Evaluation of Denaturing Gradient Gel Electrophoresis (DGGE) for the Determination of Marine Phytoplankton Genetic Diversity

Gires Usup*, Asmat Ahmad and Lee S. Jin

Marine Science Program, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia

ABSTRACT:

A study was conducted to optimize gradient gel electrophoresis (DGGE) protocols for determination of marine eukaryotic phytoplankton genetic diversity. The target genetic marker was a 330-bp fragment of the 18S subunit of the ribosomal RNA gene. PCR products incorporating a 40 base pair (bp) GC-clamp were successfully obtained from clonal cultures of eleven dinoflagellate and one diatom species. All the DGGE-PCR products denatured within a denaturant concentration range of 30% to 55%. Optimum DGGE run conditions were 10 hours at a constant 150V and a temperature of 60o C in TAE buffer. DGGE-PCR product with the lowest GC content denatured first, but not all products from different species separated out as expected. Products from Alexandrium affine and A. leei, which differed in nucleotide sequence but had identical GC content were separable but products from Cochlodinium sp., Gyrodinium instriatum and Karenia sanguinea, which differed in both nucleotide sequence and GC content, were not separable. The minimum number of cells in a mixed population required to obtain detectable DGGE-PCR product varied widely between species. Analysis of natural phytoplankton samples from six different locations by DGGE showed 28 bands, of which 13 were unique. In contrast, microscopic analysis of the samples showed the presence of at least 45 morphospecies belonging to 38 genera. These results suggest that DGGE will underestimate the diversity in a natural phytoplankton community.

KEYWORDS:

18S ribosomal RNA; DGGE; Genetic diversity; Marine phytoplankton

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