Evaluation of introgressive hybridization among Cervidae in Japan's Kinki District via two novel genetic markers developed from public NGS data.

Hybridization and backcrossing of native populations with introduced species can lead to introgression and genetic alteration. In this study, we evaluated introgression in 43 deer from a potential hybrid zone around Okinoshima Island, Kinki District, Japan. This region witnessed the migration of a hybrid population (cross between the Formosan sika deer [] and other deer species) that could potentially breed with the native Japanese sika deer (). We used an existing genetic marker for the mitochondrial gene and two novel markers for nuclear DNA, developed using publicly available next-generation sequencing data. We identified one mainland deer with a mitochondrial haplotype identical to that of the Formosan sika deer as well as nuclear heterozygous sequences identical to those of Formosan and Japanese sika deer. This suggests that the mainland deer is a hybrid offspring of the Okinoshima population and native deer. However, only Japanese sika deer sequences were found in the other 42 samples, indicating limited introgression. Nevertheless, hybridization pre- and postintroduction in the Okinoshima population could cause multispecies introgression among Japanese sika deer, negatively affecting genetic integrity. We developed a simple test based on polymerase chain reaction-restriction fragment length polymorphism to detect introgression in natural populations. Our method can accelerate genetic monitoring of Japanese sika deer in Kinki District. In conclusion, to prevent further introgression and maintain genetic integrity of Japanese sika deer, we recommend establishing fences around Okinoshima Island to limit migration, besides a continued genetic monitoring of the native deer.