Journal of Applied Phycology

, Volume 22, Issue 6, pp 709–716 | Cite as

M13-based genotyping of marine cyanobacterial strains from the Indian subcontinent and maintained in the NFMC germplasm collection



The aim of this study was to analyze marine cyanobacterial culture collections strains of the Indian subcontinent at the level below species. This is important to improve the abilities of service culture collections to provide their user community with correctly identified and clean organisms. A total of 50 marine cyanobacterial strains were genotyped with M13 polymerase chain reaction (PCR) fingerprinting to provide diagnostic fingerprints for each culture. Depending on the strains, 9 to 26 bands were observed for the primer tested. Within the species, strains representing different isolates were genetically clearly different. Data obtained from genomic fingerprinting were used to construct binary distance matrix, and the neighbor-joining tree constructed demonstrated the ability of this method to differentiate strains at the intraspecific level. An important and useful result obtained in this study is the application of the M13 PCR fingerprinting method on almost all forms of cyanobacteria for strain and species discrimination.


Culture collection Genetic diversity M13 PCR fingerprinting Marine cyanobacteria Strain differentiation 



The authors are grateful to the Ministry of Earth Sciences (MoES), Government of India, for the financial support. Furthermore, the authors express their gratitude to The Director, National Facility for Marine Cyanobacteria, Bharathidasan University, for providing cyanobacterial strains to carry out this work. The first author acknowledges the Department of Biotechnology (Govt. of India) for the fellowship.


  1. Armstrong J, Gibbs A, Peakall R, Weiller G (1994) The RAPDistance Package.
  2. Bartie KL, Williams DW, Wilson MJ, Potts AJC, Lewis MAO (2001) PCR fingerprinting of Candida albicans associated with chronic hyperplastic candidosis and other oral conditions. J Clin Microbiol 39:4066–4075CrossRefPubMedGoogle Scholar
  3. Claros MC, Gerardo SH, Citron DM, Goldstein EJC, Schönian G, Rodloff AC (1997) Use of polymerase chain fingerprinting to compare clinical isolates of Bacteriodes fragilis and Bacteriodes thetaiotaomicron from Germany and the United States. Clin Infect Dis 25:S295–S298CrossRefPubMedGoogle Scholar
  4. Comte K, Rippka R, Friedl T, Day JG, Tandeau de Marsac N, Herdman M (2004) Assessment of genotypic identity of cyanobacterial strains in culture collections using HIP1-based primers. Nova Hedwigia 79:293–311CrossRefGoogle Scholar
  5. Day JG, Benson EE, Fleck RA (1999) In vitro culture and conservation of microalgae: applications for aquaculture, biotechnology and environmental research. In Vitro Cell Dev Biol Plant 35:127–136CrossRefGoogle Scholar
  6. Desikachary TV (1959) Cyanophyta. Indian Council Agricultural Research, New Delhi, p 686Google Scholar
  7. Fewer D, Friedl T, Büdel B (2002) Chroococcidiopsis and heterocyst-differentiating cyanobacteria are each other’s closest living relatives. Mol Phylogenet Evol 23:82–90CrossRefPubMedGoogle Scholar
  8. Gibbons NE, Murray RGE (1978) Proposals concerning the higher taxa of bacteria. Int J Syst Bacteriol 28:1–6CrossRefGoogle Scholar
  9. Giovannoni SJ, Turner S, Olsen GJ, Barns S, Lane DJ, Pace NR (1988) Evolutionary relationships among cyanobacteria and green chloroplasts. J Bacteriol 170:3584–3592PubMedGoogle Scholar
  10. Grif K, Karch H, Schneider C, Daschner FD, Beutin L, Cheasty T, Smit H, Rowe B, Dierich MP, Allerberger F (1998) Comparative study of five different techniques for epidemiological typing of Escherichia coli O157. Diagn Microbiol Infect Dis 32:165–176CrossRefPubMedGoogle Scholar
  11. Ishida T, Yokota A, Sugiyama J (1997) Phylogenetic relationships of filamentous cyanobacterial taxa inferred from 16S rDNA sequence divergence. J Gen Appl Microbiol 43:237–241CrossRefPubMedGoogle Scholar
  12. Kato T, Watanabe MF, Watanabe M (1991) Allozyme divergence in Microcystis and its taxonomic inference. Arch Hydrobiol Suppl Bd 64:129–140Google Scholar
  13. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163CrossRefPubMedGoogle Scholar
  14. López-Molina N, Laconcha I, Rementeria A, Audicana A, Perales I, Garaizar J (1998) Typing of Salmonella enteritidis of different phage types of PCR fingerprinting. J Appl Microbiol 84:877–882CrossRefPubMedGoogle Scholar
  15. Meyer W, Lieckfeldt E, Kuhls K, Fredman EZ, Borner T, Mitchell TG (1993) DNA and PCR-fingerprinting in fungi. EXS 67:311–320PubMedGoogle Scholar
  16. Nadeau TL, Milbrandt EC, Castenholz RW (2001) Evolutionary relationships of cultivated Antarctic oscillatorians (Cyanobacteria). J Phycol 37:650–654CrossRefGoogle Scholar
  17. Nei M, Li WH (1979) Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci USA 76:5269–5273CrossRefPubMedGoogle Scholar
  18. Neilan BA (1995) Identification and phylogenetic analysis of toxigenic cyanobacteria by multiplex randomly amplified polymorphic DNA PCR. Appl Environ Microbiol 61:2286–2291PubMedGoogle Scholar
  19. Neilan BA (2002) The molecular evolution and DNA profiling of toxic cyanobacteria. Curr Issues Mol Biol 4:1–11PubMedGoogle Scholar
  20. Oren A (2004) A proposal for further integration of the cyanobacteria under the bacteriological code. Int J Syst Evol Microbiol 54:1895–1902CrossRefPubMedGoogle Scholar
  21. Rasmussen U, Svenning M (1998) Fingerprinting of cyanobacteria based on PCR with primers derived from short and long tandemly repeated repetitive sequences. Appl Environ Microbiol 64:265–272PubMedGoogle Scholar
  22. Rippka R (1988) Recognition and identification of cyanobacteria. Methods Enzymol 167:28–67CrossRefGoogle Scholar
  23. Rippka R, Deruelles J, Waterbury JB, Herdmann M, Stanier Y (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111:1–61Google Scholar
  24. Rouhiainen L, Sivonen K, Buikema WJ, Haselkorn R (1995) Characterisation of toxin-producing cyanobacteria by using an oligonucleotide probe containing a tandemly repeated heptamer. J Bacteriol 177:6021–6026PubMedGoogle Scholar
  25. Ryskov AP, Jincharadze AG, Pronysak MI, Ivanov PL, Limorska SA (1988) M13 phage DNA as a universal marker for DNA fingerprinting of animals, plants and microorganisms. FEBS Lett 233:388–392CrossRefPubMedGoogle Scholar
  26. Smith JK, Parry JD, Day DJ, Smith RJ (1998) A PCR technique based on Hip 1 interspersed repetitive sequence distinguishes cyanobacterial strains and species. Microbiology 144:2791–2801CrossRefPubMedGoogle Scholar
  27. Stanier RY, Sistrom WR, Hansen TA, Whitton BA, Castenholz RW, Pfennig N, Gorlenko VN, Kondratieva EN, Eimhjellen KE, Whittenbury R, Gherna RL, Truper HG (1978) Proposal to place the nomenclature of the cyanobacteria (blue-green algae) under the rules of the International Code of Nomenclature of Bacteria. Int J Syst Bacteriol 28:335–336CrossRefGoogle Scholar
  28. Taton A, Grubisic S, Brambilla E, De Witt R, Wilmotte A (2003) Cyanobacterial diversity in natural and artificial microbial mates of Lake Fryxell (McMurdo Dry Valley, Antarctica): a morphological and molecular approach. Appl Environ Microbiol 69:5157–5169CrossRefPubMedGoogle Scholar
  29. Thajuddin N (1988) Marine cyanobacteria of the southern east coast of India–survey and ecobiological studies. Ph.D. thesis, Bharathidasan University, TiruchirappalliGoogle Scholar
  30. Thajuddin N, Subramanian G (1991) New report of marine cyanobacteria from the southern east coast of India. Phykos 30:19–23Google Scholar
  31. Thajuddin N, Subramanian G (1992) Survey of cyanobacterial flora of the southern east coast of India. Bot Mar 35:305–311CrossRefGoogle Scholar
  32. Thajuddin N, Subramanian G (2005) Cyanobacterial biodiversity and potential applications in biotechnology. Curr Sci 89:47–57Google Scholar
  33. Valério E, Pereira P, Saker ML, Franca S, Tenreiro R (2005) Molecular characterization of Cylindrospermopsis raciborskii strains isolated from Portuguese freshwaters. Harmful Alg 4:1044–1052CrossRefGoogle Scholar
  34. Valério E, Chambel L, Paulino S, Faria N, Pereira P, Tenreiro R (2009) Molecular identification, typing and traceability of cyanobacteria from freshwater reservoirs. Microbiology 155:642–656CrossRefPubMedGoogle Scholar
  35. Versalovic J, Koeuth T, Lupski JR (1991) Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res 19:6823–6831CrossRefPubMedGoogle Scholar
  36. Welsh J, McClelland M (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res 18:7213–7218CrossRefPubMedGoogle Scholar
  37. Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18:6531–6535CrossRefPubMedGoogle Scholar
  38. Wilmotte A, Golubić S (1991) Morphological and genetic criteria in the taxonomy of Cyanophyta/Cyanobacteria. Algol Stud 64:1–24Google Scholar
  39. Wilmotte A, Herdman M (2001) Phylogenetic relationships among the cyanobacterial based on 16S rRNA sequences. In: Boone DR, Castenholz RW (eds) Bergey’s manual of systematic bacteriology, vol I, 2nd edn. Springer, New York, pp 487–493Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  1. 1.Department of Microbiology, School of Life SciencesBharathidasan UniversityTiruchirappalliIndia

Personalised recommendations