Abstract
Molecular cytogenetic studies were carried out for localization of 18S and 5S ribosomal DNAs on chromosomes of three cyprinid fish species viz., T. khudree, T. mussullah and T. mosal mahanadicus using two color fluorescence in situ hybridization (FISH). All the species typically possessed 100 diploid chromosomes with minor variation in karyo-morphology. The 18S rDNA signals were observed on two pair of chromosomes in T. khudree and T. mussullah, and three pairs in T. mosal mahanadicus. The location of 18S signals also showed affinity to silver nitrate and chromomycin A3 staining. Similarly, variation in localization of 5S rDNA among the three species has been detected with the presence of FISH signals on one pair of chromosome in T. khudree and T. mussullah, and on two pairs in T. mosal mahanadicus. These molecular markers could be used as species specific markers for taxonomic identification and can further add in understanding the dynamics of genome organization and karyotypic evolution of these species. The 18S rDNA region was sequenced that generated 1811, 1810 and 1776 bp long 18S sequence in T. khudree, T. mussullah and T. mosal mahanadicus, respectively. The 18S rDNA sequence showed 95–98% identity among the subject species. Similarly, 5S sequencing generated 203 bp long fragments in these species with 100% identity in coding and 9.63% variability in non-transcribed spacer regions. The nucleotide sequence variations could be used for understanding the genetic diversity and will add new informative characters in comparative genomics. These results, in general, would enhance the value and interpretation of ecological assessment data for conservation of Tor species.
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References
Phillips RB, Reed KM (2000) Localization of repetitive DNAs to zebrafish (Danio rerio) chromosomes by fluorescence in situ hybridization (FISH). Chromosome Res 8:27–35. doi:10.1023/A:1009271017998
Long EO, David ID (1980) Repeated genes in eukaryotes. Annu Rev Biochem 49:727–764. doi:10.1146/annurev.bi.49.070180.003455
Insua A, Freire R, Rios J, Mendez J (2001) The 5S rDNA of mussels Mytilus galloprovincialis and M. edulis: sequence variation and chromosomal location. Chromosome Res 9:495–505. doi:10.1023/A:1011636714052
Little RD, Braaten DC (1989) Genomic organization of human 5S rDNA and sequence of one tandem repeat. Genomics 4:376–383 PMID: 2714796
Vicari MR, Artoni RF, Moreira-Filho O, Bertollo LAC (2008) Colocalization of repetitive DNAs and silencing of major rRNA genes. A case report of the fish Astyanax janeiroensis. Cytogenet Genome Res 122:67–72. doi:10.1159/000151318
Ferro DAM, Neo DM, Moreira-Filho O, Bertollo LAC (2001) Nucleolar organizing regions, 18S and 5S rDNA in Astyanax scabripinnis (Pisces, Characidae): population distribution and functional diversity. Genetica 110:55–62
Fujiwara A, Abe S, Yamaha E, Yamazaki F, Yoshida MC (1998) Chromosomal localization and heterochromatin association of ribosomal regions in salmonid fishes. Chromosome Res 6:463–471. doi:10.1023/A:1009200428369
Martinez JL, Moran P, Garcia-Vazquez E, Pendas AM (1996) Chromosomal localization of the major and 5S rRNA genes in the European eel (Anguilla anguilla). Cytogenet Cell Genet 73:149–152. doi:10.1159/000134328
Martins C, Galetti PM (2000) Conservative distribution of 5S rDNA loci in Schizodon (Pisces, Anastomidae) chromosomes. Chromosome Res 8:353–355. doi:10.1023/A:1009243815280
Fontana F, Lanfredi M, Congiu L, Leis M, Chicca M, Rossi R (2003) Chromosomal mapping of 18S–28S and 5S rRNA genes by two color fluorescent in situ hybridization in six sturgeon species. Genome 46:473–477. doi:10.1139/g03-007
Tigano C, Rocco L, Ferrito V, Costagliola D, Pappalardo AM, Stingo V (2004) Chromosomal mapping and molecular characterization of ribosomal RNA genes in Lebias fasciata (Teleostei, Cyprinodontidae). Genetica 121:95–100. doi:10.1023/B:GENE.0000019931.89458.dc PMID: 15098742
Nelson JS (2006) Fishes of the world, 4th edn. Wiley, Hoboken, p 601
Basavaraja N, Hegde SN (2004) Cryopreservation of the endangered mahseer (Tor khudree) spermatozoa: I. Effect of extender composition, cryoprotectants, dilution ratio, and storage period on post-thaw variability. Cryobiology 49:149–156. doi:10.1016/j.cryobiol.2004.05.007
Raghavan R, Anvar Ali PH, Prasad G (2007) Need for a comprehensive re-assessment of the conservation status of critically endangered (?) freshwater fishes of Kerala. Curr Sci 92(6):721–723
David A (1953) On some new records of fish from the Damodar and the Mahanadi River systems. J Zool Soc India 5(2):243–254
Menon AGK (1992) Taxonomy of mahseer fishes of the genus Tor of gray with description of a new species from the Deccan. J Bombay Nat Hist Soc 89:210–228
Carvalho GR, Hauser L (1995) Molecular genetics and the stock concept in fisheries. In: Carvalho GR, Pitcher TJ (eds) Molecular genetics in fisheries. Chapman & Hall, London, pp 55–80
Neigel JE (1997) A comparison of alternative strategies for estimating gene flow from genetic markers. Annu Rev Ecol Syst 28:105–128. doi:10.1146/annurev.ecolsys.28.1.105
Khuda-Bukhsh AR (1982) Karyomorphology of two species of Tor (Pisces, Cyprinidae) with a high number of chromosomes. Experientia 38:82–83. doi:10.1007/BF01944540
Kushwaha B, Srivastava SK, Nagpure NS, Ogale SN, Ponniah AG (2001) Cytogenetic studies in two species of mahseer, Tor khudree and Tor mussullah (Cyprinidae, Pisces) from India. Chromosom Sci 5:47–50
Lakra WS (1996) Cytogenetic studies on endangered fish species: 1. Karyotype of three species of Mahseers. T. khudree, T. tor and T. putitora (Cyprinidae, Pisces). Cytobios 85:205–218
Lakra WS, Rishi KK (1991) Chromosomes of Indian fishes: an annotated list. Indian J Anim Sci 61:342–349
Mani I, Kumar R, Singh M, Kushwaha B, Nagpure NS, Srivastava PK, Murmu K, Rao DSK, Lakra WS (2009) Karyotypic diversity and evolution of seven mahseer species (Cyprinidae) from India. J Fish Biol 75:1079–1091. doi:10.1111/j.1095-8649.2009.02379.x
Singh M, Kumar R, Nagpure NS, Kushwaha B, Gond I, Lakra WS (2009) Chromosomal localization of 18S and 5S rDNA using FISH in the genus Tor (Pisces, Cyprinidae). Genetica 137:245–252. doi:10.1007/s10709-009-9367-x
Bertollo LAC, Takahashi CS, Moreira-Filho O (1978) Cytotaxonomic consideration on Hoplias lacrdae (Pisces, Erythrinidae). Braz J Genet 1:103–120
Howell WM, Black DA (1980) Controlled silver staining of nucleolus organizer regions with a protective colloidal developer: a one step method. Experientia 36(8):1014–1015. doi:10.1007/BF01953855
Sola L, Rossi AR, Iaselli V, Rasch EM, Monaco PJ (1992) Cytogenetics of bisexual/unisexual species of Poecilia II Analysis of heterochromatin and nucleolar organizer regions in Poecilia mexicana Mexicana by C-banding and DAPI, quinacrine, chromomycin A3, and silver staining. Cytogenet Cell Genet 60:229–235. doi:10.1159/000133346
Sambrook J, Russel DW (2001) Molecular cloning. A laboratory manual, 3rd edn. Cold Spring Harbour Laboratory Press, Plainview
Moran P, Martinez JL, Garcia-Vazquez S, Pendas AM (1996) Sex linkage of 5S rDNA in rainbow trout (Oncorhynchus mykiss). Cytogenet Cell Genet 75:145–150. doi:10.1159/000134466
Winterfeld G, Roser M (2007) Deposition of ribosomal DNAs in the chromosome of perennial oats (Poaceae: Aveneae). Bot J Linn Soc 155:193–210. doi:10.1111/j.1095-8339.2007.00690.x
Tatusova TA, Madden TL (1999) BLAST2 Sequences, a new tool for comparing protein and nucleotide sequences. FEMS Microbiol Lett 174(2):247–250. doi:10.1016/S0378-1097(99)00149-4
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402. doi:10.1093/nar/25.17.3389
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTALX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882 PMID: 9396791
Khuda-Bukhsh AR (1980) A high number of chromosomes in the hill stream cyprinid, Tor putitora (Pisces). Experientia 36:173–174. doi:10.1007/BF01953714
Khuda-Bukhsh AR, Chanda T, Barat A (1986) Karyomorphology and evolution in some Indian hillstream fishes with particular reference to polyploidy in some species. In: Uyeno T, Arai R, Taniuchi T, Matsuura K (eds) Indo Pacific Fish biology: proceedings of the second international conference on Indo Pacific Fishes. Ichthyological Society of Japan, Tokyo, pp 886–898
Manna GK (1983) Cytogenetic studies on fishes and amphibia. In: Genetical research in India. XVth International Congress of Genetics Publication and Information Division, ICAR, New Delhi, pp 242–273
Manna GK (1984) Progress in fish cytogenetics. Nucleus 27:203–231
Pendas AM, Moran P, Freije JP, Garcia-Vazquez E (1994) Chromosomal mapping and nucleotide sequence of two tandem repeats of Atlantic salmon 5S rDNA. Cytogenet Cell Genet 67:31–36. doi:10.1159/000133792
Phillips RB, Matsuoka MP, Reed KM (2002) Characterization of charr chromosomes using fluorescence in situ hybridization. Environ Biol Fish 64:223–228. doi:10.1023/A:1016053902036
Martins C, Galetti PM (1999) Chromosomal localization of 5S rDNA genes in Leporinus fish (Anostomidae, Characiformes). Chromosome Res 7:363–367. doi:0.1023/A:1009216030316
Jankun M, Ocalewicz K, Pardo BG, Martinez P, Woznicki P, Sanchez L (2003) Chromosomal characteristics of rDNA in European grayling Thymallus thymallus (Salmonidae). Genetica 119:219–224. doi:101023/A:1026022415908
Martins C, Wasko AP (2004) Organization and evolution of 5S ribosomal DNA in the fish genome. In: Williams CR (ed) Focus on genome research. Nova Science Publishers, Hauppauge, pp 289–319
Gromicho M, Coutanceau JP, Ozouf-Costaz C, Collares-Pereira MJ (2006) Contrast between extensive variation of 28S rDNA and stability of 5S rDNA and telomeric repeats in the diploidpolyploid Squalius alburnoides complex and in its maternal ancestor Squalius pyrenaicus (Teleostei, Cyprinidae). Chromosom Res 14:297–306
Santos LVDR, Foresti F, Wasko AP, Oliveira C, Martins C (2006) Nucleotide sequence, genomic organization and chromosome localization of 5S rDNA in two species of Curimatidae (Teleostei, Characiformes). Genet Mol Biol 29(2):251–256
Drouin G, Moniz De Sa M (1995) The concerted evolution of 5S ribosomal genes linked to the repeat units of other multigene families. Mol Biol Evol 12:481–493
Andrews MT, Vaughn JC, Perry BA, Bagshaw JC (1987) Interspersion of histone and 5S genes in Artemia. Gene 51:61–67
Barzotti R, Pelliccia F, Bucciarelli E, Rocchi A (2000) Organization, nucleotide sequence, and chromosomal mapping of a tandemly repeated unit containing the four core histone genes and a 5S rRNA gene in an isopod crustacean species. Genome 43:341–345
Kupriyanova NS (2000) Conservation and variation of ribosomal DNA in eukaryotes. Mol Biol 34(5):637–647. doi:10.1007/BF02759600
Das JK, Khuda-Bukhsh AR (2007) Preponderance of GC- rich sites in silver-stained nucleolus organizing regions of Rita rita (Hamilton) and Mystus gulio (Hamilton) (Bagridae, Pisces), as revealed by chromomycin A3-staining technique and scanning electron microscopic studies. Genet Mol Res 6(2):284–291
Das JK, Khuda-Bukhsh AR (2007) GC- rich heterochromatin in silver-stained nucleolar organizer (NORs) fluoresces with chromomycin A3 (CMA3) staining in three species of teleostean fishes (Pisces). Indian J Exp Biol 45:413–418
Gold JR, Zoch PK (1990) Intraspecific variation in chromosomal nucleolus organizer regions in Notropis chrysocephalus (Pisces; cyprinidae). Southwest Nat 35:211–215
Tang W, Tseng H (1999) A GC-rich sequence within the 5′ untranslated region of human basonuclin mRNA inhibits its translation. Gene 237(1):35–44. doi:10.1016/S0378-1119(99)00299-1
Hallenberg C, Nederby-Nielson J, Frederiksen S (1994) Characterization of 5S rRNA genes from mouse. Gene (Amst) 142:291–295. doi:10.1016/0378-1119(94)90277-1
Martins C, Wasko AP, Oliveira C, Wright JM (2000) Nucleotide sequence of 5S rDNA and localization of the ribosomal RNA genes to metaphase chromosomes of the Tilapiine cichlid fish, Oreochromis niloticus. Hereditas 133:39–46. doi:10.1111/j.1601-5223.2000.00039.x
Korn LJ (1982) Transcription of Xenopus 5S ribosomal RNA genes. Nature 295:101–105. doi:10.1038/295101a0
Geiduschek EP, Tocchini-Valentini GP (1988) Transcription by RNA polymerase III. Annu Rev Biochem 57:873–914. doi:10.1146/annurev.bi.57.070188.004301
Huang Y, Maraia RJ (2001) Comparison of the RNA polymerase III transcription machinery in Schizosaccharomyces pombe, Saccharomyces cerevisiae and human. Nucleic Acids Res 29:2675–2690
Sajdak SL, Reed KM, Phillips RB (1998) Intraindividual and interspecies variation in the 5S rDNA of coregonid fish. J Mol Evol 46:680–688. doi:10.1007/PL00006348
Fujiwara M, Inafuku J, Takeda A, Watanabe A, Fujiwara A, Kohno S, Kubota S (2009) Molecular organization of 5S rDNA in bitterlings (Cyprinidae). Genetica 135:355–365. doi:10.1007/s10709-008-9294-2
Cronn RC, Zhao X, Paterson AH, Wendel JF (1996) Polymorphism and concerted evolution in a tandemly repeated gene family: 5S ribosomal DNA in diploid and allopolyploid cottons. J Mol Evol 42:685–705 PMID: 8662014
Ferreira IA, Oliveira C, Venere PC, Galetti PM Jr, Martins C (2007) 5S rDNA variation and its phylogenetic inference in the genus Leporinus (Characiformes: Anostomidae). Genetica 129:253–257
Pendas AM, Moran P, Martinez JL, Garcia-Vazquez E (1995) Applications of 5S rDNA in Atlantic salmon, brown trout, and in Atlantic salmon x brown trout hybrid identification. Mol Ecol 4:275–276
Wasko AP, Martins C, Wright JM, Galetti PM Jr (2001) Molecular organization of 5S rDNA in fishes of the genus Brycon. Genome 44:893–902
Eickbush TH, Burke WD, Eickbush DG, Lathe WC III (1997) Evolution of R1 and R2 in the rDNA units of the genus Drosophila. Genetica 100:49–61
Jakubczak JL, Xiong Y, Eickbush TH (1990) Type I (R1) and type II (R2) ribosomal DNA insertions of Drosophila melanogaster are retrotransposable elements closely related to those of Bombyx mori. J Mol Biol 212:37–52
Jakubczak JL, Zenni MK, Woodruff RC, Eickbush TH (1992) Turnover of R1 (type I) and R2 (type II) retrotransposable elements in the ribosomal DNA of Drosophila melanogaster. Genetics 131:129–142
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The authors are thankful to the Department of Biotechnology (DBT), the Ministry of Science and Technology, Government of India for their financial support.
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Mani, I., Kumar, R., Singh, M. et al. Nucleotide variation and physical mapping of ribosomal genes using FISH in genus Tor (Pisces, Cyprinidae). Mol Biol Rep 38, 2637–2647 (2011). https://doi.org/10.1007/s11033-010-0405-7
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DOI: https://doi.org/10.1007/s11033-010-0405-7