, Volume 111, Issue 1–3, pp 413–422 | Cite as

Extensive polymorphism and chromosomal characteristics of ribosomal DNA in a loach fish, Cobitis vardarensis (Ostariophysi, Cobitidae) detected by different banding techniques and fluorescence in situ hybridization (FISH)

  • M. Rábová
  • P. Ráb
  • C. Ozouf-Costaz


When surveying the karyotype diversity of European loaches of the genus Cobitis to identify species involved in hybrid polyploid complexes, an extensive polymorphism in number and location of NORs was discovered in C. vardarensis using Ag-staining, C-banding, CMA3-fluorescence and fluorescence in situ hybridization (FISH). This species had 2n = 50, the karyotype contained 13 pairs of metacentric, 10 pairs of submetacentric and two pairs of subtelocentric chromosomes. The NOR-bearing chromosomes included one medium-sized metacentric pair with a large CMA3-positive heterochromatic pericentromeric block, one small metacentric as well as one large submetacentric pairs. Ribosomal sites were always located in telomeres of these chromosomes. Each of the pair of NOR-bearing chromosomes occurred in three variants – (1) presence and/or (2) absence of NORs on both homologues and (3) heterozygous combination where only one of the homologues bears NORs. Altogether, 10 different NOR cytotypes from 27 theoretically possible ones were discovered among 20 indviduals examined. The number of NORs ranged from two to five per specimen. The results regarding the number and locations of NORs as revealed by banding techniques were confirmed using FISH with rDNA probe. NOR sites were of CMA3-positive, suggesting that ribosomal sites are associated with GC-rich DNA. Very similar structural polymorphism with multiple NORs is expressed in the Danubian loach C. elongatoides indicating a close relationship between both species.

chromosome banding cytotaxonomy of Cobitis loaches FISH karyotype NOR phenotype and polymorphism rDNA 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Boroń, A., 1999. Banded karyotype of spined loach Cobitis taenia and triploid Cobitis from Poland. Genetica 105: 293–300.Google Scholar
  2. Boroń, A. & J. Kotusz, 1999. A preliminary report and karyotype of a new Cobitis species in the ichthyofauna of Poland. Cytobios 98: 59–64.Google Scholar
  3. Collares-Pereira, M.J. & P. Ráb, 1999. NOR polymorphism in the Iberian species Chondrostoma lusitanicum (Pisces:Cyprinidae) - re-examination by FISH. Genetica 105: 301–303.Google Scholar
  4. De Lucchini, S., I. Nardi, G. Barsacchi, R. Bastistoni & F. Andronico, 1993. Molecular cytogenetics of the ribosomal (18S+28S and 5S) DNA loci in primitive and advanced urodele amphibians. Genome 36: 762–773.Google Scholar
  5. Economidis, P.S. & T.T. Nalbant, 1996. A study of the loaches of the genera Cobitis and Sabanejewia (Pisces, Cobitidae) of Greece, with description of six new taxa. Trav. Mus. Natl. Hist. Nat. ‘Grigore Antipa' 36: 295–347.Google Scholar
  6. Erkanan, F., F.G. Atalay-Ekmekçi & T.T. Nalbant, 1998. Four new species and one new subspecies of the genus Cobitis (Pisces: Ostariophysi: Cobitidae) from Turkey. Tr. J. Zool. 22: 9–15.Google Scholar
  7. Erkanan, F., F.G. Atalay-Ekmekçi & T.T. Nalbant, 1999. A review of the genus Cobitis in Turkey (Pisces: Ostariophysi: Cobitidae). Hydrobiologia 403: 13–26.Google Scholar
  8. Fujiwara A., S. Abe, E. Yamaha, F. Yamazaki & M.C. Yoshida, 1998. Chromosomal localization and heterochromatin association of ribosomal regions in salmonid fishes. Chrom. Res. 6: 463–471.Google Scholar
  9. Galetti P.M. & E.M. Rasch, 1993. Chromosome studies in Poecilia latipunctata with NOR polymorphism as shown by silver nitrate and chromomycin A3 (Teleostei: Poeciliidae). Ichthyol. Explor. Freshwaters 4: 269–277.Google Scholar
  10. Galetti P.M., F. Foresti, L.A.C. Bertollo & O. Moreira-Filho,1984. Characterization of eight species of Anostomidae (Cyprini formes) fish on the basis of the nucleolar organizing region. Caryologia 37: 401–406.Google Scholar
  11. Gollmann G, Y. Bouvet, Y. Karakousis & C. Triantaphyllidis, 1997. Genetic variability in Chondrostoma from Austrian, French and Greek rivers (Teleostei, Cyprinidae). J. Zool. Syst. Evol. Res. 35: 165–169.Google Scholar
  12. Haaf, T. & M. Schmid, 1984. An early stage of ZW/ZZ sex chromosome differentiation in Poecilia sphenops var. melanistica (Poeciliidae, Cyprinodontiformes). Chromosoma 89: 37–41.Google Scholar
  13. Howell, W.M. & D.A. Black, 1980. Controlled silver staining of nucleolus organizer regions with a protective colloidal developer: a 1–step method. Experientia 36: 1014–1015.Google Scholar
  14. Jankun, M., P. Martínez, B.G. Padro, P. Ráb, M. Rábová & L. Sanchez, 2000. rRNA genes map to chromosomes 10, 11 and 12 in European whitefish (Coregonus lavaretus) and to chromosomes 1, 5, 9 and 10 in vendace (Coregonus albula). Chrom. Res. 8: 455.Google Scholar
  15. Levan, A., K. Fredga & A.A. Sandberg, 1964. Nomenclature for centromeric position on chromosomes. Hereditas 52: 201–220.Google Scholar
  16. Long, E.O. & I.D. David, 1980. Repeated genes in eucaryotes. Ann. Rev. Biochem. 49: 727–764.Google Scholar
  17. Martínez P., A. Vinas, C. Bouza, J. Arias, R. Amaro & L. Sanchez, 1991. Cytogenetical characterization of hatchery stocks and natural populations of sea and brown trout from northwestern Spain. Heredity 66: 9–17.Google Scholar
  18. Mellink C.H.M., A.A. Bosma, N.A. De Haan & A.A. Macdonald, 1992. Numerical variation of nucleolar organizer regions after silver staining in domestic and wild Suidae (Mammalia). Anim. Genet. 23: 231–239.Google Scholar
  19. Moss, T. & V.Y. Stefanovsky, 1995. Promotion and regulation of ribosomal transcription by RNA polymerase I. Prog. Nucl. Acid Res. 50: 207–250.Google Scholar
  20. Padilla, J.A., J.L. Fernández-García, A. Rabasco, M. Martinez-Trancon, I. Rodriguez de Ledesma & J.J. Perez-Regardera, 1993. Characterization of the karyotype of the tench (Tinca tinca L.) and analysis of its chromosomal heterochromatic regions by C-banding, Ag-staining, and restriction endonuclease banding. Cytogenet. Cell Genet. 62: 220–223.Google Scholar
  21. Pendás, A.M., P. Morán & E. García-Vazquez, 1993. Multichromosomal location of ribosomal RNA genes and heterochromatin associations in brown trout. Chrom. Res. 1: 63–67.Google Scholar
  22. Phillips, R.B., K.D. Zajicek & F.M. Utter, 1986. Chromosome banding in salmonid fishes: nucleolar organizer regions in Oncorhynchus. Can. J. Genet. Cytol. 28: 502–510.Google Scholar
  23. Ozouf-Costaz, C., E. Pisano, C. Bonillo & R. Williams, R. 1996: Ribosomal RNA location in the Antarctic fish Champsocephalus gunnari (Notothenioidei, Channichthyidae) using banding and fluorescence in situ hybridization. Chrom. Res. 4: 557–561.Google Scholar
  24. Ráb, P. & P. Roth, 1988. Cold-blooded vertebrates, pp. 115–124 in Methods of Chromosome Analysis, edited by P. Balíček, J.Forejt, J. & J. Rubeš., Czechoslovak Biol. Soc. Publishers, Brno.Google Scholar
  25. Ráb, P. & O. Slavík, 1996. Diploid-triploid-tetraploid complex of the spined loach, genus Cobitis in Pšovka Creek: the first evidence of new species of Cobitis in the ichthyofauna of the Czech Republic. Acta Univ. Carol - Biol. 39: 201–214.Google Scholar
  26. Ráb, P., K.M. Reed, F.A. Ponce de Leon & R.B. Phillips, 1996. A new method for detection nucleolus organizer regions in fish chromosomes using denaturation and propidium iodide staining. Biotech. Histochem. 71: 157–162.Google Scholar
  27. Ráb, P., M. Rábová, K.M. Reed & R.B. Phillips, 1999. Chromosomal characteristics of ribosomal DNA in the primitive semionotiform fish, longnose gar Lepisosteus osseus. Chrom. Res. 7: 475–480.Google Scholar
  28. Ráb, P., M. Rábová, J. Bohlen & S. Lusk, 2000a. Genetic differentiation of the two hybrid diploid-polyploid complexes of loaches, genus Cobitis (Cobitidae) involving C. taenia, C. elongatoides and C. sp. in the Czech Republic: karyotypes and cytogenetic diversity. Folia Zool. 49(Suppl. 1): 55–66.Google Scholar
  29. Ráb, P., M. Rábová, P.S. Economidis & C. Triantaphyllidis, 2000b. Banded karyotype of Greek endemic cyprinid fish Pachychilon macedonicum. Ichthyol. Res. 47: 107–110.Google Scholar
  30. Reed, K.M. & R.B. Phillips, 1995. Molecular cytogenetic analysis of the double-CMA3 chromosome of lake trout, Salvelinus namaycush. Cytogenet. Cell Genet. 70: 104–107.Google Scholar
  31. Rodrigues, E. & M.J. Collares-Pererira, 1996. NOR polymorphism in the Iberian species Chondrostoma lusitanicum (Pisces:Cyprinidae). Genetica 98: 59–63.Google Scholar
  32. Schmid M., W. Feichtinger, R. Weimer, C. Mais, F. Bolaños & P. León, 1995. Chromosome banding in Amphibia. XXI. Inversion polymorphism and multiple nucleolus organizer regions in Agalychnis callidryas (Anura, Hylidae). Cytogenet. Cell Genet. 69: 18–26.Google Scholar
  33. Sola L., A.R. Rossi, V. Iaselli, E.M. Rasch & P.J. Monaco, 1992. Cytogenetics of bisexual/unisexual species of Poecilia. II. Analysis of heterochromatin and nucleolar organizer regions in Poecilia mexicana maxicana by C-banding and DAPI, quinacrine, chromomycin A3, and silver staining. Cytogenet. Cell Genet. 60: 229–235.Google Scholar
  34. Souza I.L. & O. Moreira-Filho, 1995. Cytogenetic diversity in the Astyanax scabripinnis species complex (Pisces, Characidae). I. Allopatric distribution in a small stream. Cytologia 60: 1–11.Google Scholar
  35. Takai, A. & Y. Ojima, 1986. Some features on nucleolus organizer regions on fish chromosomes, pp: 899–909 in Indo-Pacific Fish Biology: Proc. 2nd Int. Conf. Indo-Pacific Fishes, edited by T. Uyeno, R. Arai, T. Taniuchi, T. & K. Matsura. Ichthyological Society of Japan, Tokyo.Google Scholar
  36. Volleth, M., 1987. Differences in location of nucleolus organizer regions in European vespertilinid bats. Cytogenet. Cell Genet. 44: 186–197.Google Scholar
  37. Vasil'ev, V.P., 1995. Karyotype diversity and taxonomic nonhomogeneity of Cobitistaenia’ (Pisces, Cobitidae). Dokl. Akad. Nauk. 342: 839–842.Google Scholar
  38. Zhuo L., K.M. Reed & R.B. Phillips, 1995. Hypervariability of ribosomal DNA at multiple chromosomal sites in lake trout (Salvelinus namaycush). Genome 38: 487–496.Google Scholar

Copyright information

© Kluwer Academic Publishers 2001

Authors and Affiliations

  • M. Rábová
    • 1
  • P. Ráb
    • 2
  • C. Ozouf-Costaz
    • 3
  1. 1.Laboratory of Fish Genetics, Institute of Animal Physiology and GeneticsCzech Academy of SciencesLiběchovCzech Republic
  2. 2.Laboratory of Fish Genetics, Institute of Animal Physiology and GeneticsCzech Academy of SciencesLiběchovCzech Republic
  3. 3.Service commun de Systématique moléculaire (CNRS FR 1541) & Laboratoire d’Ichtyologie generale et appliqueéMuseum national d’histoire naturelleParis Cedex 05France

Personalised recommendations