A simple and rapid determination method for zooxanthellal genetic diversity in giant clams using multiplex PCR

Abstract

Giant clams (tridacnid shellfishes) contain threatened species and represent important fishery resources. They establish a symbiotic relationship with zooxanthellae (Symbiodinium) by receiving photosynthetic products from these symbiotic algae. Symbiodinium are divided into various phylotypes (A–I); each physiological character attributed to a certain phylotype may be different and may affect infectivity with clams. However, lack of information related to Symbiodinium diversities in giant clams causes difficulties in the culture of these clams, especially during the artificial infection process of Symbiodinium sources to clam larvae, which leads to an extremely low success rate in establishing infection. In this report, we have developed a multiplex PCR method, suitable for analyzing Symbiodinium phylotypes in giant clams. We have designed new specific PCR primer sets for three phylotypes (A, C and D) that give different product sizes and thus are distinguishable from each other on an electrophoresis gel. The method is reliable, highly sensitive and most practicable for aquaculture using one PCR reaction. We also performed a trial case of the method for extracted Symbiodinium DNA from clams and revealed an unexpected heterogeneity of phylotypes even though the clams tested were kept in the same pond.