Phylogenetic classification of Japanese mtDNA assisted by complete mitochondrial DNA sequences

We investigated control and coding region polymorphisms in mitochondrial DNA (mtDNA) in 100 unrelated individuals from a Japanese population and determined the basal phylogenetic haplogroup lineages in all samples under updated information. Many of the basal phylogenetic haplogroup lineages assigned on East Asian mtDNA haplogroups corresponded to those previously established. However, new haplogroup lineages such as M7a2a, M7a2b, M7a2*, M7c1b, M11b2*, G2b*, D4c1b1a, D4g2b, A4*, A9, N9b*, B4d1, B4d2, and F1e were identified and established by complete sequencing. Although sequence comparison of the 1.15-kb control region identified 84 mitochondrial haplotypes, examination of coding region polymorphisms increased the total number of haplotypes to 91. Determination of the basal haplogroup lineages increased the discrimination power of mtDNA polymorphisms for personal identification and their usefulness in determining geographic origin in forensic casework in Japanese and other East Asian populations.     

Sequence polymorphisms of the mitochondrial DNA control region and phylogenetic analysis of mtDNA lineages in the Japanese population

Sequence polymorphisms of the hypervariable mitochondrial DNA (mtDNA) regions HVI and HVII, and coding region polymorphisms were investigated in 211 unrelated individuals from the Japanese population. Sequence comparison of the HVI and HVII regions led to the identification of 169 mitochondrial haplotypes defined by 147 variable positions. Among them 145 types were observed in only 1 individual; the other 24 types were shared by 2 or more individuals. The gene diversity was estimated at 0.9961, and the probability of two randomly selected individuals from the population having identical mtDNA types was 0.86%. We also established phylogenetic haplogroups in the Japanese population based on the coding and control region polymorphisms and compared the haplotypes with those in other Japanese, Korean and Chinese populations. As a result, three new subhaplogroups, G4a, G4b, and N9b, and several haplotypes specific for the Japanese and Korean populations were identified. The present database can be used not only for personal identification but also as an aid for geographic or phenotype (race) estimation in forensic casework in Japan.Electronic Supplementary Material Supplementary material is available for this article if you access the article at     

Forensic and genetic characterization of mtDNA from Pathans of Pakistan

Complete mitochondrial control region data were generated for 230 unrelated Pathans from North West Frontier Province and Federally Administered Tribal Areas of Pakistan. To confirm data quality and to explore the genetic structure of Pathans, mitochondrial DNA haplogroup affiliation was determined by shared haplogroup-specific polymorphisms in the control region and by the analysis of diagnostic coding region single-nucleotide polymorphisms using a multiplex system for the assignment of eight haplogroups: M, N1′5, W, R, R0, T, J, and U. Sequence comparison revealed that 193 haplotypes were defined by 215 variable sites when major insertions were ignored at nucleotide positions 16193, 309, and 573. From a phylogenetic perspective, Pathans have a heterogeneous origin, displaying a high percentage of West Eurasian haplogroups followed by haplogroups native to South Asia and a small fraction from East Asian lineages. In population comparisons, this ethnic group differed significantly from several other ethnic groups from Pakistan and surrounding countries. These results suggest that frequency estimates for mtDNA haplotypes should be determined for endogamous ethnic groups individually instead of pooling data for these subpopulations into a single dataset for the Pakistani population. Data presented here may contribute to the accuracy of forensic mtDNA comparisons in the Pathans of Pakistan.     

A one step multiplex PCR assay for rapid screening of East Asian mtDNA haplogroups on forensic samples

The mitochondrial DNA (mtDNA) haplogroup typing has become an essential tool to study human evolutionary history and to infer the matrilineal bio-geographic ancestry. In forensic field, the screening of mtDNA haplogroups by genotyping of mtDNA single nucleotide polymorphisms (SNPs) can help guarantee the quality of mtDNA sequence data as well as can reduce the need to sequence samples that do not match. Here, a multiplex mutagenically separated (MS) polymerase chain reaction (PCR) system was developed for simultaneous rapid detection of 14 coding region SNPs and one deletion motif representing common mtDNA haplogroups of East Asia. The multiplex MS PCR system we developed has the advantage of being a one step procedure that requires only a single PCR amplification with allele-specific primers and allowing straightforward designation of haplogroups along the branches of the phylogenetic tree. Therefore, it would be a simple, rapid, and reliable detection method useful for large-scale screening of mtDNA variations to determine East Asian mtDNA haplogroups.     

Recent progress in mitochondrial DNA analysis

In this review, we describe the current state of knowledge of mitochondrial genetics of East Asian populations and its application to forensic science. Recent advances in mitochondrial DNA (mtDNA) phylogeny have identified haplogroup-specific single nucleotide polymorphisms (SNPs) and the control region motifs of haplogroups. By analyzing haplogroup-specific SNPs, we can rapidly and accurately connect the mtDNA under study to the relevant haplogroup. Haplogroups are fairly continent- and/or region-specific; therefore, we can infer the ethnic background of that mtDNA. In addition, errors in hypervariable region sequences can be detected by means of haplogroup motif analysis.     

Resolving mitochondrial haplogroups B2 and B4 with next-generation mitogenome sequencing to distinguish Native American from Asian haplotypes

Mitochondrial haplogroup information can be useful in forensic contexts that rely primarily on mitochondrial DNA (mtDNA) testing, which often involve limited or degraded DNA. Due to the phylogeographic patterning of mtDNA in human populations, mitochondrial haplogroups are indicative of maternal ancestry (as mtDNA is a maternally inherited marker). In certain circumstances, maternal ancestry inferred from mitochondrial haplogrouping could be beneficial to forensic investigations. For example, ancestry information could assist in the identification of unknown service members from past conflicts, such as the World War II Battle of Tarawa involving American and Japanese forces. In this context, it could be useful to distinguish Native American mtDNA from Asian mtDNA to bolster the anthropological and circumstantial evidence leading to an identification or foreign national determination. Although most of the founding Native American haplogroups contain diagnostic variants in the mitochondrial control region (CR), haplogroup B2 does not, and this makes it more difficult to distinguish B2 from the parental B4 and closely related B4b haplogroups found in Asia. In this paper, the amount of mtDNA information required to distinguish Native American haplotypes from Asian haplotypes within haplogroup B was examined. Fifty-six samples belonging to subtypes of B2 and B4 were sequenced for the entire mitogenome. Haplogroups were estimated from three ranges of mitochondrial DNA (HV1 and 2, CR, and full mitogenome). Half of the samples could not be precisely haplogrouped without full mitogenome data, although enough variants were often provided to make an accurate B2 versus B4 distinction. Native American B2 haplotypes were distinguishable using CR data alone in 82% of samples, though the remaining samples required full mitogenome data for haplogroup B2 designation. The use of full mitogenome data consistently enables accurate haplogroup determination, and opens the possibility for gaining information on maternal ancestry.     

Haplotype diversity in mitochondrial DNA hypervariable region in a population of southeastern Brazil

Brazilian population derives from Native Amerindians, Europeans, and Africans. Southeastern Brazil is the most populous region of the country. The present study intended to characterize the maternal genetic ancestry of 290 individuals from southeastern (Brazil) population. Thus, we made the sequencing of the three hypervariable regions (HV1, HV2, and HV3) of the mitochondrial DNA (mtDNA). The statistical analyses were made using Arlequin software, and the median-joining haplotype networks were generated using Network software. The analysis of three hypervariable regios showed 230 (79.3 %) unique haplotypes and the most common haplotype was “263G” carried by 12 (4.1 %) individuals. The strikingly high variability generated by intense gene flow is mirrored in a high sequence diversity (0.9966?±?0.0010), and the probability of two random individuals showing identical mtDNA haplotypes were 0.0068. The analysis of haplogroup distribution revealed that 36.9 % (n?=?107) presented Amerindian haplogroups, 35.2 % (n?=?102) presented African haplogroups, 27.6 % (n?=?80) presented European haplogroups, and one (0.3 %) individual presented East Asian haplogroup, evidencing that the southeastern population is extremely heterogeneous and the coexistence of matrilineal lineages with three different phylogeographic origins. The genetic diversity found in the mtDNA control region in the southeastern Brazilian population reinforces the importance of increased national database in order to be important and informative in forensic cases.     

Characterization of human control region sequences for Spanish individuals in a forensic mtDNA data set

Population data on the hypervariable regions of the mitochondrial DNA (mtDNA) genome are used to convey the relative rarity of mtDNA profiles obtained from evidence samples and of profiles used to identify missing persons. In this study, mtDNA profiles of Spanish individuals (n=312) were analyzed to describe haplogroup distributions and to determine relevant single nucleotide polymorphisms (SNPs) of those haplogroups. All nine common European haplogroups were observed in the sample, and these were divided into subgroups when possible. Haplogroup H was the most common haplogroup. The haplogroups U, J, T, and V were the next most frequent groups, each occurring at a frequency of 6.4% or greater. In addition, African and Asian sequences were present though rare in the samples. The data were compared with and found to be similar to other published data sets. There were 109 SNPs observed in the data set, including 10 positions not previously reported. The most variable sites are consistent with other studies.     

Sequence polymorphisms of mtDNA HV1, HV2 and HV3 regions in eight population groups living in Taiwan

Analysis of human mitochondrial DNA (mtDNA) polymorphisms in the D-loop region has become a useful tool in forensic casework and matrilineal origin research. In this study, the mtDNA D-loop region including hypervariable region 1 (HV1), hypervariable region 2 (HV2), segment between HV1 and HV2 (7S DNA spanned region), and extended hypervariable region 3 (HV3ex) was sequenced in 539 unrelated individuals from eight population groups living in Taiwan. Combined analyses of the complete D-loop revealed a total of 383 haplotypes with 319 unique haplotypes. The probability of any two individuals sharing the same mtDNA haplotype decreased as the combination of control region segments extended and reached 0.48% with the combination of a complete D-loop region. Sequence variants in HV3ex can further discriminate the haplotypes in some population groups. Phylogenetic haplogroups of these subjects were analyzed. The multidimensional scaling plots of these population groups, constructed based on sequence of the complete D-loop, demonstrated a clear matrilineal genetic substructure in this area. In conclusion, this database of mtDNA complete D-loop sequence including HV3 can serve as a reference for forensic identification. Sequence polymorphisms of the D-loop located outside the HV1 and HV2 may be helpful in further haplogroup characterization.     

Forensic and phylogeographic characterization of mtDNA lineages from northern Thailand (Chiang Mai)

The immigration of diverse ethnic groups over the past centuries from surrounding countries into Thailand left footprints in the genetic composition of Thai mitochondrial DNA (mtDNA) lineages. The entire mtDNA control region (1,122 bp) was typed in 190 unrelated male volunteers from the northern Thailand province of Chiang Mai following highest quality standards. For a more precise haplogroup classification, selected single nucleotide polymorphisms from the mtDNA coding region were genotyped. We found several new, so far undescribed mtDNA lineages. Quasi-median networks were constructed for visualisation of character conflicts. The data were put into population-genetic relationships with other Southeast Asian populations. Although the frequencies of the Thai haplogroups were characteristic for Southeast Asia in terms of haplotype composition and genetic structure, the Thai population was significantly different from other Southeast Asian populations. This necessitates establishing regional databases, especially for forensic applications. The population data have been submitted to the EMPOP database () and will be available on publication.     

Concept for estimating mitochondrial DNA haplogroups using a maximum likelihood approach (EMMA)

The assignment of haplogroups to mitochondrial DNA haplotypes contributes substantial value for quality control, not only in forensic genetics but also in population and medical genetics. The availability of Phylotree, a widely accepted phylogenetic tree of human mitochondrial DNA lineages, led to the development of several (semi-)automated software solutions for haplogrouping. However, currently existing haplogrouping tools only make use of haplogroup-defining mutations, whereas private mutations (beyond the haplogroup level) can be additionally informative allowing for enhanced haplogroup assignment. This is especially relevant in the case of (partial) control region sequences, which are mainly used in forensics. The present study makes three major contributions toward a more reliable, semi-automated estimation of mitochondrial haplogroups. First, a quality-controlled database consisting of 14,990 full mtGenomes downloaded from GenBank was compiled. Together with Phylotree, these mtGenomes serve as a reference database for haplogroup estimates. Second, the concept of fluctuation rates, i.e. a maximum likelihood estimation of the stability of mutations based on 19,171 full control region haplotypes for which raw lane data is available, is presented. Finally, an algorithm for estimating the haplogroup of an mtDNA sequence based on the combined database of full mtGenomes and Phylotree, which also incorporates the empirically determined fluctuation rates, is brought forward. On the basis of examples from the literature and EMPOP, the algorithm is not only validated, but both the strength of this approach and its utility for quality control of mitochondrial haplotypes is also demonstrated.     

Genetic study of 12 X-STRs in Malay population living in and around Kuala Lumpur using Investigator Argus X-12 kit

We investigated 12 X-chromosomal short tandem repeat (STR) polymorphisms in 283 unrelated Malay individuals (160 males and 123 females) living in and around Kuala Lumpur using the Investigator Argus X-12 kit. Heterozygosity among the present 12 X-STRs showed a distribution of from 55.3 to 93.5 %. The diversity values of the haplotypes constructed using four closely linked groups were all higher than 0.9865. A comparison of allelic frequency in each system and haplotype variation indicated that the nature of these X-STRs in the Malay population differed from that in East Asian, European, or African populations. Several microvariant alleles found in the Malay population were characterized and compared with known sequence data. The present data may be helpful in forensic casework such as personal identification and kinship testing in the Malay population in Malaysia.     

Hierarchical analysis of 30 Y-chromosome SNPs in European populations

Analysis of Y-chromosome haplogroups defined by binary polymorphisms, has became a standard approach for studying the origin of modern human populations and for measuring the variability between them. Furthermore, the simplicity and population specificity of binary polymorphisms allows inferences to be drawn about the population origin of any male sample of interest for forensic purposes. From the 245 binary polymorphisms that can be analysed by PCR described in the Y Chromosome Consortium tree, we have selected 30 markers. The set of 30 has been grouped into 4 multiplexes in order to determine the most frequent haplogroups in Europe, using only 1 or 2 multiplexes. In this way, we avoid typing unnecessary SNPs to define the final haplogroup saving effort and cost, since we only need to type 9 SNPs in the best case and in the worst case, no more than 17 SNPs to define the haplogroup. The selected method for allele discrimination was a single base extension reaction using the SNaPshot multiplex kit. A total of 292 samples from 8 different districts of Galicia (northwest Spain) were analysed with this strategy. No significant differences were detected among the different districts, except for the population from Mariña Lucense, which showed a distant haplogroup frequency but not higher _st values.     

Control region sequences for East Asian individuals in the Scientific Working Group on DNA Analysis Methods forensic mtDNA data set

The Scientific Working Group on DNA Analysis Methods (SWGDAM) mitochondrial DNA (mtDNA) population data set is used to infer the relative rarity of mtDNA profiles obtained from evidence samples and of profiles used to identify missing persons. In this study, the East Asian haplogroup patterns in the SWGDAM data sets were analyzed in a phylogenetic context to determine relevant single nucleotide polymorphisms (SNPs) and to describe haplogroup distributions for Asians (n = 753; with a breakdown of individuals from China n = 356, Korea n = 182, Japan n = 163, and Thailand n = 52). We focus on the patterns observed in the SWGDAM Chinese data set and refer to interesting differences in the smaller subgroup data sets for the other East Asian populations (Japanese, Korean, and Thai). A total of 218 SNPs were observed in the data set, including 37 observed positions not previously reported. In the largest of the East Asian SWGDAM data sets (Chinese), these SNPs ranged from having 1 to 29 changes in the phylogenetic tree, with site 16519 being the most variable. On average there were 4.5 changes for a character on the tree. The most variable sites (with 14 or more changes each listed from fastest to slowest) observed were 16519 (L = 29), 16311 (L = 27), 152 (L = 24), 146 (L = 21), 16172 (L = 17), 16189 (L = 17), 195 (L = 16), 16362 (L = 15), 16093 (L = 14), 16129 (L = 14) and 150 (L = 14). These rapidly changing sites are consistent with other published analyses. Only 28 SNPs are needed to identify all clusters containing 1% (n = 7) or more individuals in the East Asian data set. All 36 haplogroups previously observed in East Asian populations were also seen in the SWGDAM data sets and include: A, B, B4, B4a, B4b, B5a, B5b, C, D, D4, D4a, D4b, D5, D5a, F, F1, F1a, F1b, F1c, F2a, G2, G2a, M, M7a1, M7b, M7b1, M7b2, M7c, M8a, M9, M10, N9a, R, R9a, Y, and Z. Haplogroups A, B4a, D4, and F1a were the most commonly observed clusters in the Chinese data set (the largest of the data sets) with each of these occurring in more than 6% of the samples in the data set. The next most common haplogroups in the Chinese data set include the clusters C, M7b1, and N9a with each observed at frequencies greater than or equal to 4%. European Caucasian, and African haplogroups were rarely observed within the East Asian data sets. The various analyses revealed that the data set was similar to published East Asian data sets such as those from Han Chinese.     

Genetic polymorphism of both 29 Y-STRs and 213 Y-SNPs in Han populations from Shandong Province,China

Genetic markers on the Y chromosome, including short tandem repeats (Y-STRs) and single nucleotide polymorphisms (Y-SNPs), are used widely in forensic genetics. Both Y-STR-based haplotypes and Y-SNP-based haplogroups provide information on a population’s genetic structure, which is useful for the identification of individuals. However, there are few studies on these two types of genetic markers in the various Chinese populations. In this study, 284 Han individuals from four prefecture-level cities in Shandong Province (Binzhou, Dezhou, Heze, and Weihai) were genotyped by 29 Y-STRs (from our previous study) and 213 Y-SNPs (self-designed for the Haplogroup O2 Y-SNP panel). Haplogroup O was the most predominant among the four cities. The highest haplogroup diversity (0.9745) was observed in the Heze population, with a discrimination capacity (DC) value of 0.5625. The haplotype diversity and DC values of the Binzhou and Heze populations were 1.0000. Furthermore, genetic differences were observed between the coastal and inland cities; the results of their statistical analysis are presented herein.     

mtDNA sequence diversity of Hazara ethnic group from Pakistan

The present study was undertaken to investigate mitochondrial DNA (mtDNA) control region sequences of Hazaras from Pakistan, so as to generate mtDNA reference database for forensic casework in Pakistan and to analyze phylogenetic relationship of this particular ethnic group with geographically proximal populations. Complete mtDNA control region (nt 16024-576) sequences were generated through Sanger Sequencing for 319 Hazara individuals from Quetta, Baluchistan. The population sample set showed a total of 189 distinct haplotypes, belonging mainly to West Eurasian (51.72%), East & Southeast Asian (29.78%) and South Asian (18.50%) haplogroups. Compared with other populations from Pakistan, the Hazara population had a relatively high haplotype diversity (0.9945) and a lower random match probability (0.0085). The dataset has been incorporated into EMPOP database under accession number EMP00680. The data herein comprises the largest, and likely most thoroughly examined, control region mtDNA dataset from Hazaras of Pakistan.     

Understanding the Y chromosome variation in Korea—relevance of combined haplogroup and haplotype analyses

We performed a molecular characterization of Korean Y-chromosomal haplogroups using a combination of Y-chromosomal single nucleotide polymorphisms (Y-SNPs) and Y-chromosomal short tandem repeats (Y-STRs). In a test using DNA samples from 706 Korean males, a total of 19 different haplogroups were identified by 26 Y-SNPs including the newly redefined markers (PK4, KL2, and P164) in haplogroup O. When genotyping the SNPs, phylogenetic nonequivalence was found between SNPs M117 and M133, which define haplogroup O3a3c1 (O3a2c1a according to the updated tree of haplogroup O by Yan et al. (European Journal of Human Genetics 19:1013-1015, 2011)), suggesting that the position of the M133 marker should be corrected. We have shown that the haplotypes consisted of DYS392, DYS393, DYS437, DYS438, DYS448, and DYS388 loci, which exhibit a relatively lower mutation rate, can preserve phylogenetic information and hence can be used to roughly distinguish Y-chromosome haplogroups, whereas more rapidly mutating Y-STRs such as DYS449 and DYS458 are useful for differentiating male lineages. However, at the relatively rapidly mutating DYS447, DYS449, DYS458, and DYS464 loci, unusually short alleles and intermediate alleles with common sequence structures are informative for elucidating the substructure within the context of a particular haplogroup. In addition, some deletion mutations in the DYS385 flanking region and the null allele at DYS448 were associated with a single haplogroup background. These high-resolution haplogroup and haplotype data will improve our understanding of regional Y-chromosome variation or recent migration routes and will also help to infer haplogroup background or common ancestry.     

Portuguese mitochondrial DNA genetic diversity—An update and a phylogenetic revision

In recent years a large amount of mitochondrial population data for forensic purposes has been produced. Current efforts are focused at increasing the number of studied populations while generating updated genetic information of forensic quality. However, complete mitochondrial control region sequences are still scarce for most populations and even more so for complete mitochondrial genomes. In the case of Portugal, previous population genetics studies have already revealed the general portrait of HVS-I and HVS-II mitochondrial diversity, becoming now important to update and expand the mitochondrial region analysed. Accordingly, a total of 292 complete control region sequences from continental Portugal were obtained, under a stringent experimental design to ensure the quality of data through double sequencing of each target region. Furthermore, H-specific coding region SNPs were examined to detail haplogroup classification and complete mitogenomes were obtained for all sequences belonging to haplogroups U4 and U5. In general, a typical Western European haplogroup composition was found in mainland Portugal, associated to high level of mitochondrial genetic diversity. Within the country, no signs of substructure were detected. The typing of extra coding region SNPs has provided the refinement or confirmation of the previous classification obtained with EMMA tool in 96% of the cases. Finally, it was also possible to enlarge haplogroup U phylogeny with 28 new U4 and U5 mitogenomes.     

Mitochondrial DNA variation is associated with elite athletic status in the Polish population

There is mounting evidence that genetic factors located in mitochondrial and nuclear genomes influence sport performance. Certain mitochondrial haplogroups and polymorphisms were associated with the status of elite athlete, especially in endurance performance. The aim of our study was to assess whether selected mitochondrial DNA (mtDNA) and nuclear DNA variants are associated with elite athlete performance in a group of 395 elite Polish athletes (213 endurance athletes and 182 power athletes) and 413 sedentary controls. Our major finding was that the mtDNA haplogroup H and HV cluster influence endurance performance at the Olympic/World Class level of performance (P = 0.018 and P = 0.0185, respectively). We showed that two polymorphisms located in the mtDNA control region were associated with achieving the elite performance level either in the total athlete's group as compared with controls (m.16362C, 3.8% vs 9.2%, respectively, P = 0.0025, odds ratio = 0.39, 95% confidence interval: 0.21–0.72), or in the endurance athletes as compared with controls (m.16080G, 2.35% vs 0%, respectively, P = 0.004). Our results indicate that mtDNA variability affects the endurance capacity rather than the power one. We also propose that mtDNA haplogroups and subhaplogroups, as well as individual mtDNA polymorphisms favoring endurance performance, could be population‐specific, reflecting complex cross‐talk between nuclear and mitochondrial genomes.     

Utility of haplogroup determination for forensic mtDNA analysis in the Japanese population

Sequence analysis of the hypervariable regions (HVRs) of mitochondrial DNA (mtDNA) are routinely performed in forensic casework, however, there are still issues to be resolved, such as the existence of multiple errors in published databases or the limitations of individual discrimination in certain populations. Here, we analyzed the coding region of mtDNA in detail by examining 36 haplogroup (HG)-defining single nucleotide polymorphisms (SNPs) using amplified product-length polymorphisms (APLP) method in conjunction with sequence analysis of HVR1 and HVR2 to establish a methodology for forensically reliable and practical mtDNA testing. The mtDNAs from 217 unrelated Japanese were examined and could be classified into 27 haplogroups. By combining the data of the coding region with those of HVRs, genetic diversity was slightly increased from 0.9817 to 0.9888 for HVR1/HG and from 0.9967 to 0.9970 for HVR1/HVR2/HG, as compared to the results of HVRs only. Moreover, in most cases, reliability of the HVR data could be confirmed by haplogroup motif analysis. Our mtDNA profiling method can provide reliable data in a time and cost-saving way due to the rapid and economical nature of APLP analysis.