Hypervariable Segment Research Articles

Association of Mitochondrial HVS-I Region Variants with Type 2 Diabetes in Pakistani Diabetic Subjects.

To analyse mitochondrial hypervariable segment I (HVS-I) variations in Pakistani type 2 diabetic subjects. Case-control study. Place and Duration of the Study: National Institute of Diabetes and Endocrinology, Dow University of Health Sciences, Karachi, Pakistan, between January 2019 to January 2021. DNA from whole blood was isolated, and mitochondrial HVS-I region (16024-16370) of 92 individuals, including 47 controls and 45 diabetics, was amplified, sequenced, and analysed. Ninety-two variable sites in the sequenced region were identified and individuals were classified into 56 different haplotypes according to phylotree 17.0 classifications, where major haplotype M5 was nearly 2-fold higher in diabetes. Fischer's exact test revealed variant 16189T>C significantly associated with diabetes (Odds ratio = 12.9, 95% CI = 0.6917 - 2400248) as compared to controls. The authors further analysed 1000 Genomes Project data of Pakistani Control subjects (i.e. PJL, n=96) and found that besides 16189T>C (Odds ratio = 5.875, 95% CI = 1.093 - 31.57, p<0.0339), 16264C>T (Odds ratio = 16, 95% CI = 0.8026 - 314.7, p<0.0310) also showed significant association with diabetic subjects. Comparing diabetic subject data with global control population data of the 1000 Genomes Project, significant associations of eight variants in the studied region were found. Based on the results of this case-control study, it can be concluded that specific variations in the mitochondrial hypervariable segment I (HVS-I) region are significantly associated with type 2 diabetes in the Pakistani population. The major haplotype M5 was found to be higher in diabetic subjects and variants 16189T>C and 16264C>T were significantly associated with diabetes. These findings suggest that mitochondrial DNA variations may play a role in the development of type 2 diabetes in the Pakistani population. Diabetes Mellitus, HVS-1 region, Diabetic subjects, Mitochondrial genomics, Pakistani population.

The Middle Ear Microbiota in Healthy Dogs Is Similar to That of the External Ear Canal.

Otitis media can be a consequence of chronic otitis externa and could represent a perpetuating factor. While the microbiota of the EEC in healthy dogs and in the presence of otitis externa has been described, only sparse information is available concerning the normal microbiota of the middle ear. The objective was to compare the tympanic bulla (TB) with the external ear canal (EEC) microbiota in healthy dogs. Six healthy experimental Beagle dogs were selected based on the absence of otitis externa, negative cytology and bacterial culture from the TB. Samples from the EEC and TB were collected directly after death using a total ear canal ablation and lateral bulla osteotomy. The hypervariable segment V1-V3 of the 16S rDNA was amplified and sequenced with a MiSeq Illumina. The sequences were analyzed by the Mothur software using the SILVA database. No significant differences between the EEC and TB microbiota for the Chao1 richness index (p = 0.6544), the Simpson evenness index (p = 0.4328) and the reciprocal Simpson alpha diversity (p = 0.4313) were noted (Kruskal-Wallis test). A significant difference (p = 0.009) for the Chao1 richness index between the right and left EEC was observed. The microbiota profile was similar in the EEC and the TB of the Beagles.

Diversity and distribution of mitochondrial DNA in non-Austronesian-speaking Taiwanese individuals

Many studies have described the diversity of Austronesian-speaking Taiwanese people to shed more light on their origin and their connection with the “Out of Taiwan” migrations. However, the genetic relationship between the non-Austronesian-speaking groups of Taiwan and the populations of continental Asia is still unclear. Here, we studied the diversity of mtDNA in 767 non-Austronesian speakers from 16 locations in Taiwan using partial sequencing obtained from the hypervariable segment I (HVS-I) and coding regions 8,001-9,000 and 9.801–10,900 and 85 complete mtDNA genome sequences. Bayesian analysis of population structure was used to examine their relationship with over 3662 individuals representing indigenous groups of Taiwan, continental East Asia, Japan, and Island Southeast Asia. The whole analysis identified 278 haplotypes. Complete genomes revealed 62 novel subhaplogroups, of which 31 were exclusive to Taiwan. Estimates of coalescence times of all subhaplogroups showed peaks of diversification greater than 5.0 kya, likely characterizing gene flow from continental East Asian groups but not excluding in situ Taiwanese ancestry. Furthermore, a significant number of clades exclusive to non-Austronesian speakers of Taiwan (NAN_Tw) showed coalescence peaks between 1.0 and 2.6 kya, suggesting possible late Neolithic to early metal age settlements of NAN_Tw and local expansion in Taiwan.

External Ear Canal Evaluation in Dogs with Chronic Suppurative Otitis Externa: Comparison of Direct Cytology, Bacterial Culture and 16S Amplicon Profiling.

Simple SummaryDiscrepancy between the results of cytology and bacterial culture methods is sometimes observed in canine otitis externa. The objective of this study was to compare different techniques: direct cytology, aerobic bacterial culture and 16S amplicon profiling. Samples from twenty ears with chronic suppurative otitis externa were analysed. Good correlation between cytology and bacterial culture was observed in 60% of samples. Good correlation between bacterial culture and 16S amplicon profiling was observed in only 10% of samples when the overall 16S amplicon profiling results were used. Nevertheless, the correlation improved to 70% when bacterial species with a relative abundance >10% (considered as insignificant) were taken into account. Of the total bacterial species revealed by 16S amplicon profiling with relative abundance >10%, 38.7% of bacterial species were not revealed by bacterial culture; most of the time, the offending species was a Corynebacterium. This study showed that a careful interpretation of the result of the bacterial culture should be performed, and always with the support of cytology. To assess the overall bacterial population in suppurative otitis, the 16S amplicon profiling method seems to be a more accurate method but does not provide information on bacterial susceptibility.A discrepancy between cytology and bacterial culture methods is sometimes observed in canine otitis externa. The objective of this study was to compare results from cytology, bacterial culture and 16S amplicon profiling. Twenty samples from 16 dogs with chronic suppurative otitis externa were collected. A direct cytological evaluation was carried out during the consultations. Aerobic bacterial culture and susceptibility were performed by an external laboratory used in routine practice. For 16S amplicon profiling, DNA was extracted and the hypervariable segment V1–V3 of the 16S rDNA was amplified and then sequenced with a MiSeq Illumina sequence carried out by the Mothur software using the SILVA database. A good correlation between cytology and bacterial culture was observed in 60% of the samples. Some bacterial species revealed by bacterial culture were present with low relative abundance (<10%) in 16S amplicon profiling. Some bacterial species revealed by the 16S amplicon profiling analysis were not identified with culture; most of the time, the offending species was a Corynebacterium. To conclude, a careful interpretation of the results of bacterial culture should be made and always be in agreement with the cytology. The 16S amplicon profiling method appears to be a more sensitive method for detecting strains present in suppurative otitis but does not provide information on bacterial susceptibility.

Mutational profiling of mtDNA control region reveals tumor-specific evolutionary selection involved in mitochondrial dysfunction.

SummaryBackgroundMitochondrial DNA (mtDNA) mutations alter mitochondrial function in oxidative metabolism and play an important role in tumorigenesis. A series of studies have demonstrated that the mtDNA control region (mtCTR), which is essential for mtDNA replication and transcription, represents a mutational hotspot in human tumors. However, a comprehensive pan-cancer evolutionary pattern analysis of mtCTR mutations is urgently needed.MethodsWe generated a comprehensive combined dataset containing 10026 mtDNA somatic mutations from 4664 patients, covering 20 tumor types based on public and private next-generation sequencing data.FindingsOur results demonstrated a significantly higher and much more variable mutation rate in mtCTR than in the coding region across different tumor types. Moreover, our data showed a remarkable distributional bias of tumor somatic mutations between the hypervariable segment (HVS) and non-HVS, with a significantly higher mutation density and average mutation sites in HVS. Importantly, the tumor-specific mutational pattern between mtCTR HVS and non-HVS was identified, which was classified into three evolutionary selection types (relaxed, moderate, and strict constraint types). Analysis of substitution patterns revealed that the prevalence of CH > TH in non-HVS greatly contributed to the mutational selection pattern of mtCTR across different tumor types. Furthermore, we found that the mutational pattern of mtCTR in the four tumor types was clearly associated with mitochondrial biogenesis, mitochondrial oxidative metabolism, and the overall survival of patients.InterpretationOur results suggest that somatic mutations in mtCTR may be shaped by tumor-specific selective pressure and are involved in tumorigenesis.FundingsNational Natural Science Foundation of China [grants 82020108023, 81830070, 81872302], and Autonomous Project of State Key Laboratory of Cancer Biology, China [grants CBSKL2019ZZ06, CBSKL2019ZZ27].

Mitochondrial DNA polymorphism and myocardial ischemia: Association of haplogroup H with heart failure

The pathogenesis of atherosclerosis and ischemic heart disease is associated with oxidative stress and mitochondrial dysfunction. Mitochondrial DNA encodes subunits of mitochondrial respiratory chain and is highly polymorphic in human populations. Mitochondrial DNA can be considered a candidate genetic locus for predisposition to cardiovascular diseases.Aim. To analyze the associations of the mitochondrial genome polymorphism and chronic heart failure in ischemic heart disease.Material and Methods. The study included two groups of individuals: patients with a combination of ischemic heart disease and chronic heart failure (n = 175) and a population sample of residents of Tomsk (n = 424). Percentages of patients with chronic heart failure of NYHA classes II, III, and IV were 37%, 50%, and 13%, respectively. All patients underwent echocardiographic examination; body mass index and the lipid fractions in blood serum were determined. The average was 55.4 years in patients and 47.6 years in the population sample. Polymorphism of mtDNA was studied by sequencing the hypervariable segment of D-loop of mtDNA and subsequent classification of mtDNA haplotypes into the known haplogroups. The mtDNA haplogroup frequencies were compared between the samples using the Chi-square test. The associations of genotype with quantitative trait variability were analyzed by variance analysis.Results. Male patients showed a higher frequency of haplogroup H compared to the population (45.86% in patients and 35.4% in population) and a higher total frequency of haplogroup H subgroups except the most frequent subgroup H1 (36.94% and 25.22%, respectively). The values of significance level (p-value) and odds ratio (OR) were determined as follows: p = 0.04; odds ratio OR = 1.55 (95% confidence interval (CI) 1.02–2.34) for haplogroup H as a whole; p = 0.02; OR = 1.74 (95% CI 1.12–2.70) for haplogroup H without subgroup H1. Analysis of quantitative traits revealed the associations of the same genetic marker (mtDNA haplogroup H) with the levels of high-density lipoproteins (p = 0.03) and triglycerides (p = 0.02) in blood serum of men in the population sample.Conclusion. The obtained results suggested that the most frequent European mtDNA haplogroup H may be a risk factor for the complications of ischemic heart disease in men.

Analysis of Polymorphism of Uniparental Markers in Reindeer-Herding Populations

We analyzed the data on the variability of the Y chromosome and mitochondrial DNA (mtDNA) in populations of the Tsaatans of Mongolia and the Tozhu Tuvans of Russia. The populations studied are characterized by low genetic diver¬sity for both marker systems. The analysis of Y chromosome haplogroups in the Tsaatan and Tozhu revealed three hap¬logroups in the Tsaatan and seven haplogroups in the Tozhu. The composition of the haplogroups is coherent to literature data on the Tuvans, which is explained by common origin. According to the data on mitochondrial DNA variability, 12 haplogroups were determined in 46 Tozhus, of which C4b (30.43%) and F1b1b (23.91%) are major haplogroups. According to the HVS–1 (HyperVariable Segment) data, 15 haplotypes were found in the Tozhu Tuvans and the diversity coefficient of 0.8677 turned out to be much lower than among the Torghut of Mongolia (0.9857). In 23 Tsaatans, 14 haplogroups were determined; the most common of which are C4b (22.73%) and C5a1 (18.18%). According to HVS-1, 14 haplotypes were revealed in the Tsaatan, the diversity is 0.9486. The data obtained on uniparental marker systems in the Tozhus and Tsaatans are due to the isolated and inaccessible taiga region and the manifestation of the “founder effect”. The Tsaatans are less polymorphic in terms of the variety of Y chromosome haplogroups, while the Tozhus are less polymorphic in terms of mitochondrial DNA, which is probably a consequence of a high rate of endogamic marriages in the populations studied.

Палеогенетический анализ жителей подола Московского Кремля XIV в. (по материалам раскопок в Тайницком саду)

Introduction. The article presents the results of paleogenetic studies of medieval human remains of three people found in a closed archaeological complex (building 32) revealed during the excavations in 2007 in the Taynitsky Garden of the Moscow Kremlin (supervisor of excavations: N.A. Makarov). Previous studies on the dating of the complex links it to the devastation of Moscow by the troops of Tokhtamysh Khan in August 1382. The archaeological layer was formed at a time as a result of a fire and contained the remains of two adults and a 3-4 year old child who remained unburied. The aim of this work was the genetic study of the ancient DNA of the remains of people who died in the 14th century, clarification of their gender, determination of kinship and presumptive origin. Material and methods. For genetic examination, teeth were selected (permanent for adults, primary for a child). The laboratory research algorithm included a set of measures to protect archaeological DNA from contamination, sample preparation and extraction of DNA from dental remains, analysis of STR markers of the Y chromosome in males, analysis of ALU markers of autosomal chromosomes, targeted NGS sequencing of hyper-variable segments of mitochondrial DNA. Results and conclusion. Using the methods of molecular genetic research, it was possible to confirm that a man, a young woman and a child (boy) died in the fire. Based on the analysis of autosomal markers, with a high degree of probability (99.9%), a close biological relationship between a woman and a child (mother-son) was revealed. The man was not a relative of either the woman or the child. The mtDNA haplogroups and STR markers of the male specific Y chromosome identified in all three individuals are generally characteristic of the Slavic population of modern Europe. The mt haplogroup J1c, found in mother and child, is now most characteristic of the inhabitants of Europe. The man has a mitochondrial haplogroup K2, which is found mainly in Northwestern Europe.

Mitochondrial DNA control region sequencing of the critically endangered Hainan gibbon (Nomascus hainanus) reveals two female origins and extremely low genetic diversity

The Hainan gibbon (Nomascus hainanus) is endemic to China and is the world’s rarest ape. The remaining wild population totals only 33 individuals. In the current study, we sequenced the Mitochondrial DNA control region of 12 wild Hainan gibbons representing three social groups of the five remaining groups. By conducting population genetic analyses, we found that the proportion of four nucleotides (T, C, A and G) were 29.0%, 27.2%, 31.9% and 11.9%, respectively. Hypervariable segments of the mtDNA D-loop region (1005 bp in length), indicated five variable sites (a point mutation), with only two haplotypes present among the 12 samples. We observed that the genetic diversity of Hainan gibbons is lower than that reported in any other wild primate population, and that the two haplotypes detected, represent two ancestral lineages. These findings have important implications for proposing effective conservation strategies to protect this Critically Endangered ape species.

Inconsistency Between Socio-Spatial and Genetic Structure in a Coastal Dolphin Population

Identifying population structure and boundaries among communities of wildlife exposed to anthropogenic threats is key to successful conservation management. Previous studies on the demography, social and spatial structure of Indo-Pacific bottlenose dolphins (Tursiops aduncus) suggested four nearly discrete behavioral communities in Perth metropolitan waters, Western Australia. We investigated the genetic structure of these four communities using highly polymorphic microsatellite markers and part of the hypervariable segment of the mitochondrial control region. Overall, there was no evidence of spatial genetic structure. We found significant, yet very small genetic differentiation between some communities, most likely due to the presence of highly related individuals within these communities. Our findings of high levels of contemporary migration and highly related individuals among communities point toward a panmictic genetic population with continuous gene flow among each of the communities. In species with slow life histories and fission-fusion dynamics, such asTursiopsspp., genetic and socio-spatial structures may reflect different timescales. Thus, despite genetic similarity, each social community should be considered as a distinct ecological unit to be conserved because they are exposed to different anthropogenic threats and occur in different ecological habitats, social structure being as important as genetic information for immediate conservation management. The estuarine community, in particular, is highly vulnerable and appropriate conservation measures are needed in order to maintain its connectivity with the adjacent, semi-enclosed coastal communities.

Maternal origins, population structure and demographic history of ten Chinese indigenous goat breeds from Yunnan.

Yunnan as a frontier zone that connects China with South and Southeast Asia, has 11 well-recognized goat breeds. However, the knowledge about maternal origins, population structure and demographic history of Chinese indigenous goats from Yunnan is limited. In this study, we analysed a 481-bp fragment of first hypervariable segment (HVSI) of the mitochondrial DNA (mtDNA) control region sequences of 749 individuals from 10 Yunnan indigenous goat breeds, of which 556 sequences were newly determined. There were 110 polymorphic sites that defined 158 haplotypes among all sequences. The haplotype and nucleotide diversity of these breeds ranged from 0.782±0.079 to 0.982±0.015 and from 0.028±0.003 to 0.043±0.005, respectively. Phylogenetic analysis identified two lineages A and B, of which the lineage A had higher frequency (68.1%) and distributed in all Yunnan breeds. We combined previously reported sequences with our sequences belonging to the lineage B and detected two subclades B1 and B2, in which the B1 subclade shared individuals from Eastern Asia, Southeast Asia and Southern Asia. Given higher level of diversity and more unique haplotypes, the B2 subclade probably originated from Southwestern China. The haplotype network, analysis of molecular variance (AMOVA) and a Mantel test revealed no significant phylogeographic structuring among Yunnan goat breeds. This can be explained by high gene flow and genetic admixture among these breeds from different geographic regions in Yunnan. Additionally, both the lineages A and B reflected different demographic histories. This study will provide a scientific basis for the conservation and utilization of Yunnan indigenous goats.

Mitochondrial DNA (hypervariable region I) diversity in Basrah population – Iraq

In attempt to investigate the origin of Basrah, we examined the mitochondrial DNA(mt-DNA) variations by hypervariable segment 1(HVS1) Sequencing and determination of specific site haplogroups. In Basrah, no significant differences diversity among Iraqis' HVS1 compared with other countries. The values were within the range of gene diversity across the Middle East and exhibited the unimodal pattern of differences in the pairwise sequence. Given the small genetic differences between people living in this area, phylogenetic analysis showed a large variability of the communities of Basrah; they didn't cluster on the phylogenetic tree.

Recent kuru-induced female gene flow disrupted the coevolution of genes and languages in the Papua New Guinea highlands.

The island of New Guinea was settled by modern human over 50,000 years ago, and is currently characterized by a complex landscape and contains one-seventh of the world's languages. The Eastern Highlands of New Guinea were also the home to the devastating prion disease called kuru that primarily affected Fore-speaking populations, with some 68% of cases involving adult females. We characterized the mitochondrial DNA (mtDNA) diversity of highlanders from Papua New Guinea (PNG) to: (a) gain insight into the coevolution of genes and languages in situ over mountainous landscapes; and (b) evaluate the recent influence of kuru mortality on the pattern of female gene flow. We sequenced the mtDNA hypervariable segment 1 of 870 individuals from the Eastern and Southern Highlands of PNG using serums collected in the 1950s to 1960s. These highlanders were selected from villages representing 15 linguistic groups within the Trans-New Guinea phylum. Genetic, linguistic, and geographic distances were calculated separately and correlations among those distance matrices were assessed using the Mantel test. Geographic, genetic, and linguistic patterns were independently correlated with each other (p < .05). Increased mtDNA diversity in kuru-affected populations and low Fst estimates between kuru-affected linguistic groups were observed. In general, the genetic structure among the Highland populations was shaped by both geography and language, and language is a good predictor of mtDNA affinity in the PNG Highlands. High kuru female mortality increased female gene flow locally, disrupting coevolutionary pattern among genes, languages, and geography.

Forensically relevant phylogeographic evaluation of mitogenome variation in the Basque Country

The Basque Country has been the focus of population (genetic) and evolutionary studies for decades, as it represents an interesting evolutionary feature: it is the only European country where a non-Indo-European language is still spoken today and, for which there are no known living or extinct relatives. Early studies that were based on anatomical and serological methods, along with subsequent molecular genetic investigations, contain controversial interpretations of their data. Additionally, the analysis of mitochondrial DNA, which is maternally inherited and thus suitable for the examination of the maternal phylogeny of the population, was the focus of some studies. Early mtDNA studies were however restricted to the information provided by the control region or its hypervariable segments only. These are known to harbour little phylogenetic information, particularly for haplogroup H that is dominant in Westeurasian populations including the Basques. Later studies analysed complete mitogenome sequences. Their information content is however limited, either because the number of samples was low, or because these studies only considered particular haplogroups. In this study we present the full mitogenome sequences of 178 autochthonous Basque individuals that were carefully selected based on their familial descent and discuss the observed phylogenetic signals in the light of earlier published findings. We confirm the presence of Basque-specific mtDNA lineages and extend the knowledge of these lineages by providing data on their distribution in comparison to other Basque and non-Basque populations. This dataset improves our understanding of the Basque mtDNA phylogeny and serves as a high-quality dataset that is provided via EMPOP for forensic genetic purposes.

Phylogeny and conservation priority assessment of Asian domestic chicken genetic resources

Chicken genetic resources are gradually decreasing in genetic diversity; thus, a theoretical basis for conservation is needed. The genetic diversity and phylogeography of domestic chickens from 10 countries in Asia and red jungle fowl were investigated by analyzing the mitochondrial DNA D-loop hypervariable segment (HVS). The order of domestic chickens distributed in 31 management units in Asia in terms of conservation priority was assessed based on genetic diversity and genetic contribution rate models. A total of 219 haplotypes were distributed in seven clades, with variants identified at 103 sites. Phylogenetic analysis revealed seven maternal origins of Asian domestic chickens. Multiple subspecies of red jungle fowl were involved in the domestication of chickens in the three centers of origin: the Indian subcontinent (clade D), China (clades B, C, E and F) and Southeast Asia (clade A). The domestication center of the G clade remains unknown. The order in terms of conservation priority based on genetic diversity was as follows: northwestern and northeastern Yunnan, China; Sri Lanka; Guangxi, China; Japan; etc. The order in terms of conservation priority based on the genetic contribution rate was as follows: northwestern Yunnan, China; Sri Lanka; Japan; Papua New Guinea; etc. This study provided new insights for the conservation and evaluation of native genetic resources.

Buccal Dna Collection Using Treated Paper

This study presents a domestic kit suitable for the collection of human buccal cells, and long-term storage of nucleic acids on a piece of paper. A filter paper with a density of 70 g/m2 was selected and treated with chemicals that make up the lysing solution, which contributes to the destruction of cells and prevents bacterial growth. A total of 25 buccal cell samples were collected on the treated paper cards. The samples had a median DNA concentration of 20 ng/μl (range 0.5–45 ng/μl). The collected DNA samples were tested using a real-time PCR, sequencing of the first hypervariable segment of the human mtDNA control region, and multiplex genotyping of 15 short tandem repeat loci. We conclude that the domestic pretreated cards could be used for collecting and isolating DNA from buccal epithelium for genetic and other applications.

Somatic mitochondrial DNA D-loop mutations in meningioma discovered: A preliminary data.

Meningiomas are among the most common intracranial tumors of the central nervous system. It is widely accepted that the initiation and progression of meningiomas involve the accumulation of nucleus genetic alterations, but little is known about the implication of mitochondrial genomic alterations during development of these tumors. The human mitochondrial DNA (mtDNA) contains a short hypervariable, noncoding displacement loop control region known as the D-Loop. Alterations in the mtDNA D-loop have been reported to occur in most types of human cancers. The purpose of this study was to assess the mtDNA D-loop mutations in Malaysian meningioma patients. Genomic DNA was extracted from 21 fresh-frozen tumor tissues and blood samples of the same meningioma patients. The entire mtDNA D-loop region (positions 16024-576) was polymerase chain reaction amplified using designed primers, and then amplification products were purified before the direct DNA sequencing proceeds. Overall, 10 (47.6%) patients were detected to harbor a total of 27 somatic mtDNA D-loop mutations. Most of these mtDNA mutations were identified in the hypervariable segment II (40.7%), with 33.3% being located mainly in the conserved sequence block II of the D310 sequence. Furthermore, 58 different germline variations were observed at 21 nucleotide positions. Our results suggest that mtDNA alterations in the D-loop region may be an important and early event in developing meningioma. Further studies are needed, including validation in a larger patient cohort, to verify the clinicopathological outcomes of mtDNA mutation biomarkers in meningiomas.

Genetic comparison of Altai and Gobi argali sheep (Ovis ammon) populations using mitochondrial and microsatellite markers: Implication on conservation

Argali sheep is an ungulate, which inhabits the north, west, south and central regions of Mongolia. There are two major populations (Altai and Gobi) in Mongolia, but their taxonomic classification as subspecies is often disputed among researchers. Furthermore, there is no recent study about the population genetic structure of argali sheep in Mongolia. In the present study, we have investigated genetic diversity and difference between Altai and Gobi argali populations using mitochondrial control region hyper variable segment (HVS) sequence (598bp) and 3 microsatellite markers. Mitochondrial HVS haplotype analysis showed high haplotype diversity (0.982±0.012) and low nucleotide diversity (0.02589). In microsatellite analysis, total of 9 alleles were found across all loci while mean Ho were 0.59±0.13 for Altai and 0.53±0.1 for Gobi populations, indicating low allelic diversity with moderate heterozygosity. Neighbor-joining tree separated haplotypes into two clusters, Altai and Gobi population, implying distinct genetic difference between the two subspecies. Additionally, Pairwise FST and Kimura-2 parameter showed 0.127 and 0.0413±0.0068, respectively. These genetic distance analyses hinted genetic difference between Altai and Gobi populations are in subspecies level. In summary, mitochondrial HVS and microsatellite analysis demonstrated that Altai and Gobi populations had low genetic diversity but might be genetically distinct from each other in subspecies level, suggesting conservation should be separately managed.

Evidence of restricted maternal gene flow of Purana (old) Population in the suburbs of Sigiriya, Sri Lanka

IntroductionBased on historical records suggesting that the ancestry of the Purana (old) population living in the suburbs of Sigiriya in Sri Lanka can be traced back to the times of the Sinhalese Kings of the 5th century A.D. (1,450 YBP) and studies reporting that the Purana are biometrically different from the rest of present Sri Lankans and other populations in the world, we investigate the maternally inherited Mitochondrial DNA (mtDNA) of Purana population in the suburbs of Sigiriya to better understand genetic affinities of them to present day Sri Lankans. Materials and MethodsForty six Purana inhabitants belonging to Purana pedigrees were recruited in the study. Mitochondrial DNA was extracted, amplified and sequenced. Polymorphisms of mtDNA hypervariable segment I (HVS –I) between nucleotide positions 16,004 –16,411 were identified using CLUSTALX option of MEGAVA 4.0 sequence alignment software. In order to investigate genetic affinity of Purana population, Arlequin software version 3.11 and analysis of molecular variance (AMOVA) were applied using reported similar genetic data of present Sri Lankans such as Sinhalese, Sri Lankan Tamils, Indian Tamils, Sri Lankan Moors and Vedda. Genetic relationships of Purana population with other Sri Lankans were further explored by phylogenetic analysis. Results and AnalysisGenetic dissimilarity among groups was higher (2.81%) when populations were grouped into two as modern and Purana than grouping them according to their ethnic basis (0.00%). This indicates a restricted mtDNA flow between the two groups (Purana and rest of the Sri Lankans) that made Purana population was maternally isolated from the rest of Sri Lankans. ConclusionDetailed phylogenetic analysis of the study revealed that they are maternally more related to Sri Lankan Tamil than to any other present Sri Lankans.

Impact of the sequencing method on the detection and interpretation of mitochondrial DNA length heteroplasmy

Advancements in sequencing technologies allow for rapid and efficient analysis of mitochondrial DNA (mtDNA) in forensic laboratories, which is particularly beneficial for specimens with limited nuclear DNA. Next generation sequencing (NGS) offers higher throughput and sensitivity over traditional Sanger-type sequencing (STS) as well as the ability to quantitatively analyze the data. Changes in sample preparation, sequencing method and analysis required for NGS may alter the mtDNA haplotypes compared to previously generated STS data. Thus, the present study aimed to characterize the impact of different sequencing workflows on the detection and interpretation of length heteroplasmy (LHP), a particularly complicated aspect of mtDNA analysis. Whole mtDNA genome (mitogenome) data were generated for 16 high-quality samples using well-established Illumina and Ion methods, and the NGS data were compared to previously-generated STS mtDNA control region data. Although the mitogenome haplotypes were concordant with the exception of length and low-level variants (<30 % variant frequency), LHP in the hypervariable segment (HVS) polycytosine regions (C-tracts) differed across sequencing methods. Consistent with previous studies, LHP in HVS1 was observed in samples with nine or more consecutive cytosines (Cs) and eight Cs in the HVS2 region in the STS data. The Illumina data produced a similar pattern of LHP as the STS data, whereas the Ion data were noticeably different. More complex LHP (i.e. more length molecules) was observed in the Ion data, as length variation occurred in multiple homopolymer stretches within the targeted HVS regions. Further, the STS dominant or major molecule (MM) differed from the Ion MM in 11 (37 %) of the 30 regions evaluated and six instances (20 %) in Illumina data. This is of particular interest, as the MM is used by many forensic laboratories to report the HVS C-tract in the mtDNA haplotype. In general, the STS MMs were longer than the Illumina MMs, while the Ion MMs were the shortest. The higher rate of homopolymer indels in Ion data likely contributed to these differences. Supplemental analysis with alternative approaches demonstrated that the LHP pattern may also be altered by the bioinformatic tool and workflow used for data interpretation. The broader application of NGS in forensic laboratories will undoubtedly result in the use of varying sample preparation and sequencing methods. Based on these findings, minor LHP differences are expected across sequencing workflows, and it will be important that C-tract indels continue to be ignored for forensic queries and comparisons.