Development and validation of novel SNP markers for the rapid identification of natural hybrids of the 11 closely related pufferfish species (Takifugu spp.) distributed in Japan

Accurate identification of interspecific hybrids is vital for breeding programs in hybridizing taxa, as well as for the use of hybrids in aquaculture and fisheries. The use of species-specific single nucleotide polymorphisms (SNPs) has become the standard for the molecular identification of hybrids. However, it remains challenging to use these SNPs to identify hybrids involving multiple candidate parental species. Here, we developed and validated SNP markers for the identification of hybrids of the 11 closely related pufferfish species (genus Takifugu) distributed in Japan; these species have different aquacultural characteristics, such as growth rate, physiology, behavior, and toxicity. In total, 1232 species-specific SNPs were identified through double-digest restriction-site-associated DNA sequencing. The species had 11 (T. flavipterus) to 295 (T. chrysops) species-specific SNPs. We validated 33 genome-wide species-specific SNPs using TaqMan® genotyping assays for the 11 species and their hybrids, which were identified using amplified fragment length polymorphism analyses. No false negative or positive assignments were observed in 77 and 30 specimens of pure species and F1 hybrids, respectively. This marker combination allows rapid and reliable identification of natural hybrids, which will be useful for pufferfish aquaculture and will enhance our understanding of the natural hybridization between various Takifugu species.