icacgm18th International Colloquim of Animal Cytogenetics and Gene Mapping 2008

Session VIII - Fish Cytogenetics


Fish cytogenetics

E. Pisano, C. Ozouf-Costaz, F. Foresti, B.G. Kapoor (eds): an overview of the ongoing research

Department of Biology, University of Genova, Viale Benedetto XV, 5 16132 Genova ( Italy)

Fish make up half of the extant vertebrate species, they exhibit a huge level of biological diversity and, as food resource, they play major roles in the culture and economy of human populations. Therefore, the scientists dealing with fish are spread over hundreds of countries and laboratories where they operate within different research contexts, from assessing biodiversity in a river basin to control the reproduction in a cultured species, from studying the ancestry of a given lineage to tracking down DNA sequences in a fish genome, etc. For most of these biologists, cytogenetics begins to be recognized as an essential tool to approach questions in both basic and applied ichthyology. The technical advances in molecular cytogenetic procedures, such as chromosomal mapping by FISH (Fluorescence In Situ Hybridization), have enhanced the potential applications of fish cytogenetics in the various fields. For instance the last few years witnessed the first fish species having protein-coding genes cytogenetically mapped.

By overcoming the limits imposed by the constraints of current scientific journals, the book Fish Cytogenetics (E. Pisano, C. Ozouf-Costaz, F. Foresti and B.G. Kapoor Editors, Science Publishers, Enfield, 2007) was mainly thought to put together data and opinions of fish specialists, working on different fish groups, operating in various environments and scientific contexts but sharing cytogenetics as the common tool in their research. In Fish Cytogenetics, eighteen contributions from thirteen countries are organized in four sections (Systematics and Evolution; Biodiversity and Conservation; Stock assessment and Aquaculture; Genomics) representing the major themes in current fish cytogenetic research.

By providing a comprehensive (although obviously not complete) updated picture of the ongoing research around the world, the book also stresses the wide ranging role that cytogenetics can play in the nowadays advancement of biological sciences. In particular, the advent of cytogenomics, as the strategy to integrate molecular data into the broader architectural context of chromosomes, promises to further our understanding of the fish genome architecture and function. Specifically, large-insert genomic DNA BAC clones generated for genomic-scale studies or fish genome sequencing projects can be used as probes to visually map the spatial distribution and relationship of target genes by chromosomal FISH, complementing the information from genetic and molecular approaches. This exciting advance has implications for both fundamental studies in fish and vertebrates and applications in aquaculture.


A comprehensive compendium of chaotic killifish karyotypes

Martin Völker (1), Petr Ráb (2), Harald Kullmann (3)

1. Department of Biosciences, University of Kent, Canterbury, Kent CT2 7 NJ (UK)
2. Laboratory of Fish Genetics, Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 277 21 Liběchov (Czech Republic)
3. Institute for Evolutionary Biology and Ecology, University of Bonn, 53121 Bonn (Germany)

Fish karyotypes are generally quite well conserved in evolution, with more than 50% of species having 2n=48 or 2n=50 chromosomes. However, some groups of killifishes (Cyprinodontiformes) show an enormous karyotypic variability which is most distinct in the African family Nothobranchiidae. Using a combination of cytogenetic and molecular phylogenetic methods, our studies of the nothobranchiid genus Chromaphyosemion revealed chromosome numbers ranging from 2n=20 to 2n=40, several phylogenetically independent reductions of 2n, a high diversity of NOR phenotypes, variability in the number of chromosome arms due to inversions and heterochromatin additions and possibly also independent evolutions of XX/XY sex chromosome systems. Karyotpyes varied among as well as within species and in some cases also within the same population. Karyotypic variability in other killifish genera is less well studied, but the available evidence indicates that this complex pattern of chromosomal evolution is not restricted to Chromaphyosemion alone. The causes of this high karyotypic diversity are not known, but it is very likely that both structural genomic features and characteristics of the ecology and population biology of these fishes are contributing factors. The high chromosomal variability may be involved in speciation in some genera.


Chromosomal localisation of sex-specific BACs and of selected cDNAs identified by microdissection in two tilapia species, oreochromis niloticus and oreochromis aureus

C. Ozouf-Costaz (1), O. Coriton (2), H. D’Cotta (3), A. Cnaani (4), J. P. Coutanceau (1), C. Bonillo (1), E. Pepey (3), T. Kocher (4), J. F. Baroiller (3)

1. CNRS UMR 7138, MNHN (France)
2. Plate-forme de Cytogenetique moleculaire DGAP, INRA, Rennes (France)
3. UPR20 Aquaculture et gestion des ressources aquatiques, CIRAD, Montpellier (France)
4. Dept. of Biology, University of Maryland (USA)

Cichlid fish of the Tilapia group are are an ideal model for studying sex determination. Sequencing of the Tilapia (Oreochromis niloticus) genome has started in USA and France and genetic and physical linkage maps based on high resolution fingerprints of BAC clones are produced. We have in charge the chromosomal anchoring of some sex linked BACs from the genetic mapping. Some BAC libraries constructed from a male O. niloticus have been assembled into a physical map. Generally, species with a major sex determination locus on linkage group 1 (LG1) are male heterogametic and species with a major sex determination locus on linkage group 3 (LG3) are female heterogametic. In order to confirm that LG1 and LG3 are on two different, unlinked chromosomes, we physically mapped the sex-linked markers by chromosomal FISH on the chromosomes of two closely related Tilapia species, O. niloticus and O. aureus. These experiments demonstrated that LG3 corresponds to the largest chromosome in the Tilapia karyotype, and that LG1 corresponds to a different, smaller chromosome. The chromosome pair corresponding to LG3 in O. niloticus has been manually microdissected and a series of cDNAs isolated from this microdissected product have been cloned, sequenced and selected for chromosomal FISH. Their hybridization helps to validate the results.


Mugilidae: towards a cytogenomic approach

A. K. Hett (1), L. Sola (1), M. Nirchio (2), A. R. Rossi (1)

1. Department of Animal and Human Biology, University "La Sapienza", Rome (Italy)
2. Escuela de Ciencias Aplicadas del Mar. Universidad de Oriente, Isla de Margarita, (Venezuela)

Approximately 25% among the over 70 species of Mugilidae (Teleostei) have been cytogenetically analysed. Most of them show the conservative 48 uni-armed karyotype, with small differences concerning the absence or the presence of short arms on a single subtelocentric chromosome pair. The Mugil curema species complex constitutes an exception, with karyotypes mainly or exclusively composed of bi-armed chromosomes and a conserved NF=48. Moreover, preliminary data from some species with the conservative karyotype have suggested the existence of different types of satellite DNA.

Such cytogenetic features offer a great opportunity both for cytotaxonomic and phylogenetic studies, as well as for structural, evolutionary and functional analyses of the genome, i.e. for a cytogenomic approach. In this context, on one hand, the isolation and cloning of additional satellite DNA families are being carried out, to study their chromosomal location on more than ten Mediterranean and Caribbean species. On the other, laser micro-dissection followed by DOP-PCR amplification of the smallest chromosome pair of individuals of Mugil curema with 2n=24 are being carried out to produce chromosome-specific painting probe. This probe will be used for FISH on metaphases of the source and on the other Mugilidae species, in order to investigate its specificity, inter-specific homologies and the structural changes that led to divergence of mullets chromosome complements.


Karyotype characterization and comparison in moray eels (Anguilliformes, Muraenidae)

E.Coluccia (1), A.M. Deiana (1), R. Cannas (1), G. Pichiri (2), S. Salvadori (1)

1. Dipartimento di Biologia Animale ed Ecologia;
2. Dipartimanto di Scienze e Tecnologie Biomediche, Università di Cagliari, Cagliari (Italy).

The Muraenidae family consists of a large group of species, living mainly in tropical and subtropical seas, and Gymnothorax is the genus with the largest number of species. Among Anguilliformes, molecular evidences indicated this family as the most primitive of the order and, from a cytogenetic point of view, the Muraenidae species show among the highest diploid numbers and the highest DNA/nucleus contents.

The cytogenetic analysis of the eleven studied species revealed a high conservation of the diploid number (2n=42) but an extensive variability in NF (NF=42 to 76). We carried out a comparative chromosomal analysis of the Mediterranean species Muraena helena and Gymnothorax unicolor and of the Indo-Pacific species G. tile, on the basis of replication, C- and restriction endonuclease patterns and FISH localization of ribosomal, telomeric and species-specific repetitive sequences. The comparison pointed out similarities in banding patterns of some pairs among the three species and a higher degree of chromosomal homologies between the Mediterranean species. In Muraenidae, heterochromatin addition, translocations and intracromosomal rearrangements as pericentric inversion, could be the main explanation for the extensive changes in karyotype structure.

Our results support the need of further investigations to resolve the taxonomic uncertainties within this family, already found in previous cytogenetic and molecular studies.


Population cytogenetics of native and hatchery trout from Italy (Salmotrutta complex, Salmonidae)

V. Caputo, M. Giovannotti, P. Nisi Cerioni, A. Splendiani, E. Olmo

Istituto di Biologia e Genetica, Università Politecnica delle Marche, via Ranieri 65, Ancona (Italy).

A cytogenetic analysis was carried out using conventional staining, banding techniques and FISH in Italian populations of brown trout. All the individuals analysed, belonging to the Atlantic (At), Marmoratus (Ma), Adriatic (Ad) and Mediterranean (Me) lineages, showed a remarkable karyotype stability, with a diploid complement of 80 chromosomes and an arm number of 102. Such stability was observed also with reference to the location of 5S rDNA and the active NOR sites. On the contrary, FISH with a 28S ribosomal probe and staining with CMA3 revealed that inactive NOR sites are more numerous in Ad and Me than in At and Ma lineages. PCR-based cloning of satellite DNA referring to a reported Atlantic salmon centromeric sequence was successful for brown trout samples. The sequence was characterized by a high AT content and a short consensus motif similar to other centromeric satellite repeats. The obtained satellite DNA clones were localized with FISH in the centromeric regions of the brown trout chromosomes, showing lineage-specific patterns. Because it is well known that AT-rich sequences can induce a pronounced DNA curvature, which in turn would promote faster and higher chromatin spiralization, it may be hypothesised that the wide distribution of this satellite in brown trout may have played a role in determining its karyotype stability.


FISH and DAPI staining of the synaptonemal complex of the Nile tilapia (Oreochromis niloticus) allow orientation of the unpaired region of bivalent 1 observed during early pachytene

K. Ocalewicz (1), 2, J. C. Mota-Velasco (1), R. Campos-Ramosand (1,3), D. J.Penman (1)

1. Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, Scotland (UK)
2. Departement of Ichthyology, University of Warmia and Mazury in Olsztyn, Poland, ul. Oczapowskiego 5, 10-957 Olsztyn, (Poland),
3. Centro de Investigaciones Biológicas del Noroeste S.C. (CIBNOR), La Paz. (Mexico)

Bivalent 1 of the synaptonemal complex (SC) in XY male Oreochromis niloticus shows an unpaired terminal region in early pachytene. This appears to be related to recombination suppression around a sex determination locus. To allow more detailed analysis of this, and unpaired regions in the karyotype of other Oreochromis species, we developed techniques for FISH on SC preparations, combined with DAPI staining. DAPI staining identified presumptive centromeres in SC bivalents, which appeared to correspond to the positions observed in the mitotic karyotype (the kinetochores could only be identified sporadically in silver stained EM SC images). Furthermore, two BAC clones containing Dmo and OniY227 markers that hybridize to known positions in chromosome pair 1 in mitotic spreads (near the centromere, FLpter 0.25, and the putative sex determination locus, FLpter 0.57, respectively) were used as FISH probes on SCs to verify that the presumptive centromere identified by DAPI staining was located in the expected position. Visualization of both the centromere and FISH signals on bivalent 1 allowed the unpaired region to be positioned at Flpter 0.80 to 1.00, demonstrating that the unpaired region is located in the distal part of the long arm(s). Finally, differences between mitotic and meiotic measurements are discussed.


Evidence of high levels of polymorphism in NOR-phenotypes of Iberian nases (Teleostei, Cyprinidae) in hybrid zones caused by massive introgression processes

C. Pereira (1), A. Neto (2), P. Rab (3) , M. Rabova (3) & M.J. Collares-Pereira (1)

1. Universidade Lisboa, Centro Biologia Ambiental, Lisboa (Portugal);
2. Universidade Estadual Ponta Grossa, Paraná (Brazil);
3. Inst. Animal Physiology and Genetics, Libechov (Czech Republic)

Acting as a bridge for new hybrid generations with fitter genotypes introgressive hybridization may be evolutionarily more relevant than has been acknowledged. Due to frequent hybridizing events resulting in fertile offspring, cyprinids are good model organisms. Two of such hybrid zones have been studied in the Iberian rivers involving one of the straight-mouth nases Pseudochondrostoma duriense or P. polylepis and the arched-mouth nase Achondrostoma oligolepis. MtDNA analysis revealed the existence of P. duriense specimens with haplotypes of A. oligolepis indicating introgression of the latter genome into the gene pool of the former. Screening of 10 microsatellite loci and 2 nuclear genes evidenced admixture of parental genomes in several specimens confirming introgressive events at the nuclear level. After revisiting the karyotypes of the 3 parental species, a sub-sample of hybrids was cytogenetically analyzed as regards NOR-phenotypes by sequential banding (FISH with rDNA probes, CMA3 and Ag). CMA3 showed 6 positive signals in A. oligolepis and 4 in P. duriense and P. polylepis submetacentrics. Introgressed fish exhibited higher levels of polymorphism than predicted by simple parental genomes combination. The diverse arrays of polymorphic NORs sites will be presented to evidence the result of introgression in genome restructuring.


Chromosome studies of European leuciscine fishes (Cyprinidae): interspecific homology of leuciscine cytotaxonomic marker - the largest subtelocentric chromosome pair as revealed by cross-species painting

P. Ráb (1), M. Rábová (1), C.S.A. Pereira (2), M.J. Collares-Pererira (2), Š. Pelikánová (1)

1. Laboratory of Fish Genetics, Institute of Animal Physiology and Genetics, Czech Academy of Sciences, 277 21 Liběchov (Czech Republic);
2. Universidade de Lisboa, Faculdade de Ciincias, Centro de Biologia Ambiental, Campo Grande, 1749-016 Lisboa (Portugal).

Leuciscine cyprinids possess 2n = 50 and extremely uniform karyotype with 8 pairs of m, 13 -15 pairs of sm and 2-4 pairs of st/a chromosomes. The largest pair is characteristically st/a element - "leuciscine" cytotaxonomic marker. However, the interspecific homology of this chromosome pair could not be assessed due to inability to produce serial banding patterns in fish chromosomes. In present pilot study, we used laser-microdissected (10 - 15 copies of marker chromosome) whole chromosome probe (WCP) from karyotype of roach, Rutilus rutilus to ascertain the interspecific homology of marker chromosome using cross-species in situ hybridization. WCP was hybridized to chromosomes of widely distributed (R. rutilus, Scardinius erythrophthalmus, Alburnoides bipunctatus, Alburnus alburnus, Aspius aspius, Leucaspius delineatus, Abramis brama, Blicca bjoerkna, Ballerus ballerus, Ballerus sapa, Vimba vimba, Leuciscus leuciscus, L. idus, Squalius cephalus, Phoxinus phoxinus, Chondrostoma nasus) and Iberian endemic species (S. alburnoides, S. pyrenaicus, Achondrostoma oligolepis, Iberochondrostoma almacai, I. lusitanicum, Pseudochondrostoma duriense). WCP consistently hybridized to the various proportions of distal part of longer arm indicating either sequence homology here and/or problem with production of WCP.


Molecular and cytogenetic analysis of a non-LTR retrotrasposon in Polypterid fishes

L. Rocco, B. De Felice, M.A. Morescalchi, A. Archimandritis, V. Stingo

Dipartimento di Scienze della Vita, Via Vivaldi 43, Caserta (Italy)

Polypteriformes are a group of Osteichthyan fish that have been placed into a number of widely taxonomic groups, but today most authors agree in assigning to them a peculiar place in the evolutionary history of bony fish. Little is known about Polypteriformes; in order to expand current knowledge on these vertebrates we isolated and characterized for the first time Rex3, a non-long-terminal-repeat element related to the RTE family. From all autonomous fish retrotransposons reported to date, Rex3 has the widest distribution observed in teleosts and is present in species having diverged 150-200 MYA.

We report here a large PCR- and Southern blot-based assessment of Rex3 reverse transcriptase sequences in Polypteriformes representative species, including P. ornatipinnis, P. buettikoferi, P. delhezi and E. calabaricus.

Since the literature data on chromosome morphology in the whole Polypteriformes group are poor, we performed a cytogenetic analysis to detect and localize the presence or absence of the retrotrasposon on the chromosomes of P.ornatipinnis, using a fluorescence in situ hybridization (FISH). The chromosome location of Rex3 examined evidenced that intense hybridization signals are located at subtelomeric level of three chromosome pairs. These additional information on Polypteriformes might provide important data for understanding the mechanisms of chromosome evolution and phylogenetic relationships in these ancient fish species.


Molecular characterization and chromosomal localization of spermatogenesis related sequences in Torpedo ocellata (Chondrichthyes, Torpediniformes)

L. Rocco, S. Bencivenga, A. Archimandritis, I. Liguori, V. Stingo

Dipartimento di Scienze della Vita, Seconda Università di Napoli, via Vivaldi 43, Caserta (Italy)

Cytogenetic data in cartilaginous fishes are very poor, if we consider that the karyotype morphology of only about 6% of living species is actually known. Only in the last few years different molecular approaches, first of all physical mapping on metaphase chromosomes, have been used to investigate the cytotaxonomic relationship existing among the living Elasmobranchs. The aim of the work was to characterize a molecular chromosomal marker for the sexual differentiation in the species Torpedo ocellata, since its karyotype does not exhibit sexual chromosomes. Using specific primers taken from highly conserved SRY sequences in mammals, we characterize these regions at molecular level and localize them on metaphase chromosomes. The PCR products have revealed similar patterns in specimen of both sexes of Torpedo ocellata, but only the 400 bp fragment of male amplification product showed a highly percentage of identity with human genes SPATA 16, SPATA 18 and UTY. These genes in the man carry out a crucial role in the spermatogenesis process. FISH with these sequences evidenced the presence of spots at subtelomeric level of two chromosome pairs in the male and of one pair in the female. Finally these sequences are useful as chromosome markers for the sexual differentiation between the male and the female karyotypes.


Cytogenetic characterization of the dwarf oyster (Ostreola stentina) and comparative karyological analysis within Ostreinae

J Pereira (1), A Leitão (1,2), R Chaves (1), F M Batista (1,3), H Guedes-Pinto (1)

1. Institute for Biotechnology and Bioengineering, Centre of Genetics and Biotechnology (CGB-UTAD/IBB), 5001-801 Vila Real (Portugal)
2. INRB/L-IPIMAR. Avenida 5 de Outubro, 8700-305 Olhão (Portugal)
3. School of Ocean Sciences, Bangor University, Menai Bridge, Gwynedd, LL59 5AB, (UK).

Oysters are among the best studied groups of all marine invertebrates, however, and despite their high economic importance and large geographic distribution, the current knowledge of oyster phylogenetic relationships and systematics remains scarce. This lack is particularly evident in the subfamily Ostreinae of the flat oyster species. We performed a molecular cytogenetic study of O. stentina (Payradeau, 1826), the Provence oyster or dwarf oyster. The chromosomes of O. stentina were studied using conventional Giemsa staining, chromosome measurements, C-and restriction endonuclease banding, and fluorescent in situ hybridization (FISH). Comparative analysis of the different karyotype patterns obtained for this species to those of other flat oyster for which data has been previously published was performed and provided new insights into oyster evolution and systematics within this family.

This study also demonstrates that besides offering a new approach to specific problems in cytotaxonomics, molecular cytogenetic applications are diverse and can constitute a fundamental step in gene mapping in this economic important group of bivalves.

Supported by: PhD grant SFRH/BD/27720/2006 of Science and Technology Foundation (FCT) from Portugal. We thank IPIMAR for the help in the harvesting of samples collection.


Gene mapping of 28S rDNA sites in allotriploid Cobitis females (Pisces, Cobitidae) from a diploid-polyploid population

L. Kirtiklis, A. Boroń, A. Grabowska, M. Skórczyńska

Department of Zoology, Faculty of Biology, University of Warmia and Mazury, Olsztyn (Poland)

Among many natural Cobitis populations some polyploid hybrid forms were detected. There is a little information about genome rearrangement regarding diplo- and polyploid individuals. In present study we performed cytogenetic analysis on triploid Cobitis hybrid specimens from a natural diploid-polyploid population of Bug River (Poland). Chromosomes were examined by Fluorescence in situ hybridization method (FISH) with 28S rDNA as a probe. The rDNA sites were identified in the terminal telomeric position of three submetacentric and four subtelo-acrocentric chromosomes. Moreover some of observed signals were stronger then others. One small sm/st chromosome possessing rDNA sites on both p and q arms seemed to be a marker of the karyotype of allotriploid females. Our study brings useful information about polymorphism of 28S rDNA sites regarding their number, location and size in triploid specimens. Moreover, it gives a preliminary data for comparison with similar on the parental species of Cobitis hybrid females, viz. C. taenia and C. tanaitica, and other related taxa of different ploidy level. Finally presented and further studies may increase our knowledge regarding phylogenetic examinations and ribosomal gene inheritance in natural Cobitis populations.