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

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 con- sumers 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 chryso- phyte cultures. None of the non-chrysophyte species were amplified with these primers. PCR prod- ucts 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 plank- tonic systems.