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

Abstract

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.Small phototrophic and heterotrophic eukaryotes between 0.2 and 5 m in diameter are found throughout the world's oceans at concentrations between 10 2 and 10 4 cells per ml in the upper photic zone (6, 23). They constitute an essential component of microbial food webs and play significant roles in global carbon and mineral cycles, especially in oligotrophic parts of the oceans (12, 23). However, the identities of the eukaryotic picoplankton have remained elusive due to their small size and the lack of distinctive taxonomic characteristics (43, 47). Conventional approaches based on morphological criteria, such as optical, epifluorescence, or electron microscopy (5, 36), can barely discriminate between these organisms, even at the class level. Although informative, analysis of diagnostic marker pigments by high-performance liquid chromatography (HPLC) provides information about the composition of photosynthetic picoplankton populations only at the class level (20) and appears to be a complementary method that is useful for gross characterization o...

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“…This is a further indication of the presence of phototrophic microorganisms in the analyzed samples. n-hexadecanoic acid methyl ester 6 hexadecadienoic acid methyl ester 7 octadecenoic acid methyl ester 8 cis-9-octadecenoic acid methyl ester 9 n-octadecanoic acid methyl ester 10 octadecadienoic acid methyl ester 11 octadecadienoic acid methyl ester 12 octadecadienoic acid methyl ester 13 octadecadienoic acid methyl ester 14 n-nonadecanoic acid methyl ester 15 n-eicosanoic acid methyl ester 16 n-heneicosanoic acid methyl ester 17 n-docosanoic acid methyl ester 18 n-tricosanoic acid methyl ester 19 n-tetracosanoic acid methyl ester a A, B, C, and D refer to total ion chromatograms reported in Figure 9.…”

Application of the denaturing gradient gel electrophoresis (DGGE) technique as an efficient diagnostic tool for ciliate communities in soil

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