Development of primer sets for multiplex and qPCR assays targeting Skeletonema species and their application to field samples

Skeletonema is a dominant diatom, especially in coastal waters. However, species identification and quantification in the natural environment has not yet been established, as species identification can be nearly impossible with light microscopy. In the present study, we developed primer sets for multiplex and real-time PCR to identify and enumerate the 10 Skeletonema species, and applied this technique to samples obtained from the tidal zone of the Chikugo River and its adjacent waters of the Ariake Sea from spring to summer. In total, eight Skeletonema species were detected during the investigation. Among the eight, the S. marinoi–dohrnii complex was detected at all three stations. Skeletonema potamos was the second most frequently detected, mainly in the Chikugo River. The overall number of detected species was low at the upper tidal zone of the Chikugo River, where salinity was < 2. These results reveal the diverse Skeletonema community in the brackish environment, and the primer sets designed in the present study are useful for analyzing species composition of Skeletonema.