A new strategy for species identification of planktonic larvae: PCR–RFLP analysis of the internal transcribed spacer region of ribosomal DNA detected by agarose gel electrophoresis or DHPLC

Abstract

Planktonic survey is important for understanding the dynamics and structure of populations or communities. However, the small size of early planktonic larvae, usually <500 μm, makes it difficult or impossible to discriminate closely related taxa based on morphological characters. We developed a new strategy for species identification of planktonic larvae, namely, polymerase chain reaction¯restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer (ITS) region detected by agarose gel electrophoresis or denaturing high-performance liquid chromatography (DHPLC). A total of four restriction enzymes were selected for PCR-RFLP analysis. Based on any one of four restriction maps, 12 commercial shellfish species could be differentiated from each other in agarose gel analysis. But more accurate results were obtained in DHPLC analysis. As an example of larval identification, hybrid larvae were successfully identified by their showing diagnostic peaks of both parents in DHPLC analysis. These results suggest that this strategy can meet the demands for plankton survey and studies of hybridization.