Small changes make big progress: A more efficient eDNA monitoring method for freshwater fish

Abstract Comprehensive and accurate assessments of fish composition and diversity are essential for understanding fish ecology and resource management. Traditional fish surveys generally involve capturing organisms, which is invasive for the biological community under study and conflicts with the original intention of biodiversity conservation. Environmental DNA (eDNA) metabarcoding has become an integrated method for monitoring fish species without disturbing ecosystems. However, due to the serious nonspecific amplification of primers in eDNA‐based monitoring, many non‐fish sequences, usually human sequences, are also amplified, which results in serious data wastage and an increase in monitoring costs. We designed new universal primers for freshwater fish by analyzing the whole mitochondrial genome of Chinese freshwater fish. The performance of the primers was compared using an in silico polymerase chain reaction, followed by an in vitro metabarcoding analysis using eDNA from the Yangtze River, which is the third largest river in the world and harbors many freshwater fish species. We found that the mitochondrial 12S region is the most suitable metabarcoding gene marker for both Chinese and other freshwater fish. The minor change at the 3′‐end of the primer can greatly reduce the nonspecific amplification and improve the effectiveness of eDNA metabarcoding. Even small changes in primers may have qualitative and quantitative effects on the detected biodiversity, which should be considered in experimental design and data interpretation. These results will help with primer design and selection for eDNA‐based fish surveys, and consequently support the conservation of freshwater biodiversity.