REVEALING GENETIC DIVERSITY OF EUKARYOTIC MICROORGANISMS IN AQUATIC ENVIRONMENTS BY DENATURING GRADIENT GEL-ELECTROPHORESIS

A new Eucarya-specific 18S rDNA primer set was constructed and tested using denaturing gradient gel electrophoresis to analyze the genetic d iversity of eukaryotic microorganisms in aquatic environments. All euk aryal lines of descent exhibited four or fewer nucleotide mismatches i n the forward primer sequence, except for the microspora line of desce nt. The reverse primer annealed to a more conserved region with fewer than two nucleotide mismatches. Genomic DNA from test organisms with d ifferent numbers of nucleotide mismatches were amplified to test prime r specificity. Relatively low annealing temperatures allowed the ampli fication of sequences with up to four nucleotide mismatches while stil l maintaining specificity for the eukaryal domain. Denaturing gradient gel electrophoresis was used to separate similarly sized PCR products of environmental samples, and the obtained banding patterns were conv erted to a binary format for statistical comparisons. Cluster analysis of these patterns showed similar results to a cluster analysis based on environmental variables. This approach provides an analytical tool to study the population structure and molecular ecology of eukaryotic microbial communities inhabiting aquatic environments.