A Complete Mitochondrial Genome Sequence from a Mesolithic Wild Aurochs (Bos primigenius)

Youxian sheldrake is excellent native breeds in Hunan province in China. The complete mitochondrial (mt) genome sequence plays an important role in the accurate determination of phylogenetic relationships among metazoans. This is the first study to determine the complete mitochondrial genome sequence of Youxian sheldrake using PCR-based amplification and Sanger sequencing. The characteristic of the entire mitochondrial genome was analyzed in detail, the total length of the mitogenome is 16,605 bp, with the base composition of 29.21% A, 22.18% T, 32.84% C, 15.77% G in the Youxian sheldrake. It contained 2 ribosomal RNA genes, 13 protein-coding genes, 22 transfer RNA genes and a major non-coding control region (D-loop region). The complete mitochondrial genome sequence of Youxian sheldrake provided an important data for further study of the phylogenetics of poultry, and available data for the genetics and breeding.

BackgroundThe family Camelidae that evolved in North America during the Eocene survived with two distinct tribes, Camelini and Lamini. To investigate the evolutionary relationship between them and to further understand the evolutionary history of this family, we determined the complete mitochondrial genome sequence of the wild two-humped camel (Camelus bactrianus ferus), the only wild survivor of the Old World camel.ResultsThe mitochondrial genome sequence (16,680 bp) from C. bactrianus ferus contains 13 protein-coding, two rRNA, and 22 tRNA genes as well as a typical control region; this basic structure is shared by all metazoan mitochondrial genomes. Its protein-coding region exhibits codon usage common to all mammals and possesses the three cryptic stop codons shared by all vertebrates. C. bactrianus ferus together with the rest of mammalian species do not share a triplet nucleotide insertion (GCC) that encodes a proline residue found only in the nd1 gene of the New World camelid Lama pacos. This lineage-specific insertion in the L. pacos mtDNA occurred after the split between the Old and New World camelids suggests that it may have functional implication since a proline insertion in a protein backbone usually alters protein conformation significantly, and nd1 gene has not been seen as polymorphic as the rest of ND family genes among camelids. Our phylogenetic study based on complete mitochondrial genomes excluding the control region suggested that the divergence of the two tribes may occur in the early Miocene; it is much earlier than what was deduced from the fossil record (11 million years). An evolutionary history reconstructed for the family Camelidae based on cytb sequences suggested that the split of bactrian camel and dromedary may have occurred in North America before the tribe Camelini migrated from North America to Asia.ConclusionMolecular clock analysis of complete mitochondrial genomes from C. bactrianus ferus and L. pacos suggested that the two tribes diverged from their common ancestor about 25 million years ago, much earlier than what was predicted based on fossil records.

BackgroundNext-generation DNA sequencing (NGS) technologies have made huge impacts in many fields of biological research, but especially in evolutionary biology. One area where NGS has shown potential is for high-throughput sequencing of complete mtDNA genomes (of humans and other animals). Despite the increasing use of NGS technologies and a better appreciation of their importance in answering biological questions, there remain significant obstacles to the successful implementation of NGS-based projects, especially for new users.ResultsHere we present an ‘A to Z’ protocol for obtaining complete human mitochondrial (mtDNA) genomes – from DNA extraction to consensus sequence. Although designed for use on humans, this protocol could also be used to sequence small, organellar genomes from other species, and also nuclear loci. This protocol includes DNA extraction, PCR amplification, fragmentation of PCR products, barcoding of fragments, sequencing using the 454 GS FLX platform, and a complete bioinformatics pipeline (primer removal, reference-based mapping, output of coverage plots and SNP calling).ConclusionsAll steps in this protocol are designed to be straightforward to implement, especially for researchers who are undertaking next-generation sequencing for the first time. The molecular steps are scalable to large numbers (hundreds) of individuals and all steps post-DNA extraction can be carried out in 96-well plate format. Also, the protocol has been assembled so that individual ‘modules’ can be swapped out to suit available resources.

BackgroundThe scientific literature contains many examples where DNA sequence analyses have been used to provide definitive answers to phylogenetic problems that traditional (non-DNA based) approaches alone have failed to resolve. One notable example concerns the rhinoceroses, a group for which several contradictory phylogenies were proposed on the basis of morphology, then apparently resolved using mitochondrial DNA fragments.ResultsIn this study we report the first complete mitochondrial genome sequences of the extinct ice-age woolly rhinoceros (Coelodonta antiquitatis), and the threatened Javan (Rhinoceros sondaicus), Sumatran (Dicerorhinus sumatrensis), and black (Diceros bicornis) rhinoceroses. In combination with the previously published mitochondrial genomes of the white (Ceratotherium simum) and Indian (Rhinoceros unicornis) rhinoceroses, this data set putatively enables reconstruction of the rhinoceros phylogeny. While the six species cluster into three strongly supported sister-pairings: (i) The black/white, (ii) the woolly/Sumatran, and (iii) the Javan/Indian, resolution of the higher-level relationships has no statistical support. The phylogenetic signal from individual genes is highly diffuse, with mixed topological support from different genes. Furthermore, the choice of outgroup (horse vs tapir) has considerable effect on reconstruction of the phylogeny. The lack of resolution is suggestive of a hard polytomy at the base of crown-group Rhinocerotidae, and this is supported by an investigation of the relative branch lengths.ConclusionSatisfactory resolution of the rhinoceros phylogeny may not be achievable without additional analyses of substantial amounts of nuclear DNA. This study provides a compelling demonstration that, in spite of substantial sequence length, there are significant limitations with single-locus phylogenetics. We expect further examples of this to appear as next-generation, large-scale sequencing of complete mitochondrial genomes becomes commonplace in evolutionary studies."The human factor in classification is nowhere more evident than in dealing with this superfamily (Rhinocerotoidea)." G. G. Simpson (1945)

Complete mitochondrial genome and evolutionary analysis of Turritopsis dohrnii, the “immortal” jellyfish with a reversible life-cycle

Despite more than a century of debate, the evolutionary position of turtles (Testudines) relative to other amniotes (reptiles, birds, and mammals) remains uncertain. One of the major impediments to resolving this important evolutionary problem is the highly distinctive and enigmatic morphology of turtles that led to their traditional placement apart from diapsid reptiles as sole descendants of presumably primitive anapsid reptiles. To address this question, the complete (16,787-bp) mitochondrial genome sequence of the African side-necked turtle (Pelomedusa subrufa) was determined. This molecule contains several unusual features: a (TA)n microsatellite in the control region, the absence of an origin of replication for the light strand in the WANCY region of five tRNA genes, an unusually long noncoding region separating the ND5 and ND6 genes, an overlap between ATPase 6 and COIII genes, and the existence of extra nucleotides in ND3 and ND4L putative ORFs. Phylogenetic analyses of the complete mitochondrial genome sequences supported the placement of turtles as the sister group of an alligator and chicken (Archosauria) clade. This result clearly rejects the Haematothermia hypothesis (a sister-group relationship between mammals and birds), as well as rejecting the placement of turtles as the most basal living amniotes. Moreover, evidence from both complete mitochondrial rRNA genes supports a sister-group relationship of turtles to Archosauria to the exclusion of Lepidosauria (tuatara, snakes, and lizards). These results challenge the classic view of turtles as the only survivors of primary anapsid reptiles and imply that turtles might have secondarily lost their skull fenestration.

Uruguayan beef is one of the most popular products in the export market. In this study, we report the complete mitochondrial genome sequence of Uruguayan native cattle for the first time. The total mitochondrial genome sequence is 16,339 bp in length with the base composition of 33.4% for A, 27.2% for T, 26.0% for C, and 13.4% for G. The description of all genes is similar to the typical mitochondrial genomes of cattle. The annotated mitochondrial genome of Uruguayan native cattle would serve as an important genetic data set for further study.

Deep resequencing of Trichinella spiralis reveals previously un-described single nucleotide polymorphisms and intra-isolate variation within the mitochondrial genome

Ortleppascaris sinensis (Nematoda: Ascaridida) is a dominant intestinal nematode of the captive Chinese alligator. However, the epidemiology, molecular ecology and population genetics of this parasite remain largely unexplored. In this study, the complete mitochondrial (mt) genome sequence of O. sinensis was first determined using a polymerase chain reaction (PCR)-based primer-walking strategy, and this is also the first sequencing of the complete mitochondrial genome of a member of the genus Ortleppascaris. The circular mitochondrial genome (13,828 bp) of O. sinensis contained 12 protein-coding, 22 transfer RNA and 2 ribosomal RNA genes, but lacked the ATP synthetase subunit 8 gene. Finally, phylogenetic analysis of mtDNAs indicated that the genus Ortleppascaris should be attributed to the family Heterocheilidae. It is necessary to sequence more mtNDAs of Ortleppascaris nematodes in the future to test and confirm our conclusion. The complete mitochondrial genome sequence of O. sinensis reported here should contribute to molecular diagnosis, epidemiological investigations and ecological studies of O. sinensis and other related Ascaridida nematodes.

Red algae are widely used as a source of health-promoting bioactive compounds and dietary fibers in health foods. The identification and classification of red algal species based on morphological and molecular characteristics is challenging because of the similarity of the thallus and its high degree of plasticity and because complete mitochondrial genomes have only been reported for a few species. In this study, the complete mitochondrial genome sequencing of the red macroalga Chondracanthus tenellus (Harvey) (Hommersand et al., Hydrobiologia 260:105-120, 1993)) (Rhodophyta, Gigartinales) was performed for the first time. Additionally, we aimed to reconstruct the phylogenetic relationships of the species within the order Gigartinales using complete mitochondrial genome sequences. Genomic DNA was extracted, analyzed by whole-genome sequencing (WGS), and assembled using NOVOPlasty. The mitochondrial genome sequence was annotated, and both a genome map and a phylogenetic tree were constructed using maximum likelihood analysis. The mitochondrial genome was 25,928bp in length, had strongly biased [AT] content (72.08%), and comprised 3 rRNAs, 23 tRNAs, and 24 protein-coding genes (PCGs). In comparison with the mitochondrial genome of other red algae, that of C. tenellus lacks rpl5 and rpl20. Based on a phylogenetic study of the complete mitochondrial genome, C. tenellus belongs to the family Gigartinaceae and is monophyletic with other species of the order Gigartinales. This is the first report of C. tenellus complete mitochondrial genome; its characteristics are consistent with those of other red algae. The study of genomic data will be beneficial for future comparative genomics, phylogenetics, and evolutionary studies.

Next-generation sequencing and phylogenetic signal of complete mitochondrial genomes for resolving the evolutionary history of leaf-nosed bats (Phyllostomidae)

The first complete mitochondrial genome sequencing of golden tree snake, gliding snake, ornate flying snake, golden flying snake, and flying tree snake (Chrysopelea ornate_shaw 1802) was conducted utilizing Next-generation sequencing technologies. The complete mitochondrial genome of flying tree snake is 17,252 bp long with a base composition of A-36%, T- 27%, G-12%, C-25% with a GC content of 38%. Chrysopelea ornata also shows mitogenome organization of 37 genes including 13 protein-coding genes, 22 tRNA genes, two rRNA genes and two non-coding regions. Nine genes including eight tRNAs and NAD6 were encoded. Phylogenetic investigations, utilizing the entire mitochondrial genome of chrysopelea ornata, reveal a strong association with the Colubridae family.

BackgroundThere are many advantages to the application of complete mitochondrial (mt) genomes in the accurate reconstruction of phylogenetic relationships in Metazoa. Although over one thousand metazoan genomes have been sequenced, the taxonomic sampling is highly biased, left with many phyla without a single representative of complete mitochondrial genome. Sipuncula (peanut worms or star worms) is a small taxon of worm-like marine organisms with an uncertain phylogenetic position. In this report, we present the mitochondrial genome sequence of Phascolosoma esculenta, the first complete mitochondrial genome of the phylum.ResultsThe mitochondrial genome of P.esculenta is 15,494 bp in length. The coding strand consists of 32.1% A, 21.5% C, 13.0% G, and 33.4% T bases (AT = 65.5%; AT skew = -0.019; GC skew = -0.248). It contains thirteen protein-coding genes (PCGs) with 3,709 codons in total, twenty-two transfer RNA genes, two ribosomal RNA genes and a non-coding AT-rich region (AT = 74.2%). All of the 37 identified genes are transcribed from the same DNA strand. Compared with the typical set of metazoan mt genomes, sipunculid lacks trnR but has an additional trnM. Maximum Likelihood and Bayesian analyses of the protein sequences show that Myzostomida, Sipuncula and Annelida (including echiurans and pogonophorans) form a monophyletic group, which supports a closer relationship between Sipuncula and Annelida than with Mollusca, Brachiopoda, and some other lophotrochozoan groups.ConclusionThis is the first report of a complete mitochondrial genome as a representative within the phylum Sipuncula. It shares many more similar features with the four known annelid and one echiuran mtDNAs. Firstly, sipunculans and annelids share quite similar gene order in the mitochondrial genome, with all 37 genes located on the same strand; secondly, phylogenetic analyses based on the concatenated protein sequences also strongly support the sipunculan + annelid clade (including echiurans and pogonophorans). Hence annelid "key-characters" including segmentation may be more labile than previously assumed.

Cattle spread throughout the American continent during the colonization years, originating creole breeds that adapted to a wide range of climate conditions. The population of creole cattle in Peru is decreasing mainly due to the introduction of more productive breeds in recent years. During the last 15 years, there has been significant progress in cattle genomics. However, little is known about the genetics of the Peruvian creole cattle (PCC) despite its importance to (i) improving productivity in the Andean region, (ii) agricultural labor, and (iii) cultural traditions. In addition, the origin and phylogenetic relationship of the PCC are still unclear. In order to promote the conservation of the PCC, we sequenced the mitochondrial genome of a creole bull, which also possessed exceptional fighting skills and was employed for agricultural tasks, from the highlands of Arequipa for the first time. The total mitochondrial genome sequence is 16,339 bp in length with the base composition of 31.43% A, 28.64% T, 26.81% C, and 13.12% G. It contains 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a control region. Among the 37 genes, 28 were positioned on the H-strand and 9 were positioned on the L-strand. The most frequently used codons were CUA (leucine), AUA (isoleucine), AUU (isoleucine), AUC (isoleucine), and ACA (threonine). Maximum likelihood reconstruction using complete mitochondrial genome sequences showed that the PCC is related to native African breeds. The annotated mitochondrial genome of PCC will serve as an important genetic data set for further breeding work and conservation strategies.

BackgroundOutbreaks of cyclosporiasis, a diarrheal illness caused by Cyclospora cayetanensis, have been a public health issue in the USA since the mid 1990’s. In 2018, 2299 domestically acquired cases of cyclosporiasis were reported in the USA as a result of multiple large outbreaks linked to different fresh produce commodities. Outbreak investigations are hindered by the absence of standardized molecular epidemiological tools for C. cayetanensis. For other apicomplexan coccidian parasites, multicopy organellar DNA such as mitochondrial genomes have been used for detection and molecular typing.MethodsWe developed a workflow to obtain complete mitochondrial genome sequences from cilantro samples and clinical samples for typing of C. cayetanensis isolates. The 6.3 kb long C. cayetanensis mitochondrial genome was amplified by PCR in four overlapping amplicons from genomic DNA extracted from cilantro, seeded with oocysts, and from stool samples positive for C. cayetanensis by diagnostic methods. DNA sequence libraries of pooled amplicons were prepared and sequenced via next-generation sequencing (NGS). Sequence reads were assembled using a custom bioinformatics pipeline.ResultsThis approach allowed us to sequence complete mitochondrial genomes from the samples studied. Sequence alterations, such as single nucleotide polymorphism (SNP) profiles and insertion and deletions (InDels), in mitochondrial genomes of 24 stool samples from patients with cyclosporiasis diagnosed in 2014, exhibited discriminatory power. The cluster dendrogram that was created based on distance matrices of the complete mitochondrial genome sequences, indicated distinct strain-level diversity among the 2014 C. cayetanensis outbreak isolates analyzed in this study.ConclusionsOur results suggest that genomic analyses of mitochondrial genome sequences may help to link outbreak cases to the source.