Multi-invasions Are Recombination Byproducts that Induce Chromosomal Rearrangements.

Inaccurate repair of broken chromosomes generates structural variants that can fuel evolution and inflict pathology. We describe a novel rearrangement mechanism in which translocation between intact chromosomes is induced by a lesion on a third chromosome. This multi-invasion-induced rearrangement (MIR) stems from a homologous recombination byproduct, where a broken DNA end simultaneously invades two intact donors. No homology is required between the donors, and the intervening sequence from the invading molecule is inserted at the translocation site. MIR is stimulated by increasing homology length and spatial proximity of the donors and depends on the overlapping activities of the structure-selective endonucleases Mus81-Mms4, Slx1-Slx4, and Yen1. Conversely, the 3'-flap nuclease Rad1-Rad10 and enzymes known to disrupt recombination intermediates (Sgs1-Top3-Rmi1, Srs2, and Mph1) inhibit MIR. Resolution of MIR intermediates propagates secondary chromosome breaks that frequently cause additional rearrangements. MIR features have implications for the formation of simple and complex rearrangements underlying human pathologies.

TERRA and RAD51AP1 promote alternative lengthening of telomeres through an R- to D-loop switch.

Alternative lengthening of telomeres (ALT), a telomerase-independent process maintaining telomeres, is mediated by break-induced replication (BIR). RAD52 promotes ALT by facilitating D-loop formation, but ALT also occurs through a RAD52-independent BIR pathway. Here, we show that the telomere non-coding RNA TERRA forms dynamic telomeric R-loops and contributes to ALT activity in RAD52 knockout cells. TERRA forms R-loops in vitro and at telomeres in a RAD51AP1-dependent manner. The formation of R-loops by TERRA increases G-quadruplexes (G4s) at telomeres. G4 stabilization enhances ALT even when TERRA is depleted, suggesting that G4s act downstream of R-loops to promote BIR. In vitro, the telomeric R-loops assembled by TERRA and RAD51AP1 generate G4s, which persist after R-loop resolution and allow formation of telomeric D-loops without RAD52. Thus, the dynamic telomeric R-loops formed by TERRA and RAD51AP1 enable the RAD52-independent ALT pathway, and G4s orchestrate an R- to D-loop switch at telomeres to stimulate BIR.

The Configuration of RPA, RAD51, and DMC1 Binding in Meiosis Reveals the Nature of Critical Recombination Intermediates.

Meiotic recombination proceeds via binding of RPA, RAD51, and DMC1 to single-stranded DNA (ssDNA) substrates created after formation of programmed DNA double-strand breaks. Here we report high-resolution in vivo maps of RPA and RAD51 in meiosis, mapping their binding locations and lifespans to individual homologous chromosomes using a genetically engineered hybrid mouse. Together with high-resolution microscopy and DMC1 binding maps, we show that DMC1 and RAD51 have distinct spatial localization on ssDNA: DMC1 binds near the break site, and RAD51 binds away from it. We characterize inter-homolog recombination intermediates bound by RPA in vivo, with properties expected for the critical displacement loop (D-loop) intermediates. These data support the hypothesis that DMC1, not RAD51, performs strand exchange in mammalian meiosis. RPA-bound D-loops can be resolved as crossovers or non-crossovers, but crossover-destined D-loops may have longer lifespans. D-loops resemble crossover gene conversions in size, but their extent is similar in both repair pathways.

Dynamic Processing of Displacement Loops during Recombinational DNA Repair.

Displacement loops (D-loops) are pivotal intermediates of homologous recombination (HR), a universal DNA double strand break (DSB) repair pathway. We developed a versatile assay for the physical detection of D-loops in vivo, which enabled studying the kinetics of their formation and defining the activities controlling their metabolism. Nascent D-loops are detected within 2 h of DSB formation and extended in a delayed fashion in a genetic system designed to preclude downstream repair steps. The majority of nascent D-loops are disrupted by two pathways: one supported by the Srs2 helicase and the other by the Mph1 helicase and the Sgs1-Top3-Rmi1 helicase-topoisomerase complex. Both pathways operate without significant overlap and are delineated by the Rad54 paralog Rdh54 in an ATPase-independent fashion. This study uncovers a layer of quality control of HR relying on nascent D-loop dynamics.

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.

Identification of sequence polymorphism in the D-loop region of mitochondrial DNA as a risk factor for breast cancer.

Mitochondrial DNA (mtDNA) variations affect the efficiency of the electron transport chain and production of reactive oxygen species, contributing to carcinogenesis. The D-loop region of mtDNA has emerged as a variation hotspot region in human neoplasia; however, the potential contribution of these variations in breast cancer risk prediction remains unknown. We investigated the relationship between germline single nucleotide polymorphisms (SNPs) in the entire D-loop region and breast cancer risk in Chinese women. Peripheral blood-isolated mtDNA from 2329 patients with breast cancer and 2328 cancer-free controls was examined for SNPs. In the combined cohort, we used traditional risk factors, susceptibility germline polymorphisms, and logistic regression analysis to evaluate the predictive value of susceptibility variants for breast cancer risk. We calculated the area under the receiver operating characteristic curve (AUC) as a measure. We also measured the content of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Individual polymorphisms SNP573 were significantly associated with breast cancer risk in both the discovery and validation cohorts. In the combined cohort, the AUC of the traditional risk factors was 64.3%; after adding susceptibility variants, the AUC was 64.9% (DeLong test, p = 0.007). 8-OHdG levels were significantly higher in patients with breast cancer than in controls and higher in individuals with SNP573 than in those negative for this variation. Overall, oxidative stress might be associated with the risk of breast cancer, and SNP573 might be associated with oxidative stress. Our results indicate the risk potential of polymorphisms in the D-loop region in breast cancer in Southern China.

Mitochondrial DNA D-loop SNPs unveil molecular signatures of milk production variation in Murrah buffalo.

BACKGROUND: The Murrah buffalo, pivotal in Asian agriculture, faces challenges in maximizing milk production despite significant breeding efforts. Recognizing its economic importance, this study investigates mtDNA D-loop variations in Murrah buffalo as potential indicators of milk production variability, addressing challenges in maximizing yield despite significant breeding efforts. METHODS AND RESULTS: Analyzing mtDNA D-loop sequences from 50 buffaloes, we categorized them into Low (Group 1), Medium (Group 2), and High ECM (Group 3) groups based on milk yields, fat and protein percentage of a 30-day period data. Somatic cell mtDNA D-loop analysis revealed distinct genetic variations, with significant differences among ECM groups. Group 2 showed higher SNP prevalence, group 3 had more insertions/deletions, and Group 1 exhibited the highest transition frequency. Notably, a consistent "C" deletion at the 714th position occurred in Groups 1 and 3, prevalent in 68% of Group 2. A G-A variation at the 93rd position was specific to the medium ECM group. Negative Tajima D values indicated unique variations in each group, with Group 1 having the highest number, and a specific SNP linked to Group 2 was identified. These SNPs in the D-loop region could impact mtDNA replication, influencing mitochondrial content among animals. Our results provide valuable insights into the role of mtDNA D-loop polymorphisms in milk production traits in Murrah buffalo. CONCLUSIONS: Our research highlights the potential for valuable markers of cellular energy efficiency in Murrah buffalo. Exploring diverse cytoplasmic backgrounds opens avenues for mtDNA-based selection strategies, enhancing milk production and optimizing genetic traits for the dairy industry.

Mitochondrial DNA content and methylation in sperm of patients with asthenozoospermia.

PURPOSE: The aim of the current study was to investigate the mtDNA methylation levels and mtDNA copy numbers in the sperm of patients with asthenozoospermia and compare them to those observed in controls with normozoospermia. METHODS: Pyrosequencing analysis of the methylation levels of the mitochondrial D-loop and MT-CO1/chr1:631,907-632083/chrX:26,471,887-126,472,063 (hereinafter referred to as "MT-CO1-AVG") region and quantitative PCR analysis of the mtDNA copy number were performed on sperm from 30 patients with asthenozoospermia and 30 controls with normozoospermia. RESULTS: Compared with those of controls with normozoospermia, the methylation levels of D-loop and MT-CO1-AVG regions and mtDNA copy number were significantly higher in patients with asthenozoospermia. The methylation level of the D-loop region in patients with asthenozoospermia and controls with normozoospermia and that of MT-CO1-AVG region in patients with asthenozoospermia showed a decreasing tendency with increasing total sperm motility. A significant inverse correlation between the mtDNA copy number and total sperm motility was observed in patients with asthenozoospermia but not in controls with normozoospermia. In patients with asthenozoospermia, but not in controls with normozoospermia, we observed a significant inverse correlation between D-loop methylation levels and mtDNA copy number, while no significant correlation was observed between MT-CO1-AVG methylation levels and mtDNA copy number. CONCLUSION: These results reveal the occurrence of mtDNA methylation in human sperm and altered D-loop and MT-CO1-AVG methylation levels in patients with asthenozoospermia. Additional research is needed to determine the function of these features in the etiology and course of asthenozoospermia.

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.

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.

Intraspecific Variation of Brown Rat Rattus norvegicus in Russia by D-Loop mtDNA Data.

For the first time in Russia, the intraspecific structure of the brown rat Rattus norvegicus was studied using the mitochondrial control region (D-loop) as a molecular marker. The mtDNA sequence was determined in brown rats from eight regions of European and Asian Russia. Three rat clades were identified, including one in European Russia and two in Asian Russia. The synanthropic subspecies R. n. norvegicus was found to have two, European and Asian, lineages. The European lineage included haplotypes from central and southern regions of Russia, and the Asian lineage included haplotypes from Eastern Siberia and the Russian Far East. The exoanthropic subspecies R. n. caraco from the Russian Far East formed a separate genetic lineage.

Metastasis and immunosuppression promoted by mtDNA and PD-L1 in extracellular vesicles are reversed by WGP ß-glucan in oral squamous cell carcinoma.

The suppressive regulatory T cells (Treg) are frequently upregulated in cancer patients. This study aims to demonstrate the hypothesis that arecoline could induce the secretion of mitochondrial (mt) DNA D-loop and programmed cell death-ligand 1 (PD-L1) in extracellular vesicles (EVs), and attenuate T-cell immunity by upregulated Treg cell numbers. However, the immunosuppression could be reversed by whole glucan particle (WGP) ß-glucan in oral squamous cell (OSCC) patients. Arecoline-induced reactive oxygen specimen (ROS) production and cytosolic mtDNA D-loop were analyzed in OSCC cell lines. mtDNA D-loop, PD-L1, IFN-γ, and Treg cells were also identified for the surgical specimens and sera of 60 OSCC patients. We demonstrated that higher mtDNA D-loop, PD-L1, and Treg cell numbers were significantly correlated with larger tumor size, nodal metastasis, advanced clinical stage, and areca quid chewing. Furthermore, multivariate analysis confirmed that higher mtDNA D-loop levels and Treg cell numbers were unfavorable independent factors for survival. Arecoline significantly induced cytosolic mtDNA D-loop leakage and PD-L1 expression, which were packaged by EVs to promote immunosuppressive Treg cell numbers. However, WGP ß-glucan could elevate CD4+ and CD8+ T-cell numbers, mitigate Treg cell numbers, and promote oral cancer cell apoptosis. To sum up, arecoline induces EV production carrying mtDNA D-loop and PD-L1, and in turn elicits immune suppression. However, WGP ß-glucan potentially enhances dual effects on T-cell immunity and cell apoptosis and we highly recommend its integration with targeted and immune therapies against OSCC.

Pilot study of a comprehensive resource estimation method from environmental DNA using universal D-loop amplification primers.

Many studies have investigated the ability of environmental DNA (eDNA) to identify the species. However, when individual species are to be identified, accurate estimation of their abundance using traditional eDNA analyses is still difficult. We previously developed a novel analytical method called HaCeD-Seq (haplotype count from eDNA by sequencing), which focuses on the mitochondrial D-loop sequence for eels and tuna. In this study, universal D-loop primers were designed to enable the comprehensive detection of multiple fish species by a single sequence. To sequence the full-length D-loop with high accuracy, we performed nanopore sequencing with unique molecular identifiers (UMI). In addition, to determine the D-loop reference sequence, whole genome sequencing was performed with thin coverage, and complete mitochondrial genomes were determined. We developed a UMI-based Nanopore D-loop sequencing analysis pipeline and released it as open-source software. We detected 5 out of 15 species (33%) and 10 haplotypes out of 35 individuals (29%) among the detected species. This study demonstrates the possibility of comprehensively obtaining information related to population size from eDNA. In the future, this method can be used to improve the accuracy of fish resource estimation, which is currently highly dependent on fishing catches.

Genetic diversity and population structure of wild Macrobrachium nipponense populations across China: Implication for population management.

BACKGROUND: Macrobrachium nipponense, is an important economic indigenous prawn and is widely distributed in China. However, most these genetic structure analysis researches were focused on a certain water area, systematic comparative studies on genetic structure of M. nipponense across China are not yet available. METHODS AND RESULTS: In this study, D-loop region sequences was used to investigate the genetic diversity and population structure of 22 wild populations of M. nipponense through China, containing the major rivers and lakes of China. Totally 473 valid D-loop sequences with a length of 1110 bp were obtained, and 348 variation sites and 221 haplotypes were detected. The haplotype diversity (h) was ranged from 0.1630 (Bayannur) ~ 1.0000 (Amur River) and the nucleotide diversity π value ranged from 0.001164 (Min River) ~ 0.037168 (Nen River). The pairwise genetic differentiation index (FST) ranged from 0.00344 to 0.91243 and most pair-wised FST was significant (P < 0.05). The lowest FST was displayed in Min River and Jialing River populations and the highest was between Nandu River and Nen River populations. The phylogenetic tree of genetic distance showed that all populations were divided into two branches. The Dianchi Lake, Nandu River, Jialing River and Min River populations were clustered into one branch. The neutral test and mismatch distribution results showed that M. nipponense populations were not experienced expanding and kept a steady increase. CONCLUSIONS: Taken together, a joint resources protection and management strategy for M. nipponense have been suggested based on the results of this study for its sustainable use.

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.

A new type of tandem repeats in Myotis petax (Chiroptera, Vespertilionidae) mitochondrial control region.

BACKGROUND: Tandem repeats in mitochondrial DNA control region are known to different animal taxa, including bat species of the family Vespertilionidae. The long R1-repeats in the bat ETAS-domain are often presented in a variable copy number and may exhibit both inter-individual and intra-individual sequence diversity. The function of repeats in the control region is still unclear, but it has been shown that repetitive sequences in some animal groups (shrews, cats and sheep) may include parts of ETAS1 and ETAS2 conservative blocks of mitochondrial DNA. METHODS AND RESULTS: Analysis of the control region sequences for 31 Myotis petax specimens allowed the identification of the inter-individual variability and clarification of the composition of the R1-repeats. The copy number of the R1-repeats varies from 4 to 7 in individuals. The specimens examined do not exhibit a size heteroplasmy previously described for Myotis species. The unusual short 30 bp R1-repeats have been detected in M. petax for the first time. The ten specimens from Amur Region and Primorsky Territory have one or two copies of these additional repeats. CONCLUSIONS: It was determined that the R1-repeats in M. petax control region consist of parts of the ETAS1 and ETAS2 blocks. The origin of the additional repeats seems to be related to the 51 bp deletion in the central part of the R1-repeat unit and subsequent duplication. Comparison of repetitive sequences in the control region of closely-related Myotis species identified the occurrence of incomplete repeats also resulting from the short deletions, but distinct from additional repeats of M. petax.

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.

Phylogeny and Genetic Diversity of Philippine Native Pigs (Sus scrofa) as Revealed by Mitochondrial DNA Analysis.

Philippine native pigs (PhNP) are small black pigs domesticated in rural communities in the Philippines. They are valued locally for their various sociocultural roles. Recently, considerable literature has accumulated in the field of native pig production and marketing. However, there is limited research on the genetic diversity of PhNP. No previous study has investigated the evolutionary relatedness among native pigs from various islands and provinces in Luzon and the Visayas, Philippines. In addition, a much debated question is whether the PhNP were interbreeding with or even domesticated from endemic wild pigs. This study aims to clarify some of the uncertainties surrounding the identity and classification of PhNP based on mitochondrial DNA (mtDNA) signatures. Native pig samples (n = 157) were collected from 10 provinces in Luzon and the Visayas. Approximately 650 base pairs of the mtDNA D-loop region were sequenced and analyzed together with publicly available sequences. Pairwise-distance analysis showed genetic separation of North and South Luzon (SL) and the clustering of SL with Visayan pigs. Phylogenetic analysis showed that the PhNP clustered within 3 recognized Asian pig domestication centers: D2 (East Asia), D7 (Southeast Asia) and the Cordillera clade (sister to the Lanyu). We identified 19 haplotypes (1-38 samples each), forming 4 haplogroups, i.e., North Luzon, South Luzon and Visayas, Asian mix and the Cordillera cluster. No endemic wild pig mtDNA was detected in the native pig population, but evidence of interspecific hybridization was observed. This study showed that the Philippine native pigs have originated from at least 3 Sus scrofa lineage and that they were not domesticated from the endemic wild pigs of the Philippines.

Mitochondrial DNA sequencing illuminates genetic diversity and origin of Hunagrian Nonius horse breed and his relatives - Danubian horse and Serbian Nonius.

From a historical perspective, horse breeding in Bulgaria has been very well developed since the time of the Thracians (early Bronze Age c. 3000 BCE). Archaeological discoveries from this era present us with an extremely rich type diversity, including wild and local primitive horses, the prototype of heavy draft horses, and fine riding horses.The objective of this study was to investigate the genetic structure of unexamined populations of three closely related horse breeds - the Danubian Nonius Hungarian Nonius and Serbian Nonius horses. A 608 bp long fragment of the mtDNA D-loop region was amplified and sequenced. The obtained results showed completely different genetic profiles between the investigated breeds. We identified nine of the 17 haplogroups described in modern horses. Most of the obtained sequences fell into M, L, G, and O'P lineages, which reflects the genetic profiles of the ancestral mares that were probably used at the initial stages of formation of the breeds. The population of the Danubian horse was characterized by a high prevalence of Central Asian specific haplogroup G (45%), followed by Western Eurasian specific haplogroups L and M (both about 21%). In contrast to the Danubian horse, in the Nonius breed the highest frequency of Western Eurasian haplogroup M (43.5%) was found, followed by Middle Eastern haplogroups O'P (26.1%) Central Asian specific E (13.0%) and G (13.1%). The Serbian Nonius horse showed a completely different genetic profile with a prevalence of the rare for Europe haplogroup D (66.7%), followed by Central Asian specific G (16.7%). The high mitochondrial haplotype diversity (Hd = 0.886) found in the investigated samples is evidence for multiple maternal origins in all populations.In conclusion, the obtained results demonstrated a high percentage of haplogroup sharing especially in the Danubian and Hungarian Nonius horse breeds, which reflects the possible common origins of the two breeds. In contrast to these breeds, the Serbian Nonius, despite the small number of investigated animals, showed a specific genetic profile, which could be explained by different and independent origins.

Maternal genetic diversity, differentiation and phylogeny of three white yak breeds/populations in China.

White yak is a unique economic livestock animal on the Qilian Mountains of Qinghai-Tibet Plateau (QTP). Here, 369 mitochondrial D-loop sequences from three Chinese white yak breeds/populations (Tianzhu, Menyuan and Huzhu) were comprehensively analyzed to indicate their maternal genetic diversity, differentiation and phylogenetic relationship. Our results showed that the haplotype diversity (Hd) was found to be highest in Tianzhu white yak (Hd ± SD = 0.9501 ± 0.0058), while the lowest was recorded in Huzhu white yak (Hd ± SD = 0.7178 ± 0.0474). Totally, the haplotype and nucleotide diversities of white yak were 0.9407 ± 0.0069 and 0.0187 ± 0.0094, respectively, indicating an abundant maternal genetic diversity in white yak. Estimates of FST (Fixation Index) values showed a high genetic differentiation between Tianzhu and Menyuan populations (FST = 0.2928, p < 0.05) as well as that between Tianzhu and Huzhu populations (FST = 0.2721, p < 0.05), but a moderate genetic differentiation between Menyuan and Huzhu populations (FST = 0.1352, p > 0.05) was observed. Cluster analysis based on FST values among populations indicated that the genetic relationship between Menyuan and Huzhu white yak was closer, but they had a far genetic relationship with Tianzhu white yak. Maternal phylogenetic analysis revealed that white yak represented two maternal lineages (I and II), suggesting two maternal origins. In addition, it is notable that taurine mtDNA haplotypes were detected in Tianzhu and Huzhu white yak populations, indicating taurine genetic introgression to some extent. Our study would provide useful information for the conservation and utilization of white yak.