Application of denaturing gradient gel electrophoresis (DGGE) to study the diversity of marine picoeukaryotic assemblages and comparison of DGGE with other molecular techniques - PubMed (original) (raw)

Application of denaturing gradient gel electrophoresis (DGGE) to study the diversity of marine picoeukaryotic assemblages and comparison of DGGE with other molecular techniques

B Díez et al. Appl Environ Microbiol. 2001 Jul.

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.

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Figures

FIG. 1

FIG. 1

(A) Negative image of a perpendicular DGGE gel with PCR products obtained with primer set A from different algal cultures (P. calceolata, T. pseudonana, and P. suecica) and electrophoresed at 100 V for 16 h. (B) Time course separation of PCR products obtained with primer set A from an algal culture (P. suecica) and marine sample ME-B0. Samples were electrophoresed for 3, 5, 8, 11, 14, 16, and 18 h at 100 V. (C) Same as panel A but with PCR products amplified with primer set B. The electrophoresis conditions were 200 V for 6 h. (D) Same as panel B but with PCR products amplified with primer set B. Samples were electrophoresed for 2, 3, 4, 5, 6, 7, and 8 h at 200 V.

FIG. 2

FIG. 2

DGGE fingerprints of algal cultures amplified with primer set A (A) and primer set B (B). The following cultures were tested: lane 1, P. calceolata; lane 2, N. oculata; lane 3, T. pseudonana; lane 4, D. primolecta; lane 5, P. suecica; lane 6, H. akashiwo; lane 7, Heterocapsa sp.

FIG. 3

FIG. 3

DGGE fingerprints of picoeukaryotic assemblages obtained at station ME-B (southwest Mediterranean Sea) at different times (ME-B0, 11 November 1997; ME-B3, 9 May 1998; ME-B4, 12 May 1998) and at different depths (5 to 500 m). The fingerprints were obtained with primer set A (A) and primer set B (B). Bands from sample ME-B0 that were sequenced are indicated on the left side of each gel.

FIG. 4

FIG. 4

Averages and standard errors of intensity values for DGGE bands of sample ME-B0 as quantified from separate PCR and DGGE analyses with primer set A (A) (n = 5) and primer set B (B) (n = 4). Where error bars are not visible, the error was smaller than the symbol.

FIG. 5

FIG. 5

NMDS diagrams relating the picoeukaryotic assemblages in ME-B samples on the basis of the DGGE gels shown in Fig. 3. NMDS diagrams were calculated from fingerprints obtained with primer set A using the intensity (A) and binary (C) matrices and from fingerprints obtained with primer set B using the intensity (B) and binary (D) matrices. On each diagram the grey circle corresponds to sample ME-B0, the solid circles correspond to ME-B3 samples, and the open circles correspond to ME-B4 samples. Solid and dashed lines join the data for the ME-B3 and ME-B4 samples, respectively, obtained from the surface (5 m) to a depth of 500 m. Only bands that accounted for at least 1% of the intensity in a lane were used in this analysis.

FIG. 6

FIG. 6

DGGE fingerprints obtained with primer set A for the ME-B0 sample and several clones from the genetic library obtained from the same sample. The clone names are the names of the most closely related organisms in the database (levels of similarity are given in parentheses) found in a BLAST search. The lanes to the right of the ME-B0 sample contained clones representing phylotypes that have been retrieved by sequencing DGGE bands (indicated by arrowheads).

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