AME 39:171-182 (2005)  -  doi:10.3354/ame039171

Use of group-specific PCR primers for identification of chrysophytes by denaturing gradient gel electrophoresis

Johnny Berglund1,2,*, Klaus Jürgens3,4, Iris Bruchmüller3, Mats Wedin1, Agneta Andersson1,2

1Department of Ecology and Environmental Science, Umeå University, 901 87 Umeå, Sweden
2Umeå Marine Science Centre, 910 20 Hörnefors, Sweden
3Max Planck Institute for Limnology, Department of Physiological Ecology, PO Box 165, 24302 Plön, Germany
4Baltic Sea Research Institute Warnemünde, 18119 Rostock, Germany

ABSTRACT: The chrysophytes Chrysophyceae and Synurophyceae are ecologically important groups of autotrophic, mixotrophic and heterotrophic flagellates. The smallest forms are difficult to identify by classical microscopy but have important functions both as primary producers and as consumers of bacteria in the aquatic food chain. Group-specific primers for amplification of the 18S small sub-unit rRNA gene were developed for analysis of chrysophyte diversity by denaturing gradient gel electrophoresis (DGGE). Two different primer pairs were tested. The first primer pair (EukC1-F–Chryso-R) primarily targeted Paraphysomonadaceae and Ochromonadales, which generally have heterotrophic or mixotrophic nutrition. The second primer pair (EukC2-F–Chryso-R) targeted both Chrysophyceae and Synurophyceae. The primer pairs were tested for PCR amplification of the 18S rRNA gene of 25 cultured chrysophyte species and 6 other closely related nanoplanktonic species. Both primer pairs performed well, since PCR products were obtained for the corresponding chrysophyte cultures. None of the non-chrysophyte species were amplified with these primers. PCR products of chrysophyte cultures could be separated by DGGE in a denaturing gradient from 40 to 60%. In order to test this PCR-DGGE system for natural planktonic systems, we used field samples from a brackish water area (Baltic Sea) and a freshwater lake. The most intense DGGE bands were excised, sequenced and compared to sequences in GenBank. All obtained sequences grouped within the chrysophytes. Thus, the method seems to be promising for examining chrysophyte diversity in planktonic systems.

KEY WORDS: Chrysophytes · Chrysophyceae · Synurophyceae · Heterotrophic nanoflagellates · Denaturing gradient gel electrophoresis · DGGE · PCR-primers · 18S ribosomal DNA

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