P3.187 Friday, Jan. 6 Genotyping symbionts through high-resolution melting analysis: Distinguishing clades of the sponge-specific cyanobacterial symbiont Synechococcus spongiarum BLAIR, PB*; FREEMAN, CJ; THACKER, RW; Univ. of Alabama at Birmingham; Univ. of Alabama at Birmingham; Univ. of Alabama at Birmingham email@example.com
Approximately one-third of Caribbean coral reef sponges host photosynthetic cyanobacterial symbionts classified as Synechococcus spongiarum. The diverse S. spongiarum group consists of multiple, distinct 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, cloning and DNA sequencing, we developed a high-throughput method to rapidly screen sponge hosts for the presence of particular S. spongiarum genotypes. We generated a series of standard clones for each major clade based on previously identified and sequenced specimens. We conducted high-resolution melting (HRM) analysis by using specifically designed primers to amplify a variable portion of the ITS region from these clade standards. We normalized the melting curves for each clone, and then calculated fluorescence difference plots between each normalized melting curve, standardized to a single clone. These plots differentiated clones from clades A, B, and C, which are all commonly reported in previous studies from Bocas del Toro, Panama. We are currently determining the ability of HRM analysis to discriminate mixtures of symbionts within a single host. This rapid, high-throughput method of genotyping S. spongiarum will facilitate future work with these symbionts. This approach has been successfully used to genotype coral-Symbiodinium associations and can be applied to a broad range of other ecological interactions.