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1.
Methods Mol Biol ; 2498: 363-372, 2022.
Article in English | MEDLINE | ID: mdl-35727557

ABSTRACT

The opportunity to map genes and noncoding DNA sequences on the chromosomes by fluorescence in situ hybridization (FISH) has greatly enhanced the potential for fish karyotyping and comparative cytogenetics. The use of FISH allowed for significant advances in our understanding of the fish genome architecture, especially when applied to the study of the repetitive component of the genome, that is generally underestimated in the bioinformatic assembly. Here we provide a step-by-step protocol for FISH of repeated sequences onto chromosomes of fish species.


Subject(s)
Chromosomes , Repetitive Sequences, Nucleic Acid , Chromosome Mapping , Chromosomes/genetics , In Situ Hybridization, Fluorescence/methods , Karyotyping
2.
Genes (Basel) ; 11(12)2020 12 18.
Article in English | MEDLINE | ID: mdl-33352937

ABSTRACT

The ice cod Arctogadus glacialis (Peters, 1872) is one of the few fish species endemic to the Arctic. With a circumpolar distribution, the species is confined to the fjords and shelves of the Arctic seas. Biological information on A. glacialis is scarce, with genomic information restricted to microsatellites. Within the frame of the TUNU-Programme: Arctic Ocean Fishes-Diversity, Adaptation and Conservation, we studied A. glacialis at the chromosomal level to explore fish diversity and evolutionary aspects. The analysis of over 50 individuals from the Northeast Greenland fjords between latitudes 71°09' N and 76°42' N revealed a remarkable intraspecific diversity epitomized by chromosome numbers spanning from 28 to 33, the occurrence of putative B chromosomes, and diversified patterns of distribution of heterochromatin and rDNAs. The number of B chromosomes followed a latitudinal gradient from 0-2 in the north to 2-5 in the south. Considering the benthic and rather stationary life history of this species, the observed chromosomal differences might have arisen independently, possibly driven and/or fostered by the dynamics of repetitive sequences, and are being fixed in relatively isolated fjord populations. The resulting latitudinal cline we observe today might have repercussions on the fate of local populations facing the ongoing climate-driven environmental changes.


Subject(s)
Chromosomes , Gadiformes/genetics , Adaptation, Physiological , Animals , Arctic Regions , Chromosome Mapping , Chromosomes/ultrastructure , Climate Change , DNA, Ribosomal/genetics , Diploidy , Female , Genetic Drift , Genome , Greenland , Heterochromatin/genetics , Karyotype , Male , Mitosis
3.
Mar Genomics ; 52: 100737, 2020 Aug.
Article in English | MEDLINE | ID: mdl-31892467

ABSTRACT

This paper describes the cytogenetic features of the Antarctic silverfish Pleuragramma antarctica (Boulenger 1902), a keystone species of the Antarctic coastal marine ecosystem. Conventional cytogenetic analyses and physical mapping of repetitive DNA sequences were performed on metaphase plates obtained through direct chromosome preparation from P. antarctica early larvae. The Antarctic silverfish have a diploid number (2n) = 48, and a karyotype made up of a majority of two-armed chromosomes (karyotype formula36m/sm + 10st + 2a, fundamental number = 94). Major ribosomal gene repeats were detected on three chromosome pairs (20, 21, and 23), in correspondence of dim DAPI stained regions. Long Interspersed Nuclear Elements (LINEs) were abundant and wide spread over all chromosomes. Overall, the cytogenetic data presented herein are consistent with a long independent cytogenetic and evolutionary history for the species. The large number of two-armed chromosomes, indicative of highly-rearranged karyotype, coupled with a diploid number of 48, a presumed primitive character for this fish group, and the spread of the major ribosomal genes on three chromosome pairs, make the Antarctic silverfish distinct from all other notothenioid species.


Subject(s)
Base Sequence , Chromosomes/genetics , Perciformes/genetics , Repetitive Sequences, Nucleic Acid/genetics , Animals , Chromosome Mapping , Cytogenetic Analysis , Mitosis
4.
Int J Mol Sci ; 20(3)2019 Feb 06.
Article in English | MEDLINE | ID: mdl-30736325

ABSTRACT

By their faculty to transpose, transposable elements are known to play a key role in eukaryote genomes, impacting both their structuration and remodeling. Their integration in targeted sites may lead to recombination mechanisms involved in chromosomal rearrangements. The Antarctic fish family Nototheniidae went through several waves of species radiations. It is a suitable model to study transposable element (TE)-mediated mechanisms associated to genome and chromosomal diversifications. After the characterization of Gypsy (GyNoto), Copia (CoNoto), and DIRS1 (YNoto) retrotransposons in the genomes of Nototheniidae (diversity, distribution, conservation), we focused on their chromosome location with an emphasis on the three identified nototheniid radiations (the Trematomus, the plunderfishes, and the icefishes). The strong intrafamily TE conservation and wide distribution across species of the whole family suggest an ancestral acquisition with potential secondary losses in some lineages. GyNoto and CoNoto (including Hydra and GalEa clades) mostly produced interspersed signals along chromosomal arms. On the contrary, insertion hot spots accumulating in localized regions (mainly next to centromeric and pericentromeric regions) highlighted the potential role of YNoto in chromosomal diversifications as facilitator of the fusions which occurred in many nototheniid lineages, but not of the fissions.


Subject(s)
Chromosomes , Fishes/genetics , Retroelements , Animals , Antarctic Regions , Evolution, Molecular , Fishes/classification , Genome , In Situ Hybridization, Fluorescence , Phylogeny , Species Specificity , Translocation, Genetic
5.
Mar Genomics ; 39: 73-84, 2018 Jun.
Article in English | MEDLINE | ID: mdl-29510906

ABSTRACT

Antifreeze glycoproteins (AFGPs) are a novel evolutionary innovation in members of the northern cod fish family (Gadidae), crucial in preventing death from inoculative freezing by environmental ice in their frigid Arctic and sub-Arctic habitats. However, the genomic origin and molecular mechanism of evolution of this novel life-saving adaptive genetic trait remained to be definitively determined. To this end, we constructed large insert genomic DNA BAC (bacterial artificial chromosome) libraries for two AFGP-bearing gadids, the high-Arctic polar cod Boreogadus saida and the cold-temperate Atlantic tomcod Microgadus tomcod, to isolate and sequence their AFGP genomic regions for fine resolution evolutionary analyses. The BAC library construction encountered poor cloning efficiency initially, which we resolved by pretreating the agarose-embedded erythrocyte DNA with a cationic detergent, a method that may be of general use to BAC cloning for teleost species and/or where erythrocytes are the source of input DNA. The polar cod BAC library encompassed 92,160 clones with an average insert size of 94.7 kbp, and the Atlantic tomcod library contained 73,728 clones with an average insert size of 89.6 kbp. The genome sizes of B. saida and M. tomcod were estimated by cell flow cytometry to be 836 Mbp and 645 Mbp respectively, thus their BAC libraries have approximately 10- and 9.7-fold genome coverage respectively. The inclusiveness and depth of coverage were empirically confirmed by screening the libraries with three housekeeping genes. The BAC clones that mapped to the AFGP genomic loci of the two gadids were then isolated by screening the BAC libraries with gadid AFGP gene probes. Eight minimal tiling path (MTP) clones were identified for B. saida, sequenced, and assembled. The B. saida AFGP locus reconstruction produced both haplotypes, and the locus comprises three distinct AFGP gene clusters, containing a total of 16 AFGP genes and spanning a combined distance of 512 kbp. The M. tomcod AFGP locus is much smaller at approximately 80 kbp, and contains only three AFGP genes. Fluorescent in situ hybridization with an AFGP gene probe showed the AFGP locus in both species occupies a single chromosomal location. The large AFGP locus with its high gene dosage in B. saida is consistent with its chronically freezing high Arctic habitats, while the small gene family in M. tomcod correlates with its milder habitats in lower latitudes. The results from this study provided the data for fine resolution sequence analyses that would yield insight into the molecular mechanisms and history of gadid AFGP gene evolution driven by northern hemisphere glaciation.


Subject(s)
Antifreeze Proteins/genetics , Chromosomes, Artificial, Bacterial , Fish Proteins/genetics , Gadiformes/genetics , Gene Library , Genome , Animals , Cloning, Molecular , In Situ Hybridization, Fluorescence
6.
BMC Evol Biol ; 16(1): 142, 2016 06 29.
Article in English | MEDLINE | ID: mdl-27356756

ABSTRACT

BACKGROUND: Over the past 40 million years water temperatures have dramatically dropped in the Southern Ocean, which has led to the local extinction of most nearshore fish lineages. The evolution of antifreeze glycoproteins in notothenioids, however, enabled these ancestrally benthic fishes to survive and adapt as temperatures reached the freezing point of seawater (-1.86 °C). Antarctic notothenioids now represent the primary teleost lineage in the Southern Ocean and are of fundamental importance to the local ecosystem. The radiation of notothenioids has been fostered by the evolution of "secondary pelagicism", the invasion of pelagic habitats, as the group diversified to fill newly available foraging niches in the water column. While elaborate craniofacial modifications have accompanied this adaptive radiation, little is known about how these morphological changes have contributed to the evolutionary success of notothenioids. RESULTS: We used a 3D-morphometrics approach to investigate patterns of morphological variation in the craniofacial skeleton among notothenioids, and show that variation in head shape is best explained by divergent selection with respect to foraging niche. We document further an accelerated rate of morphological evolution in the icefish family Channichthyidae, and show that their rapid diversification was accompanied by the evolution of relatively high levels of morphological integration. Whereas most studies suggest that extensive integration should constrain phenotypic evolution, icefish stand out as a rare example of increased integration possibly facilitating evolutionary potential. Finally, we show that the unique feeding apparatus in notothenioids in general, and icefish in particular, can be traced to shifts in early developmental patterning mechanisms and ongoing growth of the pharyngeal skeleton. CONCLUSION: Our work suggests that ecological opportunity is a major factor driving craniofacial variation in this group. Further, the observation that closely related lineages can differ dramatically in integration suggests that this trait can evolve quickly. We propose that the evolution of high levels of phenotypic integration in icefishes may be considered a key innovation that facilitated their morphological evolution and subsequent ecological expansion.


Subject(s)
Biological Evolution , Extreme Environments , Fishes/anatomy & histology , Adaptation, Biological , Animals , Antarctic Regions , Ecosystem , Perciformes/anatomy & histology , Phylogeny
7.
PLoS One ; 9(3): e90512, 2014.
Article in English | MEDLINE | ID: mdl-24598889

ABSTRACT

Visual observations and videos of Chionodraco hamatus icefish at the "Acquario di Genova" and histological analyses of congeneric species C. hamatus and C. rastrospinosus adults sampled in the field provided new anatomical and behavioral information on the reproductive biology of these white blooded species that are endemic to the High-Antarctic region. During the reproductive season, mature males of both species, which are different from females and immature males, display fleshy, club-like knob modifications of their anal fin that consisted of a much thicker epithelium. Histology indicated that the knobs were without any specialized glandular or sensorial organization, thus suggesting a mechanical and/or ornamental role of the modified anal fin. In addition, the occurrence of necrotic regions at the base of the thickened epithelium and the detachment of the knobs in post-spawning C. hamatus males indicated the temporary nature of the knobs. The role of these structures was confirmed as mechanical and was clarified using visual observations and videos of the behavior of two C. hamatus during a reproductive event that occurred in an exhibit tank at the "Acquario di Genova". The reproductive process included pre-spawning activity, preparation of the nest, egg guarding and successfully ended with egg hatching. When the spawning event approached, the male prepared the nest. The nest was constructed on an accurately selected bottom surface, which was flattened and maintained free from sand or debris by a combination of radial body movements and continuous anal fin sweeping, thus demonstrating the important mechanical/abrasive function of the anal fin knobs. The present data are the first records of active nesting in icefish and clarify the meaning of dimorphic temporary structures, whose function would have been difficult to obtain in the field.


Subject(s)
Animal Fins/anatomy & histology , Nesting Behavior , Perciformes/physiology , Animals , Antarctic Regions , Female , Male , Perciformes/anatomy & histology , Sex Characteristics , Video Recording
8.
Biomed Res Int ; 2013: 243938, 2013.
Article in English | MEDLINE | ID: mdl-23509694

ABSTRACT

Two genes, that is, 5S ribosomal sequences and antifreeze glycoprotein (AFGP) genes, were mapped onto chromosomes of eight Antarctic notothenioid fish possessing a X1X1X2X2/X1X2Y sex chromosome system, namely, Chionodraco hamatus and Pagetopsis macropterus (family Channichthyidae), Trematomus hansoni, T. newnesi, T. nicolai, T. lepidorhinus, and Pagothenia borchgrevinki (family Nototheniidae), and Artedidraco skottsbergi (family Artedidraconidae). Through fluorescence in situ hybridization (FISH), we uncovered distinct differences in the gene content of the Y chromosomes in the eight species, with C. hamatus and P. macropterus standing out among others in bearing 5S rDNA and AFGP sequences on their Y chromosomes, respectively. Both genes were absent from the Y chromosomes of any analyzed species. The distinct patterns of Y and non-Y chromosome association of the 5S rDNA and AFGP genes in species representing different Antarctic fish families support an independent origin of the sex heterochromosomes in notothenioids with interesting implications for the evolutionary/adaptational history of these fishes living in a cold-stable environment.


Subject(s)
Chromosome Mapping , Evolution, Molecular , Fishes/genetics , Sex Chromosomes/genetics , Animals , Antarctic Regions , Antifreeze Proteins/genetics , Chromosome Banding , Female , Fishes/physiology , In Situ Hybridization, Fluorescence , Male , RNA, Ribosomal, 5S/metabolism , Species Specificity
9.
Genomics ; 98(3): 194-201, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21684327

ABSTRACT

To investigate the genomic architecture underlying the quintessential adaptive phenotype, antifreeze glycoprotein (AFGP) that enables Antarctic notothenioid survival in the frigid Southern Ocean, we isolated the AFGP genomic locus from a bacterial artificial chromosome library for Dissostichus mawsoni. Through extensive shotgun sequencing of pertinent clones and sequence assembly verifications, we reconstructed the highly repetitive AFGP genomic locus. The locus comprises two haplotypes of different lengths (363.6 kbp and 467.4 kbp) containing tandem AFGP, two TLP (trypsinogen-like protease), and surprisingly three chimeric AFGP/TLP, one of which was previously hypothesized to be a TLP-to-AFGP evolutionary intermediate. The ~100 kbp haplotype length variation results from different AFGP copy number, suggesting substantial dynamism existed in the evolutionary history of the AFGP gene family. This study provided the data for fine resolution sequence analyses that would yield insight into the molecular mechanisms of notothenioid AFGP gene family evolution driven by Southern Ocean glaciation.


Subject(s)
Antifreeze Proteins/genetics , Genetic Loci , Perciformes/genetics , Trypsinogen/genetics , Animals , Antarctic Regions , Chromosome Mapping , Chromosomes, Artificial, Bacterial/genetics , Cloning, Molecular , Gene Dosage , Haplotypes , Molecular Sequence Data , Phenotype , Phylogeny , Sequence Alignment
10.
BMC Evol Biol ; 10: 4, 2010 Jan 06.
Article in English | MEDLINE | ID: mdl-20053275

ABSTRACT

BACKGROUND: Pedomorphism is the retention of ancestrally juvenile traits by adults in a descendant taxon. Despite its importance for evolutionary change, there are few examples of a molecular basis for this phenomenon. Notothenioids represent one of the best described species flocks among marine fishes, but their diversity is currently threatened by the rapidly changing Antarctic climate. Notothenioid evolutionary history is characterized by parallel radiations from a benthic ancestor to pelagic predators, which was accompanied by the appearance of several pedomorphic traits, including the reduction of skeletal mineralization that resulted in increased buoyancy. RESULTS: We compared craniofacial skeletal development in two pelagic notothenioids, Chaenocephalus aceratus and Pleuragramma antarcticum, to that in a benthic species, Notothenia coriiceps, and two outgroups, the threespine stickleback and the zebrafish. Relative to these other species, pelagic notothenioids exhibited a delay in pharyngeal bone development, which was associated with discrete heterochronic shifts in skeletal gene expression that were consistent with persistence of the chondrogenic program and a delay in the osteogenic program during larval development. Morphological analysis also revealed a bias toward the development of anterior and ventral elements of the notothenioid pharyngeal skeleton relative to dorsal and posterior elements. CONCLUSIONS: Our data support the hypothesis that early shifts in the relative timing of craniofacial skeletal gene expression may have had a significant impact on the adaptive radiation of Antarctic notothenioids into pelagic habitats.


Subject(s)
Bone and Bones/anatomy & histology , Evolution, Molecular , Perciformes/growth & development , Animals , Calcification, Physiologic , Gene Expression Regulation, Developmental , Larva/anatomy & histology , Larva/genetics , Larva/growth & development , Perciformes/anatomy & histology , Perciformes/genetics , Phylogeny , Sequence Analysis, DNA
11.
Mar Genomics ; 2(1): 75-80, 2009 Mar.
Article in English | MEDLINE | ID: mdl-21798175

ABSTRACT

This mini-review makes a survey and a summary of some major issues concerning the chromosomal organisation of ribosomal genes in fish genomes, by using Notothenioidei as the model. The increasing body of information, published during the last two decades on the chromosomal mapping of the two ribosomal genes classes (45S rDNA and 5S rDNA) in notothenioids, makes it possible to recognise the main evolutionary trends across the phylogeny of the group. As one of the major features, the rDNA clusters are organised in a single chromosomal locus in most of the species. This locus is located at different positions along the chromosomes in the basal groups (non-Antarctic Clade), whereas it maintains a strongly conserved location in the cold-adapted species (Antarctic Clade). Important structural changes, leading to the co-localisation of the two ribosomal gene classes, occurred early in the notothenioid phylogeny, perhaps in the common ancestor of the Eleginopidae and Nototheniidae. The cytogenetic evidences indicate that an increased amount of ribosomal genes, organised in two large chromosomal loci, is present in the giant Antarctic fish Dissostichus mawsoni. This gain in rRNA genes is an important genomic change, having possible implications for the fitness of this notothenioid fish that combines large size, pelagic lifestyle and cold-adaptation.

12.
Methods Enzymol ; 436: 511-38, 2008.
Article in English | MEDLINE | ID: mdl-18237652

ABSTRACT

Sequencing genomes of model organisms is a great challenge for biological sciences. In the past decade, scientists have developed a large number of methods to align and compare sequenced genomes. The analysis of a given sequence provides much information on the genome structure but to a lesser extent on the function. Comparative genomics are a useful tool for functional and evolutionary annotation of genomes. In principle, comparison of genomic sequences may allow for identification of the evolutionary selection (negative or positive) that the functional sequences have been subjected to over time. Positively selected genome regions are the most important ones for evolution, because most changes are adaptive and often induce biological differences in organisms. The draft genomes of five fish species have recently become available. We herewith review and discuss some new insights into comparative genomics in fish globin genes. Special attention will be given to a complementary methodological approach to comparative genomics, fluorescence in situ hybridization (FISH). Internet resources for analyzing sequence alignments and annotations and new bioinformatic tools to address critical problems are thoroughly discussed.


Subject(s)
Fish Proteins/genetics , Fishes/genetics , Globins/genetics , Animals , Chromosome Mapping , Computational Biology , Evolution, Molecular , Genomics , In Situ Hybridization, Fluorescence , Models, Genetic , Phylogeny , Sequence Alignment , Software
13.
Genet. mol. biol ; 31(1,suppl): 180-187, 2008. ilus, tab
Article in English | LILACS | ID: lil-484583

ABSTRACT

The capelin, Mallotus villosus (Osmeriformes, Osmeridae), is an ecological and commercial key component of the sub-arctic ichthyofauna. Here, we provide the first cytogenetic information on the species based on both conventional karyotyping and chromosomal mapping of 45S and 5S ribosomal genes through fluorescence in situ hybridization (FISH). The capelin genome displayed a diploid number of 54 with the karyotypic formula 26m/sm+28st/a and a fundamental number (FN) = 80. Both classes of ribosomal genes appeared to be spread out to multiple chromosomal locations, i.e. the 45S and 5S rDNA clusters were detected on six and seven chromosome pairs, respectively. A linked chromosomal organization of the major and minor ribosomal genes classes has been visualized in most of the rDNAs chromosomal locations. A comparative analysis of the available cytogenetic data for the family Osmeridae reveals diploid numbers higher than 48 and high fundamental numbers. This suggests that a rearranged karyotype is a shared feature within this family.


Subject(s)
Animals , DNA, Ribosomal , In Situ Hybridization, Fluorescence , Fishes/genetics , Chromosome Mapping , Cytogenetics , Karyotyping , Fishes/classification
14.
Genetica ; 130(1): 9-17, 2007 May.
Article in English | MEDLINE | ID: mdl-16909334

ABSTRACT

The chromosomal location of the IgH locus has been analyzed in several bony fish of the Antarctic perciform group Notothenioidei. Two IgH probes were prepared from the species Trematomus bernacchii (family Nototheniidae, tribe Trematominae) and mapped onto the chromosomes of ten species belonging to the same genus (Trematomus) and in two outgroups, through one-color and two-color FISH. A single location of the IgH locus was found in the majority of the species examined, including the outgroups, whereas in four of them the IgH genes splited to two chromosomal loci. RT-PCR experiments revealed the presence of three allelic sequences in T. newnesi, a species in which the IgH genes were organized in two chromosomal loci. Possible pathways leading to IgH genes duplication during the diversification of trematomine fishes were inferred from the analysis of the FISH patterns in a phylogenetic context. The present work provides the first comprehensive picture of IgH genes organization at chromosomal level in a bony fish group.


Subject(s)
Chromosome Mapping , Genes, Immunoglobulin Heavy Chain , In Situ Hybridization, Fluorescence , Perciformes/genetics , Animals , Antarctic Regions , DNA, Complementary/analysis , Perciformes/immunology , Phylogeny
15.
Chromosome Res ; 11(6): 633-40, 2003.
Article in English | MEDLINE | ID: mdl-14516071

ABSTRACT

The pathways and mechanisms of genomic change that have led to the peculiar haemoglobinless phenotype of the white-blooded Antarctic icefishes (16 species in the family Channichthyidae) constitute an important model for understanding the rapid diversification of the Antarctic notothenioid fish flock. To provide complementary structural information on genomic change at globin-gene loci in Antarctic fish species, cytogenetic studies and in-situ chromosomal mapping have been undertaken. Using a DNA probe containing one alpha- and one beta-globin gene from the embryonic/juvenile globin gene cluster of the red-blooded species Notothenia coriiceps, we mapped the cluster on the chromosomes of Antarctic teleosts by fluorescence in-situ hybridization. As anticipated on the basis of its molecular organization, the cluster was located on a single chromosome pair in all of the red-blooded fish species probed (N. coriiceps, N. angustata, Trematomus hansoni, T. pennellii). In contrast, the alpha/beta-globin probe did not recognize complementary sequences on the chromosomes of the white-blooded species Chionodraco hamatus and Channichthys rhinoceratus. These results represent the first example of chromosomal mapping of embryonic/juvenile globin genes in teleostean fishes. Beyond its relevance to the evolutionary history of Antarctic notothenioids, this work contributes to our understanding of the evolution of the chromosomal loci of globin genes in fishes and other vertebrates.


Subject(s)
Chromosome Mapping , Globins/genetics , Perciformes/genetics , Animals , Antarctic Regions , DNA Probes/genetics , Gene Order/genetics , In Situ Hybridization, Fluorescence , Species Specificity , Telomere/genetics
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