Indian Journal of Experimental Biology Vol. 47, July 2009, pp. 597-601 Physical mapping of rRNA gene in endangered fish Osteobrama belangeri (Valenciennes, 1844) (Family: Cyprinidae) Ravindra Kumar*, B Kushwaha, N S Nagpure, B K Behera † , S K Srivastava & W S Lakra National Bureau of Fish Genetic Resources, Canal Ring Road, P.O. Dilkusha, Lucknow 226 002, India † ICAR Complex for NEH Region, Manipur Center, Lamphelpat, Imphal 795 004, India Received 1 September 2008; revised 25 March 2009 Physical mapping of the 18S ribosomal RNA gene (rDNA) was carried out by fluorescent in situ hybridization (FISH) in the endangered freshwater fish O. belangeri. The specimens were collected from Imphal valley, Manipur, India and metaphase chromosome preparation was made using standard hypotonic treatment, methanol-acetic acid fixation and flame- drying technique followed by Giemsa, silver, CMA 3 staining for complete cytogenetic characterization of the species. The diploid chromosome number was found to be 50 and the karyotype composed of 6m+16sm+12st+16t (FN=72). One active rDNA site, located on short arm of 3 rd submetacentric chromosome, was mapped by FISH and confirmed by silver and CMA 3 staining. The karyomorphology, chromosomal location of rDNA loci in this species and the utility of these cytogenetic markers have been discussed in the paper. Keywords: Ag-NORs, Chromosome, CMA 3 , FISH, Osteobrama belangeri Genetic characterization of fish species particularly that of threatened or economically important species is useful for planning their conservation strategies for safeguarding biodiversity. The fish species, Osteobrama belangeri (Valenciennes, 1844; family: Cyprinidae; order: Cypriniformes), locally known as pengba, is a medium carp found in rivers and lakes of India (Manipur), Myanmar and China. It is moderate to highly vulnerable and categorized as ‘Not Evaluated’ by FAO (www.fishbase.org), as ‘Extinct in wild’ in the Conservation Assessment and Management Plan (CAMP) workshop conducted at National Bureau of Fish Genetic Research, Lucknow during 1997 1 , as ‘Threatened’ 2 and as ‘Endangered’ 3,4 . In the past, Osteobrama belangeri formed a big fishery in Loktak lake but now this species has become rare or almost exterminated there because of the introduction of common carp. This species has not been investigated genetically so far and no information is available on the karyomorphology. Cytogenetic markers have been considered as reliable tools for characterization of fish species as well as to screen putative hybrids 5 . Some of the classical cytogenetic markers have been utilized earlier for characterization of fish species and also have application in revealing phylogenetic relationship and resolving taxonomic ambiguity among related species by comparison of chromosome morphology and staining (like G-, NORs, CMA 3 ) pattern 6,7 . The physical maps of genes by fluorescence in situ hybridization (FISH) represent a potentially new source of chromosomal characters that may be cytogentically informative. So far, physical mapping has been focused mainly on highly repetitive DNA or multigene families because of the technical difficulties encountered for mapping low-copy genes 8 . Repetitive DNAs have been applied extensively as physical chromosome markers in comparative genome, chromosome evolution and characterization studies, identification of chromosome rearrangements and sex chromosomes and also in applied genetics 9 . Ribosomal DNA, the most frequently mapped repetitive gene, has emerged as a good marker for species characterization and population genetic studies. Mapping of 18S rRNA gene on the chromosome of O. belangeri using FISH has been undertaken to determine the number and location of the rDNA loci in this species and to characterize the species ______________ *Correspondent author Téléphone: +91-522 -2442440, 2441735, 2442441 Fax: +91-522-2442403. E-mail: ravindrakumar@scientist.com; rkumar@nbfgr.res.in