Showing papers on "Ribosomal DNA published in 2020"

Long-read metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity.

Abstract: High-throughput DNA metabarcoding of amplicon sizes below 500 bp has revolutionized the analysis of environmental microbial diversity. However, these short regions contain limited phylogenetic signal, which makes it impractical to use environmental DNA in full phylogenetic inferences. This lesser phylogenetic resolution of short amplicons may be overcome by new long-read sequencing technologies. To test this idea, we amplified soil DNA and used PacBio Circular Consensus Sequencing (CCS) to obtain an ~4500-bp region spanning most of the eukaryotic small subunit (18S) and large subunit (28S) ribosomal DNA genes. We first treated the CCS reads with a novel curation workflow, generating 650 high-quality operational taxonomic units (OTUs) containing the physically linked 18S and 28S regions. To assign taxonomy to these OTUs, we developed a phylogeny-aware approach based on the 18S region that showed greater accuracy and sensitivity than similarity-based methods. The taxonomically annotated OTUs were then combined with available 18S and 28S reference sequences to infer a well-resolved phylogeny spanning all major groups of eukaryotes, allowing us to accurately derive the evolutionary origin of environmental diversity. A total of 1,019 sequences were included, of which a majority (58%) corresponded to the new long environmental OTUs. The long reads also allowed us to directly investigate the relationships among environmental sequences themselves, which represents a key advantage over the placement of short reads on a reference phylogeny. Together, our results show that long amplicons can be treated in a full phylogenetic framework to provide greater taxonomic resolution and a robust evolutionary perspective to environmental DNA.

Intragenomic polymorphisms in the ITS region of high-quality genomes of the Hypoxylaceae (Xylariales, Ascomycota)

Abstract: The internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) has been established (and is generally accepted) as a primary “universal” genetic barcode for fungi for many years, but the actual value for taxonomy has been heavily disputed among mycologists. Recently, twelve draft genome sequences, mainly derived from type species of the family Hypoxylaceae (Xylariales, Ascomycota) and the ex-epitype strain of Xylaria hypoxylon have become available during the course of a large phylogenomic study that was primarily aimed at establishing a correlation between the existing multi-gene-based genealogy with a genome-based phylogeny and the discovery of novel biosynthetic gene clusters encoding for secondary metabolites. The genome sequences were obtained using combinations of Illumina and Oxford nanopore technologies or PacBio sequencing, respectively, and resulted in high-quality sequences with an average N50 of 3.2 Mbp. While the main results will be published concurrently in a separate paper, the current case study was dedicated to the detection of ITS nrDNA copies in the genomes, in an attempt to explain certain incongruities and apparent mismatches between phenotypes and genotypes that had been observed during previous polyphasic studies. The results revealed that all of the studied strains had at least three copies of rDNA in their genomes, with Hypoxylon fragiforme having at least 19 copies of the ITS region, followed by Xylaria hypoxylon with at least 13 copies. Several of the genomes contained 2–3 copies that were nearly identical, but in some cases drastic differences, below 97% identity were observed. In one case, ascribable to the presence of a pseudogene, the deviations of the ITS sequences from the same genome resulted in only ca. 90% of overall homology. These results are discussed in the scope of the current trends to use ITS data for species recognition and segregation of fungi. We propose that additional genomes should be checked for such ITS polymorphisms to reassess the validity of this non-coding part of the fungal DNA for molecular identification.

Treacle controls the nucleolar response to rDNA breaks via TOPBP1 recruitment and ATR activation

Abstract: Induction of DNA double-strand breaks (DSBs) in ribosomal DNA (rDNA) repeats is associated with ATM-dependent repression of ribosomal RNA synthesis and large-scale reorganization of nucleolar architecture, but the signaling events that regulate these responses are largely elusive. Here we show that the nucleolar response to rDNA breaks is dependent on both ATM and ATR activity. We further demonstrate that ATM- and NBS1-dependent recruitment of TOPBP1 in the nucleoli is required for inhibition of ribosomal RNA synthesis and nucleolar segregation in response to rDNA breaks. Mechanistically, TOPBP1 recruitment is mediated by phosphorylation-dependent interactions between three of its BRCT domains and conserved phosphorylated Ser/Thr residues at the C-terminus of the nucleolar phosphoprotein Treacle. Our data thus reveal an important cooperation between TOPBP1 and Treacle in the signaling cascade that triggers transcriptional inhibition and nucleolar segregation in response to rDNA breaks.

Transcriptional suppression of ribosomal DNA with phase separation.

Abstract: The nucleolus is a nuclear body with multiphase liquid droplets for ribosomal RNA (rRNA) transcription. How rRNA transcription is regulated in the droplets remains unclear. Here, using single-molecule tracking of RNA polymerase I (Pol I) and chromatin-bound upstream binding factor (UBF), we reveal suppression of transcription with phase separation. For transcription, active Pol I formed small clusters/condensates that constrained rDNA chromatin in the nucleolus fibrillar center (FC). Treatment with a transcription inhibitor induced Pol I to dissociate from rDNA chromatin and to move like a liquid within the nucleolar cap that transformed from the FC. Expression of a Pol I mutant associated with a craniofacial disorder inhibited transcription by competing with wild-type Pol I clusters and transforming the FC into the nucleolar cap. The cap droplet excluded an initiation factor, ensuring robust silencing. Our findings suggest a mechanism of rRNA transcription suppression via phase separation of intranucleolar molecules governed by Pol I.

Super-resolution in situ analysis of active ribosomal DNA chromatin organization in the nucleolus.

Abstract: Ribosomal RNA (rRNA) transcription by RNA polymerase I (Pol I) is the first key step of ribosome biogenesis. While the molecular mechanisms of rRNA transcription regulation have been elucidated in great detail, the functional organization of the multicopy rRNA gene clusters (rDNA) in the nucleolus is less well understood. Here we apply super-resolution 3D structured illumination microscopy (3D-SIM) to investigate the spatial organization of transcriptionally competent active rDNA chromatin at size scales well below the diffraction limit by optical microscopy. We identify active rDNA chromatin units exhibiting uniformly ring-shaped conformations with diameters of ~240 nm in mouse and ~170 nm in human fibroblasts, consistent with rDNA looping. The active rDNA chromatin units are clearly separated from each other and from the surrounding areas of rRNA processing. Simultaneous imaging of all active genes bound by Pol I and the architectural chromatin protein Upstream Binding Transcription Factor (UBF) reveals a random spatial orientation of regular repeats of rDNA coding sequences within the nucleoli. These observations imply rDNA looping and exclude potential formation of systematic spatial assemblies of the well-ordered repetitive arrays of transcription units. Collectively, this study uncovers key features of the 3D organization of active rDNA chromatin units and their nucleolar clusters providing a spatial framework of nucleolar chromatin organization at unprecedented detail.

Recent advances in the nucleolar responses to DNA double-strand breaks

Abstract: DNA damage poses a serious threat to human health and cells therefore continuously monitor and repair DNA lesions across the genome. Ribosomal DNA is a genomic domain that represents a particular challenge due to repetitive sequences, high transcriptional activity and its localization in the nucleolus, where the accessibility of DNA repair factors is limited. Recent discoveries have significantly extended our understanding of how cells respond to DNA double-strand breaks (DSBs) in the nucleolus, and new kinases and multiple down-stream targets have been identified. Restructuring of the nucleolus can occur as a consequence of DSBs and new data point to an active regulation of this process, challenging previous views. Furthermore, new insights into coordination of cell cycle phases and ribosomal DNA repair argue against existing concepts. In addition, the importance of nucleolar-DNA damage response (n-DDR) mechanisms for maintenance of genome stability and the potential of such factors as anti-cancer targets is becoming apparent. This review will provide a detailed discussion of recent findings and their implications for our understanding of the n-DDR. The n-DDR shares features with the DNA damage response (DDR) elsewhere in the genome but is also emerging as an independent response unique to ribosomal DNA and the nucleolus.

A taxonomic revision of the genus Conidiobolus (Ancylistaceae, Entomophthorales): four clades including three new genera.

Abstract: The genus Conidiobolus is an important group in entomophthoroid fungi and is considered to be polyphyletic in recent molecular phylogenies. To re-evaluate and delimit this genus, multi-locus phylogenetic analyses were performed using the large and small subunits of nuclear ribosomal DNA (nucLSU and nucSSU), the small subunit of the mitochondrial ribosomal DNA (mtSSU) and the translation elongation factor 1-alpha (EF-1α). The results indicated that the Conidiobolus is not monophyletic, being grouped into a paraphyletic grade with four clades. Consequently, the well-known Conidiobolus is revised and three new genera Capillidium, Microconidiobolus and Neoconidiobolus are proposed along with one new record and 22 new combinations. In addition, the genus Basidiobolus is found to be basal to the other entomophthoroid taxa and the genus Batkoa locates in the Entomophthoraceae clade.

Comparative Studies on the Polymorphism and Copy Number Variation of mtSSU rDNA in Ciliates (Protista, Ciliophora): Implications for Phylogenetic, Environmental, and Ecological Research.

Abstract: While nuclear small subunit ribosomal DNA (nSSU rDNA) is the most commonly-used gene marker in studying phylogeny, ecology, abundance, and biodiversity of microbial eukaryotes, mitochondrial small subunit ribosomal DNA (mtSSU rDNA) provides an alternative. Recently, both copy number variation and sequence variation of nSSU rDNA have been demonstrated for diverse organisms, which can contribute to misinterpretation of microbiome data. Given this, we explore patterns for mtSSU rDNA among 13 selected ciliates (representing five classes), a major component of microbial eukaryotes, estimating copy number and sequence variation and comparing to that of nSSU rDNA. Our study reveals: (1) mtSSU rDNA copy number variation is substantially lower than that for nSSU rDNA; (2) mtSSU rDNA copy number ranges from 1.0 × 104 to 8.1 × 105; (3) a most common sequence of mtSSU rDNA is also found in each cell; (4) the sequence variation of mtSSU rDNA are mainly indels in poly A/T regions, and only half of species have sequence variation, which is fewer than that for nSSU rDNA; and (5) the polymorphisms between haplotypes of mtSSU rDNA would not influence the phylogenetic topology. Together, these data provide more insights into mtSSU rDNA as a powerful marker especially for microbial ecology studies.

Increasing methylation of sperm rDNA and other repetitive elements in the aging male mammalian germline.

Abstract: In somatic cells/tissues, methylation of ribosomal DNA (rDNA) increases with age and age‐related pathologies, which has a direct impact on the regulation of nucleolar activity and cellular metabolism. Here, we used bisulfite pyrosequencing and show that methylation of the rDNA transcription unit including upstream control element (UCE), core promoter, 18S rDNA, and 28S rDNA in human sperm also significantly increases with donor's age. This positive correlation between sperm rDNA methylation and biological age is evolutionarily conserved among mammals with widely different life spans such as humans, marmoset, bovine, and mouse. Similar to the tandemly repeated rDNA, methylation of human α‐satellite and interspersed LINE1 repeats, marmoset α‐satellite, bovine alpha‐ and testis satellite I, mouse minor and major satellite, and LINE1‐T repeats increases in the aging male germline, probably related to their sperm histone packaging. Deep bisulfite sequencing of single rDNA molecules in human sperm revealed that methylation does not only depend on donor's age, but also depend on the region and sequence context (A vs. G alleles). Both average rDNA methylation of all analyzed DNA molecules and the number of fully (>50%) methylated alleles, which are thought to be epigenetically silenced, increase with donor's age. All analyzed CpGs in the sperm rDNA transcription unit show comparable age‐related methylation changes. Unlike other epigenetic aging markers, the rDNA clock appears to operate in similar ways in germline and soma in different mammalian species. We propose that sperm rDNA methylation, directly or indirectly, influences nucleolar formation and developmental potential in the early embryo.

NORs on human acrocentric chromosome p-arms are active by default and can associate with nucleoli independently of rDNA

Abstract: Nucleoli, the sites of ribosome biogenesis and the largest structures in human nuclei, form around nucleolar organizer regions (NORs) comprising ribosomal DNA (rDNA) arrays. NORs are located on the p-arms of the five human acrocentric chromosomes. Defining the rules of engagement between these p-arms and nucleoli takes on added significance as describing the three-dimensional organization of the human genome represents a major research goal. Here we used fluorescent in situ hybridization (FISH) and immuno-FISH on metaphase chromosomes from karyotypically normal primary and hTERT-immortalized human cell lines to catalog NORs in terms of their relative rDNA content and activity status. We demonstrate that a proportion of acrocentric p-arms in cell lines and from normal human donors have no detectable rDNA. Surprisingly, we found that all NORs with detectable rDNA are active, as defined by upstream binding factor loading. We determined the nucleolar association status of all NORs during interphase, and found that nucleolar association of acrocentric p-arms can occur independently of rDNA content, suggesting that sequences elsewhere on these chromosome arms drive nucleolar association. In established cancer lines, we characterize a variety of chromosomal rearrangements involving acrocentric p-arms and observe silent, rDNA-containing NORs that are dissociated from nucleoli. In conclusion, our findings indicate that within human nuclei, positioning of all 10 acrocentric chromosomes is dictated by nucleolar association. Furthermore, these nucleolar associations are buffered against interindividual variation in the distribution of rDNA.

Four new species of Trichoderma in the Harzianum clade from northern China.

Abstract: The Harzianum clade of Trichoderma comprises many species, which are associated with a wide variety of substrates. In this study, four new species of Trichoderma, namely T. lentinulae, T. vermifimicola, T. xixiacum, and T. zelobreve, were encountered from a fruiting body and compost of Lentinula, soil, and vermicompost. Their colony and mycelial morphology, including features of asexual states, were described. For each species, their DNA sequences were obtained from three loci, the internal transcribed spacer (ITS) regions of the ribosomal DNA, the gene encoding the second largest nuclear RNA polymerase subunit (RPB2), the translation elongation factor 1-α encoding gene (TEF1-α). The analysis combining sequences of the three gene regions distinguished four new species in the Harzianum clade of Trichoderma. Among them, T. lentinulae and T. xixiacum clustered with T. lixii, from which these new species differ in having shorter phialides and smaller conidia. Additionally, T. lentinulae differs from T. xixiacum in forming phialides with inequilateral to a strongly-curved apex, cultural characteristics, and slow growth on PDA. Trichoderma vermifimicola is closely related to T. simmonsii, but it differs from the latter by producing phialides in verticillate whorls and smaller conidia. Trichoderma zelobreve is the sister species of T. breve but is distinguished from T. breve by producing shorter and narrower phialides, smaller conidia, and by forming concentric zones on agar plates. This study updates our knowledge of species diversity of Trichoderma.

The Utility of Graph Clustering of 5S Ribosomal DNA Homoeologs in Plant Allopolyploids, Homoploid Hybrids, and Cryptic Introgressants

Abstract: Introduction: Ribosomal DNA (rDNA) loci have been widely used for identification of allopolyploids and hybrids, although few of these studies employed high-throughput sequencing data. Here we use graph clustering implemented in the RepeatExplorer pipeline to analyse homoeologous 5S rDNA arrays at the genomic level searching for hybridogenic origin of species. Data were obtained from more than 80 plant genera, including several well-defined allopolyploids and homoploid hybrids of different evolutionary ages and from widely dispersed taxonomic groups. Results: (i) Diploids show simple circular-shaped graphs of their 5S rDNA clusters. In contrast, most allopolyploids and other interspecific hybrids exhibit more complex graphs composed of two or more interconnected loops representing intergenic spacers (IGS). (ii) There was a relationship between graphs complexity and locus numbers. (iii) The sequences and lengths of the 5S rDNA units reconstituted in silico from k-mers were congruent with those experimentally determined. (iv) Three-genomic comparative cluster analysis of reads from allopolyploids and progenitor diploids allowed identification of homoeologous 5S rRNA gene families even in relatively ancient (c. 1 Myr) Gossypium and Brachypodium allopolyploids which already exhibit uniparental partial loss of rDNA repeats. (v) Finally, species harbouring introgressed genomes exhibit exceptionally complex graph structures. Conclusion: We found that the cluster graph shapes and graph parameters (k-mer coverage scores and connected component index) well-reflect the organisation and intragenomic homogenity of 5S rDNA repeats. We propose that the analysis of 5S rDNA cluster graphs computed by the RepeatExplorer pipeline together with the cytogenetic analysis might be a reliable approach for the determination of the hybrid or allopolyploid plant species parentage and may also be useful for detecting historical introgression events.

Unveiling the Hidden Diversity of Rock-Inhabiting Fungi: Chaetothyriales from China.

Abstract: Rock-inhabiting fungi (RIF) are nonlichenized fungi that naturally colonize rock surfaces and subsurfaces. The extremely slow growth rate and lack of distinguishing morphological characteristics of RIF resulted in a poor understanding on their biodiversity. In this study, we surveyed RIF colonizing historical stone monuments and natural rock formations from throughout China. Among over 1000 isolates, after preliminary delimitation using the internal transcribed spacer region (ITS) sequences, representative isolates belonging to Trichomeriaceae and Herpotrichiellaceae were selected for a combined analysis of ITS and the nuclear ribosomal large subunit (nucLSU) to determine the generic placements. Eight clades representing seven known genera and one new genus herein named as Anthracina were placed in Trichomeriaceae. While, for Herpotrichiellaceae, two clades corresponded to two genera: Cladophialophora and Exophiala. Fine-scale phylogenetic analyses using combined sequences of the partial actin gene (ACT), ITS, mitochondrial small subunit ribosomal DNA (mtSSU), nucLSU, the largest subunit of RNA polymerase II (RPB1), small subunit of nuclear ribosomal RNA gene (SSU), translation elongation factor (TEF), and β-tubulin gene (TUB) revealed that these strains represented 11 and 6 new species, respectively, in Trichomeriaceae and Herpotrichiellaceae. The 17 new species were described, illustrated for their morphologies and compared with similar taxa. Our study demonstrated that the diversity of RIF is surprisingly high and still poorly understood. In addition, a rapid strategy for classifying RIF was proposed to determine the generic and familial placements through preliminary ITS and nucLSU analyses, followed by combined analyses of five loci selected from ACT, ITS, mtSSU, nucLSU, RPB1, and/or the second subunit of RNA polymerase II gene (RPB2), SSU, TEF, and TUB regions to classify RIF to the species level.

Phylogenetic Relationships of Cardiocephaloides spp. (Digenea, Diplostomoidea) and the Genetic Characterization of Cardiocephaloides physalis from Magellanic Penguin, Spheniscus magellanicus , in Chile

Abstract: Cardiocephaloides is a small genus of strigeid digeneans with an essentially cosmopolitan distribution. Most members of Cardiocephaloides are found in larid birds, however, Cardiocephaloides physalis is an exception and parasitizes penguins in some coastal regions of South America and South Africa. No prior molecular phylogenetic studies have included DNA sequence data of C. physalis. Herein, we provide molecular phylogenetic analyses of Cardiocephaloides using DNA sequences from five species of these strigeids. Adult Cardiocephaloides spp. were obtained from larid birds and penguins collected from 3 biogeographical realms (Palearctic, Nearctic and Neotropics). We have generated sequences of the complete ITS region and partial 28S gene of the nuclear ribosomal DNA, along with partial sequences of the mitochondrial CO1 gene for C. physalis, C. medioconiger and the type species of the genus, C. longicollis and used them for phylogenetic inference. Cardiocephaloides spp. appeared as a 100% supported clade in the phylogenetic tree based on 28S sequences. The position of C. physalis varied between the phylogenetic trees based on the relatively conservative 28S gene on one hand, and variable ITS1 and COI sequences on the other. Cardiocephaloides physalis was nested within the clade of Cardiocephaloides spp. in the 28S tree and appeared as the sister group to the remaining members of the genus in the ITS1 region and COI trees. We detected 0.4–1.6% interspecific divergence in 28S, 1.9–6.9% in the ITS region and 8.7–11.8% in CO1 sequences of Cardiocephaloides spp. Our 28S sequence of C. physalis from South America and a shorter sequence from Africa available in the GenBank were identical. Cardiocephaloides as represented in the currently available dataset is monophyletic with C. physalis parasitism in penguins likely resulting from a secondary host-switching event. Identical 28S sequences of C. physalis from South America and Africa cautiously confirm the broad distribution of this species, although comparison of faster mutating genes (e. g., CO1) is recommended for a better substantiated conclusion.

Integrated evidence reveals a new species of Cosmocerca (Ascaridomorpha: Cosmocercoidea) from the Asiatic toad Bufo gargarizans Cantor (Amphibia: Anura).

Abstract: Species of Cosmocerca Diesing, 1861 (Ascaridomorpha: Cosmocercoidea), are common nematode parasites of amphibians. In the present study, a new species of Cosmocerca, namely C. simile n. sp., was described using light and scanning electron microscopy, and sequencing different nuclear and mitochondrial genetic markers (i.e. small ribosomal DNA (18S), large ribosomal DNA (28S), internal transcribed spacer (ITS) and cytochrome c oxidase subunit 1 (cox1)). Cosmocerca simile n. sp. differs from its congeners based on body size, morphology and number of plectanes, relative length of spicules and gubernaculum and spicules to total body length and morphology and length of tail. Molecular analysis showed no nucleotide polymorphisms among different individuals of the new species regarding nuclear DNA. Very low intraspecific nucleotide variation (0.52-0.78%) was detected in cox1 mtDNA. In contrast, the level of interspecific nucleotide variation between C. simile n. sp. and its congeners were distinctly higher (2.74-18.1% in the partial ITS region and 10.2-13.5% in the partial cox1 region, respectively) than that of intraspecific variation. Phylogenetic analyses using maximum likelihood (ML) inference based on the partial ITS and cox1 sequence data both supported the new species to be a member of the genus Cosmocerca, and formed a sister relationship to C. japonica. The newly obtained genetic data are important for further studies of DNA-based taxonomy, population genetics and phylogenetics of the Cosmocercoidea.

Satellite DNA content of B chromosomes in the characid fish Characidium gomesi supports their origin from sex chromosomes.

Abstract: The origin of supernumerary (B) chromosomes is clearly conditioned by their ancestry from the standard (A) chromosomes. Sequence similarity between A and B chromosomes is thus crucial to determine B chromosome origin. For this purpose, we compare here the DNA sequences from A and B chromosomes in the characid fish Characidium gomesi using two main approaches. First, we found 59 satellite DNA (satDNA) families constituting the satellitome of this species and performed FISH analysis for 18 of them. This showed the presence of six satDNAs on the B chromosome: one shared with sex chromosomes and autosomes, two shared with sex chromosomes, one shared with autosomes and two being B-specific. This indicated that B chromosomes most likely arose from the sex chromosomes. Our second approach consisted of the analysis of five repetitive DNA families: 18S and 5S ribosomal DNA (rDNA), the H3 histone gene, U2 snDNA and the most abundant satDNA (CgoSat01-184) on DNA obtained from microdissected B chromosomes and from B-lacking genomes. PCR and sequence analysis of these repetitive sequences was successful for three of them (5S rDNA, H3 histone gene and CgoSat01-184), and sequence comparison revealed that DNA sequences obtained from the B chromosomes displayed higher identity with C. gomesi genomic DNA than with those obtained from other Characidium species. Taken together, our results support the intraspecific origin of B chromosomes in C. gomesi and point to sex chromosomes as B chromosome ancestors, which raises interesting prospects for future joint research on the genetic content of sex and B chromosomes in this species.

Shifts in the Active Rhizobiome Paralleling Low Meloidogyne chitwoodi Densities in Fields Under Prolonged Organic Soil Management

Abstract: Plants manipulate their rhizosphere community in a species and even a plant life stage-dependent manner. In essence plants select, promote and (de)activate directly the local bacterial and fungal community, and indirectly representatives of the next trophic level, protists and nematodes. By doing so, plants enlarge the pool of bioavailable nutrients and maximize local disease suppressiveness within the boundaries set by the nature of the local microbial community. MiSeq sequencing of specific variable regions of the 16S or 18S ribosomal DNA (rDNA) is widely used to map microbial shifts. As current RNA extraction procedures are time-consuming and expensive, the rRNA-based characterization of the active microbial community is taken along less frequently. Recently, we developed a relatively fast and affordable protocol for the simultaneous extraction of rDNA and rRNA from soil. Here, we investigated the long-term impact of three type of soil management, two conventional and an organic regime, on soil biota in fields naturally infested with the Columbian root-knot nematode Meloidogyne chitwoodi with pea (Pisum sativum) as the main crop. For all soil samples, large differences were observed between resident (rDNA) and active (rRNA) microbial communities. Among the four organismal group under investigation, the bacterial community was most affected by the main crop, and unweighted and weighted UniFrac analyses (explaining respectively 16.4% and 51.3% of the observed variation) pointed at a quantitative rather than a qualitative shift. LEfSe analyses were employed for each of the four organismal groups to taxonomically pinpoint the effects of soil management. Concentrating on the bacterial community in the pea rhizosphere, organic soil management resulted in a remarkable activation of members of the Burkholderiaceae, Enterobacteriaceae, and Pseudomonadaceae. Prolonged organic soil management was also accompanied by significantly higher densities of bacterivorous nematodes, whereas levels of M. chitwoodi had dropped drastically. Though present and active in the fields under investigation Orbiliaceae, a family harboring numerous nematophagous fungi, was not associated with the M. chitwoodi decline. A closer look revealed that a local accumulation and activation of Pseudomonas, a genus that includes a number of nematode-suppressive species, paralleled the lower M. chitwoodi densities. This study underlines the relevance of taking along both resident and active fractions of multiple organismal groups while mapping the impact of e.g. crops and soil management regimes.

Age-Dependent Ribosomal DNA Variations in Mice.

Abstract: The rRNA gene, which consists of tandem repetitive arrays (ribosomal DNA [rDNA] repeat), is one of the most unstable regions in the genome. The rDNA repeat in the budding yeast Saccharomyces cerevisiae is known to become unstable as the cell ages. However, it is unclear how the rDNA repeat changes in aging mammalian cells. Using quantitative single-cell analyses, we identified age-dependent alterations in rDNA copy number and levels of methylation in mice. The degree of methylation and copy number of rDNA from bone marrow cells of 2-year-old mice were increased by comparison to levels in 4-week-old mice in two mouse strains, BALB/cA and C57BL/6. Moreover, the level of pre-rRNA transcripts was reduced in older BALB/cA mice. We also identified many sequence variations in the rDNA. Among them, three mutations were unique to old mice, and two of them were found in the conserved region in budding yeast. We established yeast strains with the old-mouse-specific mutations and found that they shortened the life span of the cells. Our findings suggest that rDNA is also fragile in mammalian cells and that alterations within this region have a profound effect on cellular function.

Challenges and Approaches to Genotyping Repetitive DNA

Abstract: Individuals within a species can exhibit vast variation in copy number of repetitive DNA elements. This variation may contribute to complex traits such as lifespan and disease, yet it is only infrequently considered in genotype-phenotype associations. Although the possible importance of copy number variation is widely recognized, accurate copy number quantification remains challenging. Here, we assess the technical reproducibility of several major methods for copy number estimation as they apply to the large repetitive ribosomal DNA array (rDNA). rDNA encodes the ribosomal RNAs and exists as a tandem gene array in all eukaryotes. Repeat units of rDNA are kilobases in size, often with several hundred units comprising the array, making rDNA particularly intractable to common quantification techniques. We evaluate pulsed-field gel electrophoresis, droplet digital PCR, and Nextera-based whole genome sequencing as approaches to copy number estimation, comparing techniques across model organisms and spanning wide ranges of copy numbers. Nextera-based whole genome sequencing, though commonly used in recent literature, produced high error. We explore possible causes for this error and provide recommendations for best practices in rDNA copy number estimation. We present a resource of high-confidence rDNA copy number estimates for a set of S. cerevisiae and C. elegans strains for future use. We furthermore explore the possibility for FISH-based copy number estimation, an alternative that could potentially characterize copy number on a cellular level.

Genetic and Epigenetic Regulation of Skeletal Muscle Ribosome Biogenesis with Exercise

Abstract: Ribosomes are the macromolecular engines of protein synthesis. Skeletal muscle ribosome biogenesis is stimulated by exercise, but the contribution of ribosomal DNA (rDNA) copy number and methylation to exercise-induced rDNA transcription is unclear. To investigate the genetic and epigenetic regulation of ribosome biogenesis with exercise, a time course of skeletal muscle biopsies was obtained from 30 participants (18 men and 12 women; 31 {+/-}8 yrs, 25 {+/-}4 kg/m2) at rest and 30 min, 3h, 8h, and 24h after acute endurance (n=10, 45 min cycling, 70% VO2max) or resistance exercise (n=10, 4 x 7 x 2 exercises); 10 control participants underwent biopsies without exercise. rDNA transcription and dosage were assessed using qPCR and whole genome sequencing. rDNA promoter methylation was investigated using massARRAY EpiTYPER, and global rDNA CpG methylation was assessed using reduced-representation bisulfite sequencing. Ribosome biogenesis and MYC transcription were associated with resistance but not endurance exercise, indicating preferential up-regulation during hypertrophic processes. With resistance exercise, ribosome biogenesis was associated with rDNA gene dosage as well as epigenetic changes in enhancer and non-canonical MYC-associated areas in rDNA, but not the promoter. A mouse model of in vivo metabolic RNA labeling and genetic myonuclear fluorescent labeling validated the effects of an acute hypertrophic stimulus on ribosome biogenesis and Myc transcription, and corroborated rDNA enhancer and Myc-associated methylation alterations specifically in myonuclei. This study provides the first information on skeletal muscle genetic and rDNA gene-wide epigenetic regulation of ribosome biogenesis in response to exercise, revealing novel roles for rDNA dosage and CpG methylation. GRAPHICAL ABSTRACT O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

Replicative aging is associated with loss of genetic heterogeneity from extrachromosomal circular DNA in Saccharomyces cerevisiae.

Abstract: Circular DNA can arise from all parts of eukaryotic chromosomes. In yeast, circular ribosomal DNA (rDNA) accumulates dramatically as cells age, however little is known about the accumulation of other chromosome-derived circles or the contribution of such circles to genetic variation in aged cells. We profiled circular DNA in Saccharomyces cerevisiae populations sampled when young and after extensive aging. Young cells possessed highly diverse circular DNA populations but 94% of the circular DNA were lost after ∼15 divisions, whereas rDNA circles underwent massive accumulation to >95% of circular DNA. Circles present in both young and old cells were characterized by replication origins including circles from unique regions of the genome and repetitive regions: rDNA and telomeric Y' regions. We further observed that circles can have flexible inheritance patterns: [HXT6/7circle] normally segregates to mother cells but in low glucose is present in up to 50% of cells, the majority of which must have inherited this circle from their mother. Interestingly, [HXT6/7circle] cells are eventually replaced by cells carrying stable chromosomal HXT6 HXT6/7 HXT7 amplifications, suggesting circular DNAs are intermediates in chromosomal amplifications. In conclusion, the heterogeneity of circular DNA offers flexibility in adaptation, but this heterogeneity is remarkably diminished with age.

Identification of Rice Seed-Derived Fusarium Spp. and Development of LAMP Assay against Fusarium Fujikuroi.

Abstract: Fusarium species are important seedborne pathogens that cause rice bakanae disease (RBD). In this study, 421 strains were isolated from 25 rice samples collected from Zhejiang, Anhui, and Jiangxi provinces of China. Furthermore, 407 isolates were identified as F. fujikuroi (80.05% isolation frequency), F. proliferatum (8.31%), F. equiseti (5.94%), F. incarnatum (2.61%), F. andiyazi (0.95%), and F. asiaticum (0.48%) based on morphology and translation elongation factor 1-alpha (TEF1-α) gene. Phylogenetic analysis of combined sequences of the RNA polymerase II largest subunit (RPB1), RNA polymerase II second largest subunit (RPB2), TEF1-α gene, and ribosomal DNA (rDNA) internal transcribed spacer (ITS) showed that 17 representative strains were attributed to six species. Pathogenicity tests showed that representative isolates possessed varying ability to cause symptoms of bakanae on rice seedlings. Moreover, the seed germination assay revealed that six isolates had different effects, such as inhibition of seed germination, as well as seed and bud rot. The loop mediated isothermal amplification (LAMP)-based assay were developed for the detection of F. fujikuroi. According to sequences of desaturase-coding gene promoter, a species-specific marker desM231 was developed for the detection of F. fujikuroi. The LAMP assay using seeds collected from field was validated, and diagnostics developed are efficient, rapid, and sensitive.

Characterizing the ribosomal tandem repeat and its utility as a DNA barcode in lichen-forming fungi

Abstract: Regions within the nuclear ribosomal operon are a major tool for inferring evolutionary relationships and investigating diversity in fungi. In spite of the prevalent use of ribosomal markers in fungal research, central features of nuclear ribosomal DNA (nrDNA) evolution are poorly characterized for fungi in general, including lichenized fungi. The internal transcribed spacer (ITS) region of the nrDNA has been adopted as the primary DNA barcode identification marker for fungi. However, little is known about intragenomic variation in the nrDNA in symbiotic fungi. In order to better understand evolution of nrDNA and the utility of the ITS region for barcode identification of lichen-forming fungal species, we generated nearly complete nuclear ribosomal operon sequences from nine species in the Rhizoplaca melanophthalma species complex using short reads from high-throughput sequencing. We estimated copy numbers for the nrDNA operon, ranging from nine to 48 copies for members of this complex, and found low levels of intragenomic variation in the standard barcode region (ITS). Monophyly of currently described species in this complex was supported in phylogenetic inferences based on the ITS, 28S, intergenic spacer region, and some intronic regions, independently; however, a phylogenetic inference based on the 18S provided much lower resolution. Phylogenetic analysis of concatenated ITS and intergenic spacer sequence data generated from 496 specimens collected worldwide revealed previously unrecognized lineages in the nrDNA phylogeny. The results from our study support the general assumption that the ITS region of the nrDNA is an effective barcoding marker for fungi. For the R. melanophthalma group, the limited amount of potential intragenomic variability in the ITS region did not correspond to fixed diagnostic nucleotide position characters separating taxa within this species complex. Previously unrecognized lineages inferred from ITS sequence data may represent undescribed species-level lineages or reflect uncharacterized aspects of nrDNA evolution in the R. melanophthalma species complex.