Mitochondrial Assemblies Research Articles

The genome sequence of common reed, Phragmites australis (Cav.) Steud. (Poaceae).

We present a genome assembly from an individual Phragmites australis (the common reed; Streptophyta; Magnoliopsida; Poales; Poaceae). The genome sequence has a total length of 848.70 megabases. Most of the assembly is scaffolded into 24 chromosomal pseudomolecules, supporting the specimen being an allotetraploid (2 n = 4 x = 48). The three mitochondrial assemblies had lengths of 304.58, 92.24, and 76.54 kilobases and the plastid genome assembly had a length of 137.67 kilobases. Gene annotation of this assembly on Ensembl identified 47,513 protein-coding genes.

The genome sequence of black poplar, Populus nigra subsp. betulifolia L., 1753 (Salicaceae)

We present a genome assembly from an individual Populus nigra subsp. betulifola (black poplar; Tracheophyta; Malpighiales; Salicaceae). The genome sequence is 413.2 megabases in span. Most of the assembly (99.73%) is scaffolded into 19 chromosomal pseudomolecules. Mitochondrial and plastid genomes were also assembled. Three mitochondrial assemblies have lengths of 281.85, 335.57 and 186.15 kilobases, and the plastid genome has a length of 156.37 kilobases.

Mitochondrial genome diversity across the subphylum Saccharomycotina.

Eukaryotic life depends on the functional elements encoded by both the nuclear genome and organellar genomes, such as those contained within the mitochondria. The content, size, and structure of the mitochondrial genome varies across organisms with potentially large implications for phenotypic variance and resulting evolutionary trajectories. Among yeasts in the subphylum Saccharomycotina, extensive differences have been observed in various species relative to the model yeast Saccharomyces cerevisiae, but mitochondrial genome sampling across many groups has been scarce, even as hundreds of nuclear genomes have become available. By extracting mitochondrial assemblies from existing short-read genome sequence datasets, we have greatly expanded both the number of available genomes and the coverage across sparsely sampled clades. Comparison of 353 yeast mitochondrial genomes revealed that, while size and GC content were fairly consistent across species, those in the genera Metschnikowia and Saccharomyces trended larger, while several species in the order Saccharomycetales, which includes S. cerevisiae, exhibited lower GC content. Extreme examples for both size and GC content were scattered throughout the subphylum. All mitochondrial genomes shared a core set of protein-coding genes for Complexes III, IV, and V, but they varied in the presence or absence of mitochondrially-encoded canonical Complex I genes. We traced the loss of Complex I genes to a major event in the ancestor of the orders Saccharomycetales and Saccharomycodales, but we also observed several independent losses in the orders Phaffomycetales, Pichiales, and Dipodascales. In contrast to prior hypotheses based on smaller-scale datasets, comparison of evolutionary rates in protein-coding genes showed no bias towards elevated rates among aerobically fermenting (Crabtree/Warburg-positive) yeasts. Mitochondrial introns were widely distributed, but they were highly enriched in some groups. The majority of mitochondrial introns were poorly conserved within groups, but several were shared within groups, between groups, and even across taxonomic orders, which is consistent with horizontal gene transfer, likely involving homing endonucleases acting as selfish elements. As the number of available fungal nuclear genomes continues to expand, the methods described here to retrieve mitochondrial genome sequences from these datasets will prove invaluable to ensuring that studies of fungal mitochondrial genomes keep pace with their nuclear counterparts.

Nuclear and mitochondrial genome assemblies of the beetle, Zygogramma bicolorata, a globally important biocontrol agent of invasive weed Parthenium hysterophorus.

Implementing a genetic-based approach to achieve the full potential of classical biocontrol programs has been advocated for decades. The availability of genome-level information brings the opportunity to scrutinize the biocontrol traits for their efficacy and evolvability. However, implementation of this advocacy remain limited to few instances. Biocontrol of a globally noxious weed, Parthenium hysterophorus, by the leaf-feeding beetle, Zygogramma bicolorata, has been in place for more than four decades now, with varying levels of success. Towards the first step in provisioning genetic-based improvement to this biocontrol program, we describe the nuclear and mitochondrial assemblies of Z. bicolorata. We assembled the genome from the long-read sequence data, error-corrected with high throughput short-reads and checked for contaminants and sequence duplication to produce a 936 Mb nuclear genome. With 96.5% BUSCO completeness and the LAI index 12.91, we present a reference quality assembly, which appeared to be repeat-rich at 62.7% genome-wide and consists of 29437 protein-coding regions. We detected signature of NUMTs in 80 nuclear positions comprising 13 Kb out of 17.9 Kb mitochondria genome sequence. This genome, along with its annotations, provides a valuable resource to gain further insights into the biocontrol traits of Z. bicolorata in improving the control of the invasive weed Parthenium hysterophorus.

Evolutionary history of an Alpine Archaeognath (Machilis pallida): Insights from different variant.

Reconstruction of species histories is a central aspect of evolutionary biology. Patterns of genetic variation within and among populations can be leveraged to elucidate evolutionary processes and demographic histories. However, interpreting genetic signatures and unraveling the contributing processes can be challenging, in particular for non-model organisms with complex reproductive modes and genome organization. One way forward is the combined consideration of patterns revealed by different molecular markers (nuclear vs. mitochondrial) and types of variants (common vs. rare) that differ in their age, mode, and rate of evolution. Here, we applied this approach to RNAseq data generated for Machilis pallida (Archaeognatha), an Alpine jumping bristletail considered parthenogenetic and triploid. We generated de novo transcriptome and mitochondrial assemblies to obtain high-density data to investigate patterns of mitochondrial and common and rare nuclear variation in 17 M. pallida individuals sampled from all known populations. We find that the different variant types capture distinct aspects of the evolutionary history and discuss the observed patterns in the context of parthenogenesis, polyploidy, and survival during glaciation. This study highlights the potential of different variant types to gain insights into evolutionary scenarios even from challenging but often available data and the suitability of M. pallida and the genus Machilis as a study system for the evolution of sexual strategies and polyploidization during environmental change. We also emphasize the need for further research which will be stimulated and facilitated by these newly generated resources and insights.

RKOMICS: an R package for processing mitochondrial minicircle assemblies in population-scale genome projects

BackgroundThe advent of population-scale genome projects has revolutionized our biological understanding of parasitic protozoa. However, while hundreds to thousands of nuclear genomes of parasitic protozoa have been generated and analyzed, information about the diversity, structure and evolution of their mitochondrial genomes remains fragmentary, mainly because of their extraordinary complexity. Indeed, unicellular flagellates of the order Kinetoplastida contain structurally the most complex mitochondrial genome of all eukaryotes, organized as a giant network of homogeneous maxicircles and heterogeneous minicircles. We recently developed KOMICS, an analysis toolkit that automates the assembly and circularization of the mitochondrial genomes of Kinetoplastid parasites. While this tool overcomes the limitation of extracting mitochondrial assemblies from Next-Generation Sequencing datasets, interpreting and visualizing the genetic (dis)similarity within and between samples remains a time-consuming process.ResultsHere, we present a new analysis toolkit—rKOMICS—to streamline the analyses of minicircle sequence diversity in population-scale genome projects. rKOMICS is a user-friendly R package that has simple installation requirements and that is applicable to all 27 trypanosomatid genera. Once minicircle sequence alignments are generated, rKOMICS allows to examine, summarize and visualize minicircle sequence diversity within and between samples through the analyses of minicircle sequence clusters. We showcase the functionalities of the (r)KOMICS tool suite using a whole-genome sequencing dataset from a recently published study on the history of diversification of the Leishmania braziliensis species complex in Peru. Analyses of population diversity and structure highlighted differences in minicircle sequence richness and composition between Leishmania subspecies, and between subpopulations within subspecies.ConclusionThe rKOMICS package establishes a critical framework to manipulate, explore and extract biologically relevant information from mitochondrial minicircle assemblies in tens to hundreds of samples simultaneously and efficiently. This should facilitate research that aims to develop new molecular markers for identifying species-specific minicircles, or to study the ancestry of parasites for complementary insights into their evolutionary history.

Genetic diversity across the mitochondrial genome of eastern oysters (Crassostrea virginica) in the northern Gulf of Mexico.

The eastern oyster, Crassostrea virginica, is divided into four populations along the western North Atlantic, however, the only published mitochondrial genome sequence was assembled using one individual in Delaware. This study aimed to (1) assemble C. virginica mitochondrial genomes from Texas with pooled restriction-site-associated DNA sequencing (ezRAD), (2) evaluate the validity of the mitochondrial genome assemblies including comparison with Sanger sequencing data, and (3) evaluate genetic differentiation both between the Delaware and Texas genomes, as well as among three bays in Texas. The pooled-genome-assembled-genomes (PAGs) from Texas exhibited several characteristics indicating that they were valid, including elevated nucleotide diversity in non-coding and the third position of codons, placement as the sister haplotype of the genome from Delaware in a phylogenetic reconstruction of Crassostrea mitochondrial genomes, and a lack of genetic structure in the ND4 gene among the three Texas bays as was found with Sanger amplicons in samples from the same bays several years prior. In the comparison between the Delaware and Texas genome, 27 of 38 coding regions exhibited variability between the two populations, which were differentiated by 273 mutations, versus 1–13 mutations among the Texas samples. Using the full PAGs, there was no additional evidence for population structure among the three Texas bays. While population genetics is rapidly moving towards larger high-density datasets, studies of mitochondrial DNA (and genomes) can be particularly useful for comparing historic data prior to the modern era of genomics. As such, being able to reliably compile mitochondrial genomes from genomic data can improve the ability to compare results across studies.

CONSULT: accurate contamination removal using locality-sensitive hashing.

A fundamental question appears in many bioinformatics applications: Does a sequencing read belong to a large dataset of genomes from some broad taxonomic group, even when the closest match in the set is evolutionarily divergent from the query? For example, low-coverage genome sequencing (skimming) projects either assemble the organelle genome or compute genomic distances directly from unassembled reads. Using unassembled reads needs contamination detection because samples often include reads from unintended groups of species. Similarly, assembling the organelle genome needs distinguishing organelle and nuclear reads. While k-mer-based methods have shown promise in read-matching, prior studies have shown that existing methods are insufficiently sensitive for contamination detection. Here, we introduce a new read-matching tool called CONSULT that tests whether k-mers from a query fall within a user-specified distance of the reference dataset using locality-sensitive hashing. Taking advantage of large memory machines available nowadays, CONSULT libraries accommodate tens of thousands of microbial species. Our results show that CONSULT has higher true-positive and lower false-positive rates of contamination detection than leading methods such as Kraken-II and improves distance calculation from genome skims. We also demonstrate that CONSULT can distinguish organelle reads from nuclear reads, leading to dramatic improvements in skim-based mitochondrial assemblies.

Mitofusin-2 mediates doxorubicin sensitivity and acute resistance in Jurkat leukemia cells

Mitochondria oscillate along a morphological continuum from fragmented individual units to hyperfused tubular networks. Their position at the junction of catabolic and anabolic metabolism couples this morphological plasticity, called mitochondrial dynamics, to larger cellular metabolic programs, which in turn implicate mitochondria in a number of disease states. In many cancers, fragmented mitochondria engage the cell with the biosynthetic capacity of aerobic glycolysis in service of proliferation and progression. Chemo-resistant cancers, however, favor remodeling dynamics that yield fused mitochondrial assemblies utilizing oxidative phosphorylation (OXPHOS) through the electron transport chain (ETC). In this study, expression of Mitofusin-2 (MFN-2), a GTPase protein mediator of mitochondrial fusion, was found to closely correlate to Jurkat leukemia cell survival post doxorubicin (DxR) assault. Moreover, this was accompanied by dramatically increased expression of OXPHOS respiratory complexes and ATP Synthase, as well as a commensurate escalation of state III respiration and respiratory control ratio (RCR). Importantly, CRISPR knockout of MFN-2 resulted in a considerable decrease of doxorubicin (DxR) median lethal dose compared to a treated wildtype control, suggesting an important role of mitochondrial fusion in chemotherapy sensitivity and acute resistance.

299-OR: Loss of Insulin and IGF1 Receptors in Muscle Impairs Complex-I Dependent Mitochondrial Bioenergetics and Supercomplex Formation via Foxo Transcription Factors

Diabetes is associated with decreased muscle strength and mitochondrial metabolism which can lead to disability. Recently, we reported that insulin receptor (IR) and IGF-1 receptor (IGF1R) prevent muscle atrophy via suppression of FoxOs, but how FoxOs interact with mitochondrial function remains unclear. To test whether IR/IGF1R suppression of FoxOs coordinates muscle strength with mitochondrial function, we measured mitochondrial bioenergetics and RNA-Seq in mice lacking IR, IGF1R, and/or FoxO1/3/4 in muscle. Perinatal muscle-specific deletion of IR (MIRKO) showed a mild 20% decrease in pyruvate-driven respiratory capacity in soleus permeabilized fibers, whereas succinate/Rotenone respiration was not different. Deletion of both IR/IGF1R (MIGIRKO) showed a marked 60% decrease in pyruvate respiration and ATP production in soleus fibers. These mitochondrial abnormalities were reversed in muscle-specific IR/IGF1R and FoxO1/3/4 quintuple knockout (QKO), suggesting FoxO activation mediates the mitochondrial decline. FoxOs are known inducers of autophagy, but mitophagy quantification using MitoTIMER or mitoKeima biosensors was not altered in Day 21 Tamoxifen-inducible IR/IGF1R KO in adult muscle (IND-IGIRKO) or by acute in-vitro deletion of IR/IGF1R primary myotubes. Complex-I activity was decreased 50-70% in MIGIRKO and Day 21 IND-IGIRKO muscle, but other complex activities were unchanged. Furthermore, 14 complex-I subunits were decreased in MIGIRKO by RNA-Seq, and 13 of these were reversed in QKO. IND-IGIRKO also showed a decrease in pyruvate respiration, ATP production, complex-I activity, and complex-I/supercomplex content by blue-native gels 21 days after tamoxifen. Thus, loss of IR or both IR/IGF1R in muscle decreases complex-I driven mitochondrial respiration and supercomplex assemblies, at least in part by FoxO-mediated downregulation of mitochondrial genes. Disclosure G. Bhardwaj: None. C.M. Penniman: None. P.A. Suarez Beltran: None. J. Jena: None. C.M. Foster: None. K. Poro: None. T. Junck: None. A. Hinton: None. R. Souvenir: None. J. Fuqua: None. P.E. Morales: None. R. Bravo-Sagua: None. V.A. Lira: None. E.D. Abel: None. B.T. ONeill: None.

Pulse-chase SILAC–based analyses reveal selective oversynthesis and rapid turnover of mitochondrial protein components of respiratory complexes

Mammalian mitochondria assemble four complexes of the respiratory chain (RCI, RCIII, RCIV, and RCV) by combining 13 polypeptides synthesized within mitochondria on mitochondrial ribosomes (mitoribosomes) with over 70 polypeptides encoded in nuclear DNA, translated on cytoplasmic ribosomes, and imported into mitochondria. We have previously observed that mitoribosome assembly is inefficient because some mitoribosomal proteins are produced in excess, but whether this is the case for other mitochondrial assemblies such as the RCs is unclear. We report here that pulse-chase stable isotope labeling with amino acids in cell culture (SILAC) is a valuable technique to study RC assembly because it can reveal considerable differences in the assembly rates and efficiencies of the different complexes. The SILAC analyses of HeLa cells indicated that assembly of RCV, comprising F1/Fo-ATPase, is rapid with little excess subunit synthesis, but that assembly of RCI (i.e. NADH dehydrogenase) is far less efficient, with dramatic oversynthesis of numerous proteins, particularly in the matrix-exposed N and Q domains. Unassembled subunits were generally degraded within 3 h. We also observed differential assembly kinetics for individual complexes that were immunoprecipitated with complex-specific antibodies. Immunoprecipitation with an antibody that recognizes the ND1 subunit of RCI co-precipitated a number of proteins implicated in FeS cluster assembly and newly synthesized ubiquinol-cytochrome c reductase Rieske iron-sulfur polypeptide 1 (UQCRFS1), the Rieske FeS protein in RCIII, reflecting some coordination between RCI and RCIII assemblies. We propose that pulse-chase SILAC labeling is a useful tool for studying rates of protein complex assembly and degradation.

Mind the gap! The mitochondrial control region and its power as a phylogenetic marker in echinoids

BackgroundIn Metazoa, mitochondrial markers are the most commonly used targets for inferring species-level molecular phylogenies due to their extremely low rate of recombination, maternal inheritance, ease of use and fast substitution rate in comparison to nuclear DNA. The mitochondrial control region (CR) is the main non-coding area of the mitochondrial genome and contains the mitochondrial origin of replication and transcription.While sequences of the cytochrome oxidase subunit 1 (COI) and 16S rRNA genes are the prime mitochondrial markers in phylogenetic studies, the highly variable CR is typically ignored and not targeted in such analyses. However, the higher substitution rate of the CR can be harnessed to infer the phylogeny of closely related species, and the use of a non-coding region alleviates biases resulting from both directional and purifying selection. Additionally, complete mitochondrial genome assemblies utilizing next generation sequencing (NGS) data often show exceptionally low coverage at specific regions, including the CR. This can only be resolved by targeted sequencing of this region.ResultsHere we provide novel sequence data for the echinoid mitochondrial control region in over 40 species across the echinoid phylogenetic tree. We demonstrate the advantages of directly targeting the CR and adjacent tRNAs to facilitate complementing low coverage NGS data from complete mitochondrial genome assemblies. Finally, we test the performance of this region as a phylogenetic marker both in the lab and in phylogenetic analyses, and demonstrate its superior performance over the other available mitochondrial markers in echinoids.ConclusionsOur target region of the mitochondrial CR (1) facilitates the first thorough investigation of this region across a wide range of echinoid taxa, (2) provides a tool for complementing missing data in NGS experiments, and (3) identifies the CR as a powerful, novel marker for phylogenetic inference in echinoids due to its high variability, lack of selection, and high compatibility across the entire class, outperforming conventional mitochondrial markers.

Genomic insights into the mitochondria of 11 eastern North American species of Cladonia

Cladonia is among the most species-rich genera of lichens globally. Species in this lineage, commonly referred to as reindeer lichens, are ecologically important in numerous regions worldwide. In some locations, species of Cladonia can comprise the dominant groundcover, and are a major food source for caribou and other mammals. Additionally, many species are known to produce substances with antimicrobial properties or other characteristics with potentially important medical applications. This exceptional morphological and ecological variation contrasts sharply with the limited molecular divergence often observed among species. As a new resource to facilitate ongoing and future studies of these important species, we analyse here the sequences of 11 Cladonia mitochondrial genomes, including new mitochondrial genome assemblies and annotations representing nine species: C. apodocarpa, C. caroliniana, C. furcata, C. leporina, C. petrophila, C. peziziformis, C. robbinsii, C. stipitata, and C. subtenuis. These 11 genomes varied in size, intron content, and complement of tRNAs. Genes annotated within these mitochondrial genomes include 15 protein-coding genes, the large and small ribosomal subunits (mtLSU and mtSSU), and 23–26 tRNAs. All Cladonia mitochondrial genomes contained atp9, an important energy transport gene that has been lost evolutionarily in some lichen mycobiont mitochondria. Using a concatenated alignment of five mitochondrial genes (nad2, nad4, cox1, cox2, and cox3), a Bayesian phylogeny of relationships among species was inferred and was consistent with previously published phylogenetic relationships, highlighting the utility of these regions in reconstructing phylogenetic history.

Annotated mitochondrial genome assemblies for two sand lances (genus: Ammodytes) from the northwest Atlantic

Complete mitochondrial genomes of two northwest Atlantic sand lances (Ammodytes americanus and Ammodytes dubius) were sequenced, assembled, and annotated. Both genomes were 16 519 bp in length and were differentiated by a genetic distance of only 0.01. Furthermore, mitochondrial gene annotations were identical for both species. Phylogenetic analysis revealed that divergence between the two species was shallow, relative to other members of the genus.

Complete mitochondrial genome of the Macaca mulatta brevicaudus

The complete mitochondrial sequence of the Macaca mulatta brevicaudus has been determined by mapping the raw data to previously published mitochondrial assemblies of the corresponding species. The total sequence length is 16,561 bp, consisting of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and one D-loop control region. The base composition of the mtDNA genome is 31.77% A, 25.14% T, 30.33% C, and 12.76% G, with an AT content of 56.90%. The arrangement of genes in M. m. brevicaudus is identical to that of M. mulatta. All genes are encoded on the heavy strand with the exception of ND6 and eight tRNA genes. The mitochondrial genome of M. m. brevicaudus presented here will contribute to a better understanding of the population genetics, help to protect its genetic diversity and resolve phylogenetic relationships within the family.

Complete mitochondrial genome of the crested black macaque (Macaca nigra)

The complete mitochondrial sequence of the crested black macaque (Macaca nigra) has been determined by mapping the raw data to previously published mitochondrial assemblies of the corresponding species. The total sequence length is 16,564 bp and includes 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and 1 D-loop control region. The base composition of mtDNA genome is 31.76% A, 25.27% T, 30.17% C, and 12.80% G, with an AT content of 57.03%. The arrangement of genes in M. nigra is identical to that of M. mulatta. All genes are encoded on the heavy strand with the exception of ND6 and eight tRNA genes. The mitochondrial genome of M. nigra presented here will contribute to a better understanding of the population genetics, help to protect its genetic diversity and resolve phylogenetic relationships within the family.

The Organization of mitochondria in growing hyphae of Neurospora crassa

Details of mitochondrial system organization and behavior in the tip of growing N. crassa hyphae and hyphal fragments were studied by means of cell-permeable mitochondria-selective probes in glucose- and sorbitol-containing media. It was shown that filamentous mitochondria concentrate in the 30-μm apical zone of growing hyphae and hyphal fragments independently of the carbon source. The mitochondrial assemblies propagate forward with elongation of hypha, split upon apical branching, and are formed de novo when branches are formed away from the growing tip. These activities resemble microtubule behavior. Co-staining with Mitotracker Red and Mitotracker Green revealed possible functional heterogeneity in subpopulations of mitochondria. It was proposed that the observed features are governed by the microtubular network, while the primary function of mitochondria was to sustain the growth rate. The organizing role of electrical gradients on the growing tip and their influence on mitochondrial and microtubular networks are discussed.

Structural characterization of the capybara (Hydrochaeris hydrochaeris) tongue by light, scanning, and transmission electron microscopy

Capybara is the largest rodent in the world and displays a seasonally dependent herbivore feeding behavior. Here, we present an anatomical contribution for understand this fact, by light, scanning, and transmission electron microscopy methodologies for tongue tissue analysis. The histological preparations revealed filiform, fungiform, vallate, and foliate papillae on the dorsal mucosa of the capybara tongue. The epithelial layer exhibited a lining of keratinized stratified squamous epithelial cells. The lamina propria was characterized by a dense connective tissue composed of the primary and secondary papillar projections. We also revealed the original aspects of the connective papillae. The shapes of the papillae varied by region of the tongue, and filiform, fungiform, vallate, and foliate papillae and subjacent layers of muscular fibers were observed. Pyriform taste buds occupying the epithelial layer of fungiform, vallate and foliate papillae were identified and the intracellular components of the taste buds and the intracorpuscular amyelinated nerve fibers were observed. The taste buds were characterized by the distribution of granular endoplasmic reticulum throughout the perinuclear area, the Golgi apparatus, and mitochondrial assemblies of various distinct diameters. Mitochondrial accumulation was also observed in the collagen bundle-surrounded amyelinated nerve fibers beside the basal cells. Therefore, these peculiar anatomical descriptions may contribute to understanding the adaptation of the feeding behavior of capybaras in a seasonally changing environment.

Effect of carnosine on self-organization of mitochondrial assemblies in rat liver homogenate.

The effect of carnosine on self-organization of mitochondrial assemblies was studied in rat liver homogenate of quiescent and excited animals. It was shown in separate electron microscopy experiments with serial slices that under our conditions of preparation of homogenate, blocks of native mitochondrial-reticular network in the cell, assemblies of mitochondria, are kept. Carnosine was shown to prevent dissociation of assemblies during storage. Its effect is maximal for more dissociated assemblies from excited animals with decreased ability for self-organization. Prevention of disassembly of organelles by carnosine can serve as one of the mechanisms of carnosine-induced diminishing of muscle fatigue under prolonged work.