A historic religious sanctuary may have preserved ancestral genetics of Japanese sika deer (Cervus nippon).

Deer have been a major resource for human populations for thousands of years. Anthropogenic activities, such as hunting, have influenced the genetic structure and distribution of deer populations. In Japan, wild Japanese sika deer (Cervus nippon) have been hunted since ancient times but have also been historically protected as sacred animals in several sanctuaries. Sika deer have been protected for over a thousand years in the religious sanctuary around the Kasuga Taisha Shrine on the Kii Peninsula, located in the center of Japan. Here, we used short sequence repeats (SSR) and mitochondrial DNA (mtDNA) to investigate the genetic diversity, population structure, and demography of Japanese sika deer inhabiting the Kii Peninsula, Japan, and discuss possible anthropogenic influences. Using SSR, three distinct genetic groups were distinguished on the Kii Peninsula: an Eastern genetic group, a Western genetic group, and an isolated genetic group with individuals in the religious sanctuary of Kasuga Taisha Shrine in Nara city. The isolated genetic sanctuary group had only the mtDNA haplotype S4. The SSR genotype data suggested a newer divergence time of the genetic groups of the religious sanctuary than would have occurred as a result of Late Quaternary climate change. This time scale coincided with the establishment of the sanctuary with Kasuga Taisha Shrine. Thus, the religious protection conserved genetic variation over a thousand years.

Mutational effects of chronic gamma radiation throughout the life cycle of Arabidopsis thaliana: Insight into radiosensitivity in the reproductive stage.

Organisms living on Earth have always been exposed to natural sources of ionizing radiation, but following recent nuclear disasters, these background levels have often increased regionally due to the addition of man-made sources of radiation. To assess the mutational effects of ubiquitously present radiation on plants, we performed a whole-genome resequencing analysis of mutations induced by chronic irradiation throughout the life cycle of Arabidopsis thaliana grown under controlled conditions. We obtained resequencing data from 36 second generation post-mutagenesis (M2) progeny derived from 12 first generation (M1) lines grown under gamma-irradiation conditions, ranging from 0.0 to 2.0 Gray per day (Gy/day), to identify de novo mutations, including single base substitutions (SBSs) and small insertions/deletions (INDELs). The relationship between de novo mutation frequency and radiation dose rate from 0.0 to 2.0 Gy/day was assessed by statistical modeling. The increase in de novo mutations in response to irradiation dose fit the negative binomial model, which accounted for the high variability of mutation frequency observed. Among the different types of mutations, SBSs were more prevalent than INDELs, and deletions were more frequent than insertions. Furthermore, we observed that the mutational effects of chronic radiation were greater during the reproductive stage. These results will provide valuable insights into practical strategies for analyzing mutational effects in wild plants growing in environments with various mutagens.

Radiocesium accumulation and germline mutations in chronically exposed wild boar from Fukushima, with radiation doses to human consumers of contaminated meat.

Genetic effects and radioactive contamination of large mammals, including wild boar (Sus scrofa), have been studied in Japan because of dispersal of radionuclides from the Fukushima Dai-ichi Nuclear Power Plant in 2011. Such studies have generally demonstrated a declining trend in measured radiocesium body burdens in wildlife. Estimating radiation exposure to wildlife is important to understand possible long-term impacts. Here, radiation exposure was evaluated in 307 wild boar inhabiting radioactively contaminated areas (50-8000 kBq m-2) in Fukushima Prefecture from 2016 to 2019, and genetic markers were examined to assess possible germline mutations caused by chronic radiation exposures to several generations of wild boar. Internal Cs activity concentrations in boar remained high in areas near the power plant with the highest concentration of 54 kBq kg-1 measured in 2019. Total dose rates to wild boar ranged from 0.02 to 36 µGy h-1, which was primarily attributed to external radiation exposure, and dose rates to the maximally exposed animals were above the generic no-effects benchmark of 10 µGy h-1. Using the estimated age of each animal, lifetime radiation doses ranged from <0.1 mGy to 700 mGy. Despite chronic exposures, the genetic analyses showed no significant accumulation of mutation events. Because wild boar is an occasional human dietary item in Japan, effective dose to humans from ingesting contaminated wild boar meat was calculated. Hypothetical consumption of contaminated wild boar meat from radioactively contaminated areas in Fukushima, at the per capita pork consumption rate (12.9 kg y-1), would result in an average effective annual dose of 0.9 mSv y-1, which is below the annual ingestion limit of 1 mSv y-1. Additionally, a consumption rate of about 1.4 kg y-1 of the most contaminated meat in this study would not exceed annual ingestion limits.

Japanese "nameko" mushrooms (Pholiota microspora) produced via sawdust-based cultivation exhibit severe genetic bottleneck associated with a single founder.

Pholiota microspora ("nameko" in Japanese) is one of the most common edible mushrooms, especially in Japan, where sawdust-based cultivation is the most dominant method accounting for 99% of the production. The current strains for sawdust cultivation in Japan are considered to have been derived from a single wild strain collected from Fukushima, Japan, implying that commercial nameko mushrooms are derived from a severe genetic bottleneck. We tested this single founder hypothesis by developing 14 microsatellite markers for P. microspora to evaluate the genetic diversity of 50 cultivars and 73 wild strains isolated from across Japan. Microsatellite analysis demonstrated that sawdust-cultivated strains from Japan were significantly less genetically diverse than the wild strains, and the former displayed a significant bottleneck signature. Analyzing the genetic relationships among all genotypes also revealed that the sawdust-cultivated samples clustered into one monophyletic subgroup. Moreover, the sawdust-cultivated samples in Japan were more closely related than full-sibs. These results were consistent with the single founder hypothesis that suggests that all commercial nameko mushrooms produced in Japan are descendants of a single ancestor. Therefore, we conclude that cultivated P. microspora originated from a single domestication event that substantially reduced the diversity of commercial nameko mushrooms in Japan.

Development of microsatellite markers for the geographically parthenogenetic stick insect Phraortes elongatus (Insecta: Phasmatodea).

Many plant and animal species exhibit geographic parthenogenesis, wherein unisexual (= parthenogenetic) lineages are more common in their marginal habitats such as high latitude or altitudes than their closely related bisexual counterparts. The Japanese stick insect, Phraortes elongatus (Thunberg) (Insecta: Phasmatodea), is known as a geographically parthenogenetic species due to the existence of both bisexual and unisexual populations. Here, we developed microsatellite markers to infer the genetic variation among populations of P. elongatus. Totally, 13 primer pairs were developed for the species, and they were tested on 47 samples collected from both a bisexual population and a unisexual population. All 13 loci were polymorphic in the bisexual population, whereas no loci were polymorphic in the unisexual population. The loss of variation in the unisexual population implies automixis with terminal fusion or gamete duplication as the mode of parthenogenesis. The markers developed in this study will be helpful for further comprehensive analysis of the genetic diversity and gene flow between bisexual and parthenogenetic lineages of P. elongatus.

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 [Cervus nippon taiouanus] and other deer species) that could potentially breed with the native Japanese sika deer (C. n. centralis). We used an existing genetic marker for the mitochondrial cytochrome b 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.