Fingerprinting and molecular phylogeny of some heterocystous cyanobacteria using 16S rRNA, ITS regions and highly iterated palindromes as molecular markers

Document Type : Research Paper


1 Assistant Prof., Department of Biotechnology, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Assistant Prof., Department of Genetics, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran



This study aimed to investigate phylogenetic relationships of cyanobacteria based on the 16S rRNA, ITS genes and palindromic sequences of HIP, ERIC, and STRR. The use of Internal Transcribed Spacer (ITS) region secondary structures has been proposed for phylogenetic reconstructions. Sampling was done from five Ghanats of shallow aqueducts located at Gonbad Kavous villages (Golestan province, NE of Iran), and purified in Z8 culture medium. After DNA extraction, 16S rRNA and ITS genes were amplified and sequenced. The phylogenetic tree was created using the Likelihood Maximum method and the appropriate model with the help of Iqtree online web server. The secondary structure of ITS was drawn in different parts of helix D1-D1′, D2, D3, tRNAIle, tRNAAla, BOX B, BOX A, and V3 using Mfold program. Then, phylogenetic analysis of fingerprints was converted to binary information with the presence and absence of separate, and reproducible bands in each DNA fingerprint pattern generated by PCR profiles of HIP, ERIC and STRR, and binary information was used to construct a composite dendrograms. The results showed that, the studied strains belonged to four families viz. Aphanizomenonaceae, Nostocaceae, Hapalosiphonaceae, and Calotrichaceae of subsections of order Nostocales. The results of the dendrograms clusters drawn from the proliferation of palindrome sequences confirmed the clustering of phylogenetic trees. However, the results of the variable sections found in sections D1-D1′ and Box-B of the ITS gene revealed unique secondary structures that did not have a similar pattern to their close counterparts. The overall results showed that, the data obtained from genomic fingerprints, in silico and phylogenetic analysis are very useful for distinguishing closely related strains of cyanobacteria.


Article Title [فارسی]

انگشت‌نگاری و فیلوژنی مولکولی برخی از سیانوباکتری‌های هتروسیت‌دار با استفاده از مناطق 16S rRNA، ITS و پالیندروم‌های به ‌شدت تکراری به ‌عنوان مارکرهای مولکولی

Authors [فارسی]

  • بهاره نوروزی 1
  • حسین فهیمی 2
1 استادیار زیست‌شناسی مولکولی سیانوباکتری‌ها، گروه بیوتکنولوژی، دانشگاه آزاد اسلامی، واحد علوم و تحقیقات، تهران، ایران
2 استادیار گروه ژنتیک، دانشکده علوم و فناوری‌های نوین، دانشگاه علوم پزشکی آزاد اسلامی تهران، ایران
Abstract [فارسی]

هدف از این مطالعه، یافتن روابط فیلوژنتیک سیانوباکتری‌ها براساس ژن‌های 16S rRNA و ITS و توالی‌های پالیندرومی HIP، ERIC و STRR بوده است. به این منظور، نمونه‌برداری از پنج چشمه از قنات‌های کم‌عمق روستاهای گنبدکاووس (استان گلستان) انجام شد و در محیط کشت Z8 خالص‌سازی گردید. پس از استخراج DNA، ژن‌های 16S rRNA و ITS تکثیر و توالی‌یابی شدند. درخت فیلوژنتیک با استفاده از روش Likelihood Maximum و مدل مناسب به کمک وب سرور برخط Iqtree server ساخته شد. ساختار ثانویه ITS، در بخش‌های مختلف مارپیچ D1-D1′، D2، D3، tRNAIle، tRNAAla، BOX B، BOX A و V3 به کمک برنامه Mfold رسم شد. سپس، آنالیز فیلوژنتیک انگشت‌نگاری‌ها به کمک حضور و عدم حضور باندهای مجزا و قابل تکثیر در هر الگوی انگشت‌نگاری DNA تولید شده با پروفایل‌های PCR HIP، ERIC و STRR، به اطلاعات دوتایی تبدیل برای ساختن دندروگرام مرکب، استفاده شد. نتایج نشان داد که سویه‌های مورد بررسی متعلق به چهار تیره به اسامی Aphanizomenonaceae، Nostocaceae، Hapalosiphonaceae و Calotrichaceae از زیربخش راسته Nostocales بودند. نتایج کلاسترهای دندروگرام‌های رسم شده حاصل از تکثیر توالی‌های پالیندرومی تاییدکننده کلاستربندی‌های درخت‌های فیلوژنتیکی بود. به هر ‌حال، نتایج حاصل از بخش‌های متغیر در بخش‌های D1-D1′ و Box-B ژن ITS، ساختارهای ثانویه منحصر به ‌فردی یافت گردید که الگوی مشابهی با سویه‌های نزدیک به خود را نداشتند. نتایج کلی نشان داد که داده‌های حاصل از انگشت‌نگاری‌های ژنومیک، آنالیزهای درون‌رایانه‌ای و فیلوژنتیکی، برای تمایز سویه‌های نزدیک به هم سیانوباکتری‌ها بسیار مفید می‌باشند.

Keywords [فارسی]

  • آنالیزهای درون رایانه‌ای
  • Aphanizomenonaceae
  • Calotrichaceae
  • Hapalosiphonaceae
  •  Nostocaceae
Allen, M.M. & Arnon, D.I. 1955. Studies on nitrogen-fixing blue green algae. I. Growth and nitrogen fixation by Anabaena cylindrica Lemm. Plant Physiology 30: 366–372.
Anand, N., Thajuddin, N. & Dadheech, P.K. 2019. Cyanobacterial Taxonomy: Morphometry to Molecular Studies. Chapter 3. Pp. 43–64. In: Mishra, A.K., Tiwari, D.N. & Rai, A.V. (eds), Cyanobacteria. Academic Press.
Cai, F., Yang, Y., Wen, Q. & Li, R. 2018. Desmonostoc danxiaense sp. nov. (Nostocales, Cyanobacteria) from Danxia mountain in China based on polyphasic approach. Phytotaxa 367(3): 233–244.
Casamatta, D.A., Johansen, J.R., Vis, M.L. & Broadwater, S.T. 2005. Molecular and morphological characterization of ten polar and near‐polar strains within the Oscillatoriales (Cyanobacteria) 1. Journal of Phycology 41(2): 421–438.
Castenholz, R.W. 2001. Bergey’s Manual of Systematic Bacteriology, 2nd Ed. The Archaea and the Deeply Branching and Phototrophic Bacteria. Springer-Verlag, NY.
De Bruijn, F.J. 1992. Use of repetitive (repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus) sequences and the polymerase chain reaction to fingerprint the genomes of Rhizobium meliloti isolates and other soil bacteria. Applied and Environmental Microbiology 58(7): 2180–2187.
Driscoll, C.B., Meyer, K.A., Šulčius, S., Brown, N.M., Dick, G.J., Cao, H., Gasiūnas, G., Timinskas, A., Yin, Y., Landry, Z.C. & Otten, T.G. 2018. A closely-related clade of globally distributed bloom-forming cyanobacteria within the Nostocales. Harmful Algae 1(77): 93–107.
Fiore, M.F., Moon, D.H., Tsai, S.M., Lee, H. & Trevors, J.T. 2000. Miniprep DNA isolation from unicellular and filamentous cyanobacteria. Journal of Microbiological Methods 39(2): 159–169.
González‐Resendiz, L., Johansen, J.R., Escobar‐Sánchez, V., Segal-Kischinevzky, C., Jiménez‐García, L.F. & León‐Tejera, H. 2018. Two new species of Phyllonema (Rivulariaceae, Cyanobacteria) with an emendation of the genus. Journal of Phycology 54(5): 638–652.
Iteman, I., Rippka, R., de Marsac, N.T. & Herdman, M. 2000. Comparison of conserved structural and regulatory domains within divergent 16S rRNA-23S rRNA spacer sequences of cyanobacteria The GenBank accession numbers for the sequences reported in this paper are AF180968 and AF180969 for ITS-L and ITS-S, respectively. Microbiology 146(6): 1275–1286.
Kabirnataj, S., Nematzadeh, G.A., Talebi, A.F., Saraf, A., Suradkar, A., Tabatabaei, M. & Singh, P. 2020. Description of novel species of Aliinostoc, Desikacharya and Desmonostoc using a polyphasic approach. International Journal of Systematic and Evolutionary Microbiology 70(5): 3413–3426.
Komárek, J. 2013. Süßwasserflora von mitteleuropa, Bd. 19/3: cyanoprokaryota. 3. Teil/3rd part: heterocytous genera. Süßwasserflora von Mitteleuropa. Spektrum Academischer Verlag, Heidelberg.
Komárek, J. 2016. A polyphasic approach for the taxonomy of cyanobacteria: principles and applictions. European Journal of Phycology 51(3): 346–353.
Komárek, J., Kaštovský, J., Mareš, J. & Johansen, J.R. 2014. Taxonomic classification of cyanoprokaryotes (cyanobacterial genera) 2014, using a polyphasic approach. Preslia 86(4): 295–335.
Komárek, J. 2020. Quo vadis, taxonomy of cyanobacteria (2019). Fottea 20(1): 104–110.
Kotai, J. 1972. Instructions for preparation of modified nutrient solution Z8 for algae. Norwegian Institute for Water Research, Oslo 11(69): 5–15.
Liu, L., Jokela, J., Wahlsten, M., Nowruzi, B., Permi, P., Zhang, Y.Z., Xhaard, H., Fewer, D.P. & Sivonen, K. 2014. Nostosins, trypsin inhibitors isolated from the terrestrial cyanobacterium Nostoc sp. strain FSN. Journal of Natural Products 77(8):
Muhlsteinova, R., Johansen, J.R., Pietrasiak, N., Martin, M.P., Osorio-Santos, K. & Warren, S.D. 2014. Polyphasic characterization of Trichocoleus desertorum sp. nov. (Pseudanabaenales, Cyanobacteria) from desert soils and phylogenetic placement of the genus Trichocoleus. Phytotaxa 163(5): 241–261.
Moreira, C., Martins, J., Vasconcelos, V. & Antunes, A. 2020. Genomics Perspectives on Cyanobacteria Research. In: Handbook of Algal Science. Technology and Medicine 1(3): 147–159.
Negi, Y., Sharma, S., Sutradhar, N. & Adhikari, S. 2019. A study on the differentiation of filamentous cyanobacterial isolates using DNA fingerprinting approach, Indian Journal of Biotechnology 4(9): 151–163.
Nowruzi, B., Fahimi, H. & Ordodari, N. 2018. Molecular phylogenetic and morphometric evaluation of Calothrix sp. N42 and Scytonema sp. N11. Rostaniha 18(2): 210–221.
Nowruzi, B. & Soares, F. 2021. Alborzia kermanshahica gen. nov., sp. nov. (Chroococcales, Cyanobacteria), isolated from paddy fields in Iran. International Journal of Systematic and Evolutionary Microbiology 71(6): 1–13.
Nowruzi, B. & Shalygin, S. 2021. Multiple phylogenies reveal a true taxonomic position of Dulcicalothrix alborzica sp. nov. (Nostocales, Cyanobacteria). Fottea 21(2): 235–246.
Nowruzi, B. 2020. Culturing of Aquatic and terrestrial cyanobacteria. Research in Karyotic Cell and Tissue 1(1): 34–44.
Nowruzi, B. & Blanco, S. 2019. In silico identification and evolutionary analysis of candidate genes involved in the biosynthesis methylproline genes in cyanobacteria strains of Iran. Phytochemistry Letters 29: 199–211.
Osorio-Santos, K., Pietrasiak, N., Bohunická, M., Miscoe, L.H., Kováčik, L., Martin, M.P. & Johansen, J.R. 2014. Seven new species of Oculatella (Pseudanabaenales, Cyanobacteria): taxonomically recognizing cryptic diversification. European Journal of Phycology 49(4): 450–470.
Prabha, R. & Singh, D.P. 2019. Cyanobacterial phylogenetic analysis based on phylogenomics approaches render evolutionary diversification and adaptation: an overview of representative orders. Biotech 9(3): 1–16.
Rasmussen, U. & Svenning, M.M. 1998. Fingerprinting of cyanobacteria based on PCR with primers derived from short and long tandemly repeated repetitive sequences. Applied and Environmental Microbiology 64(1): 265–272.
Řeháková, K., Johansen, J.R., Casamatta, D.A., Xuesong, L. & Vincent, J. 2007. Morphological and molecular characterization of selected desert soil cyanobacteria: three species new to science including Mojavia pulchra gen. et sp. nov. Phycologia 46(5): 481–502.
Rivandi, M., Nowruzi, B. & Fahimi, H. 2021. Molecular phylogenetic study of toxic cyanobacterium Anabaena sp. strain B3 isolated from Lavasan Lake, Tehran (Iran). Rostaniha 22(1): 120–33.
Selvakumar, G. & Gopalaswamy, G. 2008. PCR based fingerprinting of Westiellopsis cultures with short tandemly repeated repetitive (STRR) and highly iterated palindrome (HIP) sequences. Biologia 63(3): 283–288.
Shokraei, R., Fahimi, H., Blanco, S. & Nowruzi, B. 2019. Genomic fingerprinting using highly repetitive sequences to differentiate close cyanobacterial strains. Microbial Bioactives 2(1): 68–75.
Sihvonen, L.M., Lyra, C., Fewer, D.P., Rajaniemi-Wacklin, P., Lehtimäki, J.M., Wahlsten, M. & Sivonen, K. 2007. Strains of the cyanobacterial genera Calothrix and Rivularia isolated from the Baltic Sea display cryptic diversity and are distantly related to Gloeotrichia and Tolypothrix. FEMS Microbiology Ecology 61(1): 74–84.
Smith, J., Parry, J., Day, J., & Smith, R. 1998. A PCR technique based on the Hipl interspersed repetitive sequence distinguishes cyanobacterial species and strains. Microbiology 144(10): 2791–2801.
Stein, J. 1973. Handbook of Phycological Methods. Culture Methods and Measurements. Cambridge University Press: 448– 510.
Taton, A., Grubisic, S., Brambilla, E., De Wit, R. & Wilmotte, A. 2003. Cyanobacterial diversity in natural and artificial microbial mats of Lake Fryxell (McMurdo Dry Valleys, Antarctica): a morphological and molecular approach. Applied and Environmental Microbiology 69(9): 5157–5169.
Thajuddin, N., Muralitharan, G., Sundaramoorthy, M., Ramamoorthy, R., Ramachandran, S., Akbarsha, M.A. & Gunasekaran, M. 2010. Morphological and genetic diversity of symbiotic cyanobacteria from cycads. Journal of Basic Microbiology 50(3): 254–65.
Whitton, B.A. 1992. Diversity, Ecology, and Taxonomy of the Cyanobacteria. Pp. 1–51. In: Photosynthetic Prokaryotes. Springer, Boston, MA.
Zuker, M. 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research 31(13): 3406–3415.
Volume 23, Issue 1 - Serial Number 63
September 2022
Pages 79-104
  • Receive Date: 06 May 2022
  • Revise Date: 17 July 2022
  • Accept Date: 19 July 2022
  • First Publish Date: 26 July 2022