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JPR Advance Access first published online on November 21, 2005
This version published online on November 23, 2005
Journal of Plankton Research, doi:10.1093/plankt/fbi082
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received May 2, 2005
Accepted October 3, 2005

Article

Picoplankton culture assessment using single strand conformation polymorphism (SSCP) and partial 18S sequencing

Klaus Valentin 1, Helga Mehl 1, and Linda Medlin 1 *

1 Alfred Wegener Institut, Am Handelshafen 12, 27570 Bremerhaven, Germany

* To whom correspondence should be addressed.
Linda Medlin, E-mail: lkmedlin{at}awi-bremerhaven.de


  Abstract

Environmental water samples were taken throughout 2001 from fractioned water samples at the Helgoland time series site. The less than 3µm fraction was inoculated into various media. Serial dilutions from these inoculations produced a large number of rough cultures from which several hundred well-growing picoplankton cultures were started. We established a combination of crude DNA extraction, SSCP fingerprinting and subsequent sequence analysis to screen these large numbers of cultures, assess their purity/clonality and determine their identity. Picoplankton species (i.e. cells smaller than 3 µm) are difficult to distinguish because of their small size and the lack of morphological characters. Therefore molecular techniques provide the most reliable method to achieve their identification. Cultures were enriched for photoautotroph cells, i.e., no carbon source was added and cultures were grown in the light. From these cultures, crude DNA was extracted, which was used for partial 18S PCR. On average, 50% of the cultures produced a PCR product. SSCP analysis of PCR products revealed the clonality of a given culture. If clonal, then there was only a single SSCP band; if not clonal, then there were multiple SSCP bands. Single SSCP bands were subsequently sequenced and sequences were used to identify the culture. For this study 300 cultures were screened resulting in the identification of 63 potentially clonal cultures. These methods proved to be a relatively easy to apply to assess the clonality and purity of the cultures.

Communicating Editor: KJ Flynn
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