The environmental DNA (eDNA) approach uses DNA recovered from environmental media, such as water and sediment, to obtain information about the species distribution based on the premise that the environmental media contains DNA released from all inhabitant organisms. This recent breakthrough in the monitoring of fauna may also permit a rapid detection of intraspecific genetic variation because it uses DNA sequences to identify target organisms. Here we aimed to develop quantitative eDNA methods which discriminate mtDNA haplotypes and nDNA genotypes using introgressed populations of common carp (Cyprinus carpio) in Japan as a model. We employed single nucleotide polymorphism (SNP) genotyping techniques, i.e., DNA-RNA-DNA probes or TaqMan MGB probes, in real-time PCR assays to discriminate and quantify Japanese native and non-native SNP alleles. The developed eDNA methods were used in the field to estimate the degree of invasion by the non-native conspecifics as well as the behavioral difference between the native and non-native common carp.
Results/Conclusions
The eDNA method successfully quantified the proportion of the native and non-native haplotypes in aquarium experiments. Then this method was applied to rivers and reservoirs in western Japan, revealing the massive invasion of non-native haplotypes. The same eDNA method was also used to monitor the habitat use of common carp in Lake Biwa for 3 years, suggesting the different seasonal migration patterns between the native and non-native individuals. The eDNA methods targeting nDNA loci also successfully quantified the proportion of the native and non-native genotypes in aquarium experiments. In the application to the field, however, the nuclear eDNA methods required more concentrated eDNA samples for successful quantification. This demerit comes from the fact that the most of nuclear polymorphic markers are single-copy in the genome. Nonetheless the use of nDNA markers would have significant advantages because it enables the analysis of multiple independent loci on both maternal and paternal chromosomes, leading to more accurate estimation of the genetic structure.