Heteroplasmy and Ancient Translocation of Mitochondrial DNA to the Nucleus in the Chinese Horseshoe Bat (Rhinolophus sinicus) Complex

The utility and reliability of mitochondrial DNA sequences in phylogenetic and phylogeographic studies may be compromised by widespread and undetected nuclear mitochondrial copies (numts) as well as heteroplasmy within individuals. Both numts and heteroplasmy are likely to be common across diverse taxa yet few studies have characterised their frequencies and variation at the intra-specific level. Here we report the presence of both numts and heteroplasmy in the mitochondrial control region of the Chinese horseshoe bat Rhinolophus sinicus. In total we generated 123 sequences from 18 bats, which contained two different numt clades (i.e. Numt-1 and Numt-2) and one mtDNA clade. The sequence divergence between Numt-1 and Numt-2 was 16.8% and each numt type was found in all four R. sinicus taxa, suggesting either two ancient translocations of mitochondrial DNA into the nucleus from the same source taxon, or a single translocation from different source taxa that occurred before the split of R. sinicus into different lineages. Within the mtDNA clade, phylogenetic relationships among the four taxa of R. sinicus were similar to those seen in previous results. Based on PCR comparisons, heteroplasmy was inferred between almost all individuals of R. sinicus with respect to sequence variation. Consistent with introgression of mtDNA between Central sinicus and septentrionalis, individuals from these two taxa exhibited similar signatures of repeated sequences in the control region. Our study highlights the importance of testing for the presence of numts and heteroplasmy when applying mtDNA markers to phylogenetic studies.     

Preventing the pollution of mitochondrial datasets with nuclear mitochondrial paralogs (numts)

Molecular tools have become prominent in ecology and evolution. A target of choice for molecular ecologists and evolutionists is mitochondrial DNA (mtDNA), whose many advantages have also convinced broad-scale, pragmatic programmes such as barcode initiatives. Of course, mtDNA is also of interest to human geneticists investigating mitochondrial diseases. Studies using mtDNA are however put at great risk by the inadvertent co-amplification or preferred amplification of nuclear pseudogenes (numts). A posteriori analysis of putative mtDNA sequences can help in removing numts but faces severe limitations (e.g. recently translocated numts will most of the time go unnoticed). Counter-measures taken a priori, i.e. explicitly designed for avoiding numt co-amplification or preferred amplification, are appealing but have never been properly assessed. Here we investigate the efficiency of four such measures (mtDNA enrichment, cDNA amplification, long-range amplification and pre-PCR dilution) on a common set of numt cases, showing that mtDNA enrichment is the worst performer while the use of pre-PCR dilution is a simple, yet robust method to prevent the pollution of putative mtDNA datasets with numts. Therefore, straightforward recommendations can be made that, if followed, will considerably increase the confidence in the mitochondrial origin of any mtDNA-like sequence.     

Mitochondrial COII Introgression into the Nuclear Genome of Gorilla gorilla

Numts are nonfunctional mitochondrial sequences that have translocated into nuclear DNA, where they evolve independently from the original mitochondrial DNA (mtDNA) sequence. Numts can be unintentionally amplified in addition to authentic mtDNA, complicating both the analysis and interpretation of mtDNA-based studies. Amplification of numts creates particular issues for studies on the noncoding, hypervariable 1 mtDNA region of gorillas. We provide data on putative numt sequences of the coding mitochondrial gene cytochrome oxidase subunit II (COII). Via polymerase chain reaction (PCR) and cloning, we obtained COII sequences for gorilla, orangutan, and human high-quality DNA and also from a gorilla fecal DNA sample. Both gorilla and orangutan samples yielded putative numt sequences. Phylogenetically more anciently transferred numts were amplified with a greater incidence from the gorilla fecal DNA sample than from the high-quality gorilla sample. Data on phylogenetically more recently transferred numts are equivocal. We further demonstrate the need for additional investigations into the use of mtDNA markers for noninvasively collected samples from gorillas and other primates.     

Few mitochondrial DNA sequences are inserted into the turkey (Meleagris gallopavo) nuclear genome: evolutionary analyses and informativity in the domestic lineage

Mitochondrial DNA (mtDNA) insertions have been detected in the nuclear genome of many eukaryotes. These sequences are pseudogenes originated by horizontal transfer of mtDNA fragments into the nuclear genome, producing nuclear DNA sequences of mitochondrial origin (numt). In this study we determined the frequency and distribution of mtDNA‐originated pseudogenes in the turkey (Meleagris gallopavo) nuclear genome. The turkey reference genome (Turkey_2.01) was aligned with the reference linearized mtDNA sequence using last . A total of 32 numt sequences (corresponding to 18 numt regions derived by unique insertional events) were identified in the turkey nuclear genome (size ranging from 66 to 1415 bp; identity against the modern turkey mtDNA corresponding region ranging from 62% to 100%). Numts were distributed in nine chromosomes and in one scaffold. They derived from parts of 10 mtDNA protein‐coding genes, ribosomal genes, the control region and 10 tRNA genes. Seven numt regions reported in the turkey genome were identified in orthologues positions in the Gallus gallus genome and therefore were present in the ancestral genome that in the Cretaceous originated the lineages of the modern crown Galliformes. Five recently integrated turkey numts were validated by PCR in 168 turkeys of six different domestic populations. None of the analysed numts were polymorphic (i.e. absence of the inserted sequence, as reported in numts of recent integration in other species), suggesting that the reticulate speciation model is not useful for explaining the origin of the domesticated turkey lineage.     

Origin of intra-individual variation in PCR-amplified mitochondrial cytochrome oxidase I of Thrips tabaci (Thysanoptera: Thripidae): mitochondrial heteroplasmy or nuclear integration?

The mitochondrial genome is increasingly being used as a species diagnostic marker in insects. Typically, genomic DNA is PCR amplified and then analysed by restriction analyses or sequencing. This analysis system may cause some serious problems for molecular diagnosis. Besides the errors introduced by the PCR process, mtDNA sequence variation of amplified fragments may originate from mtDNA heteroplasmy or from nuclear integrations of mtDNA fragments, both of which have been shown to occur in insects. Here we document abundant variation in PCR-amplified sequences of the mitochondrial cytochrome oxidase I gene of Thrips tabaci. We confirm that the most common haplotype is of mitochondrial origin. Some of the observed mutations were introduced by the amplification process. However, the occurrence of some haplotypes at elevated frequencies indicates that within-individual variation of the respective fragment exists at low levels in T. tabaci. The frequencies of these sequences are too low to negatively affect mtDNA-based molecular diagnosis of T. tabaci. The possible origin of these variant haplotypes is discussed.     

Primers for a PCR-based approach to mitochondrial genome sequencing in birds and other vertebrates.

A PCR-based approach to sequencing complete mitochondrial genomes is described along with a set of 86 primers designed primarily for avian mitochondrial DNA (mtDNA). This PCR-based approach allows an accurate determination of complete mtDNA sequences that is faster than sequencing cloned mtDNA. The primers are spaced at about 500-base intervals along both DNA strands. Many of the primers incorporate degenerate positions to accommodate variation in mtDNA sequence among avian taxa and to reduce the potential for preferential amplification of nuclear pseudogenes. Comparison with published vertebrate mtDNA sequences suggests that many of the primers will have broad taxonomic utility. In addition, these primers should make available a wider variety of mitochondrial genes for studies based on smaller data sets.     

Selective enrichment and sequencing of whole mitochondrial genomes in the presence of nuclear encoded mitochondrial pseudogenes (numts)

Numts are an integral component of many eukaryote genomes offering a snapshot of the evolutionary process that led from the incorporation of an α-proteobacterium into a larger eukaryotic cell some 1.8 billion years ago. Although numt sequence can be harnessed as molecular marker, these sequences often remain unidentified and are mistaken for genuine mtDNA leading to erroneous interpretation of mtDNA data sets. It is therefore indispensable that during the process of amplifying and sequencing mitochondrial genes, preventive measures are taken to ensure the exclusion of numts to guarantee the recovery of genuine mtDNA. This applies to mtDNA analyses in general but especially to studies where mtDNAs are sequenced de novo as the launch pad for subsequent mtDNA-based research. By using a combination of dilution series and nested rolling circle amplification (RCA), we present a novel strategy to selectively amplify mtDNA and exclude the amplification of numt sequence. We have successfully applied this strategy to de novo sequence the mtDNA of the Black Field Cricket Teleogryllus commodus, a species known to contain numts. Aligning our assembled sequence to the reference genome of Teleogryllus emma (GenBank EU557269.1) led to the identification of a numt sequence in the reference sequence. This unexpected result further highlights the need of a reliable and accessible strategy to eliminate this source of error.     

Nuclear insertions help and hinder inference of the evolutionary history of gorilla mtDNA

Numts are fragments of mitochondrial DNA (mtDNA) that have been translocated to the nucleus, where they can persist while their mitochondrial counterparts continue to rapidly evolve. Thus, numts represent 'molecular fossils' useful for comparison with mitochondrial variation, and are particularly suited for studies of the fast-evolving hypervariable segment of the mitochondrial control region (HV1). Here we used information from numts found in western gorillas (Gorilla gorilla) and eastern gorillas (Gorilla beringei) to estimate that these two species diverged about 1.3 million years ago (Ma), an estimate similar to recent calculations for the divergence of chimpanzee and bonobo. We also describe the sequence of a gorilla numt still possessing a segment lost from all contemporary gorilla mtDNAs. In contrast to that sequence, many numts of the HV1 are highly similar to authentic mitochondrial organellar sequences, making it difficult to determine whether purported mitochondrial sequences truly derive from that genome. We used all available organellar HV1 and corresponding numt sequences from gorillas in a phylogenetic analysis aimed at distinguishing these two types of sequences. Numts were found in several clades in the tree. This, in combination with the fact that only a limited amount of the extant variation in gorillas has been sampled, suggests that categorization of new sequences by the indirect means of phylogenetic comparison would be prone to uncertainty. We conclude that for taxa such as gorillas that contain numerous numts, direct approaches to the authentication of HV1 sequences, such as amplification strategies relying upon the circularity of the mtDNA molecule, remain necessary.     

Fidelity of capture-enrichment for mtDNA genome sequencing: influence of NUMTs

Enriching target sequences in sequencing libraries via capture hybridization to bait/probes is an efficient means of leveraging the capabilities of next-generation sequencing for obtaining sequence data from target regions of interest. However, homologous sequences from non-target regions may also be enriched by such methods. Here we investigate the fidelity of capture enrichment for complete mitochondrial DNA (mtDNA) genome sequencing by analyzing sequence data for nuclear copies of mtDNA (NUMTs). Using capture-enriched sequencing data from a mitochondria-free cell line and the parental cell line, and from samples previously sequenced from long-range PCR products, we demonstrate that NUMT alleles are indeed present in capture-enriched sequence data, but at low enough levels to not influence calling the authentic mtDNA genome sequence. However, distinguishing NUMT alleles from true low-level mutations (e.g. heteroplasmy) is more challenging. We develop here a computational method to distinguish NUMT alleles from heteroplasmies, using sequence data from artificial mixtures to optimize the method.     

Absolute quantitation of a heteroplasmic mitochondrial DNA deletion using a multiplex three-primer real-time PCR assay

Quantitation of wild-type and deleted mitochondrial DNA (mtDNA) coexisting within the same cell (a.k.a., heteroplasmy) is important in mitochondrial disease and aging. We report the development of a multiplex three-primer PCR assay that is capable of absolute quantitation of wild-type and deleted mtDNA simultaneously. Molecular beacons were designed to hybridize with either type of mtDNA molecule, allowing real-time detection during PCR amplification. The assay is specific and can detect down to six copies of mtDNA, making it suitable for single-cell analyses. The relative standard deviation in the threshold cycle number is approximately 0.6%. Heteroplasmy was quantitated in individual cytoplasmic hybrid cells (cybrids), containing a large mtDNA deletion, and bulk cell samples. Individual cybrid cells contained 100-2600 copies of wild-type mtDNA and 950-4700 copies of deleted mtDNA, and the percentage of heteroplasmy ranged from 43+/-16 to 95+/-16%. The average amount of total mtDNA was 3800+/-1600 copies/cybrid cell, and the average percentage of heteroplasmy correlated well with the bulk cell sample. The single-cell analysis also revealed that heteroplasmy in individual cells is highly heterogeneous. This assay will be useful for monitoring clonal expansions of mtDNA deletions and investigating the role of heteroplasmy in cell-to-cell heterogeneity in cellular models of mitochondrial disease and aging.     

PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus

The aim of this study was to enable the polymerase chain reaction (PCR) amplification of DNA fragments within endoglucanase gene(s) of Torula thermophila, by using degenerate primers so that the amplified fragment(s) could be used as homologous probe(s) for cloning of full-length endoglucanase gene(s). The design of the degenerate PCR primers was mainly based on the endoglucanase sequences of other fungi. The endoglucanase gene sequence of Humicola insolens was the only sequence from a thermophilic fungus publicly available in the literature. Therefore, the endoglucanase sequences of the two Trichoderma species, Trichoderma reesei and Trichoderma longibrachiatum, were used to generalize the primers. PCR amplification of T. thermophila genomic DNA with these primers resilied in a specific amplification. The specificity of the amplified fragment was shown by Southern hybridization analysis using egl3 gene of T. reesei as probe. This result suggested that the degenerate primers used in this study may be of value for studies aimed at cloning of endoglucanase genes from a range of related fungi.     

Assessing the effects of primer specificity on eliminating numt coamplification in DNA barcoding: a case study from Orthoptera (Arthropoda: Insecta)

DNA barcoding is a diagnostic method of species identification based on sequencing a short mitochondrial DNA fragment of cytochrome oxidase I (COI), but its ability to correctly diagnose species is limited by the presence of nuclear mitochondrial pseudogenes (numts). Numts can be coamplified with the mitochondrial orthologue when using universal primers, which can lead to incorrect species identification and an overestimation of the number of species. Some researchers have proposed that using more specific primers may help eliminate numt coamplification, but the efficacy of this method has not been thoroughly tested. In this study, we investigate the taxonomic distribution of numts in 11 lineages within the insect order Orthoptera, by analysing cloned COI sequences and further test the effects of primer specificity on eliminating numt coamplification in four lineages. We find that numts are coamplified in all 11 taxa using universal (barcoding) primers, which suggests that numts may be widespread in other taxonomic groups as well. Increased primer specificity is only effective at reducing numt coamplification in some species tested, and only eliminates it in one species tested. Furthermore, we find that a number of numts do not have stop codons or indels, making it difficult to distinguish them from mitochondrial orthologues, thus putting the efficacy of barcoding quality control measures under question. Our findings suggest that numt coamplification is a serious problem for DNA barcoding and more quality control measures should be implemented to identify and eliminate numts prior to using mitochondrial barcodes for species diagnoses.     

Development of internal COI primers to improve and extend barcoding of fruit flies (Diptera: Tephritidae: Dacini)

Accurate species-level identifications underpin many aspects of basic and applied biology;however,identifications can be hampered by a lack of discriminating morphological characters,taxonomic expertise or time.Molecular approaches,such as DNA"barcoding"of the cytochrome c oxidase(COI)gene,are argued to overcome these issues.However,nuclear encoding of mitochondrial genes(numts)and poor amplification success of suboptimally preserved specimens can lead to erroneous identifications.One insect group for which these molecular and morphological problems are significant are the dacine fruit flies(Diptera:Tephritidae:Dacini).We addressed these issues associated with COI barcoding in the dacines by first assessing several"universal"COI primers against public mitochondrial genome and numt sequences for dacine taxa.We then modified a set of four primers that more closely matched true dacine COI sequence and amplified two overlapping portions of the COI barcode region.Our new primers were tested alongside universal primers on a selection of dacine species,including both fresh preserved and decades-old dry specimens.Additionally,Bactrocera tiyoni mitochondrial and nuclear genomes were compared to identify putative numts.Four numt clades were identified,three of which were amplified using existing universal primers.In contrast,our new primers preferentially amplified the"true"mitochondrial COI barcode in all dacine species tested.The new primers also successfully amplified partial barcodes from dry specimens for which full length barcodes were unobtainable.Thus we recommend these new primers be incorporated into the suites of primers used by diagnosticians and quarantine labs for the accurate identification of dacine species.     

Analysis of nuclear copies of mitochondrial sequences in honeybee (Apis mellifera) genome

At least 0.08% of the Apis mellifera nuclear genome contains sequences that originated from mitochondria. These nuclear copies of mitochondrial sequences (numts) are scattered all over the honeybee chromosomes and have originated by multiple independent insertions of mitochondrial DNA (mtDNA) as evident by phylogenetic analysis. Apart from original insertions, moderate duplications of numts also contributed to the present pattern and distribution of mitochondrial sequences in honeybee chromosomes. Assimilation of mitochondrial genes in the nuclear genome is mediated by extensive fragmentations of the original inserts. Replication slippage seems to be a major mechanism by which small sequences are inserted or deleted from mtDNA destined to nucleus. Most of the honeybee numts (84%) are located in the nongenic regions. The majority (94%) of the numts that are located in predicted nuclear genes have originated from mitochondrial genes coding for cytochrome oxidase and NADH dehydrogenase subunits. On the other hand, the mitochondrial rRNA or tRNA gene sequences are predominantly (88%) located in nongenic regions of the genome. Evidences also support for exertion of purifying selection on numts located in specific genes. Comparative analysis of numts of European, African, and Africanized honeybees suggests that numt evolution in A. mellifera is probably not demarked by speciation time frame but may be a continuous and dynamic process.     

小麦印度腥黑粉菌线粒体DNA提取及其ATP6基因在真菌遗传发育分析中的应用

小麦印度腥黑粉菌(Tilletia indica Mitra)是一种世界范围的重要检疫性有害真菌,该病原菌和近似种之间冬孢子的形态特征极为相似,遗传关系非常相近。为了从分子水平上探讨小麦印度腥黑粉菌和近似种之间线粒体基因序列的差异,从新鲜菌丝中提取总DNA,经两次氯化铯密度梯度超速离心分离线粒体DNA(mtDNA),提取的mtDNA纯度较高,可用于克隆、酶切分析和PCR扩增等分析。选取基因ATP(adenosine triphosphate)6的序列,并结合GenBank中相关种类的ATP6基因DNA序列进行了系统发育分析。结果表明,线粒体基因ATP6可用于科属水平的分类鉴定。     

Rates of DNA Duplication and Mitochondrial DNA Insertion in the Human Genome

The hundreds of mitochondrial pseudogenes in the human nuclear genome sequence (numts) constitute an excellent system for studying and dating DNA duplications and insertions. These pseudogenes are associated with many complete mitochondrial genome sequences and through those with a good fossil record. By comparing individual numts with primate and other mammalian mitochondrial genome sequences, we estimate that these numts arose continuously over the last 58 million years. Our pairwise comparisons between numts suggest that most human numts arose from different mitochondrial insertion events and not by DNA duplication within the nuclear genome. The nuclear genome appears to accumulate mtDNA insertions at a rate high enough to predict within-population polymorphism for the presence/absence of many recent mtDNA insertions. Pairwise analysis of numts and their flanking DNA produces an estimate for the DNA duplication rate in humans of 2.2 × 10^–9 per numt per year. Thus, a nucleotide site is about as likely to be involved in a duplication event as it is to change by point substitution. This estimate of the rate of DNA duplication of noncoding DNA is based on sequences that are not in duplication hotspots, and is close to the rate reported for functional genes in other species.     

Searching for a needle in the haystack: comparing six methods to evaluate heteroplasmy in difficult sequence context

Mitochondrial DNA (mtDNA) mutations have been involved in disease, aging and cancer and furthermore exploited for evolutionary and forensic investigation. When investigating mtDNA mutations the peculiar aspects of mitochondrial genetics, such as heteroplasmy and threshold effect, require suitable approaches which must be sensitive enough to detect low-level heteroplasmy and, precise enough to quantify the exact mutational load. In order to establish the optimal approach for the evaluation of heteroplasmy, six methods were experimentally compared for their capacity to reveal and quantify mtDNA variants. Drawbacks and advantages of cloning, Fluorescent PCR (F-PCR), denaturing High Performance Liquid Chromatography (dHPLC), quantitative Real-Time PCR (qRTPCR), High Resolution Melting (HRM) and 454 pyrosequencing were determined. In particular, detection and quantification of a mutation in a difficult sequence context were investigated, through analysis of an insertion in a homopolymeric stretch (m.3571insC).     

No recombination of mtDNA after heteroplasmy for 50 generations in the mouse maternal germline

Variants of mitochondrial DNA (mtDNA) are commonly used as markers to track human evolution because of the high sequence divergence and exclusive maternal inheritance. It is assumed that the inheritance is clonal, i.e. that mtDNA is transmitted between generations without germline recombination. In contrast to this assumption, a number of studies have reported the presence of recombinant mtDNA molecules in cell lines and animal tissues, including humans. If germline recombination of mtDNA is frequent, it would strongly impact phylogenetic and population studies by altering estimates of coalescent time and branch lengths in phylogenetic trees. Unfortunately, this whole area is controversial and the experimental approaches have been widely criticized as they often depend on polymerase chain reaction (PCR) amplification of mtDNA and/or involve studies of transformed cell lines. In this study, we used an in vivo mouse model that has had germline heteroplasmy for a defined set of mtDNA mutations for more than 50 generations. To assess recombination, we adapted and validated a method based on cloning of single mtDNA molecules in the λ phage, without prior PCR amplification, followed by subsequent mutation analysis. We screened 2922 mtDNA molecules and found no germline recombination after transmission of mtDNA under genetically and evolutionary relevant conditions in mammals.     

Real-Time PCR Quantification of Heteroplasmy in a Mouse Model with Mitochondrial DNA of C57BL/6 and NZB/BINJ Strains

Mouse models are widely employed to study mitochondrial inheritance, which have implications to several human diseases caused by mutations in the mitochondrial genome (mtDNA). These mouse models take advantage of polymorphisms between the mtDNA of the NZB/BINJ and the mtDNA of common inbred laboratory (i.e., C57BL/6) strains to generate mice with two mtDNA haplotypes (heteroplasmy). Based on PCR followed by restriction fragment length polymorphism (PCR-RFLP), these studies determine the level of heteroplasmy across generations and in different cell types aiming to understand the mechanisms underlying mitochondrial inheritance. However, PCR-RFLP is a time-consuming method of low sensitivity and accuracy that dependents on the use of restriction enzyme digestions. A more robust method to measure heteroplasmy has been provided by the use of real-time quantitative PCR (qPCR) based on allelic refractory mutation detection system (ARMS-qPCR). Herein, we report an ARMS-qPCR assay for quantification of heteroplasmy using heteroplasmic mice with mtDNA of NZB/BINJ and C57BL/6 origin. Heteroplasmy and mtDNA copy number were estimated in germline and somatic tissues, providing evidence of the reliability of the approach. Furthermore, it enabled single-step quantification of heteroplasmy, with sensitivity to detect as low as 0.1% of either NZB/BINJ or C57BL/6 mtDNA. These findings are relevant as the ARMS-qPCR assay reported here is fully compatible with similar heteroplasmic mouse models used to study mitochondrial inheritance in mammals.     

线粒体DNA的异质性与检测方法

线粒体DNA的异质性在生物学、医学、法医、生物技术等领域有重要的应用。本文从自然发生(包括体细胞突变、父系渗入和杂交)和人工产生(包括转基因、细胞融合、核移植和转线粒体)两大方面系统地介绍了线粒体DNA异质性的产生机制及其遗传。并介绍了线粒体DNA异质性的检测方法,已知突变位点mtDNA异质性的检测方法有原位PCR技术、PCR-RFLP和实时荧光定量PCR技术,未知突变位点mtDNA异质性的检测方法有长PCR技术、时相温度梯度凝胶电泳(TTGE)和变性高效液相色谱(DHPLC)等。最后对克隆生物中线粒体异质性检测的应用实例作了介绍。