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Monogeneans of the genus Dactylogyrus Diesing, 1850, the largest genus in the family Dactylogyridae, mostly parasitize the gills of cyprinoid hosts; however, only 3 Dactylogyrus' mitochondrial genomes (mitogenomes) are studied so far. The aim of this research is to extend our understanding of the mitogenomes of Dactylogyrus. We sequenced the mitogenomes of D. crucifer and D. zandti isolated from Rutilus rutilus and Abramis brama orientalis in northwest China, and then we compared these mitogenomes with other monogeneans. We used Illumina NovaSeq to sequence the entire mitochondrial genomes of D. crucifer and D. zandti and characterized the mitogenomes to understand the gene structure, gene identity, the secondary structures of the 22 tRNA genes, and relative synonymous codon usage. We used the analytic Bayesian Information and Maximum Likelihood methods to determine their associated phylogenetic trees. The mitogenomes of D. crucifer and D. zandti were 14,403 and 18,584 bp, respectively. Organization and positioning of these genes were in accordance with Dactylogyrus lamellatus and Dactylogyrus tuba. The nucleotide composition of Dactylogyridae was different from other families of Monogenea, and the A+T count of genus Dactylogyrus (54 - 58.4 %) was lower than other genus species of the family Dactylogyridea (63.9 - 78.4 %) in protein-coding genes. Dactylogyrus members displayed a codon usage bias. The relative synonymous codon used by Dactylogyrus was not conserved and was lower than other monogeneans. The codon use patterns of closely-related species isolated from closely-related hosts were identical. Phylogenetic analyses using mitogenomic dataset produced Dactylogyrus isolated from host subfamily Leuciscinae formed a sister-group. Our results contributed significantly to an increased database of mitogenomes, more than 50 %, for Dactylogyrus that may help future studies of mitochondrial genes and codon uses for the analysis of monogenean phylogenetics.

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Nucleotide base composition plays an influential role in the molecular mechanisms involved in gene function, phenotype, and amino acid composition. GC content (proportion of guanine and cytosine in DNA sequences) shows a high level of variation within and among species. Many studies measure GC content in a small number of genes, which may not be representative of genome-wide GC variation. One challenge when assembling extensive genomic data sets for these studies is the significant amount of resources (monetary and computational) associated with data processing, and many bioinformatic tools have not been optimized for resource efficiency. Using a high-performance computing (HPC) cluster, we manipulated resources provided to the targeted gene assembly program, automated target restricted assembly method (aTRAM), to determine an optimum way to run the program to maximize resource use. Using our optimum assembly approach, we assembled and measured GC content of all of the protein-coding genes of a diverse group of parasitic feather lice. Of the 499 426 genes assembled across 57 species, feather lice were GC-poor (mean GC = 42.96%) with a significant amount of variation within and between species (GC range = 19.57%-73.33%). We found a significant correlation between GC content and standard deviation per taxon for overall GC and GC3, which could indicate selection for G and C nucleotides in some species. Phylogenetic signal of GC content was detected in both GC and GC3. This research provides a large-scale investigation of GC content in parasitic lice laying the foundation for understanding the basis of variation in base composition across species.

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Cyprinidae is the largest family in the order of freshwater fish Cypriniformes. Increased subfamily members of Cyprinidae have been suggested to be re-classified for decades. In this study, we sequenced the mitochondrial genomes (mitogenomes) of Leuciscus baicalensis and Rutilus rutilus collected from northwest China and compared with other closely related species to determine their associated family or subfamily. We used Illumina NovaSeq to sequence the entire mitochondrial genomes of Leuciscus baicalensis and Rutilus rutilus and characterized the mitogenomes by the gene structure, gene order, and the secondary structures of the 22 tRNA genes. We compared mitogenome features of Leuciscinae with other subfamilies in Cyprinidae. We used the analytic Bayesian Information and Maximum Likelihood methods to determine phylogenetic trees of 13 PCGs. The mitogenomes of Leuciscus baicalensis and Rutilus rutilus were 16,607 bp and 16,606 bp, respectively. Organization and location of these genes were consistent with already studied Leuciscinae fishes. Synonymous codon usage was conservative in Leuciscinae as compared with other subfamilies in Cyprinidae. Phylogenetic analysis indicated that Leuciscinae was a monophyletic group, and genus Leuciscus was a paraphyletic group. Our approach, for the first time, of studying comparative mitochondrial genomics and phylogenetics together provided a supportive platform to the analysis of population genetics and phylogeny for Leuciscinae. Our results indicated a promising potential of comparative mitochondrial genomics in the manifestation of phylogenetic relationships between fishes, leading us to a suggestion that mitogenomes should be routinely considered in clarifying phylogenetics of family and subfamily members of fish.

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A common genome composition pattern in eubacteria is an asymmetry between the leading and lagging strands resulting in opposite skew patterns in the two replichores that lie between the origin and terminus of replication. Although this pattern has been reported for a couple of isolated plastid genomes, it is not clear how widespread it is overall in this chromosome. Using a random walk approach, we examine plastid genomes outside of the land plants, which are excluded since they are known not to initiate replication at a single site, for such a pattern of asymmetry. Although it is not a common feature, we find that it is detectable in the plastid genome of species from several diverse lineages. The euglenozoa in particular show a strong skew pattern as do several rhodophytes. There is a weaker pattern in some chlorophytes but it is not apparent in other lineages. The ramifications of this for analyses of plastid evolution are discussed.

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Satellite DNA (satDNA) is a fast-evolving portion of eukaryotic genomes. The homogeneous and repetitive nature of such satDNA causes problems during the assembly of genomes, and therefore it is still difficult to study it in detail in nonmodel organisms as well as across broad evolutionary timescales. Here, we combined the use of short- and long-read data to explore the diversity and evolution of satDNA between individuals of the same species and between genera of birds spanning ~40 millions of years of bird evolution using birds-of-paradise (Paradisaeidae) and crow (Corvus) species. These avian species highlighted the presence of a GC-rich Corvoidea satellitome composed of 61 satellite families and provided a set of candidate satDNA monomers for being centromeric on the basis of length, abundance, homogeneity and transcription. Surprisingly, we found that the satDNA of crow species rapidly diverged between closely related species while the satDNA appeared more similar between birds-of-paradise species belonging to different genera.

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The complete nucleotide sequence of the mitochondrial (mt) genome of the demersal zebra seabream Diplodus cervinus (Lowe, 1838) was determined for the first time. The double stranded circular molecule is 16,559 base pairs (bp) in length and encodes for the typical 37 metazoan mitochondrial genes, and 2 non-coding regions (D-loop and L-origin). The gene arrangement of the D. cervinus mt genome follows the usual one for fishes. The nucleotide sequences of the mt protein coding and ribosomal genes of D. cervinus mt genome were aligned with orthologous sequences from representatives of the Sparidae family and phylogenetic relationships were inferred. Maximum likelihood analyses placed D. cervinus as a sister species of Diplodus sargus (Linnaeus, 1758).

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BACKGROUND: Robust analysis of DNA sequencing data needs to include a set of quality control steps to ensure that technical bias is kept to a minimum. A metric easily obtained is the frequency of each of the nucleobases for each position across all sequencing reads. Here, we explore the differences in nucleobase compositions of various library types produced by standard experimental methodologies.  Methods: We obtained the compositions of nearly 3000 publicly available datasets and subjected them to Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction for a two-dimensional representation of their composition characteristics.   Results: We find that most library types result in a specific composition profile. We use this to give an estimate of how strongly the composition of a test library resembles the profiles of previously published libraries, and how likely the test sample is to be of a particular type. We introduce Librarian, a user-friendly web application and command line tool which enables checking base compositions of test libraries against known library types.   Conclusions: Library preparation methods strongly influence the per position nucleobase content. By comparing test libraries to a database of previously published library types we can make predictions regarding the library preparation method. Librarian is a user-friendly tool to access this information for quality assurance purposes as discrepancies can flag potential irregularities very early on.

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Transposable elements exhibit a base composition that is often different from the genomic average and from hosts' genes. The most common compositional bias is towards Adenosine and Thymine, although this bias is not universal, and elements with drastically different base composition can coexist within the same genome. The AT-richness of transposable elements is apparently maladaptive because it results in poor transcription and sub-optimal translation of proteins encoded by the elements. The cause(s) of this unusual base composition remain unclear and have yet to be investigated. Here, I review what is known about the nucleotide content of transposable elements and how this content can affect the genome of their host as well as their own replication. The compositional bias of transposable elements could result from several non-exclusive processes including horizontal transfer, mutational bias, and selection. It appears that mutation alone cannot explain the high AT-content of transposons and that selection plays a major role in the evolution of the compositional bias. The reason why selection would favor a maladaptive nucleotide content remains however unexplained and is an area of investigation that clearly deserves attention.

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Cynipoidea is a medium-sized superfamily of Hymenoptera with diverse lifestyles. In this study, 16 mitochondrial genomes were newly sequenced, 11 of which were the first obtained mitochondrial genomes in the family Liopteridae and four subfamilies (Anacharitinae, Aspicerinae, Figitinae, and Parnipinae) of Figitidae. All of the newly sequenced mitogenomes have unique rearrangement types within Cynipoidea, whereas some gene patterns are conserved in several groups. nad5-nad4-nad4L-nad6-cytb was remotely inverted and two rRNA genes were translocated to nad3 downstream in Ibaliidae and three subfamilies (Anacharitinae, Eucoilinae, and Parnipinae within Figitidae); two rRNA genes in Aspicerinae, Figitinae, and Liopteridae were remotely inverted to the cytb-nad1 junction; rrnL-rrnS was translocated to the cytb-nad1 junction in Cynipidae. Phylogenetic inference suggested that Figitidae was a polyphyletic group, while the Ibaliidae nested deep within Cynipoidea and was a sister-group to the Figitidae. These results will improve our understanding of the gene rearrangement of the mitogenomes and the phylogenetic relationships in the Cynipoidea.

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BACKGROUND: Diplozoidae are monogenean (Monogenea: Polyopisthocotylea) fish parasites characterised by a unique life history: two larvae permanently fuse into an X-shaped "Siamese" organism. Taxonomy and phylogeny of Diplozoidae and Polyopisthocotylea remain unresolved due to the unavailability of molecular markers with sufficiently high resolution. Mitogenomes may be a suitable candidate, but there are currently only 12 available for the Polyopisthocotylea (three for Diplozoidae). The only available study of diplozoid mitogenomes found unique base composition patterns and elevated evolution rates in comparison with other Monogenean mitogenomes. METHODS: To further explore their evolution and generate molecular data for evolutionary studies, we sequenced the complete mitogenomes of two Diplozoidae species, Paradiplozoon homoion and Paradiplozoon yarkandense, and conducted a number of comparative mitogenomic analyses with other polyopisthocotyleans. RESULTS: We found further evidence that mitogenomes of Diplozoidae evolve at a unique, elevated rate, which was reflected in their exceptionally long branches, large sizes, unique base composition, skews, and very low gene sequence similarity levels between the two newly sequenced species. They also exhibited remarkably large overlaps between some genes. Phylogenetic analysis of Polyopisthocotylea resolved all major taxa as monophyletic, and Mazocraeidea was split into two major clades: (Diplozoidae) + (all four remaining families: Diclidophoridae, Chauhaneidae, Mazocraeidae and Microcotylidae). It also provided further confirmation that the genus Paradiplozoon is paraphyletic and requires a taxonomic revision, so the two species may have to be renamed Indodiplozoon homoion and Diplozoon yarkandense comb. nov. CONCLUSIONS: Although our findings indicate that mitogenomes may be a promising tool for resolving the phylogeny of Polyopisthocotylea, elevated evolutionary rates of Diplozoidae may cause phylogenetic artefacts, so future studies should pay caution to this problem. Furthermore, as the reason for their elevated evolution remains unknown, Diplozoidae are a remarkably interesting lineage for other types of evolutionary mitogenomic studies.

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The rate of mutations varies >100-fold across the genome, altering the rate of evolution, and susceptibility to genetic diseases. The strongest predictor of mutation rate is the sequence itself, varying 75-fold between trinucleotides. The fact that DNA sequence drives its own mutation rate raises a simple but important prediction; highly mutable sequences will mutate more frequently and eliminate themselves in favor of sequences with lower mutability, leading to a lower equilibrium mutation rate. However, purifying selection constrains changes in mutable sequences, causing higher rates of mutation. We conduct a simulation using real human mutation data to test if 1) DNA evolves to a low equilibrium mutation rate and 2) purifying selection causes a higher equilibrium mutation rate in the genome's most important regions. We explore how this simple process affects sequence evolution in the genome, and discuss the implications for modeling evolution and susceptibility to DNA damage.

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Flow cytometry (FCM) is currently the most widely-used method to establish nuclear DNA content in plants. Since simple, 1-3-parameter, flow cytometers, which are sufficient for most plant applications, are commercially available at a reasonable price, the number of laboratories equipped with these instruments, and consequently new FCM users, has greatly increased over the last decade. This paper meets an urgent need for comprehensive recommendations for best practices in FCM for different plant science applications. We discuss advantages and limitations of establishing plant ploidy, genome size, DNA base composition, cell cycle activity, and level of endoreduplication. Applications of such measurements in plant systematics, ecology, molecular biology research, reproduction biology, tissue cultures, plant breeding, and seed sciences are described. Advice is included on how to obtain accurate and reliable results, as well as how to manage troubleshooting that may occur during sample preparation, cytometric measurements, and data handling. Each section is followed by best practice recommendations; tips as to what specific information should be provided in FCM papers are also provided.

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The white seabream Diplodus sargus (Linnaeus, 1758) is a species of interest for commercial fisheries throughout its range of distribution and it is also reared using aquaculture techniques. Herein, we present the first complete sequence and annotation of the mitochondrial genome of this species. The D. sargus mitogenome is 16,515 base pairs in length and contains 13 protein-coding genes, 2 rRNA, 22 tRNA, and 2 non-coding regions (D-loop and L-origin). The overall nucleotide composition is: 27.3% A, 28.9% C, 26.8% T, and 17.0% G. Maximum likelihood analyses placed D. sargus as a sister species of Diplodus puntazzo. This study provides valuable information for further studying identification methods and evolutionary relationships of Sparidae species.

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Base composition skews (G-C/G+C) of mitochondrial genomes are believed to be primarily driven by mutational pressure, which is positively correlated with metabolic rate. In marine animals, metabolic rate is also positively correlated with locomotory capacity. Given the central role of mitochondria in energy metabolism, we hypothesised that selection for locomotory capacity should be positively correlated with the strength of purifying selection (dN/dS), and thus be negatively correlated with the skew magnitude. Therefore, these two models assume diametrically opposite associations between the metabolic rate and skew magnitude: positive correlation in the prevailing paradigm, and negative in our working hypothesis. We examined correlations between the skew magnitude, metabolic rate, locomotory capacity, and several other variables previously associated with mitochondrial evolution on 287 crustacean mitogenomes. Weakly locomotory taxa had higher skew magnitude and ω (dN/dS) values, but not the gene order rearrangement rate. Skew and ω magnitudes were correlated. Multilevel regression analyses indicated that three competing variables, body size, gene order rearrangement rate, and effective population size, had negligible impacts on the skew magnitude. In most crustacean lineages selection for locomotory capacity appears to be the primary factor determining the skew magnitude. Contrary to the prevailing paradigm, this implies that adaptive selection outweighs nonadaptive selection (mutation pressure) in crustaceans. However, we found indications that effective population size (nonadaptive factor) may outweigh the impact of locomotory capacity in sessile crustaceans (Thecostraca). In conclusion, skew magnitude is a product of the interplay between adaptive and nonadaptive factors, the balance of which varies among lineages.

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Inversions of the origin of replication (ORI) in mitochondrial genomes produce asymmetrical mutational pressures that can cause strong base composition skews. Due to skews often being overlooked, the total number of crustacean lineages that underwent ORI events remains unknown. We analysed skews, cumulative skew plots, conserved sequence motifs, and mitochondrial architecture of all 965 available crustacean mitogenomes (699 unique species). We found indications of an ORI in 159 (22.7%) species, and mapped these to 23 ORI events: 16 identified with confidence and 7 putative (13 newly proposed, and for 5 we improved the resolution). Two ORIs occurred at or above the order level: Isopoda and Copepoda. Shifts in skew plots are not a precise tool for identifying the replication mechanism. We discuss how ORIs can produce mutational bursts in mitogenomes and show how these can interfere with various types of evolutionary studies. Phylogenetic analyses were plagued by artefactual clustering, and ORI lineages exhibited longer branches, a higher number of synonymous substitutions, higher mutational saturation, and higher compositional heterogeneity. ORI events also affected codon usage and protein properties. We discuss how this may have caused erroneous interpretation of data in previous studies that did not account for skew patterns.

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The genetic material of the three domains of life (Bacteria, Archaea, and Eukaryota) is always double-stranded DNA, and their GC content (molar content of guanine plus cytosine) varies between ≈ 13% and ≈ 75%. Nucleotide composition is the simplest way of characterizing genomes. Despite this simplicity, it has several implications. Indeed, it is the main factor that determines, among other features, dinucleotide frequencies, repeated short DNA sequences, and codon and amino acid usage. Which forces drive this strong variation is still a matter of controversy. For rather obvious reasons, most of the studies concerning this huge variation and its consequences, have been done in free-living organisms. However, no recent comprehensive study of all known viruses has been done (that is, concerning all available sequences). Viruses, by far the most abundant biological entities on Earth, are the causative agents of many diseases. An overview of these entities is important also because their genetic material is not always double-stranded DNA: indeed, certain viruses have as genetic material single-stranded DNA, double-stranded RNA, single-stranded RNA, and/or retro-transcribing. Therefore, one may wonder if what we have learned about the evolution of GC content and its implications in prokaryotes and eukaryotes also applies to viruses. In this contribution, we attempt to describe compositional properties of ∼ 10,000 viral species: base composition (globally and according to Baltimore classification), correlations among non-coding regions and the three codon positions, and the relationship of the nucleotide frequencies and codon usage of viruses with the same feature of their hosts. This allowed us to determine how the base composition of phages strongly correlate with the value of their respective hosts, while eukaryotic viruses do not (with fungi and protists as exceptions). Finally, we discuss some of these results concerning codon usage: reinforcing previous results, we found that phages and hosts exhibit moderate to high correlations, while for eukaryotes and their viruses the correlations are weak or do not exist.

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The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading rapidly all over the world and has raised grave concern globally. The present research aims to conduct a robust base compositional analysis of SARS-CoV-2 to reveal adaptive intricacies to the human host. Multivariate statistical analysis revealed a complex interplay of various factors including compositional constraint, natural selection, length of viral coding sequences, hydropathicity, and aromaticity of the viral gene products that are operational to codon usage patterns, with compositional bias being the most crucial determinant. UpG and CpA dinucleotides were found to be highly preferred whereas, CpG dinucleotide was mostly avoided in SARS-CoV-2, a pattern consistent with the human host. Strict avoidance of the CpG dinucleotide might be attributed to a strategy for evading a human immune response. A lower degree of adaptation of SARS-CoV-2 to the human host, compared to Middle East respiratory syndrome (MERS) coronavirus and SARS-CoV, might be indicative of its milder clinical severity and progression contrasted to SARS and MERS. Similar patterns of enhanced adaptation between viral isolates from intermediate and human hosts, contrasted with those isolated from the natural bat reservoir, signifies an indispensable role of the intermediate host in transmission dynamics and spillover events of the virus to human populations. The information regarding avoided codon pairs in SARS-CoV-2, as conferred by the present analysis, promises to be useful for the design of vaccines employing codon pair deoptimization based synthetic attenuated virus engineering.

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Of Chargaff's four "rules" on DNA base frequencies, the functional interpretation of his second parity rule (PR2) is the most contentious. Thermophile base compositions (GC%) were taken by Galtier and Lobry (1997) as favoring Sueoka's neutral PR2 hypothesis over Forsdyke's selective PR2 hypothesis, namely that mutations improving local within-species recombination efficiency had generated a genome-wide potential for the strands of duplex DNA to separate and initiate recombination through the "kissing" of the tips of stem-loops. However, following Chargaff's GC rule, base composition mainly reflects a species-specific, genome-wide, evolutionary pressure. GC% could not have consistently followed the dictates of temperature, since it plays fundamental roles in both sustaining species integrity and, through primarily neutral genome-wide mutation, fostering speciation. Evidence for a local within-species recombination-initiating role of base order was obtained with a novel technology that masked the contribution of base composition to nucleic acid folding energy. Forsdyke's results were consistent with his PR2 hypothesis, appeared to resolve some root problems in biology and provided a theoretical underpinning for alignment-free taxonomic analyses using relative oligonucleotide frequencies (k-mer analysis). Moreover, consistent with Chargaff's cluster rule, discovery of the thermoadaptive role of the "purine-loading" of open reading frames made less tenable the Galtier-Lobry anti-selectionist arguments.

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The size of the chloroplast genome (plastome) of autotrophic angiosperms is generally conserved. However, the chloroplast genomes of some lineages are greatly expanded, which may render assembling these genomes from short read sequencing data more challenging. Here, we present the sequencing, assembly, and annotation of the chloroplast genomes of Cypripedium tibeticum and Cypripedium subtropicum. We de novo assembled the chloroplast genomes of the two species with a combination of short-read Illumina data and long-read PacBio data. The plastomes of the two species are characterized by expanded genome size, proliferated AT-rich repeat sequences, low GC content and gene density, as well as low substitution rates of the coding genes. The plastomes of C. tibeticum (197,815 bp) and C. subtropicum (212,668 bp) are substantially larger than those of the three species sequenced in previous studies. The plastome of C. subtropicum is the longest one of Orchidaceae to date. Despite the increase in genome size, the gene order and gene number of the plastomes are conserved, with the exception of an ∼75 kb large inversion in the large single copy (LSC) region shared by the two species. The most striking is the record-setting low GC content in C. subtropicum (28.2%). Moreover, the plastome expansion of the two species is strongly correlated with the proliferation of AT-biased non-coding regions: the non-coding content of C. subtropicum is in excess of 57%. The genus provides a typical example of plastome expansion induced by the expansion of non-coding regions. Considering the pros and cons of different sequencing technologies, we recommend hybrid assembly based on long and short reads applied to the sequencing of plastomes with AT-biased base composition.

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Mammalian genomes contain long domains with distinct average compositions of A/T versus G/C base pairs. In a screen for proteins that might interpret base composition by binding to AT-rich motifs, we identified the stem cell factor SALL4, which contains multiple zinc fingers. Mutation of the domain responsible for AT binding drastically reduced SALL4 genome occupancy and prematurely upregulated genes in proportion to their AT content. Inactivation of this single AT-binding zinc-finger cluster mimicked defects seen in Sall4 null cells, including precocious differentiation of embryonic stem cells (ESCs) and embryonic lethality in mice. In contrast, deletion of two other zinc-finger clusters was phenotypically neutral. Our data indicate that loss of pluripotency is triggered by downregulation of SALL4, leading to de-repression of a set of AT-rich genes that promotes neuronal differentiation. We conclude that base composition is not merely a passive byproduct of genome evolution and constitutes a signal that aids control of cell fate.

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