. Its unique display consists of a symbolic representation tightly integrated with a proportional view. The symbols highlight a region of interest and provide legibility while the proportional view simultaneously allows grasping genomic locations and complex rearrangements scattered across the genomes and occurring at different scales. A second type of display is dedicated to the analysis of the presence, absence, or multiple copies of a given set of homologs. A functionality based on filters has been implemented to facilitate the retrieval of genes of interest and allow the formulation of relevant biological questions, such as finding niche-specific or core genome genes that match a few particular functions or biological processes. Our public dataset currently consists of 389 prokaryotes genomes. Alternatively, a virtual machine can be downloaded and installed locally to visualize private data. It contains a pre-installed version of the pipeline, database and visualisation tool. Insyght is suitable for a variety of analyses: genome-wide inference of gene function, detection of evolutionary events, phylogenetic profiling and investigation of the core genome or niche-specific genes.

The identification of cancer drivers is a major goal of current cancer research. Finding driver genes within large chromosomal events is especially challenging because such alterations encompass many genes. Previously, we demonstrated that zebrafish malignant peripheral nerve sheath tumors (MPNSTs) are highly aneuploid, much like human tumors. In this study, we examined 147 zebrafish MPNSTs by massively parallel sequencing and identified both large and focal copy number alterations (CNAs). Given the low degree of conserved synteny between fish and mammals, we reasoned that comparative analyses of CNAs from fish versus human MPNSTs would enable elimination of a large proportion of passenger mutations, especially on large CNAs. We established a list of orthologous genes between human and zebrafish, which includes approximately two-thirds of human protein-coding genes. For the subset of these genes found in human MPNST CNAs, only one quarter of their orthologues were co-gained or co-lost in zebrafish, dramatically narrowing the list of candidate cancer drivers for both focal and large CNAs. We conclude that zebrafish-human comparative analysis represents a powerful, and broadly applicable, tool to enrich for evolutionarily conserved cancer drivers., Kathy and Curt Marble Cancer Research Fund, Arthur C. Merrill, National Institutes of Health (U.S.) (Grant CA106416), National Institutes of Health (U.S.) (Grant ROI RR020833), National Institutes of Health (U.S.) (Grant 1F32GM095213-01)

The number of chromosomal segments conserved during the evolution of two species can be used to measure their genomic distance. The number of conserved segments containing homologous genes can be estimated by comparing synteny relations within and between the two genomes. There are three sources of underestimation, however. The first stems from conserved segments in which genes are yet to be identified in one or both species. The second results from repeated translocations or transpositions resulting in not just one, but several conserved segments from a chromosome in one species being located on a single chromosome in the other. We characterize the bias due to both effects and propose correct measures of syntenic distance. We also discuss underestimation due to intrachromosomal rearrangements such as inversion.

Synteny defines the presence of two or more genes on the same chromosome of a given species. The status of synteny requires no information about the relative order of genes along the axis of a chromosome or the distance between genes on a chromosome. Since the availability of dense gene-based marker maps and sequenced genomes, the term synteny is predominantly used in context of comparative genomics referring to the presence of the same syntenic genes in ancestrally related linkage groups of two related species, hence describing the state of conserved synteny.

International audience; BackgroundReconciled gene trees yield orthology and paralogy relationships between genes. This information may however contradict other information on orthology and paralogy provided by other footprints of evolution, such as conserved synteny.ResultsWe explore a way to include external information on orthology in the process of gene tree construction. Given an initial gene tree and a set of orthology constraints on pairs of genes or on clades, we give polynomial-time algorithms for producing a modified gene tree satisfying the set of constraints, that is as close as possible to the original one according to the Robinson-Foulds distance. We assess the validity of the modifications we propose by computing the likelihood ratio between initial and modified trees according to sequence alignments on Ensembl trees, showing that often the two trees are statistically equivalent.AvailabilitySoftware and data available upon request to the corresponding author.

Comparative genomic studies are revealing that, in sharp contrast with the strong stability found in birds and mammals, sex determination mechanisms are surprisingly labile in cold-blooded vertebrates, with frequent transitions between different pairs of sex chromosomes. It was recently suggested that, in context of this high turnover, some chromosome pairs might be more likely than others to be co-opted as sex chromosomes. Empirical support, however, is still very limited. Here we show that sex-linked markers from three highly divergent groups of anurans map to Xenopus tropicalis scaffold 1, a large part of which is homologous to the avian sex chromosome. Accordingly, the bird sex determination gene DMRT1, known to play a key role in sex differentiation across many animal lineages, is sex linked in all three groups. Our data provide strong support for the idea that some chromosome pairs are more likely than others to be co-opted as sex chromosomes because they harbor key genes from the sex determination pathway.

Identification of intragenomic conservation of gene compositions in multiple chromosomal segments led to evidence of whole genome (WGDs) duplications. The process by which WGDs have been maintained and decayed provides us with clues for understanding how the genome evolves. In this chapter, we summarize current understanding of phylogenetic distribution and evolutionary impact of WGDs, introduce basic procedures to detect conserved synteny, and discuss typical pitfalls, as well as biological insights.

Lepidoptera, i.e. moths and butterflies, have a female heterogametic sex chromosome system, with most females having a WZ constitution while males are ZZ. Besides this predominant WZ/ZZ system, Z/ZZ, WZ(1)Z(2)/Z(1)Z(1)Z(2)Z(2) and W(1)W(2)Z/ZZ systems also occur. Sex is determined by an unknown W-linked gene or genes in Bombyx mori, but by dosage-dependent and equally unknown Z-linked genes in all Z/ZZ species. The female heterogametic sex chromosome system has been conserved for at least 180 MY in the phylogenetic branch that combines Lepidoptera and Trichoptera. The W chromosome, which is present in most lepidopteran species, was incorporated in the sex chromosome system much later, about 90-100 MY ago. The Z chromosomes are highly conserved among Lepidoptera, much like the Z in birds or the X in mammals. The W, on the other hand, is evolving rapidly. It is crammed with repetitive elements which appear to have a high turnover rate but poor in or even devoid of protein-coding genes. It has frequently undergone fusion with autosomes or sporadically lost altogether.

Prader–Willi syndrome (PWS) is a neurodevelopmental disorder with aspects of psychiatric illness caused by genetic mutations at chromosome 15q11-q13. In addition to causing PWS, this interval is also thought to be of importance more generally in the development of autism and psychotic illness. The PWS genetic interval is conserved in mammals, and consequently mice carrying genetic manipulations affecting one or all of the genes in the region of conserved synteny have been generated and used in neurobehavioural studies. Here we give an overview of these models and describe the behavioural and neurobiological analyses that have been performed, many of which have provide new insights into the molecular and neural processes influenced by genes within the PWS interval.

The compact genome of fugu (Takifugu rubripes) has been used widely as a reference genome for understanding the evolution of vertebrate genomes. However, the fragmented nature of the fugu genome assembly has restricted its use for comparisons of genome architecture in vertebrates. To extend the contiguity of the assembly to the chromosomal level, we have generated a comprehensive genetic map of fugu and anchored the scaffolds of the assembly to the 22 chromosomes of fugu. The map consists of 1,220 microsatellite markers that provide anchor points to 697 scaffolds covering 86% of the genome assembly (http://www.fugu-sg.org/). The integrated genome map revealed a higher recombination rate in fugu compared with other vertebrates and a wide variation in the recombination rate between sexes and across chromosomes of fugu. We used the extended assembly to explore recent rearrangement events in the lineages of fugu, Tetraodon, and medaka and compared them with rearrangements in three mammalian (human, mouse, and opossum) lineages. Between the two pufferfishes, fugu has experienced fewer chromosomal rearrangements than Tetraodon. The gene order is more highly conserved in the three teleosts than in mammals largely due to a lower rate of interchromosomal rearrangements in the teleosts. These results provide new insights into the distinct patterns of genome evolution between teleosts and mammals. The consolidated genome map and the genetic map of fugu are valuable resources for comparative genomics of vertebrates and for elucidating the genetic basis of the phenotypic diversity of ∼25 species of Takifugu that evolved within the last 5 My.

Introduction Many genome projects were underway before the advent of high-throughput sequencing and have thus been supported by a wealth of genome information from other technologies. Such information frequently takes the form of linkage and physical maps, both of which can provide a substantial amount of data useful in de novo sequencing projects. Furthermore, the recent abundance of genome resources enables the use of conserved synteny maps identified in related species to further enhance genome assemblies. Methods The tammar wallaby (Macropus eugenii) is a model marsupial mammal with a low coverage genome. However, we have access to extensive comparative maps containing over 14,000 markers constructed through the physical mapping of conserved loci, chromosome painting and comprehensive linkage maps. Using a custom Bioperl pipeline, information from the maps was aligned to assembled tammar wallaby contigs using BLAT. This data was used to construct pseudo paired-end libraries with intervals ranging from 5-10 MB. We then used Bambus (a program designed to scaffold eukaryotic genomes by ordering and orienting contigs through the use of paired-end data) to scaffold our libraries. To determine how map data compares to sequence based approaches to enhance assemblies, we repeated the experiment using a 0.5× coverage of unique reads from 4 KB and 8 KB Illumina paired-end libraries. Finally, we combined both the sequence and non-sequence-based data to determine how a combined approach could further enhance the quality of the low coverage de novo reconstruction of the tammar wallaby genome. Results Using the map data alone, we were able order 2.2% of the initial contigs into scaffolds, and increase the N50 scaffold size to 39 KB (36 KB in the original assembly). Using only the 0.5× paired-end sequence based data, 53% of the initial contigs were assigned to scaffolds. Combining both data sets resulted in a further 2% increase in the number of initial contigs integrated into a scaffold (55% total) but a 35% increase in N50 scaffold size over the use of sequence-based data alone. Conclusions We provide a relatively simple pipeline utilizing existing bioinformatics tools to integrate map data into a genome assembly which is available at http://www.mcb.uconn.edu/fac.php?name=paska. While the map data only contributed minimally to assigning the initial contigs to scaffolds in the new assembly, it greatly increased the N50 size. This process added structure to our low coverage assembly, greatly increasing its utility in further analyses.

We performed gene-based comparative FISH mapping between a wild silkmoth, Samia cynthia ssp. with a low number of chromosomes (2n = 25-28) and the model species, Bombyx mori (2n = 56), in order to identify the genomic components that make up the chromosomes in a low-number karyotype. Mapping of 64 fosmid probes containing orthologs of B. mori genes revealed that the homologues of either two or four B. mori chromosomes constitute the S. c. ricini (Vietnam population, 2n = 27♀/28♂, Z0/ZZ) autosomes. Where tested, even the gene order was conserved between S. c. ricini and B. mori. This was also true for the originally autosomal parts of the neo-sex chromosomes in S. c. walkeri (Sapporo population, 2n = 26♀/26♂, neo-Wneo-Z/neo-Zneo-Z) and S. cynthia subsp. indet. (Nagano population, 2n = 25♀/26♂, neo-WZ1Z2/Z1Z1Z2Z2). The results are evidence for an internal stability of lepidopteran chromosomes even when all autosomes had undergone fusion processes to form a low-number karyotype.

Rodents represent the largest order of living mammals. It comprises 5 sub-orders, among which Sciuromorpha (Sciuridae, Gliridae and Aplodontiidae) are assumed to occupy a basal position in rodent evolution. Banded karyotypes of some representatives of the Sciuridae family have been compared to each other, and comparisons with man were performed using chromosome paintings. Sciuridae karyotypes have conserved several eutherian ancestral syntenies. Like Sciuridae, Gliridae possess some chromosomes easily comparable with those of Primates. Comparisons of Gliridae and Sciuridae chromosomes with those of the presumed eutherian ancestor provide information about their chromosomal evolution and their position among Rodentia. Although both Sciuridae and Gliridae karyotypes are relatively conserved, they display many differences, indicating their early divergence. The reconstruction of their chromosomal evolution allowed us to propose the composition of their presumed ancestral karyotypes, with 2n = 48 and 2n = 38 for Gliridae and Sciuridae, respectively. Since rodent emergence, a single rearrangement is common to these 2 families. It formed a chromosome with fragments homologous to human chromosomes 4-8p-4-12-22, not detected in other rodents, and thus characteristic for the Sciuromorpha. This allowed us to reassess the chromosomal signatures of Rodentia. Finally, we show that the speed of chromosomal evolution in Gliridae is intermediate between that of Sciuridae (low) and Muridae (high).

The embedding of one gene in another as a nested gene pair is a unique phenomenon of gene clustering in the metazoan genome. A gene-centric paralogous genomic sequence comparison strategy was used in this study to align these paralogous nested pairs, Mab21l2-Lrba and Mab21l1-Nbea, to identify the associated paralogous non-coding elements (pNEs) they shared. A majority of these pNEs in the Mab21l2-Lrba locus display tissue-specific enhancer activities recapitulating the expression profiles of Mab21l2 and Mab21l1. Since these enhancers are spread into the introns of Lrba, dissociation of the two genes will likely disrupt the function of at least one of them. Phylogenetic analysis of this complex locus in different species suggests that Mab21 was probably locked in the Lrba/Nbea intron in the ancestral metazoan species, in which the cis-elements uncovered in this study may act as a selective force to prevent the dissociation of this gene pair in vertebrates.

International audience; Whole-genome duplications (WGDs) have occurred repeatedly in the vertebrate lineage, but their evolutionary significance for phenotypic evolution remains elusive. Here, we have investigated the impact of the fish-specific genome duplication (FSGD) on the evolution of pigmentation pathways in teleost fishes. Pigmentation and color patterning are among the most diverse traits in teleosts, and their pigmentary system is the most complex of all vertebrate groups. Using a comparative genomic approach including phylogenetic and synteny analyses, the evolution of 128 vertebrate pigmentation genes in five teleost genomes following the FSGD has been reconstructed. We show that pigmentation genes have been preferentially retained in duplicate after the FSGD, so that teleosts have 30% more pigmentation genes compared with tetrapods. This is significantly higher than genome-wide estimates of FSGD gene duplicate retention in teleosts. Large parts of the melanocyte regulatory network have been retained in two copies after the FSGD. Duplicated pigmentation genes follow general evolutionary patterns such as the preservation of protein complex stoichiometries and the overrepresentation of developmental genes among retained duplicates. These results suggest that the FSGD has made an important contribution to the evolution of teleost-specific features of pigmentation, which include novel pigment cell types or the division of existing pigment cell types into distinct subtypes. Furthermore, we have observed species-specific differences in duplicate retention and evolution that might contribute to pigmentary diversity among teleosts. Our study therefore strongly supports the hypothesis that WGDs have promoted the increase of complexity and diversity during vertebrate phenotypic evolution.

Computational analysis has been used to align the genetic map of white clover ( Trifolium repens L.) with the draft genome sequence of the model legume species Medicago truncatula Gaertn. In silico comparison based on white clover expressed sequence tags that contain simple sequence repeat loci revealed substantial macrosynteny between the genomes of these two species, which are closely related within the Trifolieae tribe of the Fabaceae family. Six of the eight homoeologous chromosome groups (HGs) of allotetraploid white clover show predominant relationships with single M. truncatula (Mt) chromosomes, while the two remaining groups may have participated in an evolutionary reciprocal translocation event. On this basis, a new chromosome nomenclature system for allotetraploid white clover is proposed such that HG A = 3, HG B = 8, HG C = 7, HG D = 4, HG E = 1, HG F = 2, HG G = 5, and HG H = 6. A rationalized linkage map ordering system has also been demonstrated. Improved knowledge of the relationships between agricultural and model forage legume genomes will facilitate prediction of gene location for key agronomic traits for pasture production.

Pax9 codes for a transcription factor, which is expressed in various tissues during mouse development. By the determination of the exon/intron structure of the gene, a new 5'-exon and two alternative promoters were identified. An extremely highly conserved physical association with the neighboring genes (i. e. Nkx2-9) was found in the human, mouse, and pufferfish genomes. A comparative sequencing in search for conserved non-coding sequences brought about the identification of an Nkx2-9 neural tube enhancer and of a Pax9 nasal process enhancer. However, the creation of a BAC transgenic mouse could not reproduce the complete Pax9-expression domain. The data suggest that the Pax9 regulatory elements are scattered through a very large genomic region that includes the neighboring genes. This conclusion is proposed as a biological explanation for the conserved synteny. Pax9 kodiert für einen Transkriptionsfaktor, der während der Maus-Embryogenese in verschiedenen Geweben exprimiert wird. Durch die Ermittlung der Exon/Intron-Struktur des Genes wurden ein neues 5'-Exon und zwei alternative Promotore entdeckt. Eine extrem hoch konservierte physikalische Assoziation mit den anligenden Genen (u. a. Nkx2-9) wurde in dem Menschen-, Maus- und Kugelfishgenom gefunden. Eine vergleichende Sequenzierung, auf der Suche nach konservierten nicht-kodierenden Sequenzen, hat die Identifizierung von einem Nkx2-9-Neuronalrohrelement und von einem Pax9-Nasenwulstelement ermöglicht. Allerdings konnte die Generierung einer BAC-transgenen Maus das vollständige Pax9-Expressionsmuster nicht widerspiegeln. Das deutet daruf hin, dass die sämtlichen Pax9-regulatorischen Elemente vermutlich weitverbreitet in einem sehr großen genomischen Bereich, der auch die anliegenden Gene einschliesst, sich befinden. Die Folgerung wird als biologische Erklärung der konservierten Syntenie vorgeschlagen.

International audience; Teleost fishes have evolved a unique complexity and diversity of pigmentation and colour patterning that is unmatched among vertebrates. Teleost colouration is mediated by five different major types of neural-crest derived pigment cells, while tetrapods have a smaller repertoire of such chromatophores. The genetic basis of teleost colouration has been mainly uncovered by the cloning of pigmentation genes in mutants of zebrafish Danio rerio and medaka Oryzias latipes. Many of these teleost pigmentation genes were already known as key players in mammalian pigmentation, suggesting partial conservation of the corresponding developmental programme among vertebrates. Strikingly, teleost fishes have additional copies of many pigmentation genes compared with tetrapods, mainly as a result of a whole-genome duplication that occurred 320-350 million years ago at the base of the teleost lineage, the so-called fish-specific genome duplication. Furthermore, teleosts have retained several duplicated pigmentation genes from earlier rounds of genome duplication in the vertebrate lineage, which were lost in other vertebrate groups. It was hypothesized that divergent evolution of such duplicated genes may have played an important role in pigmentation diversity and complexity in teleost fishes, which therefore not only provide important insights into the evolution of the vertebrate pigmentary system but also allow us to study the significance of genome duplications for vertebrate biodiversity.

National audience; We have developed a web application to explore synteny conservation in prokaryotes : INSYGT (INteractive SYnteny Graphics and Tools). Its graphic interfaces features AJAX web development techniques. ORIGAMI, a relational database on gene context conservation, is used as a back-end. INSYGT is aimed to be integrated as a tool for the genome annotation system AGMIAL [1].