Belgian canine population and purebred study for forensics by improved mitochondrial DNA sequencing

In canine population studies for forensics, the mitochondrial DNA is profiled by sequencing the two hyper variable regions, HV1 and HV2 of the control region. In a first effort to create a Belgian population database some samples showed partially poor sequence quality. We demonstrated that a nuclear pseudogene was co-amplified with the mtDNA control region. Using a new combination of primers this adverse result was no longer observed and sequencing quality was improved. All former samples with poor sequence data were reanalyzed. Furthermore, the forensic canine population study was extended to 208 breed and mixed dogs. In total, 58 haplotypes were identified, resulting in an exclusion capacity of 0.92. The profile distribution of the Belgian population sample was not significantly different from those observed in population studies of three other countries. In addition to the total population study 107 Belgian registered pedigree dogs of six breeds were profiled. Per breed, the obtained haplotypes were supplemented with those from population and purebred studies. The combined data revealed that some haplotypes were more or less prominent present in particular dog breeds. The statistically significant differences in haplotype distribution between breeds and population sample can have consequences on mtDNA databasing and matching probabilities in forensics.     

Identification of single nucleotide polymorphisms within the mtDNA genome of the domestic dog to discriminate individuals with common HVI haplotypes

We sequenced the entire ～16 kb canine mitochondrial genome (mtGenome) of 100 unrelated domestic dogs (Canis lupus familiaris) and compared these to 246 published sequences to assess hypervariable region I (HVI) haplotype frequencies. We then used all available sequences to identify informative single nucleotide polymorphisms (SNPs) outside of the control region for use in further resolving mtDNA haplotypes corresponding to common HVI haplotypes. Haplotype frequencies in our data set were highly correlated with previous ones (e.g., F(ST)=0.02, r=0.90), suggesting the total data set reasonably reflected the broader dog population. A total of 128 HVI haplotypes was represented. The 10 most common HVI haplotypes (n=184 dogs) represented 53.3% of the sample. We identified a total 71 SNPs in the mtGenomes (external to the control region) that resolved the 10 most common HVI haplotypes into 63 mtGenome subhaplotypes. The random match probability of the dataset based solely on the HVI sequence was 4%, whereas the random match probability of the mtGenome subhaplotypes was <1%. Thus, the panel of 71 SNPs identified in this study represents a useful forensic tool to further resolve the identity of individual dogs from mitochondrial DNA (mtDNA).     

Molecular characterization of the canine mitochondrial DNA control region for forensic applications

The canine mitochondrial DNA (mtDNA) control region of 133 dogs living in the area around Innsbruck, Austria was sequenced. A total of 40 polymorphic sites were observed in the first hypervariable segment and 15 in the second, which resulted in the differentiation of 40 distinct haplotypes. We observed five nucleotide positions that were highly polymorphic within different haplogroups, and they represent good candidates for mtDNA screening. We found five point heteroplasmic positions; all located in HVS-I and a polythymine region in HVS-II, the latter often being associated with length heteroplasmy. In contrast to human mtDNA, the canine control region contains a hypervariable 10 nucleotide repeat region, which is located between the two hypervariable regions. In our population sample, we observed eight different repeat types, which we characterized by direct sequencing and fragment length analysis. The discrimination power of the canine mtDNA control region was 0.93, not taking the polymorphic repeat region into consideration. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Evaluation of mitochondrial DNA coding region assays for increased discrimination in forensic analysis

There is an increasing trend to use mitochondrial DNA (mtDNA) analysis in criminal investigations where only limited amounts of DNA are available. However, analysis of the mtDNA control region has the drawback of low discrimination power, due to the lack of recombination that results from uniparental (maternal) inheritance. As a strategy to increase discrimination, a number of typing assays detecting variation in the mitochondrial coding region have been developed. In this study, several of these assays are evaluated for their discriminatory capacity using data obtained from 495 complete Caucasian mtDNA sequences. In order to add a local geographic perspective to this evaluation, we have also sequenced and analysed the entire mtDNA from 20 individuals of Swedish origin. We find that the coding region assays are very useful for resolving sequences with identical HVI/HVII regions. The best-performing coding region assay was able to discriminate 46% of the resolvable sequences, compared to 20–30% for the other coding region assays we evaluated.     

Single nucleotide polymorphisms over the entire mtDNA genome that increase the power of forensic testing in Caucasians

We have sequenced the entire mtDNA genome (mtGenome) of 241 individuals who match 1 of 18 common European Caucasian HV1/HV2 types, to identify sites that permit additional forensic discrimination. We found that over the entire mtGenome even individuals with the same HV1/HV2 type rarely match. Restricting attention to sites that are neutral with respect to phenotypic expression, we have selected eight panels of single nucleotide polymorphism (SNP) sites that are useful for additional discrimination. These panels were selected to be suitable for multiplex SNP typing assays, with 7–11 sites per panel. The panels are specific for one or more of the common HV1/HV2 types (or closely related types), permitting a directed approach that conserves limiting case specimen extracts while providing a maximal chance for additional discrimination. Discrimination provided by the panels reduces the frequency of the most common type in the European Caucasian population from ~7% to ~2%, and the 18 common types we analyzed are resolved to 105 different types, 55 of which are seen only once.     

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.     

Mitochondrial DNA variation in Tajiks living in Tajikistan

This study aimed to characterize mtDNA control region (positions 16,024–576) of unrelated Tajiks living in Tajikistan. DNA was isolated from saliva specimens stored on FTA cards. The mtDNA fragments were amplified and directly sequenced in forward and reverse directions. Haplogroups were determined using HaploGrep and the diagnostic polymorphisms in the coding region of mtDNA. The Tajik mtDNA pool was characterized by substantial admixture of western and eastern Eurasian haplogroups, 62.6% and 26.4% sequences, respectively. It also contained 9.9% of South Asian and 1.1% of African haplotypes. The Tajik mtDNA sequences belonged to 90 different haplotypes defined by 148 transitions and 13 transversions in 156 of 1122 nucleotide sites. The Tajik mtDNA pool demonstrated the high genetic variation with genetic diversity of 0.999 ± 0.002, nucleotide diversity of 0.014 ± 0.007, and the mean number of pairwise nucleotide differences of 15.38 ± 6.93. The random match probability and the power of discrimination were 0.0112 and 0.9888, respectively. Ethno-territorial groups of Tajiks demonstrated significant genetic differentiation with 2.67% of the genetic variance explained by differences between subpopulations. This study provides the insight into the mtDNA pool of Tajiks living in Tajikistan. The data should be taken into account in forensic identifications based on mtDNA.     

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.     

Coding region mitochondrial DNA SNPs: Targeting East Asian and Native American haplogroups

We have developed a single PCR multiplex SNaPshot reaction that consists of 32 coding region SNPs that allows (i) increasing the discrimination power of the mitochondrial DNA (mtDNA) typing in forensic casework, and (ii) haplogroup assignments of mtDNA profiles in both human population studies (e.g. anthropological) and medical research. The selected SNPs target the East Asian phylogeny, including its Native American derived branches. We have validated this multiplex assay by genotyping a sample of East Asians (Taiwanese) and Native Americans (Argentineans). In addition to the coding SNP typing, we have sequenced the complete control region for the same samples. The genotyping results (control region plus SNaPshot profiles) are in good agreement with previous human population genetic studies (based on e.g. complete sequencing) and the known mtDNA phylogeny. We observe that the SNaPshot method is reliable, rapid, and cost effective in comparison with other techniques of multiplex SNP genotyping. We discuss the advantages of our SNP genotyping selection with respect to previous attempts, and we highlight the importance of using the known mtDNA phylogeny as a framework for SNP profile interpretation and as a tool to minimize genotyping errors.     

Population and forensic genetic analyses of mitochondrial DNA control region variation from six major provinces in the Korean population

We generated complete mitochondrial DNA (mtDNA) control region sequences from 704 unrelated individuals residing in six major provinces in Korea. In addition to our earlier survey of the distribution of mtDNA haplogroup variation, a total of 560 different haplotypes characterized by 271 polymorphic sites were identified, of which 473 haplotypes were unique. The gene diversity and random match probability were 0.9989 and 0.0025, respectively. According to the pairwise comparison of the 704 control region sequences, the mean number of pairwise differences between individuals was 13.47 ± 6.06. Based on the result of mtDNA control region sequences, pairwise F_ST genetic distances revealed genetic homogeneity of the Korean provinces on a peninsular level, except in samples from Jeju Island. This result indicates there may be a need to formulate a local mtDNA database for Jeju Island, to avoid bias in forensic parameter estimates caused by genetic heterogeneity of the population. Thus, the present data may help not only in personal identification but also in determining maternal lineages to provide an expanded and reliable Korean mtDNA database. These data will be available on the EMPOP database via accession number EMP00661.     

Mitochondrial DNA variation in the Sindh population of Pakistan

The present study was undertaken to investigate the control region of mitochondrial DNA for forensic discrimination and to explore the ethno-linguistic affiliations among ethnic groups of Sindh province, Pakistan. A total of 115 individuals, from six major ethnic/isonym groups, namely, Bijarani, Chandio, Ghallu, Khoso, Nasrani and Solangi, have been studied. We identified 88 haplotypes, defined by the particular set of nucleotides; of these, 70 haplotypes were unique in the investigated population. In addition, 82% sequences were observed once, 12% twice and 5.2% thrice. The most common South Asian haplogroup in six ethnic groups of Sindh, are; M (42%) and R (6.9%), whereas West Eurasian haplogroups were N (6.9%), W (6.9%), J (1.7%), U (23.4%), H (9.5%) and T (0.86%). A random match probability between two unrelated individuals was found to be 0.06%, while genetic diversity varied from 0.991 to 0.998. The high nucleotide diversity and the low random match probability of the mtDNA control region make it a beneficial tool for forensic discrimination as well as useful to the evolutionary biologist. This work is an important contribution towards establishing a National Mitochondrial DNA Database in Pakistan. Currently, the Human Genetic department at the University of Health Sciences Lahore has the depository.     

Expanding the forensic German mitochondrial DNA control region database: genetic diversity as a function of sample size and microgeography

Mitochondrial DNA control region sequences were determined in 109 unrelated German Caucasoid individuals from north west Germany for both hypervariable regions 1 (HV1) and 2 (HV2) and 100 polymorphic nucleotide positions (nps) were found, 63 in HV1 and 37 in HV2. A total of 100 different mtDNA lineages was revealed, of which 7 were shared by 2 individuals and 1 by 3 individuals. The probability of drawing a HV1 sequence match within the north west Germans or within published sets of south Germans and west Austrians is similar (within a factor of 2) to drawing a sequence match between any two of these three population samples. Furthermore, HV1 sequences of 700 male inhabitants of one village in Lower Saxony were generated and these showed a nearly linear increase of the number of different haplotypes with increasing number of individuals, demonstrating that the commonly used haplotype diversity measure (Nei 1987) for population samples tends to underestimate mtDNA diversity in the actual population. Received: 14 January 1999 / Accepted: 15 February 1999     

Investigation of mtDNA control region sequences in an Egyptian population sample

The sequences of mitochondrial DNA (mtDNA) control region were investigated in 101 unrelated individuals living in the northern region of Nile delta (Gharbia, N = 55 and Kafrelsheikh, N = 46). DNA was extracted from blood stained filter papers or buccal swabs. HV1, HV2 and HV3 were PCR amplified and sequenced; the resulted sequences were aligned and compared with revised Cambridge sequence (rCRS). The results revealed presence of total 93 different haplotypes, 86 of them are unique and 7 are shared haplotypes, the most common haplotype, was observed with a frequency, 2.97% of population sample. High mtDNA diversity was observed with genetic diversity and power of discrimination, 0.9982 and 0.9883, respectively. In this dataset the west Eurasian haplogroups predominated over the African haplogroups. The results would be useful for forensic examinations and human genetic studies.     

A database of mitochondrial DNA hypervariable regions I and II sequences of individuals from Slovakia

In order to identify polymorphic positions and to determine their frequencies and the frequency of haplotypes in the human mitochondrial control region, two hypervariable regions (HV1 and HV2) of the mitochondrial DNA (mtDNA) of 374 unrelated individuals from Slovakia were amplified and sequenced. Sequence comparison led to the identification of 284 mitochondrial lineages as defined by 163 variable sites. Genetic diversity (GD) was estimated at 0.997 and the probability of two randomly selected individuals from population having identical mtDNA types (random match probability, RMP) for the both regions is 0.60%.     

Mitochondrial DNA control region variation in a population sample from Hong Kong,China

Entire mitochondrial control region sequences were generated from 377 unrelated individuals from urban Hong Kong. In line with other control region datasets from China, the sample from Hong Kong exhibited significant genetic diversity that was reflected in a random match probability of 0.19% and a mean pairwise difference of 13.14. A total of 305 haplotypes were identified, of which 262 were unique. These sequences will be made publicly available to serve as forensic mtDNA reference data for China.     

Helena,the hidden beauty: Resolving the most common West Eurasian mtDNA control region haplotype by massively parallel sequencing an Italian population sample

The analysis of mitochondrial (mt)DNA is a powerful tool in forensic genetics when nuclear markers fail to give results or maternal relatedness is investigated. The mtDNA control region (CR) contains highly condensed variation and is therefore routinely typed. Some samples exhibit an identical haplotype in this restricted range. Thus, they convey only weak evidence in forensic queries and limited phylogenetic information. However, a CR match does not imply that also the mtDNA coding regions are identical or samples belong to the same phylogenetic lineage. This is especially the case for the most frequent West Eurasian CR haplotype 263G 315.1C 16519C, which is observed in various clades within haplogroup H and occurs at a frequency of 3–4% in many European populations.In this study, we investigated the power of massively parallel complete mtGenome sequencing in 29 Italian samples displaying the most common West Eurasian CR haplotype – and found an unexpected high diversity. Twenty-eight different haplotypes falling into 19 described sub-clades of haplogroup H were revealed in the samples with identical CR sequences. This study demonstrates the benefit of complete mtGenome sequencing for forensic applications to enforce maximum discrimination, more comprehensive heteroplasmy detection, as well as highest phylogenetic resolution.     

Mitochondrial diversity in Amerindian Kichwa and Mestizo populations from Ecuador

This study presents mitochondrial DNA (mtDNA) data from 107 unrelated individuals from two of the major ethnic groups in Ecuador: Amerindian Kichwas (n = 65) and Mestizos (n = 42). We characterized the diversity of the matrilineal lineages of these Ecuadorian groups by analyzing the entire mtDNA control region. Different patterns of diversity were observed in the two groups as result of the unique historical and demographic events which have occurred in each population. Higher genetic diversity values were obtained for the Mestizo group than for the Amerindian group. Interestingly, only Native American lineages were detected in the two population samples, but with differences in the haplogroup distribution: Kichwa (A, 49%; B, 3%; C, 8%; and D, 40%) and Mestizo (A, 33%; B, 33%; C, 10%; and D, 24%). Analysis of the complete mtDNA control region proved to be useful to increase the discrimination power between individuals who showed common haplotypes in HVSI and HVSII segments; and added valuable information to the phylogenetic interpretation of mtDNA haplotypes.     

DNA profile of dog feces as evidence to solve a homicide

Dog fecal samples were collected at the crime scene and from the shoes of the suspect to see whether they could be linked. DNA was genotyped using a 145 bp fragment containing a 60 bp hotspot region of the mitochondrial DNA (mtDNA) control region. Once the species origin was identified, sequences were aligned with the 23 canine haplotypes defined, showing that evidence and reference had 100% identity with haplotype 5. The frequency of haplotype 5 and the exclusion power of the reference population were 0.056 and 0.89, respectively. The forensic index showed that it was 20 times more likely that the evidence belonged to the reference dog than to some other unknown animal. The results support that the mtDNA hypervariable region 1 (HV1) is a good alternative for typing in trace or degraded casework samples when the STR panel fails, and demonstrate the utility of domestic animal samples to give additional information to solve human legal cases.     

Sequence polymorphism of the mitochondrial DNA control region in the population of Vojvodina Province,Serbia

In order to generate and establish the database for forensic identification purposes in Vojvodina Province (Serbia), the sequence of the hypervariable regions 1 (HV1) and 2 (HV2) of the mtDNA control region were determined in a population of 104 unrelated individuals from Vojvodina Province, using a fluorescent-based capillary electrophoresis sequencing method. A total of 93 different haplotypes were found, of these 83 mtDNA types were unique, nine haplotypes were shared by two individuals and one haplotype by three individuals. The variation of mtDNA HV1 and HV2 regions was confined to 116 nucleotide positions, of which 72 were observed in the HV1 and 44 in the HV2. A statistical estimate of the results for this population showed the genetic diversity of 0.9977 and the random match probability of 1.18%. Haplogroup H was the most common haplogroup (43.3%). Haplogroups observed at intermediate levels included clusters U (13.5%), T (10.6%), J (8.6%) and W (5.8%).     

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.