mtDNA Hypervariable Region Research Articles

Mitochondrial DNA and Y chromosome reveal the genetic structure of the native Polish Konik horse population.

Polish Konik remains one of the most important horse breeds in Poland. The primitive, native horses with a stocky body and mouse-like coat color are protected by a conservation program, while their Polish population consists of about 3,480 individuals, representing 16 dam and six sire lines. To define the population's genetic structure, mitochondrial DNA and Y chromosome sequence variables were identified. The mtDNA whole hypervariable region analysis was carried out using the Sanger sequencing method on 233 Polish Koniks belonging to all dam lines, while the Y chromosome analysis was performed with the competitive allele-specific PCR genotyping method on 36 horses belonging to all sire lines. The analysis of the mtDNA hypervariable region detected 47 SNPs, which assigned all tested horses to 43 haplotypes. Most dam lines presented more than one haplotype; however, five dam lines were represented by only one haplotype. The haplotypes were classified into six (A, B, E, J, G, R) recognized mtDNA haplogroups, with most horses belonging to haplogroup A, common among Asian horse populations. Y chromosome analysis allocated Polish Koniks in the Crown group, condensing all modern horse breeds, and divided them into three haplotypes clustering with coldblood breeds (28 horses), warmblood breeds (two horses), and Duelmener Pony (six horses). The clustering of all Wicek sire line stallions with Duelmener horses may suggest a historical relationship between the breeds. Additionally, both mtDNA and Y chromosome sequence variability results indicate crossbreeding before the studbooks closure or irregularities in the pedigrees occurred before the DNA testing introduction.

Characteristic of Przewalski horses population from Askania-Nova reserve based on genetic markers.

Przewalski horses are considered the last living population of wild horses, however, they are secondarily feral offspring of herds domesticated ~ 5000 years ago by the Botai culture. After Przewalski horses were almost extinct at the beginning of the twentieth century, their population is about 2500 individuals worldwide, with one of the largest breeding centers in Askania-Nova Biosphere Reserve (Ukraine). The research aimed to establish the maternal variation of Przewalski horses population maintained in Askania-Nova Reserve based on mitochondrial DNA hypervariable 1 and hypervariable 2 regions profiling, as well as, analysis of Y chromosome single nucleotide polymorphism unique for Przewalski horses, and coat color markers: MC1R and TBX3. The mtDNA hypervariable regions analysis in 23 Przewalski horses allowed assigning them to three distinctly different haplotypes, showing the greatest similarity to the Equus caballus reference, the Equus przewalskii reference, and to extinct species-Haringtonhippus. The Y chromosome analysis using fluorescently labelled assays differentiated horses in terms of polymorphism (g731821T>C) characteristic of Equus przewalskii. All male individuals presented genotype C characteristics for Przewalski horses. The polymorphisms within the coat color genes indicated only native, wild genotypes. The Y chromosome and coat color analysis denied admixtures of the tested horses with other Equidae.

The role of ancestral seascape discontinuity and geographical distance in structuring rockfish populations in the Pacific Northwest

Despite the apparent absence of physical barriers in the ocean to prevent dispersal, recent studies have highlighted the importance of biological, geographical, physical, and historical barriers in the genetic structuring of marine species populations. This representation is essential for the sustainable exploitation of natural marine resources and for the setup of efficient protected area networks for the conservation of marine species. In this study, we used extensive sampling of Sebastiscus marmoratus, a commercially important inshore rockfish with high site fidelity, to characterize their population genetic structure along the China–Japan coast and to determine the effect of past geological and current biological–physical barriers on the current genetic footprint. A 461-bp fragment of the mtDNA hypervariable portion control region was sequenced for 675 individuals from 15 geographical locations. A total of 292 haplotypes were identified. The population of S. marmoratus showed high haplotype and nucleotide diversity. Pairwise fixation index (FST) and analysis of molecular variance (AMOVA) revealed significant genetic differentiation among populations. The Bayesian skyline plots and neutrality statistics showed a sudden expansion of the S. marmoratus population around the Pleistocene. The Beibu Gulf group had the lowest mean number of pairwise differences, the lowest significant genetic differentiation, and the lowest haplotype and nucleotide diversity, and should be prioritized for protection in the future. Ocean currents, seascape discontinuity, geographical distance, and ecological characteristics may play an important role in shaping the contemporary phylogeographical patterns and population structures of S. marmoratus.

Forensic Feature Exploration and Comprehensive Genetic Insights Into Yugu Ethnic Minority and Northern Han Population via a Novel NGS-Based Marker Set.

The MPS technology has expanded the potential applications of DNA markers and increased the discrimination power of the targeted loci by taking variations in their flanking regions into consideration. Here, a collection of nuclear and extranuclear DNA markers (totally six kinds of nuclear genetic markers and mtDNA hypervariable region variations) were comprehensively and systematically assessed for polymorphism detections, further employed to dissect the population backgrounds in the Yugu ethnic group from Gansu province (Yugu) and Han population from the Inner Mongolia Autonomous Region (NMH) of China. The elevated efficiencies of the marker set in separating full sibling and challenging half sibling determination cases in parentage tests (iiSNPs), as well as predicting ancestry origins of unknown individuals from at least four continental populations (aiSNPs) and providing informative characteristic-related clues for Chinese populations (piSNPs) are highlighted in the present study. To sum up, different sets of DNA markers revealed sufficient effciencies to serve as promising tools in forensic applications. Genetic insights from the perspectives of autosomal DNA, Y chromosomal DNA, and mtDNA variations yielded that the Yugu ethnic group was genetically close related to the Han populations of the northern region. But we admit that more reference populations (like Mongolian, Tibetan, Hui, and Tu) should be incorporated to gain a refined genetic background landscape of the Yugu group in future studies.

The application of machine learning to predict genetic relatedness using human mtDNA hypervariable region I sequences.

Human identification of unknown samples following disaster and mass casualty events is essential, especially to bring closure to family and friends of the deceased. Unfortunately, victim identification is often challenging for forensic investigators as analysis becomes complicated when biological samples are degraded or of poor quality as a result of exposure to harsh environmental factors. Mitochondrial DNA becomes the ideal option for analysis, particularly for determining the origin of the samples. In such events, the estimation of genetic parameters plays an important role in modelling and predicting genetic relatedness and is useful in assigning unknown individuals to an ethnic group. Various techniques exist for the estimation of genetic relatedness, but the use of Machine learning (ML) algorithms are novel and presently the least used in forensic genetic studies. In this study, we investigated the ability of ML algorithms to predict genetic relatedness using hypervariable region I sequences; that were retrieved from the GenBank database for three race groups, namely African, Asian and Caucasian. Four ML classification algorithms; Support vector machines (SVM), Linear discriminant analysis (LDA), Quadratic discriminant analysis (QDA) and Random Forest (RF) were hybridised with one-hot encoding, Principal component analysis (PCA) and Bags of Words (BoW), and were compared for inferring genetic relatedness. The findings from this study on WEKA showed that genetic inferences based on PCA-SVM achieved an overall accuracy of 80–90% and consistently outperformed PCA-LDA, PCA-RF and PCA-QDA, while in Python BoW-PCA-RF achieved 94.4% accuracy which outperformed BoW-PCA-SVM, BoW-PCA-LDA and BoW-PCA-QDA respectively. ML results from the use of WEKA and Python software tools displayed higher accuracies as compared to the Analysis of molecular variance results. Given the results, SVM and RF algorithms are likely to also be useful in other sequence classification applications, making it a promising tool in genetics and forensic science. The study provides evidence that ML can be utilized as a supplementary tool for forensic genetics casework analysis.

MtDNA Haplogroup M5 Associated with Risk of Colorectal Cancer in South India Population

The life on earth is driven by energy, supplied by the tiny organelles of the cell called mitochondria and they are usually stated as the powerhouses of the cell. In population genetics, Mitochondrial DNA (mtDNA) is used extensively to categorize individuals or populations. The mutation sites observed in human mtDNA by comparing with the reference sequence (rCRS) are termed into definite human mtDNA haplogroups. Previous studies showed that mtDNA specific haplogroups and polymorphisms were established to be linked with various human diseases, including cancer in numerous populations. Furthermore, it is also known that several mitochondrial DNA polymorphisms are implicated in enhanced production of Reactive Oxygen Species (ROS) which are known to be an increased risk cause for several cancers, including colorectal cancer in Indian patients. Hence in our study, we made high resolution examination on mtDNA hypervariable region to trace the association of specific mtDNA haplogroup with colorectal cancer in south Indian populations. We report that mtDNA Haplogroup M was observed in 60% of the colorectal cancer patients and around 55% in the studied control samples. Haplogroup M is the most frequent mtDNA cluster found among south Indian populations. We further sub-lineated macro haplogroup M and found sub haplogroups (M8, M7, M6, M5, M3 and M2) in varied frequencies. In particular, we found significant association of haplogroup M5 with colorectal cancer patients (p = 0.026). Haplogroup M5 was observed in 12% of colorectal cancer patients in south Indian patients and in 3.3% of the control populations. These results suggest that individuals with haplogroup M5 may have significant risk to develop colorectal cancer.

Reduced mitochondrial DNA copy number is associated with the haplogroup, and some clinical features of breast cancer in Mexican patients.

Mitochondrial DNA (mtDNA) copy number and mitochondrial DNA haplogroups have been associated with different types of cancer, including breast cancer, because they alter cellular energy metabolism. However, whether mtDNA copy number or haplogroups are predictors of oxidative stress-related risks in human breast cancer tissue in Mexican patients remains to be determined. Using quantitative real-time PCR assays and sequencing of the mtDNA hypervariable region, analysis of mtDNA copy numbers in 82 breast cancer tissues (BCT) and matched normal adjacent tissues (NAT) was performed to determine if copy number correlated with clinical features and Amerindian haplogroups (A2, B2, B4, C1 and D1) . The results showed that the mtDNA copy number was significantly decreased in BCT compared with NAT (p=0.010); it was significantly decreased in BCT and NAT in women>50years of age, compared with NAT in women<50years of age (p=0.032 and p=0.037, respectively); it was significantly decreased in NAT and BCT in the postmenopausal group and in BCT in the premenopausal group compared with NAT in the premenopausal group (p=0.011, p=0.010 and, p=0.018; respectively); and it was also significantly decrease in members of the BCT group classified as having invasive ductal carcinoma I-III (IDC-I, IDC-II and IDC-III) and IDC-II for NAT compared to IDC-I of NAT (p=0.025, p=0.022 and p=0.031 and p=0.020; respectively). The mtDNA copy number for BCT from patients with haplogroup B2 was decreased compared to patients with haplogroup D1 (p=0.01); for BCT from patients with haplogroup C1 was also decreased compare with their NAT counterpart (p=0.006) and with BCT patients belonging to haplogroups A2 and D1 (p=0.01 and p=0.03; respectively). In addition, the mtDNA copy number was decrease in the sequences with three deletions relative to the rCRS at nucleotide positions A249del, A290del and A291del, or C16327T polymorphism with the same p=0.019 for all four variants. Contrary, the copy number increased in sequences containing C16111T, G16319A or T16362C polymorphisms (p=0.021, =0.048, and=0.001; respectively). In conclusion, a decrease in the copy number of mtDNA in BCT compared with NAT was shown by the results, which suggests an imbalance in oxidative phosphorylation (OXPHOS) that can affect the apoptosis pathway and cancer progression. It was also observed an increase of the copy number in samples with specific polymorphisms, which may be a good sign of favourable prognosis.

Population genetics assessment model reveals priority protection of genetic resources in native pig breeds in China

A variety of native pig breeds have been developed over the long history of their domestication by human and in a complex geographical climate. Rational use and maximum protection of local genetics resources are the major concern to maintain global biodiversity. Scientific protection measures can effectively prevent the extinction of biological resources. Three genetic resource assessment models were established by analyzing the mtDNA hypervariable regions of 70 native pig breeds in China. The genetics resources of native pig breeds in China from different perspectives, such as genetic diversity, genetic contribution rate and erosion degree, were analyzed. The results show that most native pig breeds in China have low genetic diversity, especially for those distributed in Sichuan and Shandong Provinces. Tibetan pigs contribute more to the haplotype richness of native pigs and have a greater impact on the genetic resources of native pig breeds. The Meishan pig of Jiangsu Province (JSMS) are slight eroded by European pigs. The assessment models of genetic diversity, genetic contribution rate and erosion degree were established to evaluate the genetic resources of native pig breeds from different perspectives, which provide new insights for the scientific protection of native breeds.

Analysis and interpretation of mixture DNA using AS-PCR of mtDNA

In forensic medicine, mixed stains usually include body fluids, secretions of two or more individuals. The application of STR profiling of single cell capture or mixed samples requires that the existence of intact nucleated cells or the appropriate proportion of different components in the sample, respectively. Trace components cannot be genotyped, and the analysis of mixture from more than two individuals is even more difficult.There are SNPs located in the mtDNA hypervariable region and its flanking sequences. According to the differences of SNPs between individuals, allele-specific PCR (AS-PCR) may be used to obtain the individual DNA fragments containing mtDNA hypervariable regions in mixed stains. There are two assumptions in the application of our method. One assumption is that when the suspect is known, the AS-PCR can be designed according to the SNPs in the mtDNA hypervariable and flanking regions. The suspect cannot be excluded if the amplified sequence is consistent with the suspect’s. Otherwise, the suspect can be excluded. The other assumption is without the suspect. We can obtain the individual’s sequence of the high component and then perform the multiple AS-PCR according to specific SNP. If the low component’s fragment can be amplified, it is possible from the potential suspect. Our method is easy to conduct and can exclude the unrelated individuals or provide the suspect’s information, especially for the individual with the low component. To some degree, it can solve the analysis of the mixture of multiple individuals. However, it is invalid for the samples of the same maternal line and the personal identification cannot be achieved.Our study is supported by National Natural Science foundation of China (NO.81671872).

The using of massively parallel sequencing of mitochondrial DNA to assist the missing person identification: Human remains in the wild

On April 2016 a man was reported missing in Phu Khiao wildlife sanctuary, Chaiyaphum province; Thailand. In the following year, twenty pieces of evidences as well as human remains were discovered in the area and was suspected to belong to the missing person. DNA retrieved from the discovered twenty evidences and the occipital bone were extracted and amplified by Investigator IDplex Plus Kit and GlobalFiler™ PCR Amplification Kit. The identification process of the missing person was performed by kinship testing of the bone and the subject’s younger sister. Due to an extremely hot and humid environment, the bone specimen were highly degraded by the moisture and the soil microorganisms. Thus, the DNA recovered only sufficient for a partial and incomplete STR profile. The retrieval of 5 STR loci lent to the failure in the full sibling test. The method of determination of maternal lineage by mtDNA was applied to this case. As a consequence of the DNA insufficiency, the conventional sanger sequencing of the mtDNA hypervariable regions (HV1 and HV2) was shifted to the massively parallel sequencing using Ion Torrent Personal Genome Machine (PGM). With the potential and a high sensitivity technique, the mtDNA haplotype was acquired from the degraded bone and matched against the subject’s younger sister. The result showed the confirmation of the same maternal lineage. The application of the mtDNA and the massively parallel sequencing technique assisted in the identification of missing person from the degraded bone which were exposed in an inappropriate environment for a long period of time

Mitochondrial DNA study in the Shuar ethnic group from Ecuador

This study presents mitochondrial data from 55 unrelated individuals from two Ecuadorian Shuar communities: Kumbatza and Yukateis. Maternal linage was determined by analyzing the two mtDNA hypervariable regions: HRVI and HRVII. It was shown that the Shuar population exhibited the haplogroup B. This demonstrates that Shuar group is a conserved population with no mixing with the European and African diaspora populations.

DETECTION OF BOVINE LEUKOCYTE ADHESION DEFICIENCY, DEFICIENCY OF URIDINE MONOPHOSPHATE SYNTHASE, AND COMPLEX VERTEBRAL MALFORMATION IN HOLSTEIN CATTLE

This research aimed to evaluate the prevalence of the most common lethal diseases in the Lithuanian Holstein cattle population. Two hundred non-related (based on the documentation of origin) cattle (cows and heifers) were included in the study. DNA extraction from blood leukocytes was performed using the chloroform salt method. The cattle were tested for three inherited bovine disorders: bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine monophosphate synthase (DUMPS), complex vertebral malformation (CVM). The PCR-RFLP test method was used to determine the polymorphism of the CD18 gene, which is responsible for BLAD inherited disorder development. A recessive allele with point mutation A→G (383), causing BLAD, was found in the Lithuanian cattle population with 0.0025 frequency. CVM disease is determined by the missense mutation, which has been found in the SLC35A3 gene. The study was performed using a sequencing method. A recessive allele with point mutation G→T (538), causing CVM, was found in the Lithuanian cattle population with 0.005 frequency. The PCR-RFLP test method was used to determine the polymorphism of the UMPS gene, responsible for DUMPS inherited disorder development. A recessive allele with point mutation C→T (1213), causing DUMPS, was not found in the Lithuanian Holstein cattle population. Because intensive selection programmes were performed over the previous decade, the number of heritable lethal diseases carriers has significantly decreased.Key words: cattle; bovine leukocyte adhesion deficiency; deficiency of uridine monophosphate synthase; complex vertebral malformation; BLAD; DUMPS; CVM UGOTAVLJANJE POMANJKLJIVE ADHEZIJE GOVEJIH LEVKOCITOV, POMANJKANJA ENCIMA URIDIN MONOPOSFAT SINTETAZE TER KOMPLEKSA MALFORMACIJE VRETENC PRI HOLŠTAJNSKEM GOVEDUPovzetek: Namen raziskave je bil oceniti razširjenost najpogostejših smrtnih bolezni v populaciji litvanskega holštajnskega goveda. V študijo je bilo vključenih dvesto nesorodnih (na podlagi dokumentacije o poreklu) krav in telic. Izolacijo DNK iz krvnih levkocitov smo izvedli z metodo izolacije s soljo in kloroformom. V vzorcih DNK krav in telic smo preverili prisotnost mutacij v treh genih, ki povzročajo naslednje bolezni: motnjo prilepljanja govejih levkocitov (BLAD), pomanjkanje encima uridin monofosfat sintaze (DUMPS) ter kompleksno malformacije vretenc(CVM). Metoda PCR-RFLP je bila uporabljena za določanje polimorfizma gena CD18, ki je odgovoren za razvoj dedne bolezni BLAD. Recesivni alel s točkovno mutacijo A → G (383), ki povzroča bolezen BLAD, je bil ugotovljen v populaciji litvanskega goveda s frekvenco pojavljanja 0,0025. Bolezen CVM je povzročena z drugačno-pomensko mutacijo v genu SLC35A3. Prisotnost mutacije v tem genu smo izvedli z metodo sekvenciranja. Recesivni alel s točkovno mutacijo G → T (538), ki povzroča CVM, je bil ugotovljen v populaciji litvanskega goveda s frekvenco pojavljanja 0,005. Testna metoda PCR-RFLP je bila uporabljena za določanje polimorfizma gena UMPS, ki je odgovoren za razvoj dedne bolezni DUMPS. Recesivni alel s točkovno mutacijo C→ T (1213), ki povzroča DUMPS, ni bil najden v litvanski populaciji holštajnskega goveda. Ker so se v preteklem desetletju izvajali intenzivni selekcijski programi, se je število prenašalcev dednih smrtnih bolezni znatno zmanjšalo.Ključne besede: govedo; moteno prilepljanje govejih levkocitov; pomanjkanje encima uridin monofosfat sintaze; kompleksna malformacije vretenc; BLAD; DUMPS; CVM

GENETIC DIVERSITY AMONG TWO COMMON POPULATIONS OF CANIS LUPUS FAMILIARIS IN EGYPT BY USING MITOCHONDRIAL DNA HVR1 SEQUENCE

The current study aimed to investigate the variation of mtDNA hypervariable region 1 (HVR1) among Egyptian Baladi and German shepherd dogs in Egypt with respect to their phylogenetic origin. Blood samples were obtained from two dog breeds; Egyptian Baladi (n = 46) and German shepherd (n = 42) and used for genomic DNA extraction and PCR amplification of the mtDNA HVR1 using primers H15360 and L16106. The determined haplotypes were aligned to the sequences of the first published dog mitochondrial genome (Accession No. U96639). We identified 22 different haplotypes from 34 single nucleotide polymorphisms (SNPs) including 2 insertion-deletion polymorphisms among the Egyptian Baladi dogs and 12 haplotypes from 22 SNPs among the German shepherd dogs. Four haplogroups (A, B, C, and D) were identified in the two breeds, their distribution includes 78% of Egyptian Baladi dogs and 76% of German shepherd dogs, respectively were located in the haplogroup A. While 19 % of the German shepherd and 15% of Egyptian Baladi dogs were found in the haplogroup B. 5% of the detected haplotypes of the two breeds were belonged to haplogroup C. 2% of the detected haplotypes of Egyptian Baladi dogs were classified to a haplogroup D. High haplotype and nucleotide diversities were found in the two breeds indicating a lack of genetic differentiation and a recent population growth. The later was confirmed in the Egyptian dogs with the negative values of the neutrality tests and their clustering in the same clade within the phylogenetic tree.

New Evidence of Ancient Mitochondrial DNA of the Southern Andes (Calchaquí Valleys, Northwest Argentina, 3,600–1,900 Years before Present)

Genetic studies on pre-Hispanic populations of the Southern Andes have been increasing steadily in the last decade. Nevertheless, ancient DNA characterization of Formative Period archaeological human remains is particularly scant, especially for Northwest Argentina. To expand current information on genetic characterization of the first agricultural communities of the southern Calchaquí Valleys, we present and discuss the first mitochondrial ancient DNA information obtained on samples dated to ca. 3,600-1,900 years before present from the Cajón Valley, Catamarca Province. Reproducible mtDNA hypervariable region 1 (HVR-1) sequences were obtained in seven individuals. Mitochondrial HVR-1 haplotypes were assigned to three of the four founding haplogroups, D1 (57.1%), C1 (28.5%), and B2 (14.2%), with absence of A2. Our results show that the Cajón Valley sample, with predominance of D1 and C1, differs from that commonly observed in ancient and modern Andean populations, which usually show a high prevalence of haplogroup B2. The fact that the Cajón Valley and Pampa Grande (Salta Province, Argentina) share a prevalence of haplogroup D1 could provide additional evidence to support possible genetic affinities between the valleys and the eastern sub-Andean region during the Formative Period in Northwest Argentina, expanding the archaeological evidence of contact between both populations. Future complete mitogenomic analysis will provide substantial information to formulate new hypotheses about the origins and phylogenetic relationships between the individuals of the Cajón Valley and other groups from the Andes, Gran Chaco, and the Amazon.

Mitochondrial DNA haplogroups and age at onset of Machado–Joseph disease/spinocerebellar ataxia type 3: a study in patients from multiple populations

Mitochondrial dysfunction has been implicated in the pathogenesis of several neurodegenerative disorders, including Machado-Joseph disease (MJD), an autosomal dominant late-onset polyglutamine ataxia that results from an unstable expansion of a CAG tract in the ATXN3 gene. The size of the CAG tract only partially explains age at onset (AO), highlighting the existence of disease modifiers. Mitochondrial DNA (mtDNA) haplogroups have been associated with clinical presentation in other polyglutamine disorders, constituting potential modifiers of MJD phenotype. A cross-sectional study, using 235 unrelated patients from Portugal, Brazil, India and Japan, was performed to investigate if mtDNA haplogroups contribute to AO of MJD. mtDNA haplogroups were obtained after sequencing the mtDNA hypervariable region I. Patients were classified in 15 phylogenetically related haplogroup clusters. The AO was significantly different among populations, implying the existence of other non-CAG factors, which seem to be population specific. In the Portuguese population, patients classified as belonging to haplogroup JT presented the earliest onset (estimated onset 34.6years of age). Haplogroups W and X seem to have a protective effect, causing a delay in onset (estimated onset 47years of age). No significant association between haplogroup clusters and AO was detected in the other populations or when all patients were pooled. Although haplogroup JT has already been implicated in other neurodegenerative disorders, no previous reports of an association between haplogroups W and X and disease were found. These findings suggest that haplogroups JT, W and X modify AO in MJD. Replication studies should be performed in European populations, where the frequency of the candidate modifiers is similar.

Allele frequencies of mitochondrial DNA HVR III 514\u2013524 (CA)n dinucleotide repeats in the Urali Kuruman tribal population of South India

BackgroundTo evaluate the 514–524 (CA)n dinucleotide repeats/allele of the mtDNA hypervariable region III in the Urali Kuruman tribe of Southern India. Blood samples were randomly collected from 100 healthy unrelated individuals, and complete mtDNA control region was amplified and sequenced by the Sanger sequencing method.FindingsThe present study revealed the presence of nucleotide insertion and deletion of C and A (CA/AC) at position 514–524 in 86 samples. Insertion of 1AC (14%) and 2AC (20%) and deletion of 1CA (49%) and 2CA (3%) were detected. A statistical estimate for this population showed a genetic diversity of 0.6866 and random match probability of 0.3202, and the discrimination power calculated was 0.6798.ConclusionThis study will be beneficial for personal identification and in forensic investigation casework.

The Highest Mutation in mtDNA Hypervariable Region and Application of Biostatistics with Nucleotide Base X t-n in Determining the Identity of the Mutation through a Transition Intensity Matrix

Human mitochondrial DNA (mtDNA) have been used intensively in the field of forensic identification of victims or suspects of crime through biological evidence. The number of mtDNA molecules in a single cell are in the tens of thousands which enable analysis of samples very little or damaged. Till now there is no standard method for identification using mtDNA in a mass disaster victims such as natural disasters, wars and accidents so that the identification process can not run fast. This study found C16.223t variants in mtDNA sequences that can be used to divide the database into two groups so as to accelerate the process of identification through a mathematical algorithm. This variant has the highest frequency (29.7%) of the 91 polymorphic human mtDNA HVS1 along the 300 nucleotide (16,024-16,324) derived from the NCBI database as much as 142 sequences. MtDNA sequences obtained from data collection Papuan human mtDNA groups that have been published in the NCBI. The next variant that can be used as a classifier in a row in the sequence is 16,311; 16,304; 16,189; and 16,270 with the identity (T→c). For a matrix Q is reversible so the matrix and could have the opposite diagonal. Thus the above equation can be solved by using the diagonal method that can be written: U³ = Uµ רUµ aµ†1 . This equation could count the number of transitions and transversion substitution mutations that occur in a nucleotide sequence of mtDNA. With this grouping, the database can be reduced so as to accelerate the process of identification of samples. Expected method of grouping by the variant with the highest frequency can be developed in the codification database for forensic interest such as the police or the mtDNA database purposes of study anthropology and evolutionary biology.

Phylogenetic analysis of Tibetan mastiffs based on mitochondrial hypervariable region I.

Recently, the number of Tibetan mastiffs, which is a precious germplasm resource and cultural heritage, is decreasing sharply. Therefore, the genetic diversity of Tibetan mastiffs needs to be studied to clarify its phylogenetics relationships and lay the foundation for resource protection, rational development and utilization of Tibetan mastiffs. We sequenced hypervariable region I of mitochondrial DNA (mtDNA) of 110 individuals from Tibet region and Gansu province. A total of 12 polymorphic sites were identified which defined eight haplotypes of which H4 and H8 were unique to Tibetan population with H8 being identified first. The haplotype diversity (Hd: 0.808), nucleotide diversity (Pi: 0.603%), the average number of nucleotide difference (K: 3.917) of Tibetan mastiffs from Gansu were higher than those from Tibet region (Hd: 0.794; Pi: 0.589%; K: 3.831), which revealed higher genetic diversity in Gansu. In terms of total population, the genetic variation was low. The median-joining network and phylogenetic tree based on the mtDNA hypervariable region I showed that Tibetan mastiffs originated from grey wolves, as the other domestic dogs and had different history of maternal origin. The mismatch distribution analysis and neutrality tests indicated that Tibetan mastiffs were in genetic equilibrium or in a population decline.

Understanding influences of culture and history on mtDNA variation and population structure in three populations from Assam, Northeast India.

Positioned at the nexus of India, China, and Southeast Asia, Northeast India is presumed to have served as a channel for land-based human migration since the Upper Pleistocene. Assam is the largest state in the Northeast. We characterized the genetic background of three populations and examined the ways in which their population histories and cultural practices have influenced levels of intrasample and intersample variation. We examined sequence data from the mtDNA hypervariable control region and selected diagnostic mutations from the coding region in 128 individuals from three ethnic groups currently living in Assam: two Scheduled tribes (Sonowal Kachari and Rabha), and the non-Scheduled Tai Ahom. The populations of Assam sampled here express mtDNA lineages indicative of South Asian, Southeast Asian, and East Asian ancestry. We discovered two completely novel haplogroups in Assam that accounted for 6.2% of the lineages in our sample. We also identified a new subhaplogroup of M9a that is prevalent in the Sonowal Kachari of Assam (19.1%), but not present in neighboring Arunachal Pradesh, indicating substantial regional population structuring. Employing a large comparative dataset into a series of multidimensional scaling (MDS) analyses, we saw the Rabha cluster with populations sampled from Yunnan Province, indicating that the historical matrilineality of the Rabha has maintained lineages from Southern China. Assam has undergone multiple colonization events in the time since the initial peopling event, with populations from Southern China and Southeast Asia having the greatest influence on maternal lineages in the region.

MPS analysis of the mtDNA hypervariable regions on the MiSeq with improved enrichment.

The non-coding displacement (D) loop of the human mitochondrial (mt) genome contains two hypervariable regions known as HVR1 and HVR2 that are most often analyzed by forensic DNA laboratories. The massively parallel sequencing (MPS) protocol from Illumina (Human mtDNA D-Loop Hypervariable Region protocol) utilizes four sets of established PCR primer pairs for the initial amplification (enrichment) step that span the hypervariable regions. Transposase adapted (TA) sequences are attached to the 5'-end of each primer, allowing for effective library preparation prior to analysis on the MiSeq, and AmpliTaq Gold DNA polymerase is the enzyme recommended for amplification. The amplification conditions were modified by replacing AmpliTaq Gold with TaKaRa Ex Taq® HS, along with an enhanced PCR buffer system. The resulting method was compared to the recommended protocol and to a conventional non-MPS approach used in an operating forensic DNA laboratory. The modified amplification conditions gave equivalent or improved results, including when amplifying low amounts of DNA template from hair shafts which are a routine evidence type in forensic mtDNA cases. Amplification products were successfully sequenced using an MPS approach, addressing sensitivity of library preparation, evaluation of precision and accuracy through repeatability and reproducibility, and mixture studies. These findings provide forensic laboratories with a robust and improved enrichment method as they begin to implement the D-loop protocol from Illumina. Given that Ex Taq® HS is a proofreading enzyme, using this approach should allow for improved analysis of low-level mtDNA heteroplasmy.