Fugu genome does not contain mitochondrial pseudogenes

Contrary to previous observations that fish genomes are devoid of nuclear mitochondrial pseudogenes, a genome-wide survey identified a large number of "recent" and "ancient" nuclear mitochondrial DNA fragments (Numts) in the whole-genome sequences of the fugu (Takifugu rubripes), Tetraodon nigroviridis, and zebrafish (Danio rerio). We have analyzed the latest assembly (v4.0) of the fugu genome and show that, like the Anopheles genome, the fugu nuclear genome does not contain mitochondrial pseudogenes. Fugu assembly v4.0 contains a single scaffold representing the near complete sequence of the fugu mitochondria. The "recent" Numts identified by the previous study in fugu assembly v2.0 are in fact shotgun sequences of mitochondrial DNA that were misassembled with the nuclear sequences, whereas the "ancient" Numts appear to be the result of spurious matches. It is likely that the Numts identified in the genomes of Tetraodon and zebrafish are also similar artifacts. Shotgun sequences of whole genomes often include some mitochondrial sequences. Therefore, any Numts identified in shotgun-sequence assemblies should be verified by Southern hybridization or PCR amplification.     

Mitochondrial pseudogenes: evolution's misplaced witnesses

Nuclear copies of mitochondrial DNA (mtDNA) have contaminated PCR-based mitochondrial studies of over 64 different animal species. Since the last review of these nuclear mitochondrial pseudogenes (Numts) in animals, Numts have been found in 53 of the species studied. The recent evidence suggests that Numts are not equally abundant in all species, for example they are more common in plants than in animals, and also more numerous in humans than in Drosophila. Methods for avoiding Numts have now been tested, and several recent studies demonstrate the potential utility of Numt DNA sequences in evolutionary studies. As relics of ancient mtDNA, these pseudogenes can be used to infer ancestral states or root mitochondrial phylogenies. Where they are numerous and selectively unconstrained, Numts are ideal for the study of spontaneous mutation in nuclear genomes.     

单个拟穴青蟹核内多拷贝线粒体COI假基因序列的研究(简报)

线粒体DNA(mtDNA)因它的高拷贝数、易扩增、高突变率、中性、低重组和母系遗传等特征,已广泛应用在系统进化和群体遗传研究方面[1,2].但是,由于mtDNA本身的特征,如异质性.     

Genomic gigantism: DNA loss is slow in mountain grasshoppers

Several studies have shown DNA loss to be inversely correlated with genome size in animals. These studies include a comparison between Drosophila and the cricket, Laupala, but there has been no assessment of DNA loss in insects with very large genomes. Podisma pedestris, the brown mountain grasshopper, has a genome over 100 times as large as that of Drosophila and 10 times as large as that of Laupala. We used 58 paralogous nuclear pseudogenes of mitochondrial origin to study the characteristics of insertion, deletion, and point substitution in P. pedestris and Italopodisma. In animals, these pseudogenes are "dead on arrival"; they are abundant in many different eukaryotes, and their mitochondrial origin simplifies the identification of point substitutions accumulated in nuclear pseudogene lineages. There appears to be a mononucleotide repeat within the 643-bp pseudogene sequence studied that acts as a strong hot spot for insertions or deletions (indels). Because the data for other insect species did not contain such an unusual region, hot spots were excluded from species comparisons. The rate of DNA loss relative to point substitution appears to be considerably and significantly lower in the grasshoppers studied than in Drosophila or Laupala. This suggests that the inverse correlation between genome size and the rate of DNA loss can be extended to comparisons between insects with large or gigantic genomes (i.e., Laupala and Podisma). The low rate of DNA loss implies that in grasshoppers, the accumulation of point mutations is a more potent force for obscuring ancient pseudogenes than their loss through indel accumulation, whereas the reverse is true for Drosophila. The main factor contributing to the difference in the rates of DNA loss estimated for grasshoppers, crickets, and Drosophila appears to be deletion size. Large deletions are relatively rare in Podisma and Italopodisma.     

Structure and chromosomal distribution of human mitochondrial pseudogenes

Nuclear mitochondrial pseudogenes (Numts) have been found in the genome of many eukaryote species, including humans. Using a BLAST approach, we found 1105 DNA sequences homologous to mitochondrial DNA (mtDNA) in the August 2001 Goldenpath human genome database. We assembled these sequences manually into 286 pseudogenes on the basis of single insertion events and constructed a chromosomal map of these Numts. Some pseudogenes appeared highly modified, containing inversions, deletions, duplications, and displaced sequences. In the case of four randomly selected Numts, we used PCR tests on cells lacking mtDNA to ensure that our technique was free from genome-sequencing artifacts. Furthermore, phylogenetic investigation suggested that one Numt, apparently inserted into the nuclear genome 25-30 million years ago, had been duplicated at least 10 times in various chromosomes during the course of evolution. Thus, these pseudogenes should be very useful in the study of ancient mtDNA and nuclear genome evolution.     

青蟹线粒体COI假基因的分离和特征分析

线粒体DNA标记在遗传结构和系统进化研究中得到广泛应用,然而核假基因的存在对此有很大威胁。本文以中国东南沿海的青蟹(Scylla paramamosain)为研究对象,利用线粒体COI基因的通用引物和特异性引物进行扩增,分别得到34个假基因（nuclear mitochondrial pseudogenes, Numts）和5个线粒体COI基因序列。在所获得的34个假基因中共定义了29种单倍型,根据序列的相似度,这些假基因可以分为2类,每类假基因都有各自保守的核苷酸序列。第Ⅰ类假基因存在2处插入序列和1处8 bp的缺失序列,这些位点导致了整个阅读框的移位;在第Ⅱ类假基因和5个线粒体COI序列中只有碱基替换,未发现插入和缺失序列。实验结果分析表明,这两类假基因分别代表了2次核整合事件,即核转移事件的最低值。研究结果提示了     

A cryptic, intergeneric cytochrome c oxidase I pseudogene in tyrant flycatchers (family: Tyrannidae)

Nuclear mitochondrial pseudogenes, or "numts", are nonfunctional copies of mitochondrial genes that have been translocated to the nuclear genome. Numts have been used to study differences in mutation rates between the nuclear and mitochondrial genomes, but have also been implicated as troublesome for phylogenetic studies and DNA-based species identification (i.e., DNA barcoding). In this study, a suspected numt discovered during a study of mitochondrial cytochrome c oxidase I (COI) diversity in North American birds was targeted and sequenced from tyrant flycatchers (family: Tyrannidae). In total, the numt was found in five taxa representing two genera. Substitution rates were compared between COI and numt sequences. None of the numt sequences harboured stop codons nor frameshift mutations, but phylogenetic analysis revealed they had accumulated more amino acid substitutions than the mitochondrial COI sequences. Mitochondrial COI appeared to be preferentially amplified in most cases, but methods for numt detection are discussed for cases like this where sequences lack obvious features for identification. Because of its persistence across a broad taxonomic lineage, this numt could form a valuable model system for studying evolution in numts. The full size of the numt and its location within the nuclear genome are yet to be determined.     

Estimates of linkage disequilibrium and effective population size in rainbow trout

Background

Mitochondrial DNA (mtDNA) is widely used in population genetic and phylogenetic studies in animals. However, such studies can generate misleading results if the species concerned contain nuclear copies of mtDNA (Numts) as these may amplify in addition to, or even instead of, the authentic target mtDNA. The aim of this study was to determine if Numts are present in Aedes aegypti mosquitoes, to characterise any Numts detected, and to assess the utility of using mtDNA for population genetics studies in this species.

Results

BLAST searches revealed large numbers of Numts in the Ae. aegypti nuclear genome on 146 supercontigs. Although the majority are short (80% < 300 bp), some Numts are almost full length mtDNA copies. These long Numts are not due to misassembly of the nuclear genome sequence as the Numt-nuclear genome junctions could be recovered by amplification and sequencing. Numt evolution appears to be a complex process in Ae. aegypti with ongoing genomic integration, fragmentation and mutation and the secondary movement of Numts within the nuclear genome. The PCR amplification of the putative mtDNA nicotinamide adenine dinucleotide dehydrogenase subunit 4 (ND4) gene from 166 Southeast Asian Ae. aegypti mosquitoes generated a network with two highly divergent lineages (clade 1 and clade 2). Approximately 15% of the ND4 sequences were a composite of those from each clade indicating Numt amplification in addition to, or instead of, mtDNA. Clade 1 was shown to be composed at least partially of Numts by the removal of clade 1-specific bases from composite sequences following enrichment of the mtDNA. It is possible that all the clade 1 sequences in the network were Numts since the clade 2 sequences correspond to the known mitochondrial genome sequence and since all the individuals that produced clade 1 sequences were also found to contain clade 2 mtDNA-like sequences using clade 2-specific primers. However, either or both sets of clade sequences could have Numts since the BLAST searches revealed two long Numts that match clade 2 and one long Numt that matches clade 1. The substantial numbers of mutations in cloned ND4 PCR products also suggest there are both recently-derived clade 1 and clade 2 Numt sequences.

Conclusion

We conclude that Numts are prevalent in Ae. aegypti and that it is difficult to distinguish mtDNA sequences due to the presence of recently formed Numts. Given this, future population genetic or phylogenetic studies in Ae. aegypti should use nuclear, rather than mtDNA, markers.     

Incorporation of mitochondrial fragments in the nuclear genome (Numts) of the longhorned beetle Monochamus galloprovincialis (Coleoptera, Cerambycidae)

A mitochondrial DNA (mtDNA) study, based on 43 European populations (33 of them sampled in France) of Monochamus galloprovincialis , vector of the pinewood nematode, and 14 populations of its sister species Monochamus sutor was realized. Sequencing of 792 bp of the cytochrome oxidase I (COI) and 521 bp of the COII genes revealed numerous ambiguities on multiple nucleotide sites for half of M. galloprovincialis specimens studied (44.8%). Hypotheses of heteroplasmy and pseudogenes ( Numts ) were examined. The mtDNA isolation by alkaline lysis and cloning (for three successfully used individuals) both support the hypothesis that the ambiguous sequences amplified were not of mtDNA nature and validate the presence of Numts in the nuclear genome of M. galloprovincialis . Multiple copies of mtDNA-like sequences were found paralogous to COI, tRNA leucin and COII regions. Phenetic analysis placed different recently diverged mtDNA-like sequences as a close relative of mtDNA sequences, and grouped 10 closely related mtDNA-like sequences as a more basal clade, closer to ancestral states and to M. sutor . This result supports that this nuclear family of pseudogenes arose independently of the other events and may represent mitochondrial haplotypes sampled from M. galloprovincialis ancestral populations. This is the first time that Numts are proved for a longhorned beetle, whereas no Numts were found within its sister species M. sutor. The incorporation mechanism of Numts in unknown for M. galloprovincialis , however, excess of ambiguous sites corresponding to synonymous mutations placed on third codon position as well as the absence of Numts in M. sutor , conducted to the hypothesis of a recent transfer of these Numts in the nuclear genome of M. galloprovincialis .     

Reliability of mitochondrial DNA in an acanthocephalan: the problem of pseudogenes

The utility of mitochondrial DNA as a molecular marker for evolutionary studies is well recognized. However, several problems can arise when using mitochondrial DNA, one of which is the presence of nuclear mitochondrial pseudogenes, or Numts. Pseudogenes of cytochrome oxidase I were preferentially amplified from Acanthocephalus lucii (Acanthocephala) using a universal PCR approach. To verify the presence and abundance of pseudogenes, length heterogeneity analysis of the PCR fragments was performed. PCR products obtained with universal primers often contained fragments of different sizes. Cloned sequences from universal PCR products nearly always contained sequence abnormalities such as indels and/or stop codons. Based on these sequences, new primers were developed to specifically target mitochondrial DNA. Sequences obtained with these specific primers lacked abnormalities. Phylogenetic analysis produced a single most parsimonious tree in which pseudogenes obtained with universal primers grouped together as did putative mitochondrial DNA sequences obtained with specific primers. The pattern of codon bias observed in the pseudogenes suggests a single nuclear integration event from the mitochondria. This is the first reported occurrence of pseudogenes in an acanthocephalan, and it demonstrates the potential dangers associated with the use of universal primers.     

Recombination among multiple mitochondrial pseudogenes from a Passerine genus

PCR products of a fragment of the mitchondrial protein coding subunit 5 of NADH-dehydrogenase (ND5) from eight individuals representing five species of the South American bird genus Conirostrum were cloned. The 130 clones, which were subsequently sequenced, constituted 55 different sequences. Due to the observed differences in substitution patterns 58% of the cloned sequences were identified as pseudogenes. Recombination could be traced in 19% of the inferred nuclear pseudogenes, but this figure probably represents a significant underestimation of the factual recombination events. The nonrecombined pseudogenes consisted of multiple haplotypes found to diverge from 1 to 16% from the mitochondrial gene. The number of mitochondrial nuclear copies and their apparent frequent recombination suggest that pseudogenes constitute a serious potential risk in confounding phylogenetic studies and population genetic analysis.     

家马核基因组中线粒体核内插入序列分析

在各种真核生物核基因组中,存在一些由线粒体基因组转移进入核基因组中的DNA片段,这些被认为是分子化石的片段叫做线粒体核内插入序列(Numt)。由于Numt与真实的线粒体序列高度相似,因此它的存在必然会成为PCR扩增线粒体DNA的不利因素。利用已经公布的家马(Equus caballus)基因组序列(2007年9月公布,GenBank登录号为NC_009144-NC_009175)对家马Numt进行了深入分析,共发现200个可能的Numt,长度范围为29到3727bp,其中有10个的长度大于800bp。分析结果显示由于不存在线粒体控制区域的疑似Numt,因此对基于此区域的群体遗传学研究不会产生影响。本研究还发现在家马进化过程中,第1号和27号染色体更倾向于接受线粒体序列的转移。以上结果将为今后马科动物的研究提供重要的参考信息,有助于避免在线粒体DNA研究中由于Numt污染的存在而得出错误的实验结果。     

Population structure of the Indonesian giant tiger shrimp Penaeus monodon: a window into evolutionary similarities between paralogous mitochondrial DNA sequences and their genomes

Here we used both microsatellites and mtCR (mitochondrial DNA control region) sequences as genetic markers to examine the genetic diversity and population structure of Penaeus monodon shrimp from six Indonesian regions. The microsatellite data showed that shrimp from the Indian and the Pacific Ocean were genetically distinct from each other. It has been reported previously that P. monodon mtCR sequences from the Indo‐Pacific group into two major paralogous clades of unclear origin. Here we show that the population structure inferred from mtCR sequences matches the microsatellite‐based population structure for one of these clades. This is consistent with the notion that this mtCR clade shares evolutionary history with nuclear DNA and may thus represent nuclear mitochondrial pseudogenes (Numts).     

Ribosomal DNA in the grasshopper Podisma pedestris: escape from concerted evolution

Eukaryote nuclear ribosomal DNA (rDNA) typically exhibits strong concerted evolution: a pattern in which several hundred rDNA sequences within any one species show little or no genetic diversity, whereas the sequences of different species diverge. We report a markedly different pattern in the genome of the grasshopper Podisma pedestris. Single individuals contain several highly divergent ribosomal DNA groups. Analysis of the magnitude of divergence indicates that these groups have coexisted in the Podisma lineage for at least 11 million years. There are two putatively functional groups, each estimated to be at least 4 million years old, and several pseudogene groups, many of which are transcribed. Southern hybridization and real-time PCR experiments show that only one of the putatively functional types occurs at high copy number. However, this group is scarcely amplified under standard PCR conditions, which means that phylogenetic inference on the basis of standard PCR would be severely distorted. The analysis suggests that concerted evolution has been remarkably ineffective in P. pedestris. We propose that this outcome may be related to the species' exceptionally large genome and the associated low rate of deletion per base pair, which may allow pseudogenes to persist.     

Marmot phylogeny revisited: molecular evidence for a diphyletic origin of sociality

We established the phylogeny of 11 species of the genus Marmota based on the entire sequence of the mitochondrial cytochrome b ( cyt-b ) gene (1.1 kb) and a partial sequence of the NADH dehydrogenase subunit 4 ( ND4 ) gene (1.2 kb). In three species ( Marmota caligata , Marmota olympus , and Marmota bobac ) full-sized nuclear pseudogenes of the mitochondrial cyt-b were identified. The mitochondrial cyt-b genes and the three pseudogenes form separate clusters in the maximum parsimony dendrogram. This finding suggests that the pseudogenes originated from a single transfer to the nucleus that may have occurred prior to the radiation of the genus Marmota . Notably, compared with their functional mitochondrial equivalents the pseudogenes show a much lower substitution rate. In the dendrograms deduced from the mitochondrial sequences two distinct clusters become apparent: one cluster consists of the North-west American species, the other contains the Eurasian species together with the North American species Marmota monax . The position of M. monax as a member of the Eurasian clade is in accordance with the evolution of chromosome numbers. The results are of special interest with respect to the evolution of social systems in the genus that vary from solitary species ( M. monax ) to highly social species living in family groups (e.g. Marmota marmota ). The molecular phylogeny suggests a diphyletic origin of high sociality in the genus Marmota .     

Nuclear mitochondrial pseudogenes in Austinograea alayseae hydrothermal vent crabs (Crustacea: Bythograeidae): effects on DNA barcoding

Members of the brachyuran crab family, Bythograeidae, are among the most abundant and common crabs in vent fields. However, their identification based on morphological characteristics often leads to incorrect species recognition due to a lack of taxonomic factors and the existence of sibling (or cryptic) species. For these reasons, we used DNA barcoding for vent crabs using mitochondrial cytochrome c oxidase subunit 1 (CO1). However, several nuclear mitochondrial pseudogenes (Numts) were amplified from Austinograea alayseae Guinot, 1990, using universal primers (Folmer primers). The Numts were characterized in six haplotypes, with 13.58–14.11% sequence divergence from A. alayseae, a higher nonsynonymous substitution ratio than true CO1, and the formation of an independent clade in bythograeids. In a neighbour‐joining tree, the origin of the Numts would be expected to incorporate into the nucleus at an ancestral node of Austinograea, and they mutated more slowly in the nucleus than CO1 in the mitochondria. This evolutionary process may have resulted in the higher binding affinity of Numts for the Folmer primers than CO1. In the present study, we performed long PCR for the amplification of CO1 in A. alayseae. We also present evidence that Numts can introduce serious ambiguity into DNA barcoding, including overestimating the number of species in bythograeids. These results may help in conducting taxonomic studies using mitochondrial genes from organisms living in hydrothermal vent fields.     

Discovery of a large number of previously unrecognized mitochondrial pseudogenes in fish genomes

Nuclear inserted copies of mitochondrial origin (numts) vary widely among eukaryotes, with human and plant genomes harboring the largest repertoires. Numts were previously thought to be absent from fish species, but the recent release of three fish nuclear genome sequences provides the resource to obtain a more comprehensive insight into the extent of mtDNA transfer in fishes. From the sequence analyses of the genomes of Fugu rubripes, Tetraodon nigroviridis, and Danio rerio, we have identified 2, 5, and 10 recent numt integrations, respectively, which integrated into those genomes less than 0.6 million years (Myr) ago. Such results contradict the hypothesis of absence or rarity of numts in fishes, as (i) the ratio of numts to the total size of the nuclear genome in T. nigroviridis was superior to the ratio observed in several higher vertebrate species (e.g., chicken, mouse, and rat), and only surpassed by humans, and (ii) the mtDNA coverage transferred to the nuclear genome of D. rerio is exceeded only by human and mouse, within the whole range of eukaryotic genomes surveyed for numts. Additionally, 335, 336, and 471 old numts (>12.5 Myr) were detected in F. rubripes, T. nigroviridis, and D. rerio, respectively. Surprisingly, old numts are inserted preferentially into known or predicted genes, as inferred for recent numts in human. However, because in fish genomes such integrations are old, they are likely to represent evolutionary successes and they may be considered a potential important evolutionary mechanism for the enhancement of genomic coding regions.     

Amplification of multiple copies of mitochondrial Cytochrome b gene fragments in the Australian freshwater crayfish,Cherax destructor Clark (Parastacidae: Decapoda)

Non-coding copies of fragments of the mitochondrial genome translocated to the nucleus or pseudogenes are being found with increasing frequency in a diversity of organisms. As part of a study to evaluate the utility of a range of mitochondrial gene regions for population genetic and systematic studies of the Australian freshwater crayfish, Cherax destructor (the yabby), we report the first detection of Cytochrome b (Cyt b) pseudogenes in crustaceans. We amplified and sequenced fragments of the mitochondrial Cyt b gene from 14 individuals of C. destructor using polymerase chain reaction (PCR) with primers designed from conserved regions of Penaeus monodon and Drosophila melanogaster mitochondrial genomes. The phylogenetic tree produced from the amplified fragments using these primers showed a very different topology to the trees obtained from sequences from three other mitochondrial genes, suggesting one or more nuclear pseudogenes have been amplified. Supporting this conclusion, two highly divergent sequences were isolated from each of two single individuals, and a 2 base pair (bp) deletion in one sequence was observed. There was no evidence to support inadvertent amplification of parasite DNA or contamination of samples from other sources. These results add to other recent observations of pseudogenes suggesting the frequent transfer of mitochondrial DNA (mtDNA) genes to the nucleus and reinforces the necessity of great care in interpreting PCR-generated Cyt b sequences used in population or evolutionary studies in freshwater crayfish and crustaceans more generally.     

Molecular mechanisms of extensive mitochondrial gene rearrangement in plethodontid salamanders

Extensive gene rearrangement is reported in the mitochondrial genomes of lungless salamanders (Plethodontidae). In each genome with a novel gene order, there is evidence that the rearrangement was mediated by duplication of part of the mitochondrial genome, including the presence of both pseudogenes and additional, presumably functional, copies of duplicated genes. All rearrangement-mediating duplications include either the origin of light-strand replication and the nearby tRNA genes or the regions flanking the origin of heavy-strand replication. The latter regions comprise nad6, trnE, cob, trnT, an intergenic spacer between trnT and trnP and, in some genomes, trnP, the control region, trnF, rrnS, trnV, rrnL, trnL1, and nad1. In some cases, two copies of duplicated genes, presumptive regulatory regions, and/or sequences with no assignable function have been retained in the genome following the initial duplication; in other genomes, only one of the duplicated copies has been retained. Both tandem and nontandem duplications are present in these genomes, suggesting different duplication mechanisms. In some of these mitochondrial DNAs, up to 25% of the total length is composed of tandem duplications of noncoding sequence that includes putative regulatory regions and/or pseudogenes of tRNAs and protein-coding genes along with the otherwise unassignable sequences. These data indicate that imprecise initiation and termination of replication, slipped-strand mispairing, and intramolecular recombination may all have played a role in generating repeats during the evolutionary history of plethodontid mitochondrial genomes.