Cost‐Effective Quantitative PCR Assays for the eDNA ‐Based Detection of Three Sympatric Salmon Species

Abstract

As ecologically, economically, and culturally important species in the North Pacific region, salmonids are critical to freshwater and coastal ecosystems. However, the co‐occurrence of multiple congeners can complicate species‐specific population monitoring. To address this challenge, we developed the first species‐specific, dye‐based quantitative PCR (qPCR) assays targeting three sympatric salmon species in Northeast Asia: Oncorhynchus keta (chum salmon), O. masou (masu salmon), and O. gorbuscha (pink salmon). We validated each assay's ability to discriminate among these closely related species and applied the assays to environmental DNA (eDNA) collected from the Tumen River Basin, Northeast China, to characterize spatial and seasonal distributions. All three qPCR assays demonstrated strict species specificity, high amplification efficiency, strong sensitivity (LOD: 2.2–8.4 copies/reaction; LOQ: 9–21 copies/reaction), and robust inter‐batch consistency. Using these assays on eDNA samples, we detected two target species, O. keta and O. masou , during the spawning season, with O. masou exhibiting a broader distribution. In contrast, O. gorbuscha was not detected, suggesting that the local population is either extremely rare or has been extirpated from the study area. Overall, these dye‐based qPCR assays provide a reliable, cost‐effective approach for species‐specific monitoring of the spatiotemporal dynamics of sympatric salmonids, supporting their conservation and management.