Novel blood and tissue-based mitochondrial D-loop mutations detected in an Iranian NAFLD patient cohort.

Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent chronic liver disease characterized by an elusive etiology. In its advanced stages, this condition can pose life-threatening implications. Mitochondrial dysfunction due to its impact on hepatic lipid homeostasis, cytokine release, ROS production, and cell death, contributes to the pathogenesis of NAFLD. Previous research reveals a direct link between NAFLD genetic predictors and mitochondrial dysfunction. The emphasis on the D-loop stems from its association with impaired mtDNA replication, underscoring its crucial role in NAFLD progression. We included 38 Iranian NAFLD patients (comprising 16 patients with non-alcoholic fatty liver [NAFL] and 22 patients with non-alcoholic steatohepatitis [NASH]), with matched blood and liver tissue samples collected from each to compare variations in the mitochondrial D-loop sequence within samples. The mitochondrial DNA (mtDNA) D-loop region was amplified using PCR, and variations were identified through sequencing. The resultant sequences were compared with the reference sequence of human mtDNA available in the MITOMAP Database for comparative analysis. In this study, 97 somatic mutations in the mtDNA D-loop region were identified in NAFLD patients. Our study revealed significant difference between the NAFLD patients and control group in 13 detected mutations (P ≤ 0.05). Novel mutations were discovered in hepatic tissues, while mutation 16220-16221ins C was found in both tissues and blood. A significant difference was found in the distribution of D310 and mt514-mt523 (CA)n repeat variations between NAFLD patients and the control group (P < 0.001). C to T and T to C transitions were the prevalent substitution among patients. Identification of the 16220-16221ins C mutation in both blood and tissue samples from NAFLD patients holds substantial promise as a potential diagnostic marker. However, further research is imperative to corroborate these findings.

Human Rad51 Protein Requires Higher Concentrations of Calcium Ions for D-Loop Formation than for Oligonucleotide Strand Exchange.

Human Rad51 protein (HsRad51)-promoted DNA strand exchange, a crucial step in homologous recombination, is regulated by proteins and calcium ions. Both the activator protein Swi5/Sfr1 and Ca2+ ions stimulate different reaction steps and induce perpendicular DNA base alignment in the presynaptic complex. To investigate the role of base orientation in the strand exchange reaction, we examined the Ca2+ concentration dependence of strand exchange activities and structural changes in the presynaptic complex. Our results show that optimal D-loop formation (strand exchange with closed circular DNA) required Ca2+ concentrations greater than 5 mM, whereas 1 mM Ca2+ was sufficient for strand exchange between two oligonucleotides. Structural changes indicated by increased fluorescence intensity of poly(dεA) (a poly(dA) analog) reached a plateau at 1 mM Ca2+. Ca2+ > 2 mM was required for saturation of linear dichroism signal intensity at 260 nm, associated with rigid perpendicular DNA base orientation, suggesting a correlation with the stimulation of D-loop formation. Therefore, Ca2+ exerts two different effects. Thermal stability measurements suggest that HsRad51 binds two Ca2+ ions with KD values of 0.2 and 2.5 mM, implying that one step is stimulated by one Ca2+ bond and the other by two Ca2+ bonds. Our results indicate parallels between the Mg2+ activation of RecA and the Ca2+ activation of HsRad51.

D-Loop Mutations as Prognostic Markers in Glioblastoma-A Pilot Study.

Glioblastoma, a highly aggressive brain tumor, poses significant treatment challenges. A deeper investigation into its molecular complexity is essential for the identification of novel prognostic biomarkers and therapeutic strategies, potentially improving patient outcomes in terms of survival and quality of life. While nuclear DNA mutations have been extensively studied, the role of mitochondrial DNA (mtDNA) mutations, specifically in the D-loop region, remains poorly understood. This prospective case-control study aimed to assess the prognostic significance of the mtDNA D-loop m.16126T>C variant in glioblastoma patients. Immunohistochemistry and droplet digital PCR (ddPCR) were employed for mutation analysis, complemented by statistical analyses and a literature review. The study cohort comprised 22 glioblastoma patients (mean age 59.36 ± 14.17, 12 (54.55%) females), and 25 controls (59.48 ± 13.22, 12 (80%) females). The D-loop m.16126T>C variant was observed in four (18%) of the glioblastoma samples and was associated with shorter median survival (9.5 vs. 18 months; p = 0.016, log-rank test). This study underscores the importance of investigating mtDNA, especially D-loop variants, in glioblastoma, suggesting its potential as a prognostic biomarker and, therefore, its possible therapeutic targets, warranting further exploration.

Uganda chicken genetic resources: II. genetic diversity and population demographic history inferred from mitochondrial DNA D-loop sequences.

The genetic diversity of indigenous chickens, which comprise over 80% of the chicken resources in Uganda, is largely not well-characterized for their genetic contribution. This study assessed the genetic diversity and population structure of the indigenous chicken population in Uganda to serve as an essential component for improvement and conservation strategies. A set of 344 mitochondrial DNA (mtDNA) D-loop sequences among 12 Ugandan chicken populations was evaluated. Twenty-eight polymorphic sites, accounting for 4.26% of the total analyzed loci of 658 bp, defined 32 haplotypes. The haplotype diversity (Hd) was 0.437, with a nucleotide diversity (π) of 0.0169, while the average number of nucleotide differences (k) was 0.576, indicating a population that is moderately genetically diverse. Analysis of molecular variance found 98.39% (ρ < 0.01) of the total sequence variation among the chicken haplotypes within populations, 1.08% (ρ < 0.05) among populations, and 0.75% (ρ > 0.05) among populations within regions. This revealed subtle genetic differentiation among the populations, which appeared to be influenced by population fragmentation, probably due to neutral mutation, random genetic drift, and/or balancing selection. All the haplotypes showed affinity exclusively to the haplogroup-E mtDNA phylogeny, with haplotype UGA01 signaling an ancestral haplotype in Uganda. Neutrality tests Tajima's D (-2.320) and Fu's Fs (-51.369), augmented with mismatch distribution to measure signatures of recent historical demographic events, supported a population expansion across the chicken populations. The results show one matrilineal ancestry of Ugandan chickens from a lineage widespread throughout the world that began in the Indian subcontinent. The lack of phylogeographic signals is consistent with recent expansion events with extensive within-country genetic intermixing among haplotypes. Thus, the findings in this study hold the potential to guide conservation strategies and breeding programs in Uganda, given that higher genetic diversity comes from within the chicken population.

The origin of genetic diversity of indigenous cockfighting chickens of Pakistan by analyzing the mtDNA.

In Pakistan, the origin of the indigenous cockfighting chicken (ICC) or gamecock population is unknown. However, it is speculated that this might have been associated with domestication, an event linked to recreational, entertainment (cockfighting), religious or ornamental activities. This study aims to understand the origin and genetic diversity of the ICC population in Pakistan. A total of 185 ICC population and 10 captive Indian red junglefowl (Gallus gallus murghi) were analyzed for genetic diversity indices and phylogenetic reconstruction using a 397 bp of mtDNA D-loop region. It is reported that a total of 43 haplotypes from 38 polymorphic nucleotide sites. The haplotype and nucleotide diversity are also estimated in the range of 0.643-0.909, and 0.00585-0.01575, respectively. The total genetic diversity within the population was 91.52%. Four mitochondrial haplogroups A, B, C and D were identified by median-joining network analysis, two of them have high percentages, haplogroup D (81.6%) and A (15.1%). Phylogenetic analysis showed that the ICC population of Pakistan and Gallus gallus murghi shared haplogroup D. The results of this study showed that sub-haplogroup D17a05, has significantly high haplotype diversity and percentage as compared to previously published studies, this indicated that Pakistan might be one of the centres of domestication for chicken, as it is considered that Southeast Asia is the centre of domestication. Frequencies of Haplogroup A also indicate South-North indices. This research work showed that the indigenous cockfighting chicken population of Pakistan is genetically introgressed from Gallus gallus murghi, and significant variations could be attributed to the underlying differences in the geographics, selection pressures, introgression, and regional practices; and multiple origins of cockfighting chickens' populations around the world which reflected the past trading routes between human communities and civilizations.

Mitochondrial Variation of Bottlenose Dolphins (Tursiops truncatus) from the Canary Islands Suggests a Key Population for Conservation with High Connectivity within the North-East Atlantic Ocean.

In recent decades, worldwide cetacean species have been protected, but they are still threatened. The bottlenose dolphin (Tursiops truncatus) is a vulnerable keystone species and a useful bioindicator of the health and balance of marine ecosystems in oceans all over the world. The genetic structure of the species is shaped by their niche specialization (along with other factors), leading to the classification of two ecotypes: coastal and pelagic. In this study, the genetic diversity, population structure, and ecotypes of bottlenose dolphins from the Canary Islands were assessed through the analysis of 49 new samples from biopsies and from stranded animals using the 636 bp portion of the mitochondrial control region and 343 individuals from databases (n = 392). The results reveal high genetic diversity in Canarian bottlenose dolphins (Hd = 0.969 and π = 0.0165) and the apparent lack of population genetic structure within this archipelago. High genetic structure (Fst, Φst) was found between the Canary Islands and coastal populations, while little to no structure was found with the pelagic populations. These results suggest that Canarian bottlenose dolphins are part of pelagic ecotype populations in the North Atlantic. The studied Special Areas of Conservation in the Canary Islands may correspond to a hotspot of genetic diversity of the species and could be a strategic area for the conservation of the oceanic ecotype of bottlenose dolphins.

Absence of both MGME1 and POLG EXO abolishes mtDNA whereas absence of either creates unique mtDNA duplications.

Both POLG and MGME1 are needed for mitochondrial DNA (mtDNA) maintenance in animal cells. POLG, the primary replicative polymerase of the mitochondria, has an exonuclease activity (3'→5') that corrects for the misincorporation of bases. MGME1 serves as an exonuclease (5'→3'), producing ligatable DNA ends. Although both have a critical role in mtDNA replication and elimination of linear fragments, these mechanisms are still not fully understood. Using digital PCR to evaluate and compare mtDNA integrity, we show that Mgme1 knock out (Mgme1 KK) tissue mtDNA is more fragmented than POLG exonuclease-deficient "Mutator" (Polg MM) or WT tissue. In addition, next generation sequencing of mutant hearts showed abundant duplications in/nearby the D-loop region and unique 100 bp duplications evenly spaced throughout the genome only in Mgme1 KK hearts. However, despite these unique mtDNA features at steady-state, we observed a similar delay in the degradation of mtDNA after an induced double strand DNA break in both Mgme1 KK and Polg MM models. Lastly, we characterized double mutant (Polg MM/Mgme1 KK) cells and show that mtDNA cannot be maintained without at least one of these enzymatic activities. We propose a model for the generation of these genomic abnormalities which suggests a role for MGME1 outside of nascent mtDNA end ligation. Our results highlight the role of MGME1 in and outside of the D-loop region during replication, support the involvement of MGME1 in dsDNA degradation, and demonstrate that POLG EXO and MGME1 can partially compensate for each other in maintaining mtDNA.

Population genetics reveals new introgression in the nucleus herd of min pigs.

OBJECTIVE: Min pigs are a unique genetic resource among local pig breeds in China. They have more excellent characteristics in cold and stress resistance, good meat quality, and a high reproductive rate. However, the genetic structure and driving factors remain unclear in the nucleus herd. In this study, the genetic diversity of Min pigs was studied to reveal the formation mechanism of its unique genetic structure. We hope to protect and develop the genetic resources of Min pigs. METHODS: We analyzed different types of genes to identify the genetic structure and gene introgression pattern of Min pigs. The nuclear DNA dataset includes information on 21 microsatellite loci and 6 Y-chromosome genes, and the mitochondrial D-loop gene is selected to represent maternal lineages. The above genes are all from the nucleus herd of Min pigs. RESULTS: The results of genetic structure identification and analysis of potential exogenous gene introgression patterns indicate that the nucleus herd of Min pigs maintains a high level of genetic diversity (polymorphism information content = 0.713, expected heterozygosity = 0.662, observed heterozygosity = 0.612). Compared with other Asian pig breeds, the formation of Min pig breeds is more special. Gene introgression from European pig breeds to Min pigs has occurred, which is characterized by complete introgression of paternal genes and incomplete introgression of maternal genes. CONCLUSION: Gene introgression caused by cross-breeding is not the main factor leading to the formation of the current genetic structure of Min pigs, but this process has increased the level of genetic diversity in the nucleus herd. Compared with the influence of gene introgression, our research suggest that artificial selection and environmental adaptive evolution make Min pigs form unique genetic characteristics.

Mitochondrial D-loop methylation levels inversely correlate with disease duration in amyotrophic lateral sclerosis.

Aim: To correlate mitochondrial D-loop region methylation levels and mtDNA copy number with disease duration in familial amyotrophic lateral sclerosis (ALS) patients. Patients & methods: The study population included 12 ALS patients with a mutation in SOD1 and 13 ALS patients with the C9orf72 hexanucleotide repeat expansion. Methylation levels of the D-loop region and mtDNA copy number were quantified using pyrosequencing and quantitative PCR, respectively. Results: We observed that D-loop methylation levels inversely correlated while mtDNA copy number positively correlated with disease duration. Conclusion: Considering the central role played by mitochondria in ALS, this preliminary study provides new knowledge for future studies aimed at identifying biomarkers of disease progression and new targets for therapeutic interventions.

Amyotrophic lateral sclerosis is a devastating neurodegenerative disease which leads to the patient's death a few years after the onset of the first symptoms. There are currently no treatments to cure the disease, and the only drugs available are able to prolong patients' lives by only a few months. Patients may have much variability in the presentation of symptoms, including different duration of disease. This study aims to research whether mitochondrial DNA methylation, a mechanism involved in the biology of the mitochondrion, is associated with the duration of the disease. We observed that methylation of mitochondrial DNA inversely correlates with the disease duration, providing new knowledge for future studies aimed at identifying biomarkers of disease progression.

Mitochondrial DNA diversity and maternal origins of Pakistani donkey / Diversidade de DNA mitocondrial e origens maternas do burro do Paquistão

Domestic donkey plays a key role as a draft animal in rural economy of Pakistan where its population is increasing every year. The complete mtDNA control region of forty randomly sampled donkeys was PCR- amplified and sequenced bi-directionally using specific primers. Distinct mtDNA haplotypes obtained in the current study (KY446001−KY446011) were subjected to haplotype (h) and nucleotide diversity (π) measures using DnaS as well as to phylogenetic, Network, and AMOVA analyses. There were a total 27 polymorphic sites present within 11 unique mtDNA haplotypes from the studied 40 animals from different regions. Neighbor-joining network and median-joining network both illustrated the splitting of all these haplotypes into two well-defined Nubian and Somali lineages, confirming African maternal origin of Pakistani domestic donkey. Diversity parameters h (0.967± 0.037) and π (0.02917± 0.00307) were found to reveal high levels of genetic diversity in Pakistani donkeys. AMOVA demonstrated only 1% of genetic differences between two mtDNA maternal lineages, pointing to lack of population substructure in Pakistani donkeys as is the case with worldwide domestic donkey population. Pakistani donkeys have African maternal origin and high levels of mtDNA diversity. High genetic diversity may be due to non-selective breeding and heteroplasmy. We herein provide the first report on mtDNA diversity of control region in Pakistani domestic donkey.

O burro doméstico possui um papel fundamental como animal de tração na economia rural do Paquistão, onde a população desse animal está aumentando a cada ano. A região de controle de mtDNA completa de 40 burros amostrados aleatoriamente foi ampliada por PCR e sequenciada bidirecionalmente por intermédio de primers específicos. Haplótipos distintos de mtDNA obtidos no estudo atual (KY446001 − KY446011) foram submetidos a medidas de haplótipo (h) e diversidade de nucleotídeos (π) por meio de DnaS, bem como análises filogenéticas, de rede e AMOVA. Havia um total de 27 sítios polimórficos presentes em 11 haplótipos de mtDNA exclusivos dos 40 animais estudados de diferentes regiões. A rede de união de vizinhos e a rede de união mediana ilustram a divisão de todos esses haplótipos em duas linhagens núbias e somalis bem definidas, confirmando a origem materna africana do burro doméstico do Paquistão. Os parâmetros de diversidade h (0,967 ± 0,037) e π (0,02917 ± 0,00307) revelaram altos níveis de diversidade genética em burros paquistaneses. AMOVA demonstrou apenas 1% de diferenças genéticas entre as duas linhagens maternas de mtDNA, apontando a falta de subestrutura populacional em burros paquistaneses, como é o caso da população mundial de burros domésticos. Os burros paquistaneses têm origem materna africana e altos níveis de diversidade de mtDNA. A alta diversidade genética pode ser por causa da reprodução não seletiva e de heteroplasmia. Aqui, fornecemos o primeiro relatório sobre a diversidade do mtDNA da região de controle em burros domésticos do Paquistão

Mitochondrial DNA diversity and maternal origins of Pakistani donkey

Abstract Domestic donkey plays a key role as a draft animal in rural economy of Pakistan where its population is increasing every year. The complete mtDNA control region of forty randomly sampled donkeys was PCR- amplified and sequenced bi-directionally using specific primers. Distinct mtDNA haplotypes obtained in the current study (KY446001KY446011) were subjected to haplotype (h) and nucleotide diversity () measures using DnaS as well as to phylogenetic, Network, and AMOVA analyses. There were a total 27 polymorphic sites present within 11 unique mtDNA haplotypes from the studied 40 animals from different regions. Neighbor-joining network and median-joining network both illustrated the splitting of all these haplotypes into two well-defined Nubian and Somali lineages, confirming African maternal origin of Pakistani domestic donkey. Diversity parameters h (0.967± 0.037) and (0.02917± 0.00307) were found to reveal high levels of genetic diversity in Pakistani donkeys. AMOVA demonstrated only 1% of genetic differences between two mtDNA maternal lineages, pointing to lack of population substructure in Pakistani donkeys as is the case with worldwide domestic donkey population. Pakistani donkeys have African maternal origin and high levels of mtDNA diversity. High genetic diversity may be due to non-selective breeding and heteroplasmy. We herein provide the first report on mtDNA diversity of control region in Pakistani domestic donkey.

Resumo O burro doméstico possui um papel fundamental como animal de tração na economia rural do Paquistão, onde a população desse animal está aumentando a cada ano. A região de controle de mtDNA completa de 40 burros amostrados aleatoriamente foi ampliada por PCR e sequenciada bidirecionalmente por intermédio de primers específicos. Haplótipos distintos de mtDNA obtidos no estudo atual (KY446001 KY446011) foram submetidos a medidas de haplótipo (h) e diversidade de nucleotídeos () por meio de DnaS, bem como análises filogenéticas, de rede e AMOVA. Havia um total de 27 sítios polimórficos presentes em 11 haplótipos de mtDNA exclusivos dos 40 animais estudados de diferentes regiões. A rede de união de vizinhos e a rede de união mediana ilustram a divisão de todos esses haplótipos em duas linhagens núbias e somalis bem definidas, confirmando a origem materna africana do burro doméstico do Paquistão. Os parâmetros de diversidade h (0,967 ± 0,037) e (0,02917 ± 0,00307) revelaram altos níveis de diversidade genética em burros paquistaneses. AMOVA demonstrou apenas 1% de diferenças genéticas entre as duas linhagens maternas de mtDNA, apontando a falta de subestrutura populacional em burros paquistaneses, como é o caso da população mundial de burros domésticos. Os burros paquistaneses têm origem materna africana e altos níveis de diversidade de mtDNA. A alta diversidade genética pode ser por causa da reprodução não seletiva e de heteroplasmia. Aqui, fornecemos o primeiro relatório sobre a diversidade do mtDNA da região de controle em burros domésticos do Paquistão.

Mitochondrial Diversity and Phylogenetic Relationship of Eight Native Bulgarian Sheep Breeds.

The geographical, geomorphological, and climatic characteristics of Bulgaria are particularly favorable for animal breeding and, above all, for pastoral farming and sheep breeding. These conditions created prerequisites for the creation of about 30 unique local breeds of sheep. In this study we investigated the genetic diversity of eight of the most popular Bulgarian native breeds, based on the sequence analysis of a part of the mitochondrial D-loop region. An almost entire mitochondrial DNA (mtDNA) D-loop region (1180 bp) was amplified and sequenced. The obtained results showed the presence of a large number of haplotypes-225, belonging to two main haplogroups. The majority of samples showed a high prevalence of the European haplogroup B (95.2%) while the remaining individuals were assigned to haplogroup A (4.8%). None of the other reported mitochondrial haplogroups were observed. The number of polymorphic sites, nucleotide and haplotype diversity was high (240, 0.01237, and 0.9968, respectively), which is evidence for multiple maternal origins in all populations. The Tajima D-test value in all the study populations was -1.905 (p < 0.05), indicating that the abundance of rare alleles was most likely due to population expansion after a recent bottleneck. The Median joining network showed that almost all haplotypes belonging to haplogroup B formed a star-like network, which revealed a weak genetic differentiation and a large gene flow between the Bulgarian native breeds.

Tracing the genetic footprints: India's role as a gateway for pig migration and domestication across continents.

This study explored the maternal genetic diversity in the pig genetic resources of India by analyzing a mitochondrial D-loop fragment and comparing it with the corresponding sequences of previously published studies involving domestic pigs and wild boars. Sequencing of 103 samples representing different domestic pig populations revealed existence of 32 maternal haplotypes. The indices of haplotype and nucleotide diversity in Indian domestic pigs were 0.9421 and 0.015, respectively. Median-Joining network revealed that Indian pigs belong to Clade A and show conformity to 6 haplogroups reported worldwide (D1a, D1a1, D1a2, D1e, D1h and D3a). Among these, D1e and D1a2 were shared with Asian wild boars too. Interestingly, haplotype sharing was evident between Indian pigs and samples from other countries representing Africa, Asia, Europe and Oceania. This study substantiates India's contribution as a possible pig domestication center and highlights the importance of the Indian subcontinent in dispersal of the species to other continents. Additionally, genetic evidence suggested the influence of trading routes and historical interactions in shaping pig genetic exchange. Overall, this investigation provides valuable insights into the genetic diversity, historical migration, and domestication of Indian domestic pigs, contributing to the broader understanding of global pig genetic resources and their evolutionary history.

Estimating the true stability of the prehydrolytic outward-facing state in an ABC protein.

CFTR, the anion channel mutated in cystic fibrosis patients, is a model ABC protein whose ATP-driven conformational cycle is observable at single-molecule level in patch-clamp recordings. Bursts of CFTR pore openings are coupled to tight dimerization of its two nucleotide-binding domains (NBDs) and in wild-type (WT) channels are mostly terminated by ATP hydrolysis. The slow rate of non-hydrolytic closure - which determines how tightly bursts and ATP hydrolysis are coupled - is unknown, as burst durations of catalytic site mutants span a range of ~200-fold. Here, we show that Walker A mutation K1250A, Walker B mutation D1370N, and catalytic glutamate mutations E1371S and E1371Q all completely disrupt ATP hydrolysis. True non-hydrolytic closing rate of WT CFTR approximates that of K1250A and E1371S. That rate is slowed ~15-fold in E1371Q by a non-native inter-NBD H-bond, and accelerated ~15-fold in D1370N. These findings uncover unique features of the NBD interface in human CFTR.

Comparative analysis of the population diversity of black rockfish (Sebastes schlegelii) in northern China.

The nearshore marine fish known as black rockfish (Sebastes schlegelii) is found in the Yellow Sea, Bohai Sea, and East China Sea. The population structure and genetic diversity of S. schlegelii are vulnerable to the effects of artificial stocking, environmental pollution, overfishing, and climate change, so relevant studies are urgently needed. This study used comparative mtDNA loop (D-loop) analysis to examine the genetic diversity and natural population structure of 98 individuals from the northern Chinese cities of Qingdao, Jinzhou, and Dalian. A total of 22 haplotypes were identified in the three groups of samples, with the most common haplotypes being Hap-2, Hap-3, Hap-4, Hap-5, and Hap-6. The results of genetic diversity based on the D-LOOP sequence showed that the genetic diversity of S. schlegelii in the study area showed high Hd and low π type, indicating that the genetic diversity of S. schlegelii was low. Analyses of molecular variance (AMOVA) showed that the percentage of among population variation was - 0.29%, and the percentage of within population variation was 100.29%, indicating that the genetic variation was mainly from within the population. Between the three locations, the genetic differentiation index (Fst) was - 0.0113 ~ 0.0061, and there was no genetic differentiation among the populations. The results of gene flow (Nm) coefficients showed that the average Nm among the three populations was infinite (Nm = inf > > 4) and the three populations formed a stochastic unit. The results of the neutrality test (Tajima's D, Fu's Fs) and the frequency of nucleotide mismatch distribution demonstrated that the three geographic populations of S. schlegelii did not undergo a large population expansion in recent history. Based on the above conclusions, the S. schlegelii as a whole should be protected in situ.

Quality of DNA extracted from freshwater fish scales and mucus and its application in genetic diversity studies of Perca fluviatilis and Rutilus rutilus.

Studies on genetic diversity require biological material containing a reliable source of DNA that can be extracted and analyzed. Recently, non-invasive sampling has become a preferred sampling method of biological material. The suitability of a less invasive approach that involves obtaining samples by swabbing the fish skin (including live, non-anesthetized fish) should be considered. In this study, we compared the efficiency of DNA extraction, amplification, and sequencing of mtDNA fragments of two fish species Perca fluviatilis and Rutilus rutilus based on DNA collected from the scales and mucus using the modified Aljanabi and Martinez method. The results revealed a higher quality of DNA extracted from the mucus; however, the mean DNA concentration obtained from the scales of both fish species was higher. We verified the method suitable for amplification and sequencing of mtDNA fragments of both fish species using newly designed markers (D-loop, ATP6) and examined the potential risk of intraspecific cross-contamination. The DNA sequence alignment analysis revealed identical sequences attributed to the same individual when DNA, extracted from two different sources (scales and mucus), was used. We demonstrated that the quantity and quality of DNA extracted from the scales and mucus using the proposed method were high enough to carry out genetic diversity studies based on sampling of live fish with the possibility to release it after collecting samples.

Population structure of Vietnamese pigs using mitochondrial DNA.

The D-loop region on mitochondrial DNA (mtDNA) is frequently used for analyses of maternal lineages within domestic animal species. There are many native pig breeds in Vietnam, but their origins remain unclear. This study investigated maternal lineages using the D-loop region on mtDNA of 260 samples collected from native pigs in 20 provinces across Vietnam. The D-loop region of all samples was amplified and sequenced. We obtained 713 bp sequences of the D-loop region for each sample excluding the repeat region, and variants on this region were used to construct a phylogenetic tree. We detected 50 haplotypes from Vietnamese native pigs, with 27 novel haplotypes. Phylogenetic tree analysis showed two haplotype groups: one for the MTSEA group, frequently found in domestic pigs in the mountainous areas of Cambodia and Laos; and the D2 group, found in pigs originating from Chinese pigs. No European haplotype was found. Haplotypes in northeast Vietnam comprised only haplotypes of the D2 group, whereas in areas from the northwest mountains to the south, we found haplotypes belonging to both the D2 and MTSEA groups. This study suggested that both origins contributed to maternal lineages of current populations of Vietnamese native pigs.

Insights on the Evolutionary History and Genetic Patterns of Octopus vulgaris Cuvier, 1797 in the Northeastern Atlantic Using Mitochondrial DNA.

Octopus vulgaris is one of the most harvested octopus species in the world. In the Iberian Peninsula, there are several small-scale fisheries that have a long-term tradition of harvesting octopus. The Asturias fleet (in Northern Spain) has an internationally recognized MSC label for its exploitation. Of concern, genetic assessments of exploited stocks are currently scarce, which could prevent the implementation of adequate managing strategies. We use two mitochondrial regions (cytochrome oxidase subunit 1 and control region) to analyze the genetic status and evolutionary events that conditioned octopus populations' characteristics in the Northeastern Atlantic. A total of 90 individuals were sampled from three different localities in the Iberian Peninsula as well as a location in Macaronesia. Temporal genetic analyses on Asturias and Algarve populations were also performed. Results indicated the absence of fine spatial genetic structuring but showed the Canary Islands (in Macaronesia) as the most distinct population. Our analyses detected two distinct clades, already described in the literature, but, for the first time, we confirmed the presence of the α-southern haplogroup in the Northern Iberian Peninsula. This result indicates a more continuous cline for the distribution of these two haplogroups than previously reported. Temporal changes in the distribution of both haplogroups in contact zones were also detected.

Exploring Genetic Markers: Mitochondrial DNA and Genomic Screening for Biodiversity and Production Traits in Donkeys.

Donkeys (Equus asinus) play a pivotal role as essential livestock in arid and semi-arid regions, serving various purposes such as transportation, agriculture, and milk production. Despite their significance, donkey breeding has often been overlooked in comparison to other livestock species, resulting in limited genetic improvement programs. Preserving donkey genetic resources within each country necessitates the establishment of breed conservation programs, focusing on managing genetic diversity among populations. In recent years, significant strides have been made in sequencing and analyzing complete mitochondrial DNA (mtDNA) molecules in donkeys. Notably, numerous studies have honed in on the mitochondrial D-loop region, renowned for its remarkable variability and higher substitution rate within the mtDNA genome, rendering it an effective genetic marker for assessing genetic diversity in donkeys. Furthermore, genetic markers at the RNA/DNA level have emerged as indispensable tools for enhancing production and reproduction traits in donkeys. Traditional animal breeding approaches based solely on phenotypic traits, such as milk yields, weight, and height, are influenced by both genetic and environmental factors. To overcome these challenges, genetic markers, such as polymorphisms, InDel, or entire gene sequences associated with desirable traits in animals, have achieved widespread usage in animal breeding practices. These markers have proven increasingly valuable for facilitating the selection of productive and reproductive traits in donkeys. This comprehensive review examines the cutting-edge research on mitochondrial DNA as a tool for assessing donkey biodiversity. Additionally, it highlights the role of genetic markers at the DNA/RNA level, enabling the informed selection of optimal production and reproductive traits in donkeys, thereby driving advancements in donkey genetic conservation and breeding programs.

Investigating the role of mitochondrial DNA D-loop variants, haplotypes, and copy number in polycystic ovary syndrome: implications for clinical phenotypes in the Chinese population.

Background: The presence of genetic variations in mitochondrial DNA (mtDNA) has been associated with a diverse array of diseases. The objective of this study was to examine the correlations between mtDNA D-loop, its haplotypes, and polycystic ovary syndrome (PCOS) in the Chinese population, and the associations between mtDNA D-loop and symptoms of PCOS. The study also sought to determine whether the mtDNA copy number in Chinese patients with PCOS differed from that of individuals in the control group. Methods: Infertile individuals who only had tubal or male factor treatment were the focus of research by The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). mtDNA haplotypes were categorized using polymorphic D-loop sites. mtDNA D-loop, PCOS features, and mtDNA haplotypes were analyzed using R software to determine the strength of the association between the three. There are certain DNA haplotypes linked to PCOS. Microdroplet digital polymerase chain reaction (PCR) was used to determine the mtDNA copy number in a convenience sample of 168 PCOS patients and 83 controls. Results: Among the research group, the majority of D-loop mutations were infrequent (frequency< 1%), with only 45 variants displaying a minimum allele frequency (MAF) of 5% or higher. No association was found between polymorphism loci in PCOS patients and body mass index (BMI). Noteworthy, C194T, 1A200G, 523delAC, and C16234T showed positive correlations with elevated LH/FSH levels. Additionally, specific polymorphic loci G207A, 16036GGins, and 16049Gins within the D-loop region of mtDNA potentially exerted a protective role in PCOS development. Conversely, no statistical significance was observed in the expression levels of C16291T and T489C. Chinese women with mtDNA haplotype A15 exhibited a decreased risk of developing PCOS. Moreover, a significant difference in mtDNA copy number was detected, with controls averaging 25.87 (21.84, 34.81), while PCOS patients had a mean of 129.91 (99.38, 168.63). Conclusion: Certain mtDNA D-loop mutations and haplotypes appear to confer protection against PCOS in Chinese women. In addition, elevated mtDNA copy number may serve as an indicator during early stages of PCOS.