mtDNA Analysis Research Articles

Next Generation Sequencing Mitochondrial DNA Analysis in Autism Spectrum Disorder.

Autism is a complex genetic disorder where both de‐novo and inherited genetics factors play a role. Next generation sequencing approaches have been extensively used to identify rare variants associated with autism. To date, all such studies were focused on nuclear genome; thereby leaving the role of mitochondrial DNA (mtDNA) variation in autism unexplored. Recently, analytical tools have been developed to evaluate mtDNA in whole‐exome data. We have analyzed the mtDNA sequence derived from whole‐exome sequencing in 10 multiplex families. In one of the families we have identified two variants of interest in MT‐ND5 gene that were previously determined to impair mitochondrial function. In addition in a second family we have identified two VOIs; mtDNA variant in MT‐ATP6 and nuclear DNA variant in NDUFS4, where both VOIs are within mitochondrial Respiratory Chain Complex. Our findings provide further support for the role of mitochondria in ASD and confirm that whole‐exome sequencing allows for analysis of mtDNA, which sets a stage for further comprehensive genetic investigations of the role of mitochondria in autism. Autism Res 2017, 10: 1338–1343. © 2017 International Society for Autism Research, Wiley Periodicals, Inc.

Additional analysis of mtDNA from the Tommy and Mine Canyon sites

Ancient mitochondrial DNA analysis of samples from the Chaco outlier Tommy and Mine Canyon sites (dating to PII and PIII, respectively, and located near Farmington, New Mexico) originally revealed distinctly different haplogroup frequency distributions from one another. An additional twelve samples were added to those already published (Snow et al., 2010), bringing the total number of individuals with mitochondrial DNA data to 60. The additional samples diminished this difference but maintained the statistically significant distinction between the sites, pointing to a potential sampling bias at the Mine Canyon Site that might explain some, but not all, of the difference. The sites were compared with those from both modern and prehistoric populations in the desert Southwest and Mexico to better understand the regional context of the inhabitants, as well as inter-regional relationships, demonstrating that the Tommy Site fits well within the general Southwestern pattern, while the Mine Canyon Site stands out due to its higher than normal frequency of haplogroup A2. Thirteen burials from a kiva structure at the Mine Canyon Site are compared to the remainder of the excavated burials from the site in terms of mtDNA, burial position, pathology, and grave goods. The statistically significant differences of the kiva burials are discussed in terms of possible evidence of site abandonment, disease, slavery, and witchcraft.

The Mitochondrial D‐Loop Is An Error‐Prone Reference For Mitochondrial DNA Copy Number Of Common Deletion Frequency

BACKGROUNDChanges in mitochondrial DNA (mtDNA) copy number and the common deletion that affect electron transport chain function are associated with diverse age‐ and disease‐related pathologies of the heart, vasculature and other major organs. Changes in mtDNA and common deletion copy number to date have been studied as relative changes using qPCR, often using the mitochondrial D‐Loop (DL) as an internal mtDNA reference. However, the fraction of mtDNA containing triple‐stranded DNA (tsDNA) in the DL ranges from ~0 to >95% between tissue types and varying with cells cycle, such that DL copy number could be 1.0–1.5× that of mtDNA copy number. While difficult to assess by qPCR, droplet digital PCR (ddPCR) provides the absolute measurement of copy number required to assess whether changes in the fraction triple‐stranded DL significantly affects assays using the DL as a reference.OBJECTIVETo develop cost effective, duplex assays for mtDNA copy number and common deletion using DNA‐binding dyes and droplet digital PCR (ddPCR), and to test if variations in tsDNA DL affect measured changes in mtDNA copy number or common deletion frequency.METHODSTotal genomic DNA (gDNA) was isolated from rat A7r5 aorta smooth muscle cells and freshly isolated rat aorta, heart, brain, gastrocnemius, and liver using commercial kits. Primers were selected for mtDNA references (DL, ND1), mitochondrial common deletion (ND3, ND4 and ND5) and nuclear references (Eif2c1, beta‐actin). DNA copy number was analyzed using the EvaGreen dye on a Bio‐Rad QX200.RESULTSBased on bioinformatics, Eif2C1 is a single copy number gene, while beta‐actin primers detected ~3 beta‐actin genes (i.e. two pseudogenes). Counting mtDNA copy number as DL/(2*beta‐actin*beta‐actin copy #), A7r5 and aorta had 756 ±2 and 249 ± 86 (n = 9) mtDNA/cell, consistent with published values. The ND3/DL ratio was 0.7 – 0.8 in A7r5 cells and varied between rat tissues (0.4 – 0.9), giving unusually high common deletion frequencies of 20–30% and 10–60%. Using ND1 as a reference, the DL/ND1 ratio was ~1.2–1.3 in A7r5 cells, while the ratio of ND3, ND4 and ND5 to ND1 were all ~1.0. The results were consistent with DL primers were detecting tsDNA D‐Loop. Treating A7r5 cells with the replication terminator 2′,3′‐dideoxycytidine caused DL/ND1 ratios to fall (1.31 ± 0.08 to 1.21 ± 0.05), while ND3/ND1 ratios were unaffected (1.02 ± 0.01 vs 1.05 ± 0.04), consistent with inhibition of rapid DL turn‐over. In gDNA from rat tissues, DL/ND1 showed much greater variation than ND3/ND1, suggesting that variation in the fraction of DL containing tsDNA is a significant confounding factor when comparing mtDNA copy number and common deletion between tissues or experimentally imposed conditions.CONCLUSIONThe D‐Loop is an error prone reference for analyses of mtDNA, imposing up to ~60% error in estimates of mtDNA copy number and common deletion frequency. Future studies should avoid the use of the DL as a reference. Further, ddPCR provides a novel and simple method for detecting changes the frequency of tsDNA in the mitochondrial D‐Loop.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada, Canadian Foundation for Innovation

Asymmetric hybridization and introgression between sibling species of the pufferfish Takifugu that have undergone explosive speciation

Pufferfishes belonging to the genus Takifugu are a prominent example of recent adaptive radiations of marine fishes. Using amplified fragment length polymorphism (AFLP) and mitochondrial DNA (mtDNA) markers, the genetic characteristics of natural hybrids between two sibling species, Takifugu snyderi and Takifugu stictonotus, were investigated to gain insights into the role of hybridization in rapid diversification. Numerous early generations of hybrids (131 F1 hybrids and 18 first-generation backcrosses) were screened by Bayesian assignment procedures from samples collected at three sites off the Pacific coast of eastern Honshu, Japan (Ibaraki: 36°21′N, 140°37′E; Fukushima: 37°03′N, 141°03′E; Iwate: 40°02′N, 141°59′E), during 2012–2014. Analysis of mtDNA indicated that hybridization is highly directional, as the majority of the F1 hybrids (75.6%) were offspring between T. stictonotus females and T. snyderi males. Among the 18 backcrosses, 17 were toward T. snyderi and one was toward T. stictonotus. Two of 118 individuals classified as genetically pure T. snyderi based on AFLP markers were affected by mtDNA introgression from T. stictonotus. These results suggest that interspecific gene flow has been highly asymmetrical toward T. snyderi, which may partly explain the marked difference in intraspecific genetic diversity between the two species. The proportion of F1 hybrids in the Ibaraki and Fukushima areas is exceptionally high compared with that of other marine fishes, indicating the need for continuous monitoring of hybridization and its impact on integrity of each parental species under the changing marine environment.

Phlebotomine sandflies of Botswana: a taxonomic review and a faunistic update with the first record of genus Phlebotomus

The first records of phlebotomine sandflies from Botswana have been published only recently, comprising of four species of genus Sergentomyia. This update presents the first record of genus Phlebotomus, namely Ph. (Anaphlebotomus) rodhaini Parrot, which is also the first detection of a putative vector of leishmaniasis in Botswana. In addition, records of the Sergentomyia “bedfordi (Newstead) group” are reviewed, and the molecular taxonomy of all taxa known from Botswana is analysed based on three mitochondrial gene fragments (mtDNA). The presence of Se. congolensis (Bequaert and Walrveus) and Se. salisburiensis (Abonnenc) is confirmed, whereas the previously mentioned Se. caliginosa Davidson and unassigned specimens of the “bedfordi group” are proposed to belong to the tentatively named Se. bedfordi “Maun” form. The mtDNA analyses confirmed the species delimitations. For the first time, portions of the ND5 gene were used for the purpose of sandfly molecular taxonomy. This gene revealed a high inter-specific variability and may thus be applied as an alternative molecular marker for future studies.

Genetic affinities among the historical provinces of Romania and Central Europe as revealed by an mtDNA analysis

BackgroundAs a major crossroads between Asia and Europe, Romania has experienced continuous migration and invasion episodes. The precise routes may have been shaped by the topology of the territory and had diverse impacts on the genetic structure of mitochondrial DNA (mtDNA) in historical Romanian provinces. We studied 714 Romanians from all historical provinces, Wallachia, Dobrudja, Moldavia, and Transylvania, by analyzing the mtDNA control region and coding markers to encompass the complete landscape of mtDNA haplogroups.ResultsWe observed a homogenous distribution of the majority of haplogroups among the Romanian provinces and a clear association with the European populations. A principal component analysis and multidimensional scaling analysis supported the genetic similarity of the Wallachia, Moldavia, and Dobrudja groups with the Balkans, while the Transylvania population was closely related to Central European groups. These findings could be explained by the topology of the Romanian territory, where the Carpathian Arch played an important role in migration patterns. Signals of Asian maternal lineages were observed in all Romanian historical provinces, indicating gene flow along the migration routes through East Asia and Europe.ConclusionsOur current findings based on the mtDNA analysis of populations in historical provinces of Romania suggest similarity between populations in Transylvania and Central Europe, supported both by the observed clines in haplogroup frequencies for several European and Asian maternal lineages and MDS analyses.

Clonal analysis as a prognostic factor in multiple oral squamous cell carcinoma

ObjectivesA novel classification based on molecular methods to assess clonality defines three types of secondary oral squamous cell carcinoma (OSCC): second primary tumour (SPT) independent from the index tumour, local recurrence (LR), clonally related to the primary tumour, and second field tumour (SFT), derived from the same genetically altered mucosal field as the primary tumour. The present study applied mtDNA analysis in a group of patients experiencing a second loco-regional neoplastic manifestation. The purpose was to differentiate secondary tumours into LRs, SPTs and SFTs and evaluate the prognostic impact in terms of survival rate. Material and methodsThe study population comprised 23 patients who experienced a second neoplastic lesion after a surgical resection of primary OSCC. mtDNA D-loop analysis was applied in paired neoplastic lesions and in clinically and histologically normal mucosa. On the basis of mtDNA results, the second OSCC was classified as LR or SPT or SFT. Disease-free survival was defined as the duration between the appearance of the second neoplastic lesion and death of disease, or last follow-up visit. ResultsSeven secondary tumours were classified as LR, 12 as SFT, 4 as SPT. An altered mucosal field proved a variable significantly related to a better survival rate (p<0.05); 2/12 (16.6%) SFT events failed as compared to 5/7 LRs (71.4%) and 3/4 SPTs (75%). ConclusionmtDNA analysis may be considered a useful tool to differentiate secondary tumours and might influence the choice of the most appropriate treatment in patients with multiple OSCCs.

Mitophagy and mitochondrial biogenesis in atrial tissue of patients undergoing heart surgery with cardiopulmonary bypass.

Mitophagy occurs during ischemia/reperfusion (I/R) and limits oxidative stress and injury. Mitochondrial turnover was assessed in patients undergoing cardiac surgery involving cardiopulmonary bypass (CPB). Paired biopsies of right atrial appendage before initiation and after weaning from CPB were processed for protein analysis, mitochondrial DNA/nuclear DNA ratio (mtDNA:nucDNA ratio), mtDNA damage, mRNA, and polysome profiling. Mitophagy in the post-CPB samples was evidenced by decreased levels of mitophagy adapters NDP52 and optineurin in whole tissue lysate, decreased Opa1 long form, and translocation of Parkin to the mitochondrial fraction. PCR analysis of mtDNA comparing amplification of short vs. long segments of mtDNA revealed increased damage following cardiac surgery. Surprisingly, a marked increase in several mitochondria-specific protein markers and mtDNA:nucDNA ratio was observed, consistent with increased mitochondrial biogenesis. mRNA analysis suggested that mitochondrial biogenesis was traniscription independent and likely driven by increased translation of existing mRNAs. These findings demonstrate in humans that both mitophagy and mitochondrial biogenesis occur during cardiac surgery involving CPB. We suggest that mitophagy is balanced by mitochondrial biogenesis during I/R stress experienced during surgery. Mitigating mtDNA damage and elucidating mechanisms regulating mitochondrial turnover will lead to interventions to improve outcome after I/R in the setting of heart disease.

Mitochondrial control region diversity in Sindhi ethnic group of Pakistan.

The entire mitochondrial DNA control region (nt 16024-576) of 88 unrelated individuals of Sindhi ethnic group residing in different parts of Sindh province of Pakistan was sequenced. Out of 66 different observed haplotypes 50 were unique and 16 were shared by more than one individual. Results showed admixture of mtDNA pool constituting the haplogroups derived mainly from South Asia (47.6%) and West Eurasian (35.7%) whereas the contribution of the African haplogroup was very small (2.4%). High values of genetic diversity (0.992), power of discrimination (0.981) and low value of random match probability (0.018) indicates that mtDNA analysis for this population can effectively be used for forensic casework. The results are valuable contribution towards building mtDNA population variation database for this particular ethnic group from Pakistan.

Evaluation of GeneMarker® HTS for improved alignment of mtDNA MPS data, haplotype determination, and heteroplasmy assessment

Existing software has not allowed for effective alignment of mitochondrial (mt) DNA sequence data generated using a massively parallel sequencing (MPS) approach, combined with the ability to perform a detailed assessment of the data. The regions of sequence that are typically difficult to align are homopolymeric stretches, isolated patterns of SNPs (single nucleotide polymorphisms), and INDELs (insertions/deletions). A custom software solution, GeneMarker® HTS, was developed and evaluated to address these limitations, and to provide a user-friendly interface for forensic practitioners and others interested in mtDNA analysis of MPS data. GeneMarker® HTS generates an exportable consensus mtDNA sequence that produces phylogenetically correct SNP and INDEL calls using a customizable motif-based alignment algorithm. Sequence data from 500 individuals, with various alignment asymmetries and levels of heteroplasmy, were used to assess the software. Accuracy in producing mtDNA haplotypes, the ability to correctly identify low-level heteroplasmic sequence variants, and the user-based features of the software were evaluated. Analyzed sequences yielded correct mtDNA haplotypes, and heteroplasmic variants were properly identified with minimal manual interpretation. The software offers numerous user-defined parameters for filtering the data that address the interests of researchers and practitioners, and provides multiple options for viewing and navigating through the data.

Population genetic structure of the endangered Sierra Nevada yellow-legged frog (Rana sierrae) in Yosemite National Park based on multi-locus nuclear data from swab samples

The mountain yellow-legged species complex (Rana sierrae and Rana muscosa) has declined precipitously in distribution and abundance during the last century. The two primary threats are chytrid epidemic-associated population collapses and predation from the introduction of non-native trout. Widespread declines have occurred throughout the ranges of these species, including populations of R. sierrae in Yosemite National Park. A clear picture of genetic structure of remaining Yosemite R. sierrae populations is critical to short-term management and conservation. We conducted a population genetics study that included samples from 23 geographic sites distributed throughout the range of R. sierrae in Yosemite NP. We used minimally-invasive swab samples to collect genetic data from mitochondrial and nuclear DNA via sequencing (43 transcriptome-derived markers) and analyzed the distribution of genetic variation in a geographic context. Our mtDNA analysis partially confirmed previous results suggesting that two haplotype groups occur in Yosemite: one haplotype group contained high bootstrap support for monophyly while the other did not. However, increased geographic sampling demonstrated that the two haplotypes are not completely geographically partitioned into the two main drainages (Merced drainage and Tuolumne drainage) as previously postulated. Our nuclear DNA analysis revealed a general pattern of genetic isolation by distance, where genetic differentiation was correlated with geographic distance between sites. In addition, our analyses suggested that three clusters of genetically cohesive sites occur in the study area. Understanding population genetic patterns of variability will inform management strategies such as translocations, reintroductions, and monitoring for this endangered frog. Lastly, our next generation sequencing enabled approach allowed us to obtain multi-locus data from minimally-invasive swab samples. Thus researchers can now leverage extensive archives of swab samples (initially collected for pathogen testing) to study host genetics in previously surveyed amphibian populations.

Growth hormone insensitivity with immune dysfunction caused by a STAT5B mutation in the south of Brazil: evidence for a founder effect.

ABSTRACTHomozygous STAT5B mutations causing growth hormone insensitivity with immune dysfunction were described in 10 patients since 2003, including two Brazilian brothers from the south of Brazil. Our objectives were to evaluate the prevalence of their STAT5B mutation in this region and to analyze the presence of a founder effect. We obtained DNA samples from 1,205 local inhabitants, 48 relatives of the homozygous patients and four individuals of another affected family. Genotyping for STAT5B c.424_427del mutation and for two polymorphic markers around it was done through fragment analysis technique. We also determined Y-chromosome and mtDNA haplotypes and genomic ancestry in heterozygous carriers. We identified seven families with STAT5B c.424_427del mutation, with 33 heterozygous individuals. The minor allelic frequency of this mutation was 0.29% in this population (confidence interval 95% 0.08-0.5%), which is significantly higher than the frequency of other pathogenic STAT5B allele variants observed in public databases (p < 0.001). All heterozygous carriers had the same haplotype present in the homozygous patients, found in only 9.4% of non-carriers (p < 0.001), supporting the existence of a founder effect. The Y-chromosome haplotype, mtDNA and genomic ancestry analysis indicated a European origin of this mutation. Our results provide compelling evidence for a founder effect of STAT5B c.424_427del mutation.

When molecules and morphology work together: lines of evidence for the validity of Caridina buehleri Roux (Crustacea : Decapoda : Atyidae) and for C. gueryi Marquet, Keith &amp; Kalfatak as its junior synonym

The taxonomy of the freshwater shrimps Caridina (Atyidae) is very complex and confused mostly because the morphological characters that have traditionally been used for species delimitation and identification are highly plastic. There is thus a need for an integrative approach to their taxonomy. In total, 42 specimens belonging to either Caridina buehleri Roux, 1934 or Caridina gueryi Marquet, Keith &amp;amp; Kalfatak, 2009 were examined. We combined here morphological data from 12 specimens from the whole distributional range of the species, including type specimens with 16S mtDNA analyses from seven freshly sampled specimens, to verify the specific status of Caridina buehleri from Papua New-Guinea, Central Sulawesi, Western Samoa and the Solomon Islands, and C. gueryi from Vanuatu. The comparison of 24 morphological characters showed that morphological variations of character traits between C. gueryi and C. buehleri are widely overlapping and that no morphological feature can effectively split specimens into two clear groups. Molecular characters corroborated these results, as specimens from both groups were only separated by a 2% p-distance, a genetic distance that is coherent with their potentially high dispersal abilities. We thus consider C. gueryi as a junior synonym of C. buehleri. Finally, C. buehleri is characterised mainly by a styliform and characteristically indented rostrum and a long stylocerite. Detailed re-descriptions of the type specimens of C. buehleri and C. gueryi are given, as well as their geographical and ecological distribution.

Entire Mitochondrial DNA Sequencing on Massively Parallel Sequencing for the Korean Population.

Mitochondrial DNA (mtDNA) genome analysis has been a potent tool in forensic practice as well as in the understanding of human phylogeny in the maternal lineage. The traditional mtDNA analysis is focused on the control region, but the introduction of massive parallel sequencing (MPS) has made the typing of the entire mtDNA genome (mtGenome) more accessible for routine analysis. The complete mtDNA information can provide large amounts of novel genetic data for diverse populations as well as improved discrimination power for identification. The genetic diversity of the mtDNA sequence in different ethnic populations has been revealed through MPS analysis, but the Korean population not only has limited MPS data for the entire mtGenome, the existing data is mainly focused on the control region. In this study, the complete mtGenome data for 186 Koreans, obtained using Ion Torrent Personal Genome Machine (PGM) technology and retrieved from rather common mtDNA haplogroups based on the control region sequence, are described. The results showed that 24 haplogroups, determined with hypervariable regions only, branched into 47 subhaplogroups, and point heteroplasmy was more frequent in the coding regions. In addition, sequence variations in the coding regions observed in this study were compared with those presented in other reports on different populations, and there were similar features observed in the sequence variants for the predominant haplogroups among East Asian populations, such as Haplogroup D and macrohaplogroups M9, G, and D. This study is expected to be the trigger for the development of Korean specific mtGenome data followed by numerous future studies.

A new view on the European feline population from mtDNA analysis in Polish domestic cats

Domestic cats from Eastern Europe have been poorly represented in studies on mitochondrial DNA diversity for forensic purposes until now. The aim of the present study was to contribute to closing this gap. The genetic structure and the origin of a cat population in Poland were examined against the background of human migrations over the centuries. One hundred and eighty-one cats from animal shelters in seven cities were genotyped. Twenty-one mtDNA haplotypes were found, with only one haplotype present in each of the populations, at an average frequency of 63.54%, and 13 haplotypes being found only in single populations. The analysis revealed the unexpectedly high frequency of haplotype PL02, in previous studies observed only in single cats. Differences in the number of the haplotypes, from four to eight, were observed among the shelters. The findings are discussed with regard to a world-wide database of feline sequences and to the complicated history of Poland. The study underscores the necessity of creating local databases of haplotypes that are of high evidentiary value to the forensic investigations conducted in a given country.

Whole mitochondrial genome analysis in South Indian patients with Leber's hereditary optic neuropathy

Leber's hereditary optic neuropathy (LHON) is a mitochondrial DNA (mtDNA) associated neurodegenerative disorder of retinal ganglion cells. In this study, whole mitochondrial genome sequencing of 75 LHON patients and 40 controls was performed to identify the mutation frequency and haplogroup background of South Indian population. Analysis of mtDNA revealed 559 different variants in LHON patients, including 7 pathogenic mutations, 30 private, and 22 other disease associated variants. A significantly higher (p=0.0008) overall variation load per individual was noted among LHON patients versus controls. We reported for the first time, the association of M haplogroup (p=0.028) with LHON in this cohort.

A collaborative EDNAP exercise on SNaPshot™-based mtDNA control region typing

A collaborative European DNA Profiling (EDNAP) Group exercise was undertaken to assess the performance of an earlier described SNaPshot™-based screening assay (denoted mini-mtSNaPshot) (Weiler et al., 2016) [1] that targets 18 single nucleotide polymorphism (SNP) positions in the mitochondrial (mt) DNA control region and allows for discrimination of major European mtDNA haplogroups. Besides the organising laboratory, 14 forensic genetics laboratories were involved in the analysis of 13 samples, which were centrally prepared and thoroughly tested prior to shipment. The samples had a variable complexity and comprised straightforward single-source samples, samples with dropout or altered peak sizing, a point heteroplasmy and two-component mixtures resulting in one to five bi-allelic calls. The overall success rate in obtaining useful results was high (97.6%) given that some of the participating laboratories had no previous experience with the typing technology and/or mtDNA analysis. The majority of the participants proceeded to haplotype inference to assess the feasibility of assigning a haplogroup and checking phylogenetic consistency when only 18 SNPs are typed. To mimic casework procedures, the participants compared the SNP typing data of all 13 samples to a set of eight mtDNA reference profiles that were described according to standard nomenclature (Parson et al., 2014) [2], and indicated whether these references matched each sample or not. Incorrect scorings were obtained for 2% of the comparisons and derived from a subset of the participants, indicating a need for training and guidelines regarding mini-mtSNaPshot data interpretation.

Historical dimensions of population structure in a continuously distributed marine species: The case of the endemic Chilean dolphin.

The complementarity of historical and contemporary processes contributes to understanding the genetic structure of continuously distributed marine species with high dispersal capabilities. Cephalorhynchus eutropia, has a continuous coastal distribution with strong genetic differentiation identified by nuclear DNA markers. We explored the historical dimension of this genetic differentiation between northern and southern populations to evaluate phylogeographic structure. Additionally, we conducted mtDNA and microsatellite analyses to detect past and recent demographic changes. The southern population was characterized by lower genetic diversity with a signal of population expansion, likely associated with ice retreat and habitat extension after the Last Glacial Maximum (LGM). In contrast, structure within the northern population was more consistent with stable historical population size. Approximate Bayesian Computation analyses suggested that during the LGM, C. eutropia persisted in the northern area; while the south was colonized by dispersal ~11,000 years ago followed by population expansion. This study shows that Chilean dolphin population structure is consistent with predictions from the Expansion-Contraction biogeographic model, with a poleward post-glacial shift revealed in current genetic structure. The results also confirm the validity of the population units previously identified, demonstrating their historical origin and highlighting the utility of integrating genetic markers with different temporal scale resolutions.

Computed tomography examination and mitochondrial DNA analysis of Japanese wolf skull covered with skin.

A Canis skull, right half of the mandible and part of the left half of the mandible were subjected to three-dimensional (3D) computed tomography (CT) observation and mitochondrial DNA (mtDNA) analysis in order to determine whether the specimens belonged to the extinct Japanese wolf, Canis lupus hodophilax (Temminck, 1839). Osteometric analysis of the skull and right half of the mandible revealed that the material (JW275) was indeed typical of the Japanese wolf. Sequence analysis of a 600-bp mtDNA region revealed that the JW275 belonged to haplotype Group B, which is characterized by an 8-bp deletion in the mtDNA control region. The findings of this study suggest that 3D CT analysis is well suited to examining fragile and valuable biological samples, as it removes the need for destructive sampling.

Seven genes of mitochondrial genome enabling differentiation of honeybee subspecies Apis mellifera

On the basis of comparative sequence analysis of 12 honeybee mitochondrial genes, seven genes enabling us to differentiate honeybees subspecies of the A, M, C, O evolutionary lineages were found. Applying comparative sequence analysis of ND2 gene mtDNA as an example on a statistically valid sample size, we showed a high level of differentiating ability of this gene and assumed that each of these seven genes probably can be used for differentiation of the subspecies within four evolutionary lineages.