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

Abstract

Environmental water samples were taken throughout 2001 from fractioned water samples at the Helgoland time series site. The less than 3-μm 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, single strand conformation polymorphism (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 polymerase chain reaction (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 relatively easy to apply to assess the clonality and purity of the cultures. © The Author 2005. Published by Oxford University Press. All rights reserved.