Diversity of the marine picocyanobacteria Prochlorococcus and Synechococcus assessed by terminal restriction fragment length polymorphisms of 16S-23S rRNA internal transcribed spacer sequences

In order to assess the appropriateness of the use of internal transcribed spacer (ITS) sequences for the study of population genetics of marine cyanobacteria, we amplified and cloned the 16S rRNA gene plus the 16S-23S ITS regions of six strains of Prochlorococcus and Synechococcus. We analyzed them...

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Autores principales:	LAVIN,PARIS, GÓMEZ,PATRICIA, GONZÁLE,BERNARDO, ULLOA,OSVALDO
Lenguaje:	English
Publicado:	Sociedad de Biología de Chile 2008
Materias:	cyanobacteria DGGE T-RFLP ITS secondary structure
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-078X2008000400006
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spelling	oai:scielo:S0716-078X20080004000062009-06-08Diversity of the marine picocyanobacteria Prochlorococcus and Synechococcus assessed by terminal restriction fragment length polymorphisms of 16S-23S rRNA internal transcribed spacer sequencesLAVIN,PARISGÓMEZ,PATRICIAGONZÁLE,BERNARDOULLOA,OSVALDO cyanobacteria DGGE T-RFLP ITS secondary structure In order to assess the appropriateness of the use of internal transcribed spacer (ITS) sequences for the study of population genetics of marine cyanobacteria, we amplified and cloned the 16S rRNA gene plus the 16S-23S ITS regions of six strains of Prochlorococcus and Synechococcus. We analyzed them by denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphisms (T-RFLP). When using the standard application of these techniques, we obtained more than one band or terminal restriction fragment (T-RF) per strain or cloned sequence. Reports in literature have suggested that these anomalies can result from the formation of secondary structures. Secondary structures of the ITS sequences of Prochlorococcus and Synechococcus strains were computationally modelled at the different temperatures that were used during the polymerase chain reaction (PCR). Modelling results predicted the existence of hairpin loops that would still be present at the extensión temperature; it is likely that these loops produced incomplete and single stranded PCR products. We modified the standard T-RFLP procedure by adding the labelled ITS primer in the last two cycles of the PCR reaction; this resulted, in most cases, in only one T-RF per ribotype. Application of this technique to a natural picoplankton community in marine waters off northern Chile, showed that it was possible to identify the presence, and determine the relative abundance, of several phylogenetic lineages within the genera Prochlorococcus and Synechococcus inhabiting the euphotic zone. Phylogenetic analysis of ITS sequences obtained by cloning and sequencing DNA from the same sample confirmed the presence of the different genotypes. With the proposed modification, T-RFLP profiles should therefore be suitable for studying the diversity of natural populations of cyanobacteria, and should become an important tool to study the factors influencing the genetic structure and distribution of these organisms.info:eu-repo/semantics/openAccessSociedad de Biología de ChileRevista chilena de historia natural v.81 n.4 20082008-12-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-078X2008000400006en10.4067/S0716-078X2008000400006
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	cyanobacteria DGGE T-RFLP ITS secondary structure
spellingShingle	cyanobacteria DGGE T-RFLP ITS secondary structure LAVIN,PARIS GÓMEZ,PATRICIA GONZÁLE,BERNARDO ULLOA,OSVALDO Diversity of the marine picocyanobacteria Prochlorococcus and Synechococcus assessed by terminal restriction fragment length polymorphisms of 16S-23S rRNA internal transcribed spacer sequences
description	In order to assess the appropriateness of the use of internal transcribed spacer (ITS) sequences for the study of population genetics of marine cyanobacteria, we amplified and cloned the 16S rRNA gene plus the 16S-23S ITS regions of six strains of Prochlorococcus and Synechococcus. We analyzed them by denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphisms (T-RFLP). When using the standard application of these techniques, we obtained more than one band or terminal restriction fragment (T-RF) per strain or cloned sequence. Reports in literature have suggested that these anomalies can result from the formation of secondary structures. Secondary structures of the ITS sequences of Prochlorococcus and Synechococcus strains were computationally modelled at the different temperatures that were used during the polymerase chain reaction (PCR). Modelling results predicted the existence of hairpin loops that would still be present at the extensión temperature; it is likely that these loops produced incomplete and single stranded PCR products. We modified the standard T-RFLP procedure by adding the labelled ITS primer in the last two cycles of the PCR reaction; this resulted, in most cases, in only one T-RF per ribotype. Application of this technique to a natural picoplankton community in marine waters off northern Chile, showed that it was possible to identify the presence, and determine the relative abundance, of several phylogenetic lineages within the genera Prochlorococcus and Synechococcus inhabiting the euphotic zone. Phylogenetic analysis of ITS sequences obtained by cloning and sequencing DNA from the same sample confirmed the presence of the different genotypes. With the proposed modification, T-RFLP profiles should therefore be suitable for studying the diversity of natural populations of cyanobacteria, and should become an important tool to study the factors influencing the genetic structure and distribution of these organisms.
author	LAVIN,PARIS GÓMEZ,PATRICIA GONZÁLE,BERNARDO ULLOA,OSVALDO
author_facet	LAVIN,PARIS GÓMEZ,PATRICIA GONZÁLE,BERNARDO ULLOA,OSVALDO
author_sort	LAVIN,PARIS
title	Diversity of the marine picocyanobacteria Prochlorococcus and Synechococcus assessed by terminal restriction fragment length polymorphisms of 16S-23S rRNA internal transcribed spacer sequences
title_short	Diversity of the marine picocyanobacteria Prochlorococcus and Synechococcus assessed by terminal restriction fragment length polymorphisms of 16S-23S rRNA internal transcribed spacer sequences
title_full	Diversity of the marine picocyanobacteria Prochlorococcus and Synechococcus assessed by terminal restriction fragment length polymorphisms of 16S-23S rRNA internal transcribed spacer sequences
title_fullStr	Diversity of the marine picocyanobacteria Prochlorococcus and Synechococcus assessed by terminal restriction fragment length polymorphisms of 16S-23S rRNA internal transcribed spacer sequences
title_full_unstemmed	Diversity of the marine picocyanobacteria Prochlorococcus and Synechococcus assessed by terminal restriction fragment length polymorphisms of 16S-23S rRNA internal transcribed spacer sequences
title_sort	diversity of the marine picocyanobacteria prochlorococcus and synechococcus assessed by terminal restriction fragment length polymorphisms of 16s-23s rrna internal transcribed spacer sequences
publisher	Sociedad de Biología de Chile
publishDate	2008
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-078X2008000400006
work_keys_str_mv	AT lavinparis diversityofthemarinepicocyanobacteriaprochlorococcusandsynechococcusassessedbyterminalrestrictionfragmentlengthpolymorphismsof16s23srrnainternaltranscribedspacersequences AT gomezpatricia diversityofthemarinepicocyanobacteriaprochlorococcusandsynechococcusassessedbyterminalrestrictionfragmentlengthpolymorphismsof16s23srrnainternaltranscribedspacersequences AT gonzalebernardo diversityofthemarinepicocyanobacteriaprochlorococcusandsynechococcusassessedbyterminalrestrictionfragmentlengthpolymorphismsof16s23srrnainternaltranscribedspacersequences AT ulloaosvaldo diversityofthemarinepicocyanobacteriaprochlorococcusandsynechococcusassessedbyterminalrestrictionfragmentlengthpolymorphismsof16s23srrnainternaltranscribedspacersequences
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Diversity of the marine picocyanobacteria Prochlorococcus and Synechococcus assessed by terminal restriction fragment length polymorphisms of 16S-23S rRNA internal transcribed spacer sequences

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