Genetic structure and diversity of bottlenose dolphin, Tursiops truncatus, off Puerto Rico

Report Project Title: Genetic structure and diversity of bottlenose dolphin, Tursiops truncatus, off Puerto Rico Principal Invetsigator: Grisel Rodriguez Co-Principal Investigator: Nikolaos Schizas, PhD Associate Investigator: Richard Appeldoorn, PhD Methods used: The distribution of bottlenose dolphins was determined by conducting boat-based surveys in both coastal and offshore waters of Puerto Rico. Surveys included skin and fecal sampling, photo identification of dorsal fins and analysis of dolphin behavior. Collection permits were approved. Genetic analysis Skin samples collected were analyzed to determine genetic diversity (mitochondrial DNA) and sex determination (nuclear DNA). Five samples of stranded Tursiops of known sex (after the autopsy examination) were used as controls, to determine efficacy of the process. Extraction DNA was isolated using a commercial DNA extraction kit and the manufacturers’ instructions (DNeasy Tissue kit, Qiagen Inc., Valencia, California). Sex determination Gender was determined with a pattern of DNA bands amplified with the primers Zfx/Zfy and Sry (Bérubé and Palsbøll 1996). Reactions consisted of 2 µl template DNA, a 5 mm MgCl2, 0.2 mM dNTPs, 0.2 µM of primers. The amplification profile was 7 min at 95 C, then 37 cycles of 30 s at 94 °C, 30 s at 47 °C, and 30 s at 72 °C. A final 2 min elongation was performed at 72 °C, and reactions were then held at 4 °C. Amplification products were visualized using gel electrophoresis in a 1%. MtDNA The primers MtCRf and MtCRr (Hoezel et al. 1998) were used to amplify the control region of mitochondrial DNA. This region should provide enough genetic variability to distinguish between coastal and pelagic ecotypes. PCR conditions were 100µM dNTP, 1.5 mM MgCL2, 10 mM Tris HCL pH 8.0, 50 mM KCl, 200 nM of primer and 0.02 µl of Taq Polymerase. PCR profile was 4 min at 95°C, 35 cycles of 45 sec at 94°C, 1.5 min at 50 °C, 1.5 min at 72°C and 8 min at 72 °C. Results and findings The grant period of November 1, 2015 – April 15. 2016 was dedicated to sample analysis. A total of fifty (50) more samples from stranded dolphins were obtained from the Caribbean Stranding Network. This samples go back from 1937-2006.