Mitochondrial DNA Hypervariable Region Research Articles

Genetic identification of human remains from the Korean War.

The genetic identification of skeletal remains from Chinese People's Volunteers (CPVs) of the Korean War has been challenging because of the degraded DNA samples and the lack of living close relatives. This study established a workflow for identifying CPVs by combining Y-chromosome short tandem repeats (Y-STRs), mitochondrial DNA (mtDNA) hypervariable regions I and II, autosomal STRs (aSTRs), and identity-informative SNPs (iiSNPs). A total of 20 skeletal remains of CPVs and 46 samples from their alleged relatives were collected. The success rate of DNA extraction from human remains was 100%. Based on Y-STRs, six remains shared the same male lineages with their alleged relatives. Meanwhile, mtDNA genotyping supports two remains sharing the same maternal lineages with their alleged relatives. Likelihood ratios (LRs) were further obtained from 27 aSTRs and 94 iiSNPs or 1936 iiSNPs to confirm their relationship. All joint pedigree LRs were >100. Finally, six remains were successfully identified. This pilot study for the systematic genetic identification of CPVs from the Korean War can be applied for the large-scale identification of CPVs in the future.

High Mitochondrial Haplotype Diversity Found in Three Pre-Hispanic Groups from Colombia.

The analysis of mitochondrial DNA (mtDNA) hypervariable region (HVR) sequence data from ancient human remains provides valuable insights into the genetic structure and population dynamics of ancient populations. mtDNA is particularly useful in studying ancient populations, because it is maternally inherited and has a higher mutation rate compared to nuclear DNA. To determine the genetic structure of three Colombian pre-Hispanic populations and compare them with current populations, we determined the haplotypes from human bone remains by sequencing several mitochondrial DNA segments. A wide variety of mitochondrial polymorphisms were obtained from 33 samples. Our results support a high population heterogeneity among pre-Hispanic populations in Colombia.

Detection of 13 Hypervariable Region 1 (Hv1) SNPs using single-base extension (Sbe) primers in parallel with Sanger sequencing

The aim of this study was to determine whether single-base extension (SBE) chemistry can be applied to forensic practice of testing the target single nucleotide polymorphisms (SNPs) of the mitochondrial DNA (mtDNA) Hypervariable Region 1 (HV1). Despite itsweak discrimination power, high copy number of mtDNA per cell and its stability against degradation guarantee mtDNA testing a place in modern forensic genetics. In this research, buccal swab samples were obtained from 294 individuals from Bosnia and Herzegovina. Following DNA isolation using QIAamp® DNA Mini Kit, full sequencing of HV1 was completed using chain-termination Sanger sequencing method. SBE reactions were then performed by targeting 13 SNPs that were identified to be the most frequent in the study population. Uniplex SBE reactions for each individual SNP, as well as two multiplex reactions were prepared for both pure and mixed samples. The results showed complete agreement of the Sanger sequencing results with SBE reactions for both uniplex and multiplex reactions. The results obtained with SBE were encouraging in regard to multiplexing and processing of the mixed samples, since the allele of the minor contributor to the sample was observed in SBE electropherogram in all prepared mixtures. SBE method is limited by the fact that only target SNPs of interest will be analyzed, meaning that they must be carefully selected and curated for each population. However, typing with SBE protocol is accurate, as compared to the golden standard of Sanger sequencing, but was more time- and labor-efficient and simpler to analyze, therefore offering a suitable alternative when Sanger sequencing is not available, given that polymorphic SNPs are known for the population of interest.

Mitochondrial DNA (Hypervariable region II) diversity from Basrah population

Mitochondrial DNA hypervariable regions II of control region were sequenced from 139 random healthy unrelated individuals ethnic of Iraqi population in Basrah city. The aim of this study was to detect and classify the mtDNA haplotypes into mtDNA haplogroups will be better in applications of the forensic genetics and will determine the history of the Iraqi Population The variation in sequence within the D-loop control region was analyzed for the haplogroups composition which displayed a high haplogroups frequency. H, J, U, B, N and R (25%, 12%,12%, 7%, 5% and 5%, respectively, Moderate Haplogroups Frequency L, T and W was (3%) HV, I, X and M (1%), and low haplogroups frequency in pre-HV (0.7%). This study Reported absence of haplogroups V, P, Y, O, Z, Q, G, E and C.

Mitochondrial DNA diversity of the Sardinian local cattle stock

The aim of this research was to characterize the genetic diversity of the Sarda (Sa, n = 131), Sardo Bruna (SB, n = 44) and Sardo Modicana (SM, n = 26) cattle breeds, reared in the island of Sardinia (Italy). A portion of the mitochondrial DNA hypervariable region was sequenced, in order to identify a potential signature of African introgression. The FST coefficients among populations ranged between 0.056 for Sa vs SB and 0.167 for SB vs SM. AMOVA analysis indicated there was a significant differentiation of the three breeds, although most of diversity was gathered at the within-breed level. The Median Joining Network of the Sardinian sequences showed a potential founder effect signature. A MJ network including Sardinian cattle plus African, Italian, Iberian and Asian sequences, revealed the presence of haplogroup T3, already detected in Sa cattle, and the presence of Hg T1 and Hg T1′2′3, in Sa and SB. The presence of a private haplotype belonging to haplogroup T1, which is characteristic of African taurine breeds, may be due to the introgression of Sardinian breeds with African cattle, either directly (most probable source: North African cattle) or indirectly (through a Mediterranean intermediary already introgressed with African blood).

Forensic Genetic Analysis of Mitochondrial DNA Hypervariable Region III Sequences in Muslims from South India

The absence of recombination, high rate of mutation, and maternal inheritance of the mitochondrial DNA (mtDNA) genome makes it a valuable tool in many fields including evolutionary anthropology, genetic genealogy, population history, and forensic science. The mtDNA genome can be separated into two parts: a large coding region and a smaller fragment called the control region or non-coding region that generally referred to as displacement loop (D loop). The mtDNA D-loop region was highly polymorphic and has proven a precious marker in forensic identification. The study aims to examine and suggests polymorphism of the HVRIII region as a power marker along with HVRI and HVRII in forensic investigations. Within the control region of the mtDNA genome, the sequences of hypervariable region III (HVR III) (nucleotide position 438-574) were obtained from 60 unrelated Muslims of Shrirangapattana town, located in Karnataka state of South India. The complete mtDNA control region was amplified and sequenced by the Sanger sequencing method. The study provided the identification of 18 different haplotypes and 17 polymorphic nucleotide positions. The most common haplotype (H.18) was consistent with the Anderson sequence which occurred fourteen times. The distribution of nucleotide substitutions, insertions, and deletions was computed and determined that transitions made up the majority of the variations (58%) in this region. The genetic diversity was estimated at 0.89939 and the random match probability at 0.1155. The power of discrimination was found to be 0.8844 and the rest of the statistical parameters such as the mean of pair-wise differences and nucleotide diversity were established as 2.255932 ± 1.25884 and 0.010071± 0.00623, respectively. Consequently, the discovery of high genetic, haplotype, and nucleotide diversity, and high power of discrimination impart the use of hypervariable region III (HVR III) as an important marker in forensic investigations.

Origin and Genetic Diversity of Nepalese Indigenous Goats (Capra hircus)

Background: A very little study has been conducted on the phylogenetic diversity of Nepalese indigenous goats where four breeds, Khari, Chyangra, Terai and Sinhal, have been identified. Methods: The 625-bp long sequences of the mitochondrial DNA hyper-variable region obtained from 93 goats in this study revealed high haplotype diversity among breeds, which come under four haplogroups (A-D).Result: The study demonstrated a certain level of gene flow among the neighboring goat populations exhibiting no correspondence between the geographic regions of origin and relationships among breeds. The complex mtDNA diversity and structure identified among indigenous Nepalese goats can be explained by gene flow through ancient trading and the current ‘free’ movement of goats across the geographic vicinities in India and China. Furthermore, HapG B showed the southward direction of gene flow which does not cross the Himalayas, whereas HapG B1 revealed the South-West gene flow from the claimed domestication center for HapG B, China, to Nepal.

Phylogeny and genetic diversity reveal the influence of Qinghai-Tibet Plateau uplift on the divergence and distribution of Gymnocypris species

The complex environments and climatic conditions of the Qinghai-Tibet Plateau (QTP) create unique patterns of species diversity. However, the effect of uplift of the QTP on the distribution of Gymnocypris species (Gymnocypris) remain unclear. We found that the Qilian Mountain (QL) population has the highest genetic diversity by mitochondrial DNA hypervariable region of Gymnocypris species, the Tibetan (T) population has the lowest genetic diversity. The T population differs significantly from the Qinghai Lake (QH) and QL populations based on single nucleotide polymorphisms. A phylogeny and network distribution of this species on the QTP reveal differences in its genetic structure and support its taxonomic status. Neutrality tests reveal that Gymnocypris species has experienced different degrees of expansion, even secondary expansion. A divergence timetree estimates that the time of separation of different populations is possible related to uplift of the QTP and is consistent with the time of corresponding geological events. The QL population was separated from the other populations during the third uplift of the QTP (approximately 0.14 million years ago), and the QH and T populations were separated in the late Pleistocene to the Holocene (approximately 0.03 million years ago). In conclusion, this study provides different insights into the distribution of Gymnocypris species due to uplift of the QTP. Gymnocypris species was separated into different populations by this uplift, and each population exhibits unique characteristics, especially in terms of genetic structure and the degree of expansion. Moreover, the divergence time is consistent with the time of uplift of the QTP.

Mitochondrial DNA screening by melting curve analysis using peptide nucleic acid probes

Analysis of single nucleotide polymorphisms (SNPs) in mitochondrial (mt)DNA hypervariable regions (HV) 1/2 is valuable in forensic investigations. We developed a method for mtDNA screening of the HV1 and HV2 regions by melting curve analysis, using peptide nucleic acid (PNA) probes. This method focuses on melting peak patterns obtained by thermal dissociation of PNA/DNA duplexes in amplified mtDNA products. Five PNA probe sets were designed to detect 25 SNPs in the two HV regions. We also detected non-target SNPs based on unexpected melting temperature (Tm) shifts. In fact, 62 SNPs (42 SNPs in HV1 and 20 in HV2) were identified, including the 25 target SNPs. Using this method, 46 melting peak patterns, including 8 pattern groups, were obtained in 60 unrelated individuals. The peak patterns were compared to 55 haplotypes identified by Sanger sequencing. The results obtained from analysis of target mtDNA SNPs were entirely consistent with those obtained by Sanger sequencing. Screening the HV1 and HV2 regions of mtDNA by this method may help minimize unnecessary recourse to full sequence analysis, allows to rapidly exclude samples that do not match evidence and reference samples, and may reduce turnaround times and analysis costs. Overall, this method may be effective and helpful in forensic investigations.

Nucleotide sequence analysis of mitochondrial DNA hypervariable region II and inter HVR in Thais

ABSTRACT In this study, the nucleotide sequences of the mitochondrial DNA hypervariable region II (HVRII) and inter HVR in the Thai population were investigated. Buccal swab samples were randomly collected from 100 unrelated healthy individuals residing in Thailand. The analysis of HVRII revealed a total of 65 different haplotypes of which 47 were unique and 18 were found in more than one individual. The most frequent haplotype observed was A73G-A249D-A263G-309.1C–315.1C (7%). Nucleotide transition from adenine (A) to guanine (G) was the most frequent substitution. Nucleotide transversions were also observed but with a lower frequency compared to nucleotide transitions. Insertions of C (57%), CC (14%), and CCC (1%) were found at position 309. Twenty-six samples were found to exhibit nucleotide deletion at position 249. The haplotype diversity, random match probability, and discrimination power were calculated to be 0.9859, 0.0240, and 0.976 respectively. In addition, the outside HVR fragment within the control region at positions 16381–72 (inter HVRI/HVRII) and 341–437 (inter HVRII/HVRIII) were sequenced in order to examine the polymorphic sites. High polymorphism was found at positions 16519, 150, 249, and 309. It was concluded that the nucleotide sequence variations in HVRII and inter HVRI/HVRII would be useful for forensic identification.

An innovative DNA extraction method: Water versus commercial buffers

The main objective of DNA extraction is to obtain good quality genetic material in order to carry out its amplification, and corresponding analysis. Most laboratories tend to resort to commercial extraction buffers, which allow a simple and rigorous DNA extraction, with limited handling of the sample, but with high financial cost.As an alternative, we proposed to use water as reagent to extract DNA from blood spots, collected on Whatman® FTA® Cards (Sigma-Aldrich®). We propose to take advantage of hypotonic solutions, in order to obtain a better efficiency in the extraction of genetic material (DNA).On this sense, 219 extractions were performed with Whatman® FTA® Cards (SigmaAldrich®) stained with blood, based on Garcia-Palacios et al., extraction method, with some modifications. Extractions were performed with two types of water (sterile (Lonza™) and distilled (Milli-Q, MilliporeSigma®), as well as, with a commercial extraction kit (Prep-n-Go™, ThermoFisher™ Scientific, Foster City, USA). To evaluate and compare each reagent effectiveness, we amplified DNA extract with mitochondrial DNA Hypervariable regions I and II, and with nuclear DNA markers (autosomal STRs and X-InDels).Our preliminary results suggest that distilled water allows an extraction as effective, or even better, as that obtained with the commercial buffer. These preliminary results offer the possibility for laboratories to choose an inexpensive protocol for DNA extraction, avoiding expensive commercial buffers. Further research is needed to evaluate the performance of each method in blood samples, but also in other biological samples.

Unexpected low genetic variation in the South American hystricognath rodent Lagostomus maximus (Rodentia: Chinchillidae).

The South American plains vizcacha, Lagostomus maximus inhabits primarily the Pampean and adjoining Espinal, Monte and Chaquenean regions of Argentina. In order to study the population genetic structure of L. maximus, a fragment of 560 bp of the mitochondrial DNA hypervariable region 1from 90 individuals collected from the 3 subspecies and 8 groups along Argentina was amplified and analyzed. We found 9 haplotypes. The haplotype network did not show an apparent phylogeographical signal. Although low levels of genetic variation were found in all the subspecies and groups analyzed, a radiation of L. maximus would have occurred from the North and Center of the Pampean region toward the rest of its geographic range in Argentina. Low levels of genetic diversity, the existence of a single genetically distinct population in Argentina and changes of its effective size indicate that metapopulation processes and changes in human population dynamics during the late-Holocene were important factors shaping the population genetic structure of L. maximus in Argentina.

Evidence for prehistoric origins of the G2019S mutation in the North African Berber population.

The most common cause of the monogenic form of Parkinson’s disease known so far is the G2019S mutation of the leucine-rich repeat kinase 2 (LRRK2) gene. Its frequency varies greatly among ethnic groups and geographic regions ranging from less than 0.1% in Asia to 40% in North Africa. This mutation has three distinct haplotypes; haplotype 1 being the oldest and most common. Recent studies have dated haplotype 1 of the G2019S mutation to about 4000 years ago, but it remains controversial whether the mutation has a Near-Eastern or Moroccan-Berber ancestral origin. To decipher this evolutionary history, we genotyped 10 microsatellite markers spanning a region of 11.27 Mb in a total of 57 unrelated Moroccan PD patients carrying the G2019S mutation for which the Berber or Arab origin was established over 3 generations based on spoken language. We estimated the age of the most recent common ancestor for the 36 Arab-speaking and the 15 Berber-speaking G2019S carriers using the likelihood-based method with a mutation rate of 10−4. Data analysis suggests that the shortest haplotype originated in a patient of Berber ethnicity. The common founder was estimated to have lived 159 generations ago (95% CI 116–224) for Arab patients, and 200 generations ago (95% CI 123–348) for Berber patients. Then, 29 native North African males carrying the mutation were assessed for specific uniparental markers by sequencing the Y-chromosome (E-M81, E-M78, and M-267) and mitochondrial DNA (mtDNA) hypervariable regions (HV1 and HV2) to examine paternal and maternal contributions, respectively. Results showed that the autochthonous genetic component reached 76% for mtDNA (Eurasian and north African haplogroups) and 59% for the Y-chromosome (E-M81 and E-M78), suggesting that the G2019S mutation may have arisen in an autochthonous DNA pool. Therefore, we conclude that LRRK2 G2019S mutation most likely originated in a Berber founder who lived at least 5000 years ago (95% CI 3075–8700).

Fine-scale genetic structure analyses reveal dispersal patterns in a critically endangered primate, Trachypithecus leucocephalus.

Dispersal is a critically important life history trait of social organisms that has a major impact on the population genetic structure and social relationships within groups. Primates exhibit highly diversified dispersal and philopatry patterns, but knowledge of these patterns is difficult to obtain and usually limited to observations of a small number of focal social groups or individuals. Here, we investigated the dispersal pattern of a critically endangered colobine monkey, the white-headed langur (Trachypithecus leucocephalus), using molecular approaches, and sex-specific population genetic structure analyses at fine geographical scales. We non-invasively collected 403 fecal samples from 41 social groups across 90% of the langur's range in Fusui (FS) and Chongzuo (CZ) in southwestern Guangxi Province, China. We identified 214 unique individuals from the samples by genotyping 15 polymorphic autosomal microsatellite loci, a sex-specific marker, and sequencing the mitochondrial DNA (mtDNA) hypervariable region I (HVRI). We found higher intragroup than intergroup genetic relatedness in males and females in both populations. A significant positive correlation between genetic distance and geographical distance, that is a pattern of isolation-by-distance, was detected in females from the FS population, but not in males. Spatial autocorrelation analyses revealed high within-group relatedness in both sexes and populations, as well as an additional positive correlation at the 0.5-km distance class in females from the FS population. Furthermore, we inferred first-generation migrants using genetic assignment tests. Our results suggest that male T. leucocephalus disperse at random distances within habitat areas, whereas dispersal of females may mainly occur among adjacent groups near their home site. Our study provides the first genetic evidence for sex-biased dispersal in T. leucocephalus, which has important management and conservation implications for the species.

Effects of Hair dyes on Sequence Analysis of Hair Mitochondrial DNA Hypervariable Region 1

Hair can be a valuable source of DNA especially in forensic casework and for noninvasive studies of human, when blood samples not available. This study emphasizes the impact of hair dyes on DNA sequence analysis. Samples collected from forty Iraqi families; each sample was divided in to two parts hair follicles and hair shaft. DNA extracted by using two different techniques, Phenol-chloroform (organic) method and prepFiler forensic DNA extraction kit. After quantification of DNA by real time PCR to confirm the exact DNA yield, Mitochondrial DNA (MtDNA) hypervariable region 1 successfully amplified from (50% of samples include hair follicle and 20% samples include shaft only), which all extracted by organic method. Whereas by using prepFiler kit the ratio of amplification success reach to 95% of samples included hair follicles, but there wais no DNA outcome from hair shaft by using this kit. Our results demonstrate that treated hair by dyed or henna had a significant influence on the sequence analysis results. Organic method was an appropriate method for extraction DNA from hair shaft, since this method used for extracting the old and degraded samples. While prepFiler DNA extraction kit was more convenient for isolation DNA from, hair samples included follicles only with excellent result.

Research Article Mitochondrial DNA control region analysis of three ethnic populations in lower Northern part of Thailand.

The lower northern part of Thailand contains various genetically diverse ethnic populations. The sequences of the mitochondrial DNA hypervariable region were studied in three ethnic populations inhabiting Phitsanulok Province. One hundred and nine nucleotide sequences - 53, 29, and 27 from Hmongs (Hill tribe), Lao Songs, and Thai-Siams, respectively - were collected. The haplotypes were generated from 1130 nucleotides of the entire control region. Eighty-six haplotypes were found in the three ethnic populations, and no shared haplotypes were found between populations. Point heteroplasmy was noted at position 311 (C→Y). Haplotypes with ACAC-insertion at position 512 were observed in immigrant individuals from the Lao Song population. The Thai-Siam population showed higher genetic diversity than the other populations. The Hmong and Lao Song populations showed less genetic diversity than those living in their original area. The neutrality testing suggested that the result might be influenced by genetic drift.

Mitochondrial DNA hypervariable region 1 diversity in Nigerian goats

SummaryGoats make up the largest group of ruminant livestock in Nigeria and are strategic in bridging animal protein supply gap and improving the economy of rural households. The hypervariable region 1 (HVR1) of the caprine mitochondrial genome was investigated to better understand genetic diversity important for improving selection for animal breeding and conservation programs. We sequenced and analysed the mitochondrial DNA (mtDNA) HVR1 in 291 unrelated indigenous Nigerian goats (West African Dwarf (WAD), Red Sokoto (RSO) and Sahel (SAH)), randomly sampled from around the country, and compared them with the HVR1 sequences of 336 Indian goats and 12 other sequences in five different species in the genusCapra(C. falconeri, C. ibex nubiana, C. aegagrus, C. cylindricornisandC. sibirica). A total of 139 polymorphic sites from 291 individuals were captured in 204 haplotypes. Within and among population variations were 77.25 and 22.74 percent, respectively. Nigerian goats showed high genetic diversity (0.87) and high FST values, and separate from Indian goats and other wild species. Haplogroups in WAD separates it from RSO and SAH concomitant with a different demographic history. Clear genetic structure was found among Nigerian goat breeds with appreciable variation in mtDNA HVR1 region. This study grouped Nigerian goat breeds into two major groups suggesting two different demographic origins for Northern and Southern breeds. High genetic admixing denotes different maternal origins and in contrast to evidence from goats from Levant and Central Asia, where goats were originally domesticated.

Effect of migration patterns on maternal genetic structure: a case of Tai-Kadai migration from China to Thailand.

The migration of the Tai-Kadai speaking people from southern China to northern Thailand over the past hundreds of years has revealed numerous patterns that have likely been influenced by routes, purposes and periods of time. To study the effects of different migration patterns on Tai-Kadai maternal genetic structure, mitochondrial DNA hypervariable region I sequences from the Yong and the Lue people having well-documented histories in northern Thailand were analyzed. Although the Yong and Lue people were historically close relatives who shared Xishuangbanna Dai ancestors, significant genetic differences have been observed among them. The Yong people who have been known to practice mass migration have exhibited a closer genetic affinity to their Dai ancestors than have the Lue people. Genetic heterogeneity and a sudden reduced effective population size within the Lue group is likely a direct result of the circumstances of the founder effect.

Defect of the mitochondrial DNA hypervariable region as a risk factor for canine mammary tumour

The aim of this study was to identify mutations in the hypervariable region of mitochondrial DNA in canine mammary tumours and to determine their association with the process of neoplastic transformation. A total of 93 biological samples, including blood as well as normal and neoplastic tissue samples from 31 dogs with diagnosed malignant canine mammary tumours were analysed. DNA extraction, amplification and sequencing of the D-loop as well as bioinformatic and statistical analyses were performed. In the mitochondrial D-loop sequence, 26 polymorphic loci and 5 mutations were identified. For the first time, D-loop length heteroplasmy was detected in dogs with mammary tumours. The malignancy grade exerted no effect on the presence of nucleotide changes. A statistically significant association between the presence of mutations and polymorphisms and the size of dogs was demonstrated. The 100% frequency of length heteroplasmy may imply that this is a hotspot mutation of canine mammary tumour.

Mutations and Polymorphisms in the Hypervariable Region of Mitochondrial DNA in Canine Mammary Cancer

Mutations and Polymorphisms in the Hypervariable Region of Mitochondrial DNA in Canine Mammary Cancer