European Journal of Histochemistry 2011; volume 55:e12 Identification of two new repetitive elements and chromosomal mapping of repetitive DNA sequences in the fish Gymnothorax unicolor (Anguilliformes: Muraenidae) E. Coluccia,1 G. Pichiri,2 M. Nieddu,2 P. Coni,3 S. Manconi,2 A. M. Deiana,1 S. Salvadori,1 R. Mezzanotte2 1Dipartimento di Biologia Animale ed Ecologia, Università di Cagliari, Italy; 2Dipartimento di Scienze e Tecnologie Biomediche, Sez. Biologia e Genetica, Università di Cagliari, Italy; 3Dipartimento di Citomorfologia, Sez. Anatomia Patologica, Università di Cagliari, Italy Abstract Muraenidae is a species-rich family, with relationships among genera and species and taxonomy that have not been completely clari- fied. Few cytogenetic studies have been con- ducted on this family, and all of them showed the same diploid chromosome number (2n=42) but with conspicuous karyotypic vari- ation among species. The Mediterranean moray eel Gymnothorax unicolor was previ- ously cytogenetically studied using classical techniques that allowed the characterization of its karyotype structure and the constitutive heterochromatin and argyrophilic nucleolar organizer regions (Ag-NORs) distribution pat- tern. In the present study, we describe two new repetitive elements (called GuMboI and GuDdeI) obtained from restricted genomic DNA of G. unicolor that were characterized by Southern blot and physically localized by in situ hybridization on metaphase chromo- somes. As they are highly repetitive DNA sequences, they map in heterochromatic regions. However, while GuDdeI was localized in the centromeric regions, the GuMboI frac- tion was distributed on some centromeres and was co-localized with the nucleolus organizer region (NOR). Comparative analysis with other Mediterranean species such as Muraena helena pointed out that these DNA fractions are species-specific and could potentially be used for species discrimination. As a new con- tribution to the karyotype of this species, we found that the major ribosomal genes are local- ized on acrocentric chromosome 9 and that the telomeres of each chromosome are composed of a tandem repeat derived from a poly- TTAGGG DNA sequence, as it occurs in most vertebrate species. The results obtained add new information useful in comparative genomics at the chromosomal level and con- tribute to the cytogenetic knowledge regarding this fish family, which has not been extensive- ly studied. Introduction A large portion of eukaryotic genomes is composed of repetitive DNA sequences, that include tandem repeats such as satellites, min- isatellites, microsatellites, and interspersed repeats as transposable elements.1,2 In particu- lar, satellite DNA consists of the same sequence, ranging in length from a few base pairs (bp) to thousands of bp, repeated thou- sands or millions of times in tandem.3,4 Repetitive DNA makes up the predominant part of heterochromatin and is located mainly in pericentromeric and/or telomeric chromo- some regions.3,5 This fraction of the genome seems to escape the selective pressure actings on the non-repetitive segments, representing good evolutionary marker in studies of species evolution, chromosome structure and function, and in the detection of chromosomal rearrangements, supernumerary and sex chro- mosomes.6-8 The molecular organization and chromosome location of repetitive DNAs have been analyzed in a large number of fish species.9-13 These studies have demonstrated the enormous potential that the investigation of repetitive DNAs offers in extending our knowledge of karyotype differentiation in fish. Muraenidae is a species-rich family (185 species) of tropical and subtropical fish, dis- tributed worldwide and especially common on coral reefs.14 The current classification is based mainly on morphological and anatomical characteristics, but the taxonomy remains incomplete; in particular, recent molecular phylogenetic and cytogenetic data do not sup- port clear separation between the Muraena and Gymnothorax genera.14-18 Cytogenetically, only approximately 7% of the Muraenidae species has been studied, and for most of them, only karyotype morphology and heterochromatin distribution are known. They show a conserved chromosome number, genome size and AT-content, with the highest values among Anguilliformes.19-21 Chromosome numerical similarity, with a 42 diploid number reported for all species, is accompanied by great karyotype variation [fundamental num- bers (FN) = 42 to 84]; in this group, pericen- tric inversions may have played an important role in karyotype evolution.22,23 The pattern of heterochromatin distribution is known for eight Muraenidae species; these few available Correspondence: Dr. Elisabetta Coluccia, Dipartimento di Biologia Animale ed Ecologia, Università di Cagliari, via T. Fiorelli, 1, 09126 Cagliari, Italy. Tel: +39.070.6758003 - Fax: +39.070.6758022. E-mail: coluccia@unica.it Key words: anguilliformes, muraenidae, hete- rochromatin, nucleolus organizer region, repeti- tive DNA, restriction enzymes. Acknowledgements: the authors wish to thank the anonymous referees for helpful suggestions and critical review of the manuscript and Mrs. Mary Ann Groeneweg for checking the English style. Contributions: all authors contributed equally to the manuscript conception, design, revision and final approval. Conflict of interest: the authors report no con- flicts of interest. Received for publication: 15 December 2010. Accepted for publication: 23 February 2011. This work is licensed under a Creative Commons Attribution 3.0 License (by-nc 3.0). ©Copyright E. Coluccia et al., 2011 Licensee PAGEPress, Italy European Journal of Histochemistry 2011; 55:e12 doi:10.4081/ejh.2011.e12 data indicate the presence of centromeric het- erochromatic blocks in almost all species; fur- thermore, large bands in pericentromeric and/or interstitial position are present in some species.20,22,24 The nucleolus organizer regions (NOR) were localized by silver staining on one chromosome pair in all species studied; fur- thermore, fluorescence in situ hybridization (FISH) mapping of major ribosomal genes and telomeric sequences was carried out in M. helena and G. tile.18,25,26 In addition, two repeti- tive DNA fractions from the M. helena genome, were isolated, physically mapped and com- pared to G. unicolor.25,27 Previous cytogenetic studies characterized the karyotype of the Mediterranean moray eel Gymnothorax unicol- or (Delaroche, 1809) by heterochromatic (C-, restriction enzyme- and CMA3-) and replica- tion- banding and Ag-staining.20,23,28 Cytoge- netic comparison with the other Medi- terranean species Muraena helena and with the congeneric species G. tile pointed out many chromosomal banding similarities, as well as the occurrence of pericentric inver- sions and changes in heterochromatin amount.18 In the present study, we carried out the iso- lation, molecular characterization and chromo- somal mapping of two repeated DNA fractions in the G. unicolor genome and localized the [page 68] [European Journal of Histochemistry 2011; 55:e12]