Cytogenet Cell Genet 88:50–55 (2000) Karyotype and chromosome location of characteristic tandem repeats in the pufferfish Tetraodon nigroviridis C. Fischer, a C. Ozouf-Costaz, b H. Roest Crollius, a C. Dasilva, a O. Jaillon, a L. Bouneau, a C. Bonillo, b J. Weissenbach, a and A. Bernot a a Genoscope/Centre National de Séquençage, Evry, and b Muséum national d’histoire naturelle, Service commun de Systématique moléculaire (CNRS FR 1541) et Laboratoire d’Ichtyologie générale et appliquée, Paris (France) Received 29 July 1999; revision accepted 30 October 1999. Request reprints from Dr. Cécile Fischer, Genoscope/Centre National de Séquençage, 2 rue Gaston Crémieux, CP 5706, F-91057 Evry Cedex (France); telephone: +33-1-60-87-25-64; fax: +33-1-60-87-25-89; e-mail: fischer@genoscope.cns.fr. ABC Fax + 41 61 306 12 34 E-mail karger@karger.ch www. karger.com © 2000 S. Karger AG, Basel 0301–0171/00/0882–0050$17.50/0 Accessible online at: www. karger.com/journals/ccg Abstract. Karyotype analysis of Tetraodon nigroviridis, a pufferfish of the family Tetraodontidae with a small compact genome (385 Mb) which is currently being investigated in our laboratory, indicates that this species has 2n = 42 chromo- somes. The small chromosome size (the largest pair measuring less than 3 Ìm) has complicated accurate chromosome pairing based on morphology alone. DAPI staining, however, provides a banding-like pattern. Because of quantitative variations of some heterochromatin classes, the chromosome formula can not be established precisely, but is estimated to include approx- imately 20 meta- or submetacentric chromosomes and 22 sub- telocentric chromosomes. A centromeric satellite, telomeric repeats, and the major and minor rRNA clusters have been localized unequivocally by FISH. As a result, the 28S and 5S rDNA sequences can be used as chromosome-specific probes. Copyright © 2000 S. Karger AG, Basel Tetraodontiform fishes of the family Tetraodontidae exhibit the lowest DNA content per haploid genome of all teleosts so far tested (Hinegardner, 1972). Moreover, this low DNA content is associated with an extremely compact genome (Angrist, 1998; Villard et al., 1998; Chou et al., 1998). These features make puf- ferfish, such as Fugu rubripes and Tetraodon species, suitable for vertebrate genome analysis. However, since Tetraodon can easi- ly be obtained from aquarium fish suppliers, which is not the case with Fugu, we have chosen T. nigroviridis for a large-scale DNA sequence analysis of a compact genome. Such studies must start with a firm knowledge of cytogenetic characteristics. To our knowledge, previous karyological studies conducted on tetraodontiform species were done on Giemsa-stained meta- phase plates, without applying banding techniques (reviewed by Brum et al., 1995). All these studies emphasized the fact that chromosomes of tetraodontiform fishes are difficult to prepare and not easy to recognize because of their small size and other structural features. Within the genus Tetraodon, only the karyo- types of T. cutcutia (Khuda-Bukhsh and Barat, 1987) and T. fluviatilis (Barat and Khuda-Bukhsh, 1984) have been pub- lished. According to Hinegardner (1972), T. fluviatilis is the teleost with the smallest DNA content of all fishes so far tested (0.39 pg DNA per cell). With a genome size of 385 Mb (F. Quetier, unpublished results) and highly similar molecular features (C. Fischer, unpublished results), T. nigroviridis is close to T. flu- viatilis. Nevertheless, little or no cytogenetic or molecular infor- mation on T. nigroviridis have been published to date. As a prerequisite to more detailed investigations involving cytogenetic techniques, we first determined the karyotype of T. nigroviridis. Fluorescence in situ hybridization (FISH) of some classes of repeated sequences commonly used in cytogenetics were useful in refining the karyotype and provided interesting results that will serve as an initial basis for further mapping studies. Materials and methods Animals Ten specimens (two females, two males, and six immature fish) were provided by commercial suppliers. All of the specimens were identified as T. nigroviridis based on morphological and molecular characteristics (Fischer et al., in preparation), but their geographic origin is unknown. One voucher specimen has been deposited in the collections of the Muséum national d’histoire naturelle, Paris, with the reference number MNHN 1999-493 and under the name Tetraodon (Chelonodon) nigroviridis