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Applied and Environmental Microbiology, July 2001, p. 2942-2951, Vol. 67, No. 7
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.7.2942-2951.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Application of Denaturing Gradient Gel Electrophoresis (DGGE) To Study the Diversity of Marine Picoeukaryotic Assemblages and Comparison of DGGE with Other Molecular Techniques

Beatriz Díez,1 Carlos Pedrós-Alió,1,* Terence L. Marsh,2 and Ramon Massana1

Departament de Biologia Marina, Institut de Ciències del Mar, CSIC, E-08039 Barcelona, Catalunya, Spain,1 and Center of Microbial Ecology and Department of Microbiology, Michigan State University, East Lansing, Michigan 488242

Received 12 January 2001/Accepted 11 April 2001

We used denaturing gradient gel electrophoresis (DGGE) to study the diversity of picoeukaryotes in natural marine assemblages. Two eukaryote-specific primer sets targeting different regions of the 18S rRNA gene were tested. Both primer sets gave a single band when used with algal cultures and complex fingerprints when used with natural assemblages. The reproducibility of the fingerprints was estimated by quantifying the intensities of the same bands obtained in independent PCR and DGGE analyses, and the standard error of these estimates was less than 2% on average. DGGE fingerprints were then used to compare the picoeukaryotic diversity in samples obtained at different depths and on different dates from a station in the southwest Mediterranean Sea. Both primer sets revealed significant differences along the vertical profile, whereas temporal differences at the same depths were less marked. The phylogenetic composition of picoeukaryotes from one surface sample was investigated by excising and sequencing DGGE bands. The results were compared with an analysis of a clone library and a terminal restriction fragment length polymorphism fingerprint obtained from the same sample. The three PCR-based methods, performed with three different primer sets, revealed very similar assemblage compositions; the same main phylogenetic groups were present at similar relative levels. Thus, the prasinophyte group appeared to be the most abundant group in the surface Mediterranean samples as determined by our molecular analyses. DGGE bands corresponding to prasinophytes were always found in surface samples but were not present in deep samples. Other groups detected were prymnesiophytes, novel stramenopiles (distantly related to hyphochytrids or labyrinthulids), cryptophytes, dinophytes, and pelagophytes. In conclusion, the DGGE method described here provided a reasonably detailed view of marine picoeukaryotic assemblages and allowed tentative phylogenetic identification of the dominant members.

* Corresponding author. Mailing address: Departament de Biologia Marina, Institut de Ciències del Mar, CSIC, Passeig Joan de Borbó s/n, E-08039 Barcelona, Catalunya, Spain. Phone: 34-932216416. Fax: 34-932217340. E-mail: cpedros{at}icm.csic.es.

Applied and Environmental Microbiology, July 2001, p. 2942-2951, Vol. 67, No. 7
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.7.2942-2951.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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