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1.
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.  相似文献   

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We have determined the complete mitochondrial DNA (mtDNA) sequences of three chytridiomycete fungi, Monoblepharella15, Harpochytrium94 and Harpochytrium105. Our phylogenetic analysis based on concatenated mitochondrial protein sequences confirms the placement of Mono blepharella15 together with Harpochytrium spp. and Hyaloraphidium curvatum within the taxonomic order Monoblepharidales, with overwhelming support. These four mtDNA sequences encode the standard fungal mitochondrial gene complement and, like certain other chytridiomycete fungi, encode a reduced complement of 7–9 tRNAs, some of which require 5′-tRNA editing to be functional. Highly conserved sequence elements were identified upstream of almost all protein-coding genes in the mtDNAs of Monoblepharella15 and both Harpochytrium species. Finally, a guanosine residue is conserved upstream of the predicted ATG or GTG start codons of almost every protein-coding gene in these genomes. The appearance of this G residue correlates with the presence of a non-canonical cytosine residue at position 37 in the anticodon loop of the mitochondrial initiator tRNAs. Based on the unorthodox features in these four genomes, we propose that a 4 bp interaction between the CAUC anticodon of these tRNAs and GAUG/GGUG codons is involved in translation initiation in monoblepharidalean mitochondria. Intriguingly, a similar interaction may also be involved in mitochondrial translation initiation in the sea anemone Metridium senile.  相似文献   

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DNA sequences from orthologous loci can provide universal characters for taxonomic identification. Molecular taxonomy is of particular value for groups in which distinctive morphological features are difficult to observe or compare. To assist in species identification for the little known family Ziphiidae (beaked whales), we compiled a reference database of mitochondrial DNA (mtDNA) control region (437 bp) and cytochrome b (384 bp) sequences for all 21 described species in this group. This mtDNA database is complemented by a nuclear database of actin intron sequences (925 bp) for 17 of the 21 species. All reference sequences were derived from specimens validated by diagnostic skeletal material or other documentation, and included four holotypes. Phylogenetic analyses of mtDNA sequences confirmed the genetic distinctiveness of all beaked whale species currently recognized. Both mitochondrial loci were well suited for species identification, with reference sequences for all known ziphiids forming robust species-specific clades in phylogenetic reconstructions. The majority of species were also distinguished by nuclear alleles. Phylogenetic comparison of sequence data from "test" specimens to these reference databases resulted in three major taxonomic discoveries involving animals previously misclassified from morphology. Based on our experience with this family and the order Cetacea as a whole, we suggest that a molecular taxonomy should consider the following components: comprehensiveness, validation, locus sensitivity, genetic distinctiveness and exclusivity, concordance, and universal accessibility and curation.  相似文献   

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Background

The orders Ascaridida, Oxyurida, and Spirurida represent major components of zooparasitic nematode diversity, including many species of veterinary and medical importance. Phylum-wide nematode phylogenetic hypotheses have mainly been based on nuclear rDNA sequences, but more recently complete mitochondrial (mtDNA) gene sequences have provided another source of molecular information to evaluate relationships. Although there is much agreement between nuclear rDNA and mtDNA phylogenies, relationships among certain major clades are different. In this study we report that mtDNA sequences do not support the monophyly of Ascaridida, Oxyurida and Spirurida (clade III) in contrast to results for nuclear rDNA. Results from mtDNA genomes show promise as an additional independently evolving genome for developing phylogenetic hypotheses for nematodes, although substantially increased taxon sampling is needed for enhanced comparative value with nuclear rDNA. Ultimately, topological incongruence (and congruence) between nuclear rDNA and mtDNA phylogenetic hypotheses will need to be tested relative to additional independent loci that provide appropriate levels of resolution.

Results

For this comparative phylogenetic study, we determined the complete mitochondrial genome sequences of three nematode species, Cucullanus robustus (13,972 bp) representing Ascaridida, Wellcomia siamensis (14,128 bp) representing Oxyurida, and Heliconema longissimum (13,610 bp) representing Spirurida. These new sequences were used along with 33 published nematode mitochondrial genomes to investigate phylogenetic relationships among chromadorean orders. Phylogenetic analyses of both nucleotide and amino acid sequence datasets support the hypothesis that Ascaridida is nested within Rhabditida. The position of Oxyurida within Chromadorea varies among analyses; in most analyses this order is sister to the Ascaridida plus Rhabditida clade, with representative Spirurida forming a distinct clade, however, in one case Oxyurida is sister to Spirurida. Ascaridida, Oxyurida, and Spirurida (the sampled clade III taxa) do not form a monophyletic group based on complete mitochondrial DNA sequences. Tree topology tests revealed that constraining clade III taxa to be monophyletic, given the mtDNA datasets analyzed, was a significantly worse result.

Conclusion

The phylogenetic hypotheses from comparative analysis of the complete mitochondrial genome data (analysis of nucleotide and amino acid datasets, and nucleotide data excluding 3rd positions) indicates that nematodes representing Ascaridida, Oxyurida and Spirurida do not share an exclusive most recent common ancestor, in contrast to published results based on nuclear ribosomal DNA. Overall, mtDNA genome data provides reliable support for nematode relationships that often corroborates findings based on nuclear rDNA. It is anticipated that additional taxonomic sampling will provide a wealth of information on mitochondrial genome evolution and sequence data for developing phylogenetic hypotheses for the phylum Nematoda.
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Recent molecular studies addressing the phylogenetic relationships of avian orders have had conflicting results. While studies using nuclear DNA sequences tend to support traditional taxonomic views, also supported by morphological data [(paleognaths (galloanseres (all other birds)))], with songbirds forming a clade within Neoaves (all other birds), analyses with complete mtDNA genomes have resulted in topologies that place songbirds as one of the earliest-diverging avian lineages. Considering that over half of the extant bird species are songbirds, these different results have very different implications for our understanding of avian evolution. We analyzed data sets comprising nearly 4 kb of mitochondrial DNA (mtDNA) (complete 12S, ND1, ND2, and cytochrome b) plus 600 bp of the nuclear gene c-mos for 15 birds that were chosen to represent all major avian clades and to minimize potential long-branch attraction problems; we used a partition-specific maximum likelihood approach. Our results show congruence with respect to the ingroup among phylogenies obtained with mtDNA and the nuclear gene c-mos, separately or combined. The data sets support a traditional avian taxonomy, with paleognaths (ratites and tinamous) occupying a basal position and with songbirds more derived and forming a monophyletic group. We also show that, for mtDNA studies, turtles may be a better outgroup for birds than crocodilians because of their slower rate of sequence evolution.  相似文献   

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Mitochondrial DNA sequences from Georgians and Kurds were analyzed in order to test the possible correlation between female lineages and languages in these two neighboring West Eurasian groups. Mitochondrial sequence pools in both populations are very similar despite their different linguistic and prehistoric backgrounds. Both populations present mtDNA lineages that clearly belong to the European gene pool, as shown by 1) similar nucleotide and sequence diversities; 2) a large number of sequences shared with the rest of European samples; 3) nonsignificant genetic distances; and 4) classification of the present lineages into the major European mtDNA haplogroups already described. The outlier position of the populations from the Caucasus according to classical genetic markers is not recognized in the present Georgian mtDNA sequence pool. This result suggests that the differentiation of mtDNA sequences in West Eurasia and the outlier features of Caucasian populations should be attributed to different processes. Moreover, the putative linguistic relationship between Caucasian groups and the Basques, another outlier population within Europe for classical genetic markers, is not detected by the analysis of mtDNA sequences.  相似文献   

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Several plant mitochondrial genomes contain repeated sequences that are postulated to be sites of homologous intragenomic recombination (1-3). In this report, we have used filter hybridizations to investigate sequence relationships between the cloned mitochondrial DNA (mtDNA) recombination repeats from turnip, spinach and maize and total mtDNA isolated from thirteen species of angiosperms. We find that strong sequence homologies exist between the spinach and turnip recombination repeats and essentially all other mitochondrial genomes tested, whereas a major maize recombination repeat does not hybridize to any other mtDNA. The sequences homologous to the turnip repeat do not appear to function in recombination in any other genome, whereas the spinach repeat hybridizes to reiterated sequences within the mitochondrial genomes of wheat and two species of pokeweed that do appear to be sites of recombination. Thus, although intragenomic recombination is a widespread phenomenon in plant mitochondria, it appears that different sequences either serve as substrates for this function in different species, or else surround a relatively short common recombination site which does not cross-hybridize under our experimental conditions. Identified gene sequences from maize mtDNA were used in heterologous hybridizations to show that the repeated sequences implicated in recombination in turnip and spinach/pokeweed/wheat mitochondria include, or are closely linked to genes for subunit II of cytochrome c oxidase and 26S rRNA, respectively. Together with previous studies indicating that the 18S rRNA gene in wheat mtDNA is contained within a recombination repeat (3), these results imply an unexpectedly frequent association between recombination repeats and plant mitochondrial genes.  相似文献   

11.
A survey of bacterial insertion sequences using IScan   总被引:4,自引:0,他引:4  
Bacterial insertion sequences (ISs) are the simplest kinds of bacterial mobile DNA. Evolutionary studies need consistent IS annotation across many different genomes. We have developed an open-source software package, IScan, to identify bacterial ISs and their sequence elements—inverted and target direct repeats—in multiple genomes using multiple flexible search parameters. We applied IScan to 438 completely sequenced bacterial genomes and 20 IS families. The resulting data show that ISs within a genome are extremely similar, with a mean synonymous divergence of Ks = 0.033. Our analysis substantially extends previously available information, and suggests that most ISs have entered bacterial genomes recently. By implication, their population persistence may depend on horizontal transfer. We also used IScan's ability to analyze the statistical significance of sequence similarity among many IS inverted repeats. Although the inverted repeats of insertion sequences are evolutionarily highly flexible parts of ISs, we show that this ability can be used to enrich a dataset for ISs that are likely to be functional. Applied to the thousands of genomes that will soon be available, IScan could be used for many purposes, such as mapping the evolutionary history and horizontal transfer patterns of different ISs.  相似文献   

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Mitochondrial genomes of spermatophytes are the largest of all organellar genomes. Their large size has been attributed to various factors; however, the relative contribution of these factors to mitochondrial DNA (mtDNA) expansion remains undetermined. We estimated their relative contribution in Malus domestica (apple). The mitochondrial genome of apple has a size of 396 947 bp and a one to nine ratio of coding to non-coding DNA, close to the corresponding average values for angiosperms. We determined that 71.5% of the apple mtDNA sequence was highly similar to sequences of its nuclear DNA. Using nuclear gene exons, nuclear transposable elements and chloroplast DNA as markers of promiscuous DNA content in mtDNA, we estimated that approximately 20% of the apple mtDNA consisted of DNA sequences imported from other cell compartments, mostly from the nucleus. Similar marker-based estimates of promiscuous DNA content in the mitochondrial genomes of other species ranged between 21.2 and 25.3% of the total mtDNA length for grape, between 23.1 and 38.6% for rice, and between 47.1 and 78.4% for maize. All these estimates are conservative, because they underestimate the import of non-functional DNA. We propose that the import of promiscuous DNA is a core mechanism for mtDNA size expansion in seed plants. In apple, maize and grape this mechanism contributed far more to genome expansion than did homologous recombination. In rice the estimated contribution of both mechanisms was found to be similar.  相似文献   

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Background

Large-scale genome sequencing poses enormous problems to the logistics of laboratory work and data handling. When numerous fragments of different genomes are PCR amplified and sequenced in a laboratory, there is a high immanent risk of sample confusion. For genetic markers, such as mitochondrial DNA (mtDNA), which are free of natural recombination, single instances of sample mix-up involving different branches of the mtDNA phylogeny would give rise to reticulate patterns and should therefore be detectable.

Methodology/Principal Findings

We have developed a strategy for comparing new complete mtDNA genomes, one by one, to a current skeleton of the worldwide mtDNA phylogeny. The mutations distinguishing the reference sequence from a putative recombinant sequence can then be allocated to two or more different branches of this phylogenetic skeleton. Thus, one would search for two (or three) near-matches in the total mtDNA database that together best explain the variation seen in the recombinants. The evolutionary pathway from the mtDNA tree connecting this pair together with the recombinant then generate a grid-like median network, from which one can read off the exchanged segments.

Conclusions

We have applied this procedure to a large collection of complete human mtDNA sequences, where several recombinants could be distilled by our method. All these recombinant sequences were subsequently corrected by de novo experiments – fully concordant with the predictions from our data-analytical approach.  相似文献   

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The analysis of mitochondrial DNA (mtDNA) sequences has been a potent tool in our understanding of human evolution. However, almost all studies of human evolution based on mtDNA sequencing have focused on the control region, which constitutes less than 7% of the mitochondrial genome. The rapid development of technology for automated DNA sequencing has made it possible to study the complete mtDNA genomes in large numbers of individuals, opening the field of mitochondrial population genomics. Here we describe a suitable methodology for determining the complete human mitochondrial sequence and the global mtDNA diversity in humans. Also, we discuss the implications of the results with respect to the different hypotheses for the evolution of modern humans.  相似文献   

16.
Lilly JW  Havey MJ 《Genetics》2001,159(1):317-328
Closely related cucurbit species possess eightfold differences in the sizes of their mitochondrial genomes. We cloned mitochondrial DNA (mtDNA) fragments showing strong hybridization signals to cucumber mtDNA and little or no signal to watermelon mtDNA. The cucumber mtDNA clones carried short (30-53 bp), repetitive DNA motifs that were often degenerate, overlapping, and showed no homology to any sequences currently in the databases. On the basis of dot-blot hybridizations, seven repetitive DNA motifs accounted for >13% (194 kb) of the cucumber mitochondrial genome, equaling >50% of the size of the Arabidopsis mitochondrial genome. Sequence analysis of 136 kb of cucumber mtDNA revealed only 11.2% with significant homology to previously characterized mitochondrial sequences, 2.4% to chloroplast DNA, and 15% to the seven repetitive DNA motifs. The remaining 71.4% of the sequence was unique to the cucumber mitochondrial genome. There was <4% sequence colinearity surrounding the watermelon and cucumber atp9 coding regions, and the much smaller watermelon mitochondrial genome possessed no significant amounts of cucumber repetitive DNAs. Our results demonstrate that the expanded cucumber mitochondrial genome is in part due to extensive duplication of short repetitive sequences, possibly by recombination and/or replication slippage.  相似文献   

17.
Evolution operates on whole genomes through direct rearrangements of genes, such as inversions, transpositions, and inverted transpositions, as well as through operations, such as duplications, losses, and transfers, that also affect the gene content of the genomes. Because these events are rare relative to nucleotide substitutions, gene order data offer the possibility of resolving ancient branches in the tree of life; the combination of gene order data with sequence data also has the potential to provide more robust phylogenetic reconstructions, since each can elucidate evolution at different time scales. Distance corrections greatly improve the accuracy of phylogeny reconstructions from DNA sequences, enabling distance-based methods to approach the accuracy of the more elaborate methods based on parsimony or likelihood at a fraction of the computational cost. This paper focuses on developing distance correction methods for phylogeny reconstruction from whole genomes. The main question we investigate is how to estimate evolutionary histories from whole genomes with equal gene content, and we present a technique, the empirically derived estimator (EDE), that we have developed for this purpose. We study the use of EDE on whole genomes with identical gene content, and we explore the accuracy of phylogenies inferred using EDE with the neighbor joining and minimum evolution methods under a wide range of model conditions. Our study shows that tree reconstruction under these two methods is much more accurate when based on EDE distances than when based on other distances previously suggested for whole genomes. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Martin Kreitman]  相似文献   

18.
Abstract.  We describe a new species of Halys Fabricius (Pentatomidae: Pentatominae: Halyini) based on morphological and DNA sequence data, and demonstrate the value of DNA sequences for taxonomic problems that are difficult to resolve on the basis of morphology alone. Halys sindillus Memon, Meier & Manan, sp.n. varies with regard to characters that are usually constant within the genus (spermathecal bulb of females; blade of male clasper; ratio between the second and third antennomeres; length of labium). The surprising levels of variation raised the question as to how many species were represented in three series of specimens from Pakistan. Because the morphological variability was largely continuous, we hypothesized the presence of one new species, and confirm this result here using sequence data from two mitochondrial markers. The data reveal very little molecular variation within the newly described species (COI: 730 bp: 0–0.16%; COI/tRNALeu/COII: 563 bp: 0–0.36%), that is, morphology and DNA sequences show very different patterns of variability. The new species is compared with the closely related Halys sulcatus (Thunberg) whose sequences are distinctly different and whose spermathecal bulbs are largely invariable (I: 2.87–3.28%; II: 2.13–2.49%). We discuss the shortcomings of mitochondrial data in taxonomy and compare the genetic distances in Halys with frequency distributions of intra- and interspecific distances obtained for all 878 Hemiptera COI sequences in GenBank. We conclude that the observed distances for Halys are consistent with our taxonomic conclusions, thus demonstrating the usefulness of DNA sequences for Halys taxonomy. However, the observed overlap between intra- and interspecific sequence variability in Hemiptera is so wide that it questions the feasibility of approaches to taxonomy based predominantly on DNA sequences (e.g. DNA taxonomy, DNA barcoding).  相似文献   

19.
The 18S ribosomal DNA (rDNA) and cytochrome oxidase I region of mitochondrial DNA (mtDNA) were sequenced for 24 Xiphinema americanum-group populations sourced from a number of geographically disparate locations. Sequences were subjected to phylogenetic analysis and compared. 18S rDNA strongly suggested that only X. pachtaicum, X. simile (two populations) and a X. americanum s.l. population from Portugal were different from the other 20 populations studied, whereas mtDNA indicated some heterogeneity between populations. Phylogenetically, based on mtDNA, an apparent dichotomy existed amongst X. americanum-group populations from North America and those from Asia, South America and Oceania. Analyses of 18S rDNA and mtDNA sequences underpin the classical taxonomic issues of the X. americanum-group and cast doubt on the degree of speciation within the X. americanum-group.  相似文献   

20.
Few botanical studies have explored the potential of nuclear ribosomal DNA (nrDNA) and mitochondrial DNA (mtDNA) data obtained through genome skimming for phylogeny reconstruction. Here, we analyzed the phylogenetic information included in the nrDNA and mtDNA of 44 species of the “Adenocalymma‐Neojobertia” clade (Bignoniaceae). To deal with intraindividual polymorphisms within the nrDNA, different coding schemes were explored through the analyses of four datasets: (i) “nrDNA contig,” with base call following the majority rule; (ii) “nrDNA ambiguous,” with ambiguous base calls; (iii) “nrDNA informative,” with ambiguities converted to multistate characters; and, (iv) “mitochondrial,” with 39 mitochondrial genes. Combined analyses using the nrDNA and mtDNA data and previously published “plastid” datasets were also conducted. Trees were obtained using Maximum Likelihood and Bayesian criteria. The congruence among genomes was assessed. The nrDNA datasets were shown to be highly polymorphic within individuals, while the “mitochondrial” dataset was the least informative, with 0.36% of informative bases within the ingroup. The topologies inferred using the nrDNA and mtDNA datasets were broadly congruent with the tree derived from the analyses of the “plastid” dataset. The topological differences recovered were generally poorly supported. The topology that resulted from the analyses of the “combined” dataset largely resembles the “plastid” tree. These results highlight limitations of nuclear ribosomal DNA and mitochondrial genes for phylogeny reconstruction obtained through genome skimming and the need to include more data from both genomes. The different topologies observed among genomes also highlight the importance of exploring data from various genomes in plant phylogenetics.  相似文献   

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