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

Abstract

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.

Environmental DNA (eDNA) Monitoring of Noble Crayfish Astacus astacus in Lentic Environments Offers Reliable Presence-Absence Surveillance – But Fails to Predict Population Density

“…It is commonly reported that ddPCR overcomes the challenges connected to inhibitory substances to a higher extent than qPCR, and displays both higher sensitivity and higher quantification accuracy (Dingle et al, 2013;Rački et al, 2014;Zhao et al, 2016), and several studies have highlighted the benefits of ddPCR for eDNA detection (e.g., Doi et al, 2015a;Hunter et al, 2017;Hamaguchi et al, 2018;Mauvisseau et al, 2019a;Brys et al, 2020). However, while ddPCR offers absolute quantification, the variation in estimated copy numbers increases at low numbers, therefore the LOQ of ddPCR is often within the same range as LOQ for qPCR (Dobnik et al, 2015).…”

Advances in DNA Barcoding of Toxic Marine Organisms

“…Droplet digital PCR (ddPCR) confirmed ribavirin, harringtonine, and 2-hydroxytetradecanoic acid (2-HOM) were effective at inhibiting SGIV infection [ 94 ]. In addition, ddPCR was also considered more suitable for the detection of Z. marina DNA from marine sediments [ 95 ].…”

Toward a Coordinated Global Observing System for Seagrasses and Marine Macroalgae

“…In the marine environment, eDNA has been used primarily to detect microbes and viruses, eukaryotic phytoplankton, zooplankton, macro-invertebrates, and vertebrates (Djurhuus et al, 2018;Goodwin et al, 2019). But eDNA has also been used to confirm the historic presence of seagrass at locations where it no longer exists (Hamaguchi et al, 2018). The only other study using eDNA from marine plants of which we are aware estimated the contribution of seagrasses and macroalgae to carbon stocks in sediments (Reef et al, 2017).…”