Integrating mitochondrial and nuclear genomic data to decipher the evolutionary history of Eubranchipus species in Japan.

Understanding the genetic diversity and evolutionary history of species is crucial for their conservation and management. In this study, we investigated the genetic diversity and phylogenetic relationships among Eubranchipus species occurring in Japan. Phylogenetic analyses revealed that nuclear and mitochondrial data yield incompatible results. In E. uchidai, nuclear data support the monophyly of the Shimokita area, while mitochondrial data indicate a clustering of Higashidori2 individuals with Hokkaido (Ishikari and Wakkanai) E. uchidai. Similar incongruences were observed in E. hatanakai, where nuclear data favor the monophyly of the Chokai area, while mitochondrial data cluster some Chokai pool 3 individuals with Aizu individuals. These incompatibilities might be caused by mitochondrial gene flow. The findings emphasize the importance of considering both nuclear and mitochondrial data during phylogenetic studies and provide valuable insights into the complex dynamics of migration and genetic exchange in Eubranchipus species.

Complete mitochondrial genomes of three fairy shrimps from snowmelt pools in Japan.

BACKGROUND: Fairy shrimps belong to order Anostraca, class Branchiopoda, subphylum Crustacea, and phylum Arthropoda. Three fairy shrimp species (Eubranchipus uchidai, E. asanumai, and E. hatanakai) that inhabit snowmelt pools are currently known in Japan. Whole mitochondrial genomes are useful genetic information for conducting phylogenetic analyses. Mitochondrial genome sequences for Branchiopoda members are gradually being collated. RESULTS: Six whole mitochondrial genomes from the three Eubranchipus species are presented here. Eubranchipus species share the anostracan pattern of gene arrangement in their mitochondrial genomes. The mitochondrial genomes of the Eubranchipus species have a higher GC content than those of other anostracans. Accelerated substitution rates in the lineage of Eubranchipus species were observed. CONCLUSION: This study is the first to obtain whole mitochondrial genomes for Far Eastern Eubranchipus species. We show that the nucleotide sequences of cytochrome oxidase subunit I and the 16S ribosomal RNA of E. asanumai presented in a previous study were nuclear mitochondrial DNA segments. Higher GC contents and accelerated substitution rates are specific characteristics of the mitochondrial genomes of Far Eastern Eubranchipus. The results will be useful for further investigations of the evolution of Anostraca as well as Branchiopoda.

Identification and Characterization of Genes for the Allo A Lectins in Japanese Rhinoceros Beetle (Trypoxylus dichotomus [Allomyrina dichotoma]).

The Japanese rhinoceros beetle (Trypoxylus dichotomus [Allomyrina dichotoma]) produces the lectins allo A-I and allo A-II, which have strong N-acetyllactosamine (Galß1-4GlcNAc)-binding activity. It has been suggested that the two lectins are formed from three subunits (α, ß, and γ), with allo A-I comprising α and γ subunits and allo A-II comprising ß and γ subunits. Here, we determined the cDNA sequences of these subunits using both conventional polymerase chain reaction (PCR)-cloning-sequencing and transcriptome-sequencing analyses. For the α and ß subunits, one gene (locus) for each was predicted, whereas for the γ subunit, two types of cDNA sequences were obtained, which we named γ1 and γ2. These two types probably have distinct loci. Average nucleotide sequence identities among the subunits ranged from 87.6% (between α and γ1) to 92.6% (between γ1 and γ2), suggesting that they form a gene family. Although no homology was found between the sequences of allo A and other known lectin proteins in a protein database search, some unknown proteins containing the DUF3421 domain were identified. Those DUF3421 domain-encoding proteins are upregulated in the insect larval midgut. Thus, we infer that allo A genes also play an important role in larvae and that their lectin activity may have been obtained collaterally.

Nutrient Imbalance of the Host Plant for Larvae of the Pale Grass Blue Butterfly May Mediate the Field Effect of Low-Dose Radiation Exposure in Fukushima: Dose-Dependent Changes in the Sodium Content.

The pale grass blue butterfly Zizeeria maha is sensitive to low-dose radioactive pollution from the Fukushima nuclear accident in the field but is also highly tolerant to radioactive cesium (137Cs) in an artificial diet in laboratory experiments. To resolve this field-laboratory paradox, we hypothesize that the butterfly shows vulnerability in the field through biochemical changes in the larval host plant, the creeping wood sorrel Oxalis corniculata, in response to radiation stress. To test this field-effect hypothesis, we examined nutrient contents in the host plant leaves from Tohoku (mostly polluted areas including Fukushima), Niigata, and Kyushu, Japan. Leaves from Tohoku showed significantly lower sodium and lipid contents than those from Niigata. In the Tohoku samples, the sodium content (but not the lipid content) was significantly negatively correlated with the radioactivity concentration of cesium (137Cs) in leaves and with the ground radiation dose. The sodium content was also correlated with other nutrient factors. These results suggest that the sodium imbalance of the plant may be caused by radiation stress and that this nutrient imbalance may be one of the reasons that this monophagous butterfly showed high mortality and morphological abnormalities in the field shortly after the accident in Fukushima.

Japaneseplex: A forensic SNP assay for identification of Japanese people using Japanese-specific alleles.

It is sometimes necessary to determine whether a forensic biological sample came from a Japanese person. In this study, we developed a 60-locus SNP assay designed for the differentiation of Japanese people from other East Asians using entirely and nearly Japanese-specific alleles. This multiplex assay consisted of 6 independent PCR reactions followed by single nucleotide extension. The average number and standard deviation of Japanese-specific alleles possessed by an individual were 0.81 ±â€¯0.93 in 108 Koreans from Seoul, 8.87 ±â€¯2.89 in 103 Japanese from Tottori, 17.20 ±â€¯3.80 in 88 Japanese from Okinawa, and 0 in 220 Han Chinese from Wuxi and Changsha. The Koreans had 0-4 Japanese-specific alleles per individual, whereas the Japanese had 4-26 Japanese-specific alleles. Almost all Japanese were distinguished from the Koreans and other people by the factorial correspondence and principal component analyses. The Snipper program was also useful to estimate the degree of Japaneseness. The method described here was successfully applied to the differentiation of Japanese from non-Japanese people in forensic cases. This Japanese-specific SNP assay was named Japaneseplex.

Brown hagfish from the northwest and east coasts of Honshu, Japan are genetically different.

The brown hagfish (Eptatretus atami) is one of several known hagfish species occurring in Japanese coastal waters. To date, there has been no research studying genetic polymorphisms in the species. In the present study, we analyzed differences in nucleotide sequences between two populations: one from Suruga Bay on the Pacific coast of Honshu, Japan, and the other from the Sea of Japan, off Akita on the northwest coast of Honshu. We sequenced part of the cytochrome oxidase subunit 1 gene (COX1) from the mitochondrial genome, and three G protein-coupled receptor genes from the nuclear genome. Phylogenetic networks of all four genes showed divergence between the two populations. Further, comparison of the COX1 data using a phylogenetic tree for a range of hagfish species indicated clear differences between the populations, suggesting that they differ at the species level. The numbers of their teeth, in particular of fused cusps (anterior/posterior multicusps), also supported these findings. Individuals of the Suruga Bay population had 3/3 fused cusps, as described for E. atami, whereas individuals of the Akita population had 3/2 fused cusps. These results suggest that the brown hagfish from the Sea of Japan, off the northwest coast of Honshu, is a distinct species from E. atami.

Genotyping of the c.1423C>T (p.P475S) polymorphism in the ADAMTS13 gene by APLP and HRM assays: Northeastern Asian origin of the mutant.

ADAMTS13 is a von Willebrand factor-cleaving protease. The mutant types of p.P475S (c.1423C>T) polymorphism in ADAMTS13 have a reduced activity in comparison with the wild type. In the present study, we investigated the frequency of the C-to-T substitution in 2584 genomic DNA samples from 25 Asian, European, and African populations using APLP (amplified product length polymorphism) and/or HRM (high-resolution melting) assays. Allele T (ADAMTS13(∗)T) was detected only in Asian populations and its frequency was observed to decrease gradually from north to south in 24 East Asian populations. Almost all ADAMTS13(∗)T were associated with ABO(∗)O. These results suggested that ADAMTS13(∗)T had occurred on a chromosome with ABO(∗)O in a northern part of East Asia. This SNP is useful as an ancestry-informative marker, and the present genotyping techniques are applicable to the investigation of an association between this SNP and aortic dissection (Kobayashi et al., 2012).

Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs.

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10-13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs.

The art of traditional native PAGE: The APLP 48-ID assay for human identification.

When full STR profiles cannot be obtained, further DNA analyses targeting single nucleotide polymorphisms (SNPs) may occasionally yield valuable information. Although the discrimination power of each SNP is relatively low, combined analysis of many SNPs can improve the personal identification ability to a level as high as that of commercial STR typing kits. In this study, we developed a new SNP typing method, named the amplified-product length polymorphism (APLP) 48-ID assay, for genotyping of 47 autosomal SNPs and two X and Y chromosomal markers for sex typing. Forty-seven SNPs were selected from all 22 autosomes, showing high diversity in European, Nigerian, Han Chinese, and Japanese population in the HapMap data. PCR primers were designed to generate amplicons 40-100 bp in length to increase the robustness of the PCR. The APLP 48-ID assay consisted of four independent PCR reactions followed by electrophoretic run on four lanes in a polyacrylamide gel. Complete profiles were obtained when more than 1.2 ng of DNA was used. We applied this assay for genotyping of 236 Japanese individuals. The random matching probability was 3.3E-20, and the power of exclusion was greater than 0.9999999. This method is a rapid, robust, and cost-effective approach for human identification and paternity testing.

The global distribution of the p.R1193Q polymorphism in the SCN5A gene.

The SCN5A (sodium channel, voltage-gated, type V, alpha subunit) gene encodes the cardiac sodium channel, a member of the voltage-gated sodium channel family. The p.R1193Q (c.3578G>A) polymorphism in SCN5A is known to accelerate inactivation of the sodium channel current, and has been identified in patients with Brugada and long QT syndromes. In the present study, we investigated the frequency of the p.R1193Q substitution in more than 4000 genomic DNA samples from 34 Asian, European, and African populations using TaqMan and/or APLP (amplified product length polymorphism) assays. Allele A (p.1193Q) was detected in most Asian populations, but was sporadically observed or absent in European and African populations. These results demonstrated that the p.R1193Q substitution is characteristic of Asian populations.

Investigation of Japanese-specific alleles: most are of Jomon lineage.

Japanese-specific alleles are expected to be powerful markers for the differentiation of the Japanese from other people. In this study, three single nucleotide polymorphisms (SNPs) in the GALNT11, H19, and PLA2G12A genes were analyzed in 2396 DNA samples from 25 global populations, and the derived alleles suggested that Japanese-specific alleles exist on autosomes. To identify new Japanese-specific alleles, candidate SNPs obtained from the HapMap database were investigated using 875 DNA samples from nine populations. A total of 67 (nearly) Japanese-specific derived alleles were observed. Of them, 57 showed higher frequencies in the Ryukyuans, living in the southernmost part of the Japanese Archipelago, than in the Wajins living in mainland Japan, and 43 were also present in Koreans at low frequencies. Jomon skeletons excavated from Hokkaido, the northernmost island of Japan, showed higher frequencies of the three derived alleles in the GALNT11, H19, and PLA2G12A genes than the Ryukyuans, suggesting that most of the 57 derived alleles observed at the high frequencies in the Ryukyuans originated from the Jomon lineage. These novel markers will be useful in the field of forensics.

An N-acetyllactosamine-specific lectin, PFA, isolated from a moth (Phalera flavescens), structurally resembles an invertebrate-type lysozyme.

PFA (Phalera flavescens agglutinin) lectin purified from larvae of the lobster moth (P. flavescens) shows a strong binding ability specific to the N-acetyllactosamine (Galß1-4GlcNAc) site. We determined the genomic and cDNA sequences of the PFA gene, which consists of five exons and spans approximately 5 kb of a genomic region. Surprisingly, the amino acid sequence (149 amino acids) was similar to invertebrate-type lysozymes and related proteins. The predicted tertiary structure of the PFA protein was similar to the lysozymes of clams such as the common orient clam (Meretrix lusoria) and Japanese littleneck (Venerupis philippinarum (Tapes japonica)). The PFA, however, lacks a catalytically essential amino acid, an Asp (D), which is one of the two important amino acids (Glu (E) and D) express the function of lysozyme. As a result, lysozyme activity assays indicated that PFA does not have lysozyme activity. Results suggest that the PFA gene evolved from a lysozyme gene through the loss of lysozyme activity sites and the acquisition of lectin activity during evolution of the genus Phalera.

Forensic strategy to ensure the quality of sequencing data of mitochondrial DNA in highly degraded samples.

Mitochondrial DNA (mtDNA) is widely used for DNA analysis of highly degraded samples because of its polymorphic nature and high number of copies in a cell. However, as endogenous mtDNA in deteriorated samples is scarce and highly fragmented, it is not easy to obtain reliable data. In the current study, we report the risks of direct sequencing mtDNA in highly degraded material, and suggest a strategy to ensure the quality of sequencing data. It was observed that direct sequencing data of the hypervariable segment (HVS) 1 by using primer sets that generate an amplicon of 407 bp (long-primer sets) was different from results obtained by using newly designed primer sets that produce an amplicon of 120-139 bp (mini-primer sets). The data aligned with the results of mini-primer sets analysis in an amplicon length-dependent manner; the shorter the amplicon, the more evident the endogenous sequence became. Coding region analysis using multiplex amplified product-length polymorphisms revealed the incongruence of single nucleotide polymorphisms between the coding region and HVS 1 caused by contamination with exogenous mtDNA. Although the sequencing data obtained using long-primer sets turned out to be erroneous, it was unambiguous and reproducible. These findings suggest that PCR primers that produce amplicons shorter than those currently recognized should be used for mtDNA analysis in highly degraded samples. Haplogroup motif analysis of the coding region and HVS should also be performed to improve the reliability of forensic mtDNA data.

A hypervariable STR polymorphism in the CFI gene: southern origin of East Asian-specific group H alleles.

Previous studies of four populations revealed that a hypervariable short tandem repeat (iSTR) in intron 7 of the human complement factor I (CFI) gene on chromosome 4q was unique, with 17 possible East Asian-specific group H alleles observed at relatively high frequencies. To develop a deeper anthropological and forensic understanding of iSTR, 1161 additional individuals from 11 Asian populations were investigated. Group H alleles of iSTR and c.1217A allele of a SNP in exon 11 of the CFI gene were associated with each other and were almost entirely confined to East Asian populations. Han Chinese in Changsha, southern China, showed the highest frequency for East Asian-specific group H alleles (0.201) among 15 populations. Group H alleles were observed to decrease gradually from south to north in 11 East Asian populations. This expansion of group H alleles provides evidence that southern China and Southeast Asia are a hotspot of Asian diversity and a genetic reservoir of Asians after they entered East Asia. The expected heterozygosity values of iSTR ranged from 0.927 in Thais to 0.874 in Oroqens, higher than those of an STR in the fibrinogen alpha chain (FGA) gene on chromosome 4q. Thus, iSTR is a useful marker for anthropological and forensic genetics.

A founder haplotype of APOE-Sendai mutation associated with lipoprotein glomerulopathy.

Lipoprotein glomerulopathy (LPG) is a hereditary disease characterized by lipoprotein thrombi in the glomerulus, hyperlipoproteinemia, and a marked increase in serum apolipoprotein E (APOE). More than 12 APOE mutations have been identified as causes of LPG, and APOE-Sendai (Arg145Pro) mutation was frequently detected in patients from the eastern part of Japan including Yamagata prefecture. Recently, effective therapy with intensive lipid-lowering agents was established, and epidemiologic data are required for early diagnosis. We determined the haplotype structure of APOE-Sendai in 13 patients from 9 unrelated families with LPG, and found that the haplotype of all APOE-Sendai mutations was identical, suggesting that APOE-Sendai mutation is common in Japanese patients probably through a founder effect. We also studied the gene frequency of APOE-Sendai in 2023 control subjects and 418 patients receiving hemodialysis in Yamagata prefecture using the TaqMan method, but did not identify any subjects carrying the mutation, indicating that it is very rare in the general population even in the eastern part of Japan. In addition to APOE mutation, other genetic and/or epigenetic factors are considered to be involved in the pathogenesis of LPG because of its low penetrance. The patients did not have a common haplotype of the counterpart APOE allele, and some patients had the same haplotype of the counterpart APOE allele as the asymptomatic carriers. These results suggest that the counterpart APOE allele is not likely associated with the onset of LPG. Further study is required to clarify the pathogenesis of LPG.

A hypervariable STR polymorphism in the CFI gene: mutation rate and no linkage disequilibrium with FGA.

A hypervariable short tandem repeat (STR) polymorphism in intron 7 of the human complement factor I gene (CFI) was investigated to estimate the mutation rate in Japanese samples and to test linkage disequilibrium (LD) with an STR in the fibrinogen alpha chain gene (FGA). The expected heterozygosity and the mutation rate of CFI were estimated to be 0.917 and 0.002, respectively. No LD was observed between CFI and FGA. CFI is a useful supplementary marker for forensic science.

The history of human populations in the Japanese Archipelago inferred from genome-wide SNP data with a special reference to the Ainu and the Ryukyuan populations.

The Japanese Archipelago stretches over 4000 km from north to south, and is the homeland of the three human populations; the Ainu, the Mainland Japanese and the Ryukyuan. The archeological evidence of human residence on this Archipelago goes back to >30 000 years, and various migration routes and root populations have been proposed. Here, we determined close to one million single-nucleotide polymorphisms (SNPs) for the Ainu and the Ryukyuan, and compared these with existing data sets. This is the first report of these genome-wide SNP data. Major findings are: (1) Recent admixture with the Mainland Japanese was observed for more than one third of the Ainu individuals from principal component analysis and frappe analyses; (2) The Ainu population seems to have experienced admixture with another population, and a combination of two types of admixtures is the unique characteristics of this population; (3) The Ainu and the Ryukyuan are tightly clustered with 100% bootstrap probability followed by the Mainland Japanese in the phylogenetic trees of East Eurasian populations. These results clearly support the dual structure model on the Japanese Archipelago populations, though the origins of the Jomon and the Yayoi people still remain to be solved.

Mitochondrial DNA analysis of Hokkaido Jomon skeletons: remnants of archaic maternal lineages at the southwestern edge of former Beringia.

To clarify the colonizing process of East/Northeast Asia as well as the peopling of the Americas, identifying the genetic characteristics of Paleolithic Siberians is indispensable. However, no genetic information on the Paleolithic Siberians has hitherto been reported. In the present study, we analyzed ancient DNA recovered from Jomon skeletons excavated from the northernmost island of Japan, Hokkaido, which was connected with southern Siberia in the Paleolithic period. Both the control and coding regions of their mitochondrial DNA (mtDNA) were analyzed in detail, and we confidently assigned 54 mtDNAs to relevant haplogroups. Haplogroups N9b, D4h2, G1b, and M7a were observed in these individuals, with N9b being the predominant one. The fact that all these haplogroups, except M7a, were observed with relatively high frequencies in the southeastern Siberians, but were absent in southeastern Asian populations, implies that most of the Hokkaido Jomon people were direct descendants of Paleolithic Siberians. The coalescence time of N9b (ca. 22,000 years) was before or during the last glacial maximum, implying that the initial trigger for the Jomon migration in Hokkaido was increased glaciations during this period. Interestingly, Hokkaido Jomons lack specific haplogroups that are prevailing in present-day native Siberians, implying that diffusion of these haplogroups in Siberia might have been after the beginning of the Jomon era, about 15,000 years before present.

Distribution of OCA2∗481Thr and OCA2∗615Arg, associated with hypopigmentation, in several additional populations.

Two mutants, OCA2∗481Thr (c.1441G>A, p.Ala481Thr) and OCA2∗615Arg (c.1844A>G, p.His615Arg), in the OCA2 (oculocutaneous albinism type II) gene are associated with hypopigmentation in East Asians. Here, these two alleles were studied to assess the frequencies in five different populations. In addition, the allele frequency of OCA2∗615Arg was investigated in seven populations. Among a total of 24 global populations investigated, Oroqens in Heihe showed the highest frequency for OCA2∗481Thr (0.519), and among 26 populations, Han Chinese in Changsha showed the highest frequency for OCA2∗615Arg (0.673). This study confirmed that these two East Asian-specific alleles are characteristic of northern and central-southern East Asian populations.