Application of denaturing gradient gel electrophoresis (DGGE) to study thediversity of marine picoeukaryotic assemblages and comparison of DGGE withother molecular techniques

We used denaturing gradient gel electrophoresis (DGGE) to study the diversi ty of picoeukaryotes in natural marine assemblages. Two eukaryote-specific primer sets targeting different regions of the 18S rRNA gene were tested. B oth primer sets gave a single band when used with algal cultures and comple x fingerprints when used with natural assemblages. The reproducibility of t he fingerprints was estimated by quantifying the intensities of the same ba nds obtained in independent PCR and DGGE analyses, and the standard error o f these estimates was less than 2% on average. DGGE fingerprints were then used to compare the picoeukaryotic diversity in samples obtained at differe nt depths and on different dates from a station in the southwest Mediterran ean Sea, Both primer sets revealed significant differences along the vertic al profile, whereas temporal differences at the same depths were less marke d, The phylogenetic composition of picoeukaryotes from one surface sample w as investigated by excising and sequencing DGGE bands. The results were com pared with an analysis of a clone library and a terminal restriction fragme nt length polymorphism fingerprint obtained from the same sample, The three PCR-based methods, performed with three different primer sets, revealed ve ry similar assemblage compositions; the same main phylogenetic groups were present at similar relative levels. Thus, the prasinophyte group appeared t o be the most abundant group in the surface Mediterranean samples as determ ined by our molecular analyses, DGGE bands corresponding to prasinophytes w ere always found in surface samples but were not present in deep samples. O ther groups detected were prymnesiophytes, novel stramenopiles (distantly r elated to hyphochytrids or labyrinthulids), cryptophytes, dinophytes, and p elagophytes, In conclusion, the DGGE method described here provided a reaso nably detailed view of marine picoeukaryotic assemblages and allowed tentat ive phylogenetic identification of the dominant members.