Control Region Haplotypes Research Articles

Molecular phylogenetic status of the Bulgarian marbled polecat (Vormela peregusna, Mustelidae, Carnivora), revealed by Y chromosomal genes and mitochondrial DNA sequences

In this study, we investigated molecular phylogenetic status of the marbled polecat (Vormela peregusna) from Bulgaria, using sequences of two Y-chromosomal genes (SRY and ZFY). The phylogenetic tree inferred using combined sequences of both genes indicated that the marbled polecat was split from genera Lutra, Neovison and Mustela after genus Martes was diverged in family Mustelidae. In addition, we analyzed molecular phylogeography of the Bulgarian population of the marbled polecat, using cytochrome b and control region sequences of mitochondrial DNA (mtDNA). The phylogenetic tree of cytochrome b indicated that the haplotypes of the Bulgarian population comprised two haplogroups, which were the most ancestral clades. Additionally, the control region phylogeny showed that the haplotypes of Bulgaria formed two haplogroups: one was the most ancestral clade, and the other was the derivative clade. One individual with the most ancestral cytochrome b clade had a control region haplotype of the derivative clade. Thus, this study revealed that the most ancestral lineages of the marbled polecat are included in the population of Bulgaria. The Bulgarian population could be a remnant lineage from a basal for the species, which in Pleistocene occupied a relatively large area related to the Balkan-Caucasian.

Genetic structure of the hawksbill turtle rookery and foraging aggregation in Tobago, West Indies

Mitochondrial DNA control region haplotypes of immature hawksbills feeding in Tobago waters and adult females nesting on the beaches of Tobago were characterized. Eleven haplotypes were documented among foraging aggregations and six haplotypes among nesting females, including two which were previously of unknown natal origin. The Tobago rookery is genetically distinct from all other rookeries in the region characterized to date. Significant genetic differentiation was found between the foraging aggregations on the windward and leeward sides of Tobago. Within the rookery, greater diversity was detected among samples from the North-East coast compared to samples from the South-West coast, however no significant difference was detected based on pairwise FST. Mixed stock analysis was conducted to estimate the natal origins of immature hawksbills foraging in Tobago waters and the contribution of the Tobago rookery to regional foraging aggregations using rookery size as a constraint. The analysis estimated that the Tobago foraging aggregation is mostly composed of animals originating from Cuba, Barbados (Leeward Coast) and Puerto Rico, though this represents only a small fraction of the production from these rookeries. The impact of harvest of juveniles in Tobago is likely to be distributed across the region and not concentrated on any particular rookery. A significant proportion of individuals from the Tobago rookery contributes to the foraging aggregation of the Cayman Islands, and a large proportion of both the Cayman Islands and Puerto Rico foraging aggregations are derived from the Tobago rookery.

Phylogeography of the Koala, (Phascolarctos cinereus), and Harmonising Data to Inform Conservation

The Australian continent exhibits complex biogeographic patterns but studies of the impacts of Pleistocene climatic oscillation on the mesic environments of the Southern Hemisphere are limited. The koala (Phascolarctos cinereus), one of Australia’s most iconic species, was historically widely distributed throughout much of eastern Australia but currently represents a complex conservation challenge. To better understand the challenges to koala genetic health, we assessed the phylogeographic history of the koala. Variation in the maternally inherited mitochondrial DNA (mtDNA) Control Region (CR) was examined in 662 koalas sampled throughout their distribution. In addition, koala CR haplotypes accessioned to Genbank were evaluated and consolidated. A total of 53 unique CR haplotypes have been isolated from koalas to date (including 15 haplotypes novel to this study). The relationships among koala CR haplotypes were indicative of a single Evolutionary Significant Unit and do not support the recognition of subspecies, but were separated into four weakly differentiated lineages which correspond to three geographic clusters: a central lineage, a southern lineage and two northern lineages co-occurring north of Brisbane. The three geographic clusters were separated by known Pleistocene biogeographic barriers: the Brisbane River Valley and Clarence River Valley, although there was evidence of mixing amongst clusters. While there is evidence for historical connectivity, current koala populations exhibit greater structure, suggesting habitat fragmentation may have restricted female-mediated gene flow. Since mtDNA data informs conservation planning, we provide a summary of existing CR haplotypes, standardise nomenclature and make recommendations for future studies to harmonise existing datasets. This holistic approach is critical to ensuring management is effective and small scale local population studies can be integrated into a wider species context.

Genetic structure of the beaked whale genusBerardiusin the North Pacific, with genetic evidence for a new species

AbstractThere are two recognized species in the genusBerardius, Baird's and Arnoux's beaked whales. In Japan, whalers have traditionally recognized two forms of Baird's beaked whales, the common “slate‐gray” form and a smaller, rare “black” form. Previous comparison of mtDNAcontrol region sequences from three black specimens to gray specimens around Japan indicated that the two forms comprise different stocks and potentially different species. We have expanded sampling to include control region haplotypes of 178 Baird's beaked whales from across their range in the North Pacific. We identified five additional specimens of the black form from the Aleutian Islands and Bering Sea, for a total of eight “black” specimens. The divergence between mtDNAhaplotypes of the black and gray forms of Baird's beaked whale was greater than their divergence from the congeneric Arnoux's beaked whale found in the Southern Ocean, and similar to that observed among other congeneric beaked whale species. Taken together, genetic evidence from specimens in Japan and across the North Pacific, combined with evidence of smaller adult body size, indicate presence of an unnamed species ofBerardiusin the North Pacific.

Mitochondrial DNA control region haplotype and haplogroup diversity in South Eastern Turkey

Despite its large geographic and population size only little is known about the mitochondrial (mt)DNA make up of Turkey.orensically relevant data are almost completely absent in the literature. We analyzed the mtDNA control region of 224 volunteers from South Eastern Turkey and compared the data to populations from neighboring countries. The haplotypes will be made available via the EMPOP database (EMP00670) and contribute to the body of forensic mtDNA data.

Analysis of uni and bi-parental markers in mixture samples: Lessons from the 22nd GHEP-ISFG Intercomparison Exercise

Since 1992, the Spanish and Portuguese-Speaking Working Group of the ISFG (GHEP-ISFG) has been organizing annual Intercomparison Exercises (IEs) coordinated by the Quality Service at the National Institute of Toxicology and Forensic Sciences (INTCF) from Madrid, aiming to provide proficiency tests for forensic DNA laboratories. Each annual exercise comprises a Basic (recently accredited under ISO/IEC 17043: 2010) and an Advanced Level, both including a kinship and a forensic module. Here, we show the results for both autosomal and sex-chromosomal STRs, and for mitochondrial DNA (mtDNA) in two samples included in the forensic modules, namely a mixture 2:1 (v/v) saliva/blood (M4) and a mixture 4:1 (v/v) saliva/semen (M8) out of the five items provided in the 2014 GHEP-ISFG IE. Discrepancies, other than typos or nomenclature errors (over the total allele calls), represented 6.5% (M4) and 4.7% (M8) for autosomal STRs, 15.4% (M4) and 7.8% (M8) for X-STRs, and 1.2% (M4) and 0.0% (M8) for Y-STRs. Drop-out and drop-in alleles were the main cause of errors, with laboratories using different criteria regarding inclusion of minor peaks and stutter bands. Commonly used commercial kits yielded different results for a micro-variant detected at locus D12S391. In addition, the analysis of electropherograms revealed that the proportions of the contributors detected in the mixtures varied among the participants. In regards to mtDNA analysis, besides important discrepancies in reporting heteroplasmies, there was no agreement for the results of sample M4. Thus, while some laboratories documented a single control region haplotype, a few reported unexpected profiles (suggesting contamination problems). For M8, most laboratories detected only the haplotype corresponding to the saliva. Although the GHEP-ISFG has already a large experience in IEs, the present multi-centric study revealed challenges that still exist related to DNA mixtures interpretation. Overall, the results emphasize the need for further research and training actions in order to improve the analysis of mixtures among the forensic practitioners.

Taxonomic status and origin of the Egyptian weasel (Mustela subpalmata) inferred from mitochondrial DNA.

The Egyptian weasel (Mustela subpalmata) is a small mustelid with a distribution restricted to the lower Nile Valley and the Nile Delta. Traditionally considered a subspecies of the least weasel (M. nivalis), it is currently recognized as a separate species based on morphology. Here we present the first genetic assessment of the taxonomic status of the Egyptian weasel by comparing mitochondrial DNA (Cytochrome b gene and control region) sequences to those of least weasels from the western Palearctic, with a focus on the Mediterranean region. Our results provide no evidence to support the view that the Egyptian weasel is genetically distinct from the least weasel, as we found that, for both Cytochrome b and control region, haplotypes were shared between the two taxa. Specifically, the Cytochrome b and control region haplotypes detected in the Egyptian weasel were also present in M. nivalis from Turkey and Malta, two populations genetically analysed here for the first time. Our results suggest that the Egyptian weasel is distinct from the least weasel populations currently living in the Maghreb, which were inferred to be the result of an earlier colonization of North Africa, but the genetic data alone do not allow us to determine whether the Egyptian weasel is native or introduced. Nevertheless, the observed genetic patterns, together with the weasel fossil record in Israel and the unique commensal lifestyle of the Egyptian weasel, are consistent with the hypothesis that the Egyptian population is a relict of past range expansion from the Levant into Egypt. We suggest that the large size and characteristic sexual dimorphism of the Egyptian weasel are likely to represent ecotypic variation, but genomic studies are required to clarify the extent of its functional genetic divergence.

The Genetic History of Peruvian Quechua-Lamistas and Chankas: Uniparental DNA Patterns among Autochthonous Amazonian and Andean Populations.

SummaryThis study focuses on the genetic history of the Quechua‐Lamistas, inhabitants of the Lamas Province in the San Martin Department, Peru, who speak their own distinct variety of the Quechua family of languages. It has been suggested that different pre‐Columbian ethnic groups from the Peruvian Amazonia, like the Motilones or “shaven heads”, assimilated the Quechua language and then formed the current native population of Lamas. However, many Quechua‐Lamistas claim to be direct descendants of the Chankas, a famous pre‐Columbian indigenous group that escaped from Inca rule in the Andes. To investigate the Quechua‐Lamistas and Chankas’ ancestries, we compared uniparental genetic profiles (17 STRs of Q‐M3 Y‐chromosome and mtDNA complete control region haplotypes) among autochthonous Amazonian and Andean populations from Peru, Bolivia and Ecuador. The phylogeographic and population genetic analyses indicate a fairly heterogeneous ancestry for the Quechua‐Lamistas, while they are closely related to their neighbours who speak Amazonian languages, presenting no direct relationships with populations from the region where the ancient Chankas lived. On the other hand, the genetic profiles of self‐identified Chanka descendants living in Andahuaylas (located in the Apurimac Department, Peru, in the Central Andes) were closely related to those living in Huancavelica and the assumed Chanka Confederation area before the Inca expansion.

Using a multi-disciplinary approach to identify a critically endangered killer whale management unit

A key goal for wildlife managers is identifying discrete, demographically independent conservation units. Previous genetic work assigned killer whales that occur seasonally in the Strait of Gibraltar (SoG) and killer whales sampled off the Canary Islands (CI) to the same population. Here we present new analyses of photo-identification and individual genotypes to assess the level of contemporary gene flow and migration between study areas, and analyses of biomarkers to assess ecological differences. We identified 47 different individuals from 5 pods in the SoG and 16 individuals in the CI, with no matches found between the areas. Mitochondrial DNA control region haplotype was shared by all individuals sampled within each pod, suggesting that pods have a matrifocal social structure typical of this species, whilst the lack of shared mitogenome haplotypes between the CI and SoG individuals suggests that there was little or no female migration between groups. Kinship analysis detected no close kin between CI and SoG individuals, and low to zero contemporary gene flow. Isotopic values and organochlorine pollutant loads also suggest ecological differences between study areas. We further found that one individual from a pod within the SoG not seen in association with the other four pods and identified as belonging to a potential migrant lineage by genetic analyses, had intermediate isotopic values and contaminant between the two study areas. Overall our results suggest a complex pattern of social and genetic structuring correlated with ecological variation. Consequently at least CI and SoG should be considered as two different management units. Understanding this complexity appears to be an important consideration when monitoring and understanding the viability of these management units. Understand the viability will help the conservation of these threatened management units.

The mitochondrial landscape of African Americans: An examination of more than 2500 control region haplotypes from 22 U.S. locations

The mitochondrial DNA (mtDNA) control region (16024-576) was Sanger-sequenced for a total of 2563 self-identified African Americans, using automated processing techniques and data review standards exceeding guidelines for forensic applications. Genetic diversity ranged from 0.9952 to 0.9998 in 22 population samples from 20 different states.Haplogroups of African ancestry, found in 82.48% of individuals overall, were most concentrated in the Southeast U.S. and decreased to the north and west. West African and West Central African haplotypes were well-represented in the population samples, especially in the southern U.S. states, while East African haplogroups were observed in low-frequency clusters in a handful of locations across the country. East Asian, Native American, and West Eurasian admixture was present in 3.16%, 2.93%, and 11.43% of samples, respectively. While some geographic substructure was detected across the population samples as clines in admixture frequencies, 20 of the 22 population samples were found to be statistically indistinguishable by pairwise comparisons and AMOVA calculations. Datasets from Hawaii and Idaho, however, were clear outliers. Overall, these more than 2500 control region sequences represent the most comprehensive regional sampling of African American mtDNA diversity to date, and are suitable for use in a forensic mtDNA database. The population data are made available via EMPOP (www.empop.org) and GenBank.

Deeper Mitochondrial Sequencing Reveals Cryptic Diversity and Structure in Brazilian Green Turtle Rookeries

Abstract Genetic markers are often used to designate population units for management and conservation, but widespread sharing of mitochondrial DNA control-region haplotypes defined from short (< 500 base-pair [bp]) sequences often limits inferences of population connectivity in marine turtles. Haplotype CM-A8, defined from 490-bp sequences, dominated the haplotype profiles of the 3 major green turtle (Chelonia mydas) rookeries in Brazil. Previous analyses based on 490-bp haplotypes did not detect differentiation between the northern rookeries of Atol das Rocas and Fernando de Norohna, but did indicate differentiation of the northern rookeries from Trindade Island in the south. We reexamined the stock structure of the Brazilian green turtle rookeries using 817-bp control region and mitochondrial short tandem repeat (mtSTR) sequences. Nine 490-bp haplotypes were subdivided into 41 haplotypes by combining 817-bp and mtSTR sequences. Eight of the 14 CM-A8 turtles from Fernando de Noronha carried mtSTR haploty...

Genetic diversity loss in a biodiversity hotspot: ancient DNA quantifies genetic decline and former connectivity in a critically endangered marsupial.

The extent of genetic diversity loss and former connectivity between fragmented populations are often unknown factors when studying endangered species. While genetic techniques are commonly applied in extant populations to assess temporal and spatial demographic changes, it is no substitute for directly measuring past diversity using ancient DNA (aDNA). We analysed both mitochondrial DNA (mtDNA) and nuclear microsatellite loci from 64 historical fossil and skin samples of the critically endangered Western Australian woylie (Bettongia penicillata ogilbyi), and compared them with 231 (n = 152 for mtDNA) modern samples. In modern woylie populations 15 mitochondrial control region (CR) haplotypes were identified. Interestingly, mtDNA CR data from only 29 historical samples demonstrated 15 previously unknown haplotypes and detected an extinct divergent clade. Through modelling, we estimated the loss of CR mtDNA diversity to be between 46% and 91% and estimated this to have occurred in the past 2000-4000 years in association with a dramatic population decline. In addition, we obtained near-complete 11-loci microsatellite profiles from 21 historical samples. In agreement with the mtDNA data, a number of 'new' microsatellite alleles was only detected in the historical populations despite extensive modern sampling, indicating a nuclear genetic diversity loss >20%. Calculations of genetic diversity (heterozygosity and allelic rarefaction) showed that these were significantly higher in the past and that there was a high degree of gene flow across the woylie's historical range. These findings have an immediate impact on how the extant populations are managed and we recommend the implementation of an assisted migration programme to prevent further loss of genetic diversity. Our study demonstrates the value of integrating aDNA data into current-day conservation strategies.

The complete mitogenome of a 500-year-old Inca child mummy

In 1985, a frozen mummy was found in Cerro Aconcagua (Argentina). Archaeological studies identified the mummy as a seven-year-old Inca sacrifice victim who lived >500 years ago, at the time of the expansion of the Inca Empire towards the southern cone. The sequence of its entire mitogenome was obtained. After querying a large worldwide database of mitogenomes (>28,000) we found that the Inca haplotype belonged to a branch of haplogroup C1b (C1bi) that has not yet been identified in modern Native Americans. The expansion of C1b into the Americas, as estimated using 203 C1b mitogenomes, dates to the initial Paleoindian settlements (~18.3 thousand years ago [kya]); however, its internal variation differs between Mesoamerica and South America. By querying large databases of control region haplotypes (>150,000), we found only a few C1bi members in Peru and Bolivia (e.g. Aymaras), including one haplotype retrieved from ancient DNA of an individual belonging to the Wari Empire (Peruvian Andes). Overall, the results suggest that the profile of the mummy represents a very rare sub-clade that arose 14.3 (5–23.6) kya and could have been more frequent in the past. A Peruvian Inca origin for present-day C1bi haplotypes would satisfy both the genetic and paleo-anthropological findings.

Phylogeography of the Golden Jackal (Canis aureus) in India.

The golden jackal (Canis aureus) is one of the most common and widely distributed carnivores in India but phylogeographic studies on the species have been limited across its range. Recent studies have observed absence of mitochondrial (mt) DNA diversity in European populations while some North African populations of golden jackal were found to carry gray wolf (Canis lupus lupaster) mtDNA lineages. In the present study, we sequenced 440 basepairs (bp) of control region (CR) and 412 bp of cytochrome b (cyt b) gene of mtDNA from 62 golden jackals sampled from India (n = 55), Israel (n = 2) and Bulgaria (n = 5), to obtain a total of eighteen haplotypes, comprising sixteen from India and one each from Israel and Bulgaria. Except for three previously described haplotypes represented by one cyt b and one CR haplotype both from India, and one CR haplotype from Bulgaria, all haplotypes identified in this study are new. Genetic diversity was high in golden jackals compared to that reported for other canids in India. Unlike the paraphyletic status of African conspecifics with the gray wolf, the Indian (and other Eurasian) golden jackal clustered in a distinct but shallow monophyletic clade, displaying no evidence of admixture with sympatric and related gray wolf and domestic dog clades in the region. Phylogeographic analyses indicated no clear pattern of genetic structuring of the golden jackal haplotypes and the median joining network revealed a star-shaped polytomy indicative of recent expansion of the species from India. Indian haplotypes were observed to be interior and thus ancestral compared to haplotypes from Europe and Israel, which were peripheral and hence more derived. Molecular tests for demographic expansion confirmed a recent event of expansion of golden jackals in the Indian subcontinent, which can be traced back ~ 37,000 years ago during the late Pleistocene. Our results suggest that golden jackals have had a potentially longer evolutionary history in India than in other parts of the world, although further sampling from Africa, the Middle East and south-east Asia is needed to test this hypothesis.

First data on genetic diversity of burbot (Lota lota L.) in the Western Siberian

Analysis of genetic diversity of burbot (Lota lota Linneus, 1758) mitochondrial control region (mtCR) haplotypes from geographically distant localities in the Ob-Irtysh River basin in comparison with distribution of known burbot haplotypes was conducted. mtCR fragments from burbot samples, obtained in two localities (longitudinal part of the Irtysh near Tobolsk and the Sob River, a left-bank tributary of the Ob River), were sequenced.

Morphological and genetic characterization of Sardinian trout Salmo cettii Rafinesque, 1810 and their conservation implications

The morphological and genetic structure of Western Mediterranean trout Salmo cettii inhabiting basins in Sardinia was completed to assist the design of its conservation programmes. Genetic analysis of protein-coding LDH-C1 plus sequencing mitochondrial control region gene and analyses of morphological characters described 253 specimens from seven localities in two basins in Southwest Sardinia. Nuclear and mitochondrial analyses revealed all of the fish were pure-bred native S. cettii, with no introgression from allochthonous S. trutta. The novel 18 mtDNA control region haplotypes were clustered in an ‘insular’ clade, strictly related to the Adriatic haplogroup, and depicted a radial network around two ancestral haplotypes. Completion of discriminant analysis using data on body pigmentation and quantitative morphologic parameters revealed three phenotypic groups within the fish. Each population and phenotype, characterised by high values of nucleotide and haplotype diversity, were not genetically differentiated and not geographically structured according to the two hydrological basins. Geometric morphometric analysis, based on 15 landmarkers, revealed pronounced and highly significant differences in body shape morphology between populations, suggesting S. cettii is locally adapting to extreme environmental conditions and so future management plans for these populations should treat the two basins as distinct morphological units.

Polyphyletic ancestry of historic gray wolves inhabiting U.S. Pacific states

Given the recent re-colonization of gray wolves (Canis lupus) to the Pacific northwest, USA, and subsequent migration into northern California, understanding how well natural migration has restored historic diversity can inform management decisions. In this study, we report the mitochondrial DNA control region haplotypes of nine museum specimens that curators identified as C. lupus from Oregon, Nevada, and California. Among the nine samples currently available for genetic analysis of historic genetic diversity of C. l. spp. in the U.S. Pacific states, we found six previously described haplotypes including two domestic dog (C. l. familiaris) haplotypes. Notably, we present the first evidence of Mexican wolf (C. l. baileyi) ancestry in southern California while the northern Californian specimen, as well as one individual from Nevada, present a haplotype common to wolves from the historic American West and extant Canadian wolf populations. Finally, the three Oregon specimens shared a haplotype that is only observed in extant wolves from coastal British Columbia (the “coastal rainforest” wolf ecotype), indicating that the historical range of this haplotype reached as far south as southwestern Oregon. In conclusion, our results indicate that the genetic composition of historic wolf populations in the Pacific northwest and southwest states was polyphyletic and included wolves that share maternal ancestry with current populations from adjacent regions. These findings, in addition with future nuclear analyses, reveal more accurate historic range delimitations, which is critical information when designing appropriate management plans for wolves naturally re-colonizing the U.S. Pacific northwest and southwest regions.

Genetic diversity of the Critically Endangered Philippine Eagle Pithecophaga jefferyi (Aves: Accipitridae) and notes on its conservation

The Philippine Eagle Pithecophaga jefferyi is a diurnal raptor endemic to the Philippines. Its distribution is restricted to remaining forests on the islands of Luzon, Samar, Leyte and Mindanao. The Philippine Eagle is classified as a Critically Endangered species under the IUCN Red List, with a high end estimated population of only 500 breeding pairs in the wild. Population decline has been attributed to continuing deforestation, particularly since the mid-1900s, and hunting. This study aimed to identify the effects of population decline on the genetic structure of the present population of the Philippine Eagle by sequencing 1132bp of the mitochondrial control region from 22 individuals. Control region haplotype diversity (h = 0.8960±0.05590) and nucleotide diversity (π = 0.006194±0.003372) are comparable with other accipitrid species. Maximum likelihood trees and network analysis show that the Luzon and Samar individuals come from different lineages, but both shared a common ancestral population with the Mindanao population. The genetic diversity, multimodal mismatch distribution for the control region and high frequency of lower class modes all indicate a recent bottleneck for the Philippine Eagle population. Possible strategies for conservation are discussed.

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.

Coding region SNP analysis to enhance dog mtDNA discrimination power in forensic casework.

The high population frequencies of three control region haplotypes contribute to the low discrimination power of the dog mtDNA control region. It also diminishes the evidential power of a match with one of these haplotypes in forensic casework. A mitochondrial genome study of 214 Belgian dogs suggested 26 polymorphic coding region sites that successfully resolved dogs with the three most frequent control region haplotypes. In this study, three SNP assays were developed to determine the identity of the 26 informative sites. The control region of 132 newly sampled dogs was sequenced and added to the study of 214 dogs. The assays were applied to 58 dogs of the haplotypes of interest, which confirmed their suitability for enhancing dog mtDNA discrimination power. In the Belgian population study of 346 dogs, the set of 26 sites divided the dogs into 25 clusters of mtGenome sequences with substantially lower population frequency estimates than their control region sequences. In case of a match with one of the three control region haplotypes, using these three SNP assays in conjunction with control region sequencing would augment the exclusion probability of dog mtDNA analysis from 92.9% to 97.0%.