Quantitative real-time polymerase chain reaction (PCR) and droplet digital PCR duplex assays for detecting Zostera marina DNA in coastal sediments

摘要

The sequestration of atmospheric CO_2 in seagrass meadows as organic carbon (OC) has been attracting more attention as a means for climate change mitigation and adaptation. A direct method to detect seagrass DNA in coastal sediments, which is essential to unravel long-term seagrass-derived OC accumulation, was developed based on environmental DNA (eDNA) detection techniques. Quantitative real-time polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR) were applied to quantify Zostera marina DNA in coastal sediments, using species-specific primers and dual-labeled probes for one nuclear and one chloroplast gene. Suitable pretreatments and methods for extracting Z. marina DNA from coastal sediments were examined and their applicability to environmental samples was demonstrated. Surface sediments collected from Z. marina meadows contained about 2000 times more Z. marina DNA than the unvegetated tidal-flats in the Seto Inland Sea. Moreover, both qPCR and ddPCR successfully detected Z. marina DNA in ancient sediments (up to 5000 calibrated years before present), evidencing that Z. marina DNA can be preserved in temperate coastal sediments for several millennia. In addition, qPCR and ddPCR results obtained in the present study were highly correlated, although the latter was more accurate than qPCR, particularly at low eDNA concentrations in ancient sediments. This work opens avenues to explore and clarify the process of the sequestration of OC produced by Z. marina and demonstrate the presence of past seagrass meadows from several millennia.