Meeting Abstract

P2.218  Saturday, Jan. 5  Distinguishing clades of the sponge-specific cyanobacterial symbiont Synechococcus spongiarum through high-resolution melting analysis and denaturing/temperature gradient gel electrophoresis BLAIR, P. B.*; FREEMAN, C. F.; THACKER, R. W.; Univ. of Alabama at Birmingham; Univ. of Alabama at Birmingham; Univ. of Alabama at Birmingham pbblair@uab.edu

Approximately one-third of coral reef sponges in the nutrient poor Caribbean host photosynthetic cyanobacterial symbionts classified as Synechococcus spongiarum. The diverse S. spongiarum group consists of multiple, diverse clades that are distinguished using DNA sequences of the 16S-23S ribosomal RNA internal transcribed spacer (ITS) region. Since this task requires labor-intensive PCR-based amplification, bacterial plasmid cloning, and DNA sequencing, we developed high-throughput methods to rapidly screen sponge hosts for the presence of particular S. spongiarum genotypes. For these genotyping trials we generated a series of standard clones for each major clade based on previously identified and sequenced specimens. High-resolution melting (HRM) and Denaturing/Temperature Gradient Gel Electrophoresis (D/TGGE) was carried out using specifically designed primers to amplify a variable portion of the ITS region from these clade standards. Clade profiling using HRM and DGGE techniques failed to resolve the clade standards as well as TGGE. It was also found that TGGE analysis could be used to resolve individual clades amplified from whole genomic DNA, even when these samples included mixes of multiple clades within a sample. Thus, we suggest that TGGE is the most appropriate method for rapid and accurate genotyping to discriminate mixtures of symbionts within a single host. These methods of genotyping S. spongiarum will facilitate future work with these symbionts and can be applied to a broad range of other ecological interactions.