Comparison of Different Denaturing Gradient Gel Electrophoresis Primer Sets for the Study of Marine Bacterioplankton Communities

An annual seasonal cycle of composition of a bacterioplankton community in an oligotrophic coastal system was studied by denaturing gradient gel electrophoresis (DGGE) using five different primer sets. Analysis of DGGE fingerprints showed that primer set 357fGC-907rM grouped samples according to seasons. Additionally, we used the set of 16S rRNA genes archived in the RDPII database to check the percentage of perfect matches of each primer for the most abundant bacterial groups inhabiting coastal plankton communities. Overall, primer set 357fGC-907rM was the most suitable for the routine use of PCR-DGGE analyses in this environment.     

PCR–DGGE method for analyzing the bacterial community in a high temperature petroleum reservoir

Different PCR–denaturing gradient gel electrophoresis (DGGE) protocols were employed to investigate bacterial communities in a high temperature and water flooded petroleum reservoir in Dagang oil field, China. Bacterial universal primers sets frequently used in PCR–DGGE were evaluated. Three primers sets P1 (341F-GC and 534R), P2 (341F-GC and 907R) and P3 (1055F and 1406R-GC) showed different DGGE patterns. Good separation and quality of patterns were obtained in DGGE analysis with the set P3. A total of 12 DNA fragments were excised from the DGGE gels and their sequences were determined. Clustering analysis of the DGGE profiles showed that bacteria in this petroleum reservoir belonged to four clusters. These results indicate that the procedure of DGGE analysis with the primer P3 (1055F and 1406R-GC) is suitable for investigating microbial community in petroleum reservoirs.     

Community Dynamics of Free-living and Particle-associated Bacterial Assemblages during a Freshwater Phytoplankton Bloom

Bacterial community dynamics were followed in a 19-day period during an induced diatom bloom in two freshwater mesocosms. The main goal was to compare diversity and succession among free-living (<10 MM) AND PARTICLE-ASSOCIATED (>10 mm) bacteria. Denaturing gradient gel electrophoresis (DGGE) of PCR amplified 16S rDNA showed the highest number of bands among free-living bacteria, but with a significant phylogenetic overlap in the two size fractions indicating that free-living bacteria were also important members of the particle-associated bacterial assemblage. Whereas the number of bands in the free-living fraction decreased during the course of the bloom, several phylotypes unique to particles appeared towards the end of the experiment. Besides the primer set targeting Bacteria, a primer set targeting most members of the Cytophaga-Flavobacterium (CF)-cluster of the Cytophaga-Flavobacterium-Bacteroides group and a primer set mainly targeting a-Proteobacteria were applied. PCR-DGGE analyses revealed that a number of phylotypes targeted by those primer sets were found solely on particles. Almost all sequenced bands from the bacterial DGGE gel were related to phylogenetic groups commonly found in freshwater: a-Proteobacteria, CF, and Firmicutes. Despite the use of primers intended to be specific mainly for a-Proteobacteria most bands sequenced from the a-proteobacterial DGGE gel formed a cluster within the Verrucomicrobiales subdivision of the Verrucomicrobia division and were not related to a-Proteobacteria. Bands sequenced from the CF DGGE gel were related to members of the CF cluster. From the present study, we suggest that free-living and particle-associated bacterial communities should not be perceived as separate entities, but rather as interacting assemblages, where the extent of phylogenetic overlap is dependent on the nature of the particulate matter.     

Evaluating Primers for Profiling Anaerobic Ammonia Oxidizing Bacteria within Freshwater Environments

Anaerobic ammonia oxidizing (anammox) bacteria play an important role in transforming ammonium to nitrogen gas and contribute to fixed nitrogen losses in freshwater environments. Understanding the diversity and abundance of anammox bacteria requires reliable molecular tools, and these are not yet well established for these important Planctomycetes. To help validate PCR primers for the detection of anammox bacteria within freshwater ecosystems, we analyzed representative positive controls and selected samples from Grand River and groundwater sites, both from Ontario, Canada. The objectives of this study were to identify a suitable anammox denaturing gradient gel electrophoresis (DGGE) fingerprint method by using GC-clamp modifications to existing primers, and to verify the specificity of anammox-specific primers used for DGGE, cloning and qPCR methods. Six primer combinations were tested from four published primer sets (i.e. A438f/A684r, Amx368f/Amx820r, An7f/An1388r, and Pla46/1392r) for both direct and nested PCR amplifications. All PCR products were run subsequently on DGGE gels to compare the resulting patterns. Two anammox-specific primer combinations were also used to generate clone libraries and quantify anammox bacterial 16S rRNA genes with qPCR. The primer set A438f/A684r was highly specific to anammox bacteria, provided reliable DGGE fingerprints and generated a high proportion of anammox-related clones. A second primer set (Amx368f/Amx820r) was anammox specific, based on clone library analysis, but PCR products from different candidate species of anammox bacteria resolved poorly using DGGE analysis. Both DGGE and cloning results revealed that Ca. Brocadia and an uncharacterized anammox bacterial cluster represented the majority of anammox bacteria found in Grand River sediment and groundwater samples, respectively. Together, our results demonstrate that although Amx368f/Amx820r was useful for anammox-specific qPCR and clone library analysis, A438f/A684r was the most suitable primer set for multiple molecular assessments of anammox bacteria in freshwater environments.     

Advantage of using inosine at the 3' termini of 16S rRNA gene universal primers for the study of microbial diversity

To overcome the shortcomings of universal 16S rRNA gene primers 8F and 907R when studying the diversity of complex microbial communities, the 3' termini of both primers were replaced with inosine. A comparison of the clone libraries derived using both primer sets showed seven bacterial phyla amplified by the altered primer set (8F-I/907R-I) whereas the original set amplified sequences belonging almost exclusively to Proteobacteria (95.8%). Sequences belonging to Firmicutes (42.6%) and Thermotogae (9.3%) were more abundant in a library obtained by using 8F-I/907R-I at a PCR annealing temperature of 54 degrees C, while Proteobacteria sequences were more frequent (62.7%) in a library obtained at 50 degrees C, somewhat resembling the result obtained using the original primer set. The increased diversity revealed by using primers 8F-I/907R-I confirms the usefulness of primers with inosine at the 3' termini in studying the microbial diversity of environmental samples.     

Advantage of Using Inosine at the 3′ Termini of 16S rRNA Gene Universal Primers for the Study of Microbial Diversity

To overcome the shortcomings of universal 16S rRNA gene primers 8F and 907R when studying the diversity of complex microbial communities, the 3′ termini of both primers were replaced with inosine. A comparison of the clone libraries derived using both primer sets showed seven bacterial phyla amplified by the altered primer set (8F-I/907R-I) whereas the original set amplified sequences belonging almost exclusively to Proteobacteria (95.8%). Sequences belonging to Firmicutes (42.6%) and Thermotogae (9.3%) were more abundant in a library obtained by using 8F-I/907R-I at a PCR annealing temperature of 54°C, while Proteobacteria sequences were more frequent (62.7%) in a library obtained at 50°C, somewhat resembling the result obtained using the original primer set. The increased diversity revealed by using primers 8F-I/907R-I confirms the usefulness of primers with inosine at the 3′ termini in studying the microbial diversity of environmental samples.     

Evaluation of Marine Bacteroidetes-Specific Primers for Microbial Diversity and Dynamics Studies

Assumptions on the matching specificity of group-specific bacterial primers may bias the interpretation of environmental microbial studies. As available sequence data continue growing, the performance of primers and probes needs to be reevaluated. Here, we present an evaluation of several commonly used and one newly designed Bacteroidetes-specific primer (CF418). First, we revised the in silico primer coverage and specificity with the current SILVA and RDP databases. We found minor differences with previous studies, which could be explained by the chosen databases, taxonomies, and matching criteria. We selected eight commonly used Bacteroidetes primers and tested them with a collection of assorted marine bacterial isolates. We also used the denaturing gradient gel electrophoresis (DGGE) approach in environmental samples to evaluate their ability to yield clear and diverse band patterns corresponding to Bacteroidetes phylotypes. Among the primers tested, CF968R did not provide satisfactory results in DGGE, although it exhibited the highest in silico coverage for Flavobacteria. Primers CFB560 and CFB555 presented undesirable features, such as requiring nested protocols or presence of degeneracies. Finally, the new primer CF418 and primer CF319a were used to explore the Bacteroidetes dynamics throughout a 1-year cycle in Mediterranean coastal waters (Blanes Bay Microbial Observatory). Both primers provided clear and diverse banding patterns, but the low specificity of CF319a was evidenced by 83.3?% of the bands sequenced corresponding to nontarget taxa. The satisfactory DGGE banding patterns and the wide diversity of sequences retrieved from DGGE bands with primer CF418 prove it to be a valuable alternative for the study of Bacteroidetes communities, recovering a wide range of phylotypes within the group.     

Molecular analyses of the methane-oxidizing microbial community in rice field soil by targeting the genes of the 16S rRNA, particulate methane monooxygenase, and methanol dehydrogenase

Rice field soil with a nonsaturated water content induced CH4 consumption activity when it was supplemented with 5% CH4. After a lag phase of 3 days, CH4 was consumed rapidly until the concentration was less than 1.8 parts per million by volume (ppmv). However, the soil was not able to maintain the oxidation activity at near-atmospheric CH4 mixing ratios (i.e., 5 ppmv). The soil microbial community was monitored by performing denaturing gradient gel electrophoresis (DGGE) during the oxidation process with different PCR primer sets based on the 16S rRNA gene and on functional genes. A universal small-subunit (SSU) ribosomal DNA (rDNA) primer set and 16S rDNA primer sets specifically targeting type I methylotrophs (members of the gamma subdivision of the class Proteobacteria [gamma-Proteobacteria]) and type II methylotrophs (members of the alpha-Proteobacteria) were used. Functional PCR primers targeted the genes for particulate methane monooxygenase (pmoA) and methanol dehydrogenase (mxaF), which code for key enzymes in the catabolism of all methanotrophs. The yield of PCR products amplified from DNA in soil that oxidized CH4 was the same as the yield of PCR products amplified from control soil when the universal SSU rDNA primer set was used but was significantly greater when primer sets specific for methanotrophs were used. The DGGE patterns and the sequences of major DGGE bands obtained with the universal SSU rDNA primer set showed that the community structure was dominated by nonmethanotrophic populations related to the genera Flavobacterium and Bacillus and was not influenced by CH4. The structure of the methylotroph community as determined with the specific primer sets was less complex; this community consisted of both type I and type II methanotrophs related to the genera Methylobacter, Methylococcus, and Methylocystis. DGGE profiles of PCR products amplified with functional gene primer sets that targeted the mxaF and pmoA genes revealed that there were pronounced community shifts when CH4 oxidation began. High CH4 concentrations stimulated both type I and II methanotrophs in rice field soil with a nonsaturated water content, as determined with both ribosomal and functional gene markers.     

Evaluation of PCR primers for denaturing gradient gel electrophoresis analysis of fungal communities in compost

AIMS: Three previously published fungal specific PCR primer sets, referred to as the NS, EF and NL primer sets, were evaluated for use in compost microbial community analysis by PCR and denaturing gradient gel electrophoresis (DGGE). METHODS AND RESULTS: Primers were first evaluated based on their tolerance to PCR inhibitors. Due to its sensitivity to inhibitors, the NS primer set was determined to require a 10-fold smaller volume addition of compost DNA to PCR than the EF and NL primer sets, based on a logistic regression model for a 75% PCR success rate. Further evaluation of the EF and NL primer sets involved testing the resolution of PCR products from pure fungal cultures on DGGE. The NL primer set, which targets the more variable 28S rDNA, resulted in multiple bands for each pure culture. Thus, the EF primer set was used to monitor the microbial community during compost colonization studies, where three fungi were inoculated onto autoclaved grape pomace and rice straw compost. CONCLUSIONS: Of the three primer sets evaluated, the EF primer set was determined to be the best for PCR-DGGE of compost fungal populations; however, concerns with the EF primer set included the lack of sequence divergence in the targeted region of 18S rDNA and PCR artifacts which interfered with detection of inoculated fungi in the colonization studies. SIGNIFICANCE AND IMPACT OF THE STUDY: There are many factors related to PCR primers that need to be assessed prior to applying PCR-DGGE to fungal communities in complex environments such as compost.     

PCR primers for assessing community structure of aquatic fungi including Chytridiomycota and Cryptomycota

Fungi are diverse and have the potential for material cycling in freshwater ecosystems. Species composition of aquatic fungi and their seasonal dynamics are often key to their role in the functioning of the ecosystems. However, community structure of aquatic fungi, especially of Chytridiomycota (Chytrids) and Cryptomycota, is still limited because few primer sets are available to examine species composition. In this study, we validated six primer sets for the detection of aquatic fungi by denaturing gradient gel electrophoresis (DGGE) analysis and found that FF390W/EF3r showed the highest specificity among the primer sets tested. We detected both Chytrids and Cryptomycota from Lake Inba by DGGE analysis using the FF390W/EF3r-GC primer set. Further study with our established analysis revealed community structures of aquatic fungi and their seasonal succession patterns in the lake. Results of our study are useful for selection of the primer set for studying community structures of aquatic fungi and their seasonal succession.     

High-Resolution Differentiation of Cyanobacteria by Using rRNA-Internal Transcribed Spacer Denaturing Gradient Gel Electrophoresis

For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE). We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS). A forward primer specific for cyanobacteria, targeted at the 3′ end of the 16S rRNA gene, was designed. The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other bacteria. DGGE profiles produced from the shortest section of rRNA-ITS consisted of one band for all but one cyanobacterial genera, and those generated from longer stretches of rRNA-ITS yielded DGGE profiles containing one to four bands. The suitability of DGGE for detecting intrageneric and intraspecific variation was tested by using strains of the genus Microcystis. Many strains could be discriminated by means of rRNA-ITS DGGE, and the resolution of this method was strikingly higher than that obtained with previously described methods. The applicability of the developed DGGE assays for analysis of cyanobacteria in field samples was demonstrated by using samples from freshwater lakes. The advantages and disadvantages associated with the use of each developed primer set are discussed.     

High-resolution differentiation of Cyanobacteria by using rRNA-internal transcribed spacer denaturing gradient gel electrophoresis

For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE). We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS). A forward primer specific for cyanobacteria, targeted at the 3' end of the 16S rRNA gene, was designed. The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other bacteria. DGGE profiles produced from the shortest section of rRNA-ITS consisted of one band for all but one cyanobacterial genera, and those generated from longer stretches of rRNA-ITS yielded DGGE profiles containing one to four bands. The suitability of DGGE for detecting intrageneric and intraspecific variation was tested by using strains of the genus Microcystis: Many strains could be discriminated by means of rRNA-ITS DGGE, and the resolution of this method was strikingly higher than that obtained with previously described methods. The applicability of the developed DGGE assays for analysis of cyanobacteria in field samples was demonstrated by using samples from freshwater lakes. The advantages and disadvantages associated with the use of each developed primer set are discussed.     

Viruses and flagellates sustain apparent richness and reduce biomass accumulation of bacterioplankton in coastal marine waters

To gain a better understanding of the interactions among bacteria, viruses and flagellates in coastal marine ecosystems, we investigated the effect of viral lysis and protistan bacterivory on bacterial abundance, production and diversity [determined by 16S rRNA gene polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE)] in three coastal marine sites with different nutrient supplies in Hong Kong. Six experiments were set up using filtration and dilution methods to develop virus, flagellate and virus+flagellate treatments for natural bacterial populations. All three predation treatments had significant repressing effects on bacterial abundance. Bacterial production was significantly repressed by flagellates and both predators (flagellates and viruses). Bacterial apparent species richness (indicated as the number of DGGE bands) was always significantly higher in the presence of viruses, flagellates and both predators than in the predator-free control. Cluster analysis of the DGGE patterns showed that the effects of viruses and flagellates on bacterial community structure were relatively stochastic while the co-effects of predators caused consistent trends (DGGE always showed the most similar patterns when compared with those of in situ environments) and substantially increased the apparent richness. Overall, we found strong evidence that viral lysis and protist bacterivory act additively to reduce bacterial production and to sustain diversity. This first systematic attempt to study the interactive effects of viruses and flagellates on the diversity and production of bacterial communities in coastal waters suggests that a tight control of bacterioplankton dominants results in relatively stable bacterioplankton communities.     

PCR‐DGGE Fingerprinting Analysis of Plankton Communities and Its Relationship to Lake Trophic Status

Plankton communities in eight lakes of different trophic status near Yangtze, China were charac‐terized by using denatured gradient gel electrophoresis (DGGE). Various water quality parameters were also measured at each collection site. Following extraction of DNA from plankton communi‐ties, 16S rRNA and 18S rRNA genes were amplified with specific primers for prokaryotes and eu‐karyotes, respectively; DNA profiles were developed by DGGE. The plankton community of each lake had its own distinct DNA profile. The total number of bands identified at 34 sampling stations ranged from 37 to 111. Both prokaryotes and eukaryotes displayed complex fingerprints composed of a large number of bands: 16 to 59 bands were obtained with the prokaryotic primer set; 21 to 52 bands for the eukaryotic primer set. The DGGE‐patterns were analyzed in relation to water quality parameters by canonical correspondence analysis (CCA). Temperature, pH, alkalinity, and the con‐centration of COD, TP and TN were strongly correlated with the DGGE patterns. The parameters that demonstrated a strong correlation to the DGGE fingerprints of the plankton community differed among lakes, suggesting that differences in the DGGE fingerprints were due mainly to lake trophic status. Results of the present study suggest that PCR‐DGGE fingerprinting is an effective and precise method of identifying changes to plankton community composition, and therefore could be a useful ecological tool for monitoring the response of aquatic ecosystems to environmental perturbations. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

DGGE method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil

A denaturing gradient gel electrophoresis (DGGE) method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil is presented. Five specific primers for 16S rDNA of methanogenic archaea, which were modified from the primers for archaea, were first evaluated by polymerase chain reaction and DGGE using genomic DNAs of 13 pure culture strains of methanogenic archaea. The DGGE analysis was possible with two primer pairs (0348aF-GC and 0691R; 0357F-GC and 0691R) of the five pairs tested although 16S rDNA of some non-methanogenic archaea was amplified with 0348aF-GC and 0691R. These two primer pairs were further evaluated for use in analysis of methanogenic archaeal community in Japanese paddy field soil. Good separation and quality of patterns were obtained in DGGE analysis with both primer pairs. A total of 41 DNA fragments were excised from the DGGE gels and their sequences were determined. All fragments belonged to methanogenic archaea. These results indicate that the procedure of DGGE analysis with the primer pair 0357F-GC and 0691R is suitable for investigating methanogenic archaeal community in paddy field soil.     

益生菌DGGE Marker的制备及验证

目的 制备指示益生菌标准菌株的DGGE marker并对其可靠性进行验证.方法 分别利用乳杆菌、双歧杆菌特异性引物和细菌V3区通用引物对选取的乳杆菌、双歧杆菌标准菌株DNA进行扩增,利用DGGE检测每个标准菌株条带位置是否与利用这些标准菌株制备的DGGE marker条带相对应.结果 DGGE图谱显示,乳杆菌和双歧杆菌特异性引物或V3区通用引物扩增后的每个标准菌株优势条带,与乳杆菌、双歧杆菌DGGE marker均有对应关系.结论 常见益生菌菌株的DGGE marker可以指示相应菌株的存在;其研制成功,可为微生物生态学中应用DGGE技术检测特定微生物种类的动态变化,提供新的思路.     

Molecular diversity analysis of rumen methanogenic Archaea from goat in eastern China by DGGE methods using different primer pairs

Aims:  To screen a pair of primers suitable for denaturing gradient gel electrophoretic (DGGE) analysis of ruminal methanogenic Archaea and to detect the archaeal communities in the rumen of goat.
Methods and Results:  Nine primer pairs for 16S rDNA of methanogenic Archaea , including six for directed polymerase chain reaction (PCR) and three for nested PCR were first evaluated by PCR amplification of the total DNA from rumen fluids and bacteria. The DGGE analysis of rumen fluids was then conducted with three primer sets (344fGC/915r, 1106fGC/1378r and 519f/915rGC) of the nine pairs tested. Good separation and quality of patterns were obtained in DGGE analysis with primer pairs 1106fGC/1378r and 519f/915rGC. A total of 40 DNA fragments were excised from the DGGE gels and their sequences were determined. All fragments belonged to methanogenic Archaea while primer pair 519f/915rGC had better amplification ranges than the other two primer pairs.
Conclusions:  The procedure of DGGE analysis with primer pair 519f/915rGC was more suitable for investigating methanogenic archaeal community in the rumen. The dominant methanogenic Archaea in the rumen of goat was Methanobrevibacter sp. and an unidentified methanogenic Archaea .
Significance and Impact of the Study:  One pair of primers suitable for DGGE analysis of ruminal methanogenic Archaea was obtained and the molecular diversity of ruminal methanogenic Archaea in goat was investigated by PCR-DGGE.     

Characterization of the bacterial community structure of Sydney Tar Ponds sediment

The Sydney Tar Ponds is one of the largest toxic waste sites in Canada. The bacterial diversity and abundance in the Sydney Tar Ponds sediment was examined using a 16S rRNA gene clone library and denaturing gradient gel electrophoresis (DGGE) with four different primer sets. The clone library was grouped into 19 phylotypes that could be divided into five phyla: Proteobacteria (56.9%), Actinobacteria (35%), Acidobacteria (4.9%), Firmicutes (2.4%), and Verrucomicrobia (0.8%). Members of the phyla Actinobacteria (represented mainly by Mycobacterium spp.) and Alphaproteobacteria (represented by Acidocella spp.) comprised the majority of the clone library. This study also revealed that the phylogenetic results obtained from clone library analysis and from DGGE analysis, with all the primer sets, showed some variability. However, similar Mycobacterium spp. and Acidocella spp. were found in all the different DGGE analyses, again suggesting that these two genera are dominant in the Sydney Tar Ponds sediment. In addition, DGGE analysis indicated that primer sets targeting the V3 region produced results that were the most similar to those obtained with the clone library.     

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

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.     

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

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.