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At least 127 records · Page 7

Advanced urothelial cancer: a radiology update

The recent genomic characterization of urothelial carcinoma by the Cancer Genome Atlas Project, made possible by the introduction of high throughput, reduced cost, and sequence analysis, has shed new insights on the biology of advanced disease. In addition, studies on imaging of advanced urothelial carcinoma have widened the knowledge on disease presentation and on pattern of metastatic spread and their correlation with the underlying biology of urothelial carcinoma. The wide range of treatments for advanced urothelial cancer, including combined chemotherapy regimens and immune checkpoint inhibitors, each result in treatment class-specific patterns of response and adverse events. Results of studies point to the need for a reliable biomarker, perhaps with imaging, that predicts prognosis and treatment response to systemic treatment, and can be used to select the most effective treatment while minimizing toxicity. This review of advanced urothelial cancer introduces the latest advances in genetic profiling, the current role of imaging, the radiographic appearance of treatment response and their toxicities, and details potential future areas of imaging research.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

Efficient Production of Polymalic Acid by a Novel Isolated Aureobasidium pullulans Using Metabolic Intermediates and Inhibitors

Polymalic acid (PMA) is a linear anionic polyester composed of L-malic acid monomers, which have potential applications as drug carriers, surgical suture, and biodegradable plastics. In this study, a novel strain of Aureobasidium pullulans var. melanogenum GXZ-6 was isolated and identified according to the morphological observation and deoxyribonucleic acid internal-transcribed spacer sequence analysis, and the product of PMA was characterized by FT-IR, {sup 13}C-NMR, and {sup 1}H-NMR spectra. The PMA titer of GXZ-6 reached 62.56 ± 1.18 g L{sup −1} with productivity of 0.35 g L{sup −1} h{sup −1} using optimized medium with addition of metabolic intermediates (citrate and malate) and inhibitor (malonate) by batch fermentation in a 10-L fermentor. Besides that the malate for PMA synthesis in GXZ-6 might mainly come from the glyoxylate cycle, based on results, citrate, malate, malonate, and maleate increased while succinate and fumarate inhibited the production of PMA, which was different from that of other A. pullulans. This study provided a potential strain and a simple metabolic control strategy for high-titer production of PMA and shared novel information on the biosynthesis pathway of PMA in A. pullulans.

60 APPLIED LIFE SCIENCES↗

Spectroradiometric detection of competitor diatoms and the grazer Poteriochromonas in algal cultures

To address challenges in early detection of pond pests, we have extended a spectroradiometric monitoring method, initially demonstrated for measurement of pigment optical activity and biomass, to the detection of algal competitors and grazers. The method relies upon measurement and interpretation of pond reflectance spectra spanning from the visible into the near-infrared. Reflectance spectra are acquired every 5 min with a multi-channel, fiber-coupled spectroradiometer, providing monitoring of algal pond conditions with high temporal frequency. The spectra are interpreted via numerical inversion of a reflectance model, in which the above-water reflectance is expressed in terms of the absorption and backscatter coefficients of the cultured species, with additional terms accounting for the pigment fluorescence features and for the water-surface reflection of sunlight and skylight. With this method we demonstrate detection of diatoms and the predator Poteriochromonas in outdoor cultures of Nannochloropsis oceanica and Chlorella vulgaris, respectively. Further, the relative strength of these signatures is compared to microscopy and sequencing analysis. Spectroradiometric detection of diatoms is then further assessed on beaker-contained mixtures of Microchloropsis salina with Phaeodactylum tricornutum, Thalassiosira weissflogii, and Thalassiosira pseudonana, respectively, providing an initial evaluation of the sensitivity and specificity of detecting pond competitors.

09 BIOMASS FUELS↗

Identification and characterization of circular RNAs in rapid atrial pacing dog atrial tissue

Highlights: • Revealed the expression characterization of circRNAs in dog atrial tissue. • Compared differentially expressed circRNAs between atrial fibrillation and control dog. • Found extensive interaction among circRNAs and AF related miRNAs and mRNAs. Circular RNAs (circRNAs) have emerged as novel molecules of interest in gene regulation as other noncoding RNAs, and participating in the process of many diseases. However, the expression and functions of circRNAs in Rapid atrial pacing (RAP) dog atrial tissue still unknown. 12 canines were randomly assigned to control and pacing group. RAP at 500 beats per minute was maintained 14 days in the pacing group. The expression characterization of circRNAs were revealed by high-throughput sequencing. We totally predicted 15,990 circRNAs in dog atrial tissues. Moreover, we found 146 differentially expressed circRNAs between control and RAP dogs. Five circRNAs were selected for subsequent RT-PCR validation, and four circRNAs confirmed with the high throughput sequencing analysis. GO analysis showed that the differentially expressed circRNAs might involve in the process of “structural constituent of cytoskeleton, ion channel activity”. We explored the circRNA-miRNA interaction network, and found extensive interaction among differentially expressed circRNAs and AF related miRNAs and mRNAs. Our work firstly identified the characterization of circRNAs in the dog atrial, and revealed the differentially expressed circRNAs in the RAP dog, this might lay a solid foundation on the function of circRNA in the mechanisms of AF.

60 APPLIED LIFE SCIENCES↗

Targeting ninjurin 2 by miR-764 regulates hydrogen peroxide (H2O2)-induced neuronal cell death

Highlights: • MicroRNA-764 (miR-764) directly regulates ninjurin2 (NINJ2) expression. • Forced overexpression of miR-764 downregulates NINJ2 in human neuronal cells. • miR-764 inhibitor upregulates NINJ2 in human neuronal cells. • NINJ2 downregulation by miR-764 is cytotoxic to human neuronal cells. • NINJ2 overexpression by lentivirus or mir-764 inhibitor protects neuronal cells from H{sub 2}O{sub 2}. Ninjurin 2 (NINJ2) is a novel adhesion molecule expressed in neurons and glial cells. The current study determined if specific microRNA (miRNA) can regulate NINJ2 expression in human neuronal cells. Sequence analysis of NINJ2 mRNA 3′-untranslated region (3′-UTR) revealed that microRNA-764 (miR-764) putatively targets NINJ2. In SH-SY5Y/SK-N-BE neuronal cells and primary human neurons, lentivirus-mediated overexpression of miR-764 conferred significant repression on NINJ2 3′-UTR luciferase activity and downregulation of NINJ2 mRNA/protein. Conversely, transfection of the miR-764 inhibitor increased NINJ2 mRNA and protein expression in the neuronal cells. Function studies show that NINJ2 downregulation by miR-764 induced significant viability reduction and apoptosis in the neuronal cells. Further, CRISPR/Cas9-mediated NINJ2 knockout mimicked and abolished miR-764-induced actions in SH-SY5Y cells. Conversely, lentivirus-mediated NINJ2 overexpression or transfection of miR-764 inhibitor protected neuronal cells from hydrogen peroxide (H{sub 2}O{sub 2})-induced cell death and apoptosis. Collectively, these results show that NINJ2 is a pro-survival factor in human neuronal cells. miR-764 regulates NINJ2 expression and neuron functions.

60 APPLIED LIFE SCIENCES↗

Lpp positions peptidoglycan at the AcrA-TolC interface in the AcrAB-TolC multidrug efflux pump

The multidrug efflux pumps of Gram-negative bacteria are a class of complexes that span the periplasm, coupling both the inner and outer membranes to expel toxic molecules. The best-characterized example of these tripartite pumps is the AcrAB-TolC complex of Escherichia coli. However, how the complex interacts with the peptidoglycan (PG) cell wall, which is anchored to the outer membrane (OM) by Braun’s lipoprotein (Lpp), is still largely unknown. In this work, we present molecular dynamics simulations of a complete, atomistic model of the AcrAB-TolC complex with the inner membrane, OM, and PG layers all present. We find that the PG localizes to the junction of AcrA and TolC, in agreement with recent cryo-tomography data. Free-energy calculations reveal that the positioning of PG is determined by the length and conformation of multiple Lpp copies anchoring it to the OM. The distance between the PG and OM measured in cryo-electron microscopy images of wild-type E. coli also agrees with the simulation-derived spacing. Finally, sequence analysis of AcrA suggests a conserved role for interactions with PG in the assembly and stabilization of efflux pumps, one that may extend to other trans-envelope complexes as well.

59 BASIC BIOLOGICAL SCIENCES↗

FTO regulates myoblast proliferation by controlling CCND1 expression in an m6A-YTHDF2-dependent manner

Highlights: • Silencing FTO delays cell cycle and inhibits cell proliferation. • FTO regulates cell proliferation depending on its m{sup 6}A demethylation activity. • Silencing FTO suppresses CCND1 expression via YTHDF2-mediated mRNA degradation. • Knockdown YTHDF2 eliminates the cell cycle arrest in FTO-deficient cells. N6-Methyladenosine (m{sup 6}A) modification is the most abundant chemical modification in mRNA, and it participates in various biological processes, such as cell differentiation and proliferation. However, little is known about the function of m{sup 6}A demethylase fat mass and obesity-associated (FTO) in myoblast proliferation. Here, we demonstrated that knockdown of FTO can significantly inhibit myoblast proliferation and promote apoptosis. RNA sequencing analysis revealed that a lot of downregulated genes in FTO knockdown cells are associated with cell cycle and apoptosis. Furthermore, silencing FTO drastically decreased cyclin D1 (CCND1) expression through YTHDF2-mediated mRNA degradation, thereby delaying the progression of G1 phase, and leading to impaired myoblast proliferation. These findings unraveled that FTO regulates myoblast proliferation by controlling CCND1 expression in an m{sup 6}A-YTHDF2-dependent manner, which highlights the critical roles of m{sup 6}A modification in myoblast proliferation.

60 APPLIED LIFE SCIENCES↗

Genomic insights into the origin, domestication and diversification of Brassica juncea

Despite early domestication around 3000 BC, the evolutionary history of the ancient allotetraploid species Brassica juncea (L.) Czern & Coss remains uncertain. Here, we report a chromosome-scale de novo assembly of a yellow-seeded B. juncea genome by integrating long-read and short-read sequencing, optical mapping and Hi-C technologies. Nuclear and organelle phylogenies of 480 accessions worldwide supported that B. juncea is most likely a single origin in West Asia, 8,000–14,000 years ago, via natural interspecific hybridization. Subsequently, new crop types evolved through spontaneous gene mutations and introgressions along three independent routes of eastward expansion. Selective sweeps, genome-wide trait associations and tissue-specific RNA-sequencing analysis shed light on the domestication history of flowering time and seed weight, and on human selection for morphological diversification in this versatile species. Our data provide a comprehensive insight into the origin and domestication and a foundation for genomics-based breeding of B. juncea.

59 BASIC BIOLOGICAL SCIENCES↗

Lyophilization of ASFV vaccine candidate ASFV-G-ΔI177L offers long term stability

Abstract For over a century African swine fever (ASF) has been causing outbreaks leading to devastating losses for the swine industry. The current pandemic of ASF has shown no signs of stopping and continues to spread causing outbreaks in additional countries. Currently control relies mostly on culling infected farms, and strict biosecurity procedures. Recently a vaccine, ASFV-G-ΔI177L was approved for use in Vietnam. In this study we evaluate the long-term stability of lyophilized ASFV-G-ΔI177L. Understanding the stability of different formulations of vaccines is information necessary for deployment of vaccines to ASF outbreak areas, particularly those that do not have a reliable well established cold chain to ensure conservation of vaccine quality. In this report, we determined that ASFV-G-ΔI177L, when lyophilized under specific conditions, is stable for up to one year at 4 °C, with similar vaccine titers after storage. Next-generation sequencing analysis also determined that lyophilization and long-term storage under these conditions had no effect on the genome of ASFV as the genome remained genetically identical to the original non-lyophilized form.

Science & Technology - Other Topics↗

Protein cost minimization promotes the emergence of coenzyme redundancy

Metabolism relies on a small class of molecules (coenzymes) that serve as universal donors and acceptors of key chemical groups and electrons. Although metabolic networks crucially depend on structurally redundant coenzymes [e.g., NAD(H) and NADP(H)] associated with different enzymes, the criteria that led to the emergence of this redundancy remain poorly understood. Our combination of modeling and structural and sequence analysis indicates that coenzyme redundancy may not be essential for metabolism but could rather constitute an evolved strategy promoting efficient usage of enzymes when biochemical reactions are near equilibrium. Our work suggests that early metabolism may have operated with fewer coenzymes and that adaptation for metabolic efficiency may have driven the rise of coenzyme diversity in living systems.

59 BASIC BIOLOGICAL SCIENCES↗

Validation of Continuous-Energy ENDF/B-VIII.0 16 O, 56 Fe, and 63,65 Cu Cross Sections for Nuclear Criticality Safety Applications

Recently completed cross-section evaluations sponsored in part by the Nuclear Criticality Safety Program were incorporated into the 2018 release of the ENDF/B-VIII.0 cross-section library. Evaluated isotopes of interest to the nuclear data and criticality safety community include 16 O, 56 Fe, and 63,65 Cu. For performance validation, benchmark models defined in the International Criticality Safety Benchmark Evaluation Project Handbook were selected based on energy-integrated k eff sensitivities to total cross sections of interest and compared with experimental values. Of the 102 benchmark configurations that were utilized, 63 are sensitive to 16 O, 32 sensitive to 63,65 Cu, and 25 sensitive to 56 Fe. Selected benchmarks were modeled in SCALE 6.2.3 Criticality Safety Analysis Sequence (CSAS) continuous-energy Monte Carlo k eff calculations with ENDF/B-VII.1, with a hybrid ENDF/B-VII.1 with ENDF/B-VIII.0 data substituted for individual isotopes of interest, and with ENDF/B-VIII.0. ENDF/B-VIII.0 showed improved agreement with experimental k eff for 56 Fe, 63 Cu, elemental copper, and full library substitution while producing lessened agreement for 16 O and 65 Cu. Furthermore, with full library and isotope-specific ENDF/B-VIII.0 performance, a best-case ENDF library was formed by excluding underperforming isotopes’ ENDF/B-VIII.0 data, reverting 16 O and 65 Cu cross sections to ENDF/B-VII.1. This resulted in the average relative deviation between calculated and experimental data improving from 1.45σ for the ENDF/B-VIII.0 library to 1.32σ for the best-case library, relative to benchmark uncertainty.

73 NUCLEAR PHYSICS AND RADIATION PHYSICS↗

Continuous-Energy ENDF/B-VIII.0 Cross Section and SCALE 6.2.4 Performance for Nuclear Criticality Safety Applications: 1 H, C, 58,60 Ni, 182,183,184,186 W, 235,238 U, 239 Pu

As part of the nuclear data evaluation and validation cycle, the ENDF/B-VIII.0 cross-section library released in 2018 requires testing to determine areas of improvement and deterioration. Previous work by the authors investigated the performance of 16 O, 56 Fe, and 63,65 Cu cross sections, with this study acting as an extension of the prior work. In addition to the isotopes and nuclear criticality safety benchmarks of interest to the prior work, benchmarks from the International Criticality Safety Benchmark Evaluation Project Handbook were selected for their k eff sensitivity to 1 H, C, 58,60 Ni, 182,183,184,186 W, 235,238U, or 239 Pu cross sections and were modeled in the SCALE code system maintained by Oak Ridge National Laboratory. In total, 253 benchmark configurations were selected for their sensitivities and modeled using SCALE 6.2.4 Criticality Safety Analysis Sequences (CSAS) continuous-energy Monte Carlo k eff calculations. This collection includes and expands upon the 99 benchmarks in the prior work. The AMPX-processed ENDF/B-VIII.0 library was decomposed into individual ENDF/B-VIII.0 datum libraries for each isotope of interest. Doing so allowed for the individual substitution of an ENDF/B-VIII.0 cross section in the place of ENDF/B-VII.1, determining isotope-specific effects of ENDF/B-VIII.0 relative to ENDF/B-VII.1. Full library calculations with entirely ENDF/B-VII.1 data or entirely ENDF/B-VIII.0 data were also executed. As a measure of performance, the average relative deviation was determined as the ratio of the deviation between calculated and experimental keff to the propagated calculational and experimental uncertainty. With calculated full library and isotope-specific ENDF/B-VIII.0 k eff ’s, an optimized combination of data libraries was estimated and confirmed with SCALE calculations. This showed that reverting 239 Pu, 58 Ni, 16 O, and 65 Cu cross sections to ENDF/B-VII.1 resulted in improved performance relative to the full ENDF/B-VIII.0 library. Across all 253 benchmarks, the average relative deviation was 1.29σ for the full ENDF/B-VII.1 library, 1.17σ for the full ENDF/B-VIII.0 library, and 0.97σ for the optimized combination. The reversion of 239 Pu, 58 Ni, 16 O, and 65 Cu cross sections to ENDF/B-VII.1 in the 99 benchmarks of the prior work resulted in further improved experimental agreement compared to the previously reported improvement from 16 O and 65 Cu alone. Therefore, it is suggested that applications with significant sensitivities to 239 Pu, 58 Ni, 16 O, and 65 Cu consider their choice of nuclear data library.

98 NUCLEAR DISARMAMENT, SAFEGUARDS, AND PHYSICAL P↗

Hypermut 3: identifying specific mutational patterns in a defined nucleotide context that allows multistate characters

Abstract Motivation The detection of APOBEC3F- and APOBEC3G-induced mutations in virus sequences is useful for identifying hypermutated sequences. These sequences are not representative of viral evolution and can therefore alter the results of downstream sequence analyses if included. We previously published the software Hypermut, which detects hypermutation events in sequences relative to a reference. Two versions of this method are available as a webtool. Neither of these methods consider multistate characters or gaps in the sequence alignment. Results Here, we present an updated, user-friendly web and command-line version of Hypermut with functionality to handle multistate characters and gaps in the sequence alignment. This tool allows for straightforward integration of hypermutation detection into sequence analysis pipelines. As with the previous tool, while the main purpose is to identify G to A hypermutation events, any mutational pattern and context can be specified. Availability and implementation Hypermut 3 is written in Python 3. It is available as a command-line tool at https://github.com/MolEvolEpid/hypermut3 and as a webtool at https://www.hiv.lanl.gov/content/sequence/HYPERMUT/hypermutv3.html.

59 BASIC BIOLOGICAL SCIENCES↗

Draft genome sequences of strains CBS6241 and CBS6242 of the basidiomycetous yeast Filobasidium floriforme

The Tremellomycetes are a species-rich group within the basidiomycete fungi; however, most analyses of this group to date have focused on pathogenic Cryptococcus species within the order Tremellales. Recent genome-assisted studies of other Tremellomycetes have identified interesting features with respect to biotechnological applications as well as the evolution of genes involved in mating and sexual development. Here, we report genome sequences of two strains of Filobasidium floriforme, a species from the order Filobasidiales, which branches basally to the Tremellales, Trichosporonales, and Holtermanniales. The assembled genomes of strains CBS6241 and CBS6242 are 27.4 Mb and 26.4 Mb in size, respectively, with 8314 and 7695 predicted protein-coding genes. Overall sequence identity at nucleic acid level between the strains is 97%. Among the predicted genes are pheromone precursor and pheromone receptor genes as well as two genes encoding homedomain (HD) transcription factors, which are predicted to be part of the mating type (MAT) locus. Sequence analysis indicates that CBS6241 and CBS6242 carry different alleles for both the pheromone/receptor genes as well as the HD transcription factors. Orthology inference identified 1482 orthogroups exclusively found in F. floriforme, some of which were involved in carbohydrate transport and metabolism. Subsequent CAZyme repertoire characterization identified 267 and 247 enzymes for CBS6241 and CBS6242, respectively, the second highest number of CAZymes among the analyzed Tremellomycete species. In addition, F. floriforme contains five CAZymes absent in other species and several plant-cell-wall degrading CAZymes with the highest copy number in Tremellomycota, indicating the biotechnological potential of this species.

59 BASIC BIOLOGICAL SCIENCES↗

Occurrence and molecular characterization of Enterocytozoon bieneusi in wild and domestic animal species in Portugal

Abstract The phylum Microsporidia encompasses a diverse group of obligate, intracellular, and spore-forming organisms able to infect a wide range of animal hosts. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and animals. Little is known about the presence and epidemiology of E. bieneusi in wildlife. We investigated E. bieneusi occurrence and genetic diversity in wild and domestic mammals, through molecular-detection methods, from different regions across Portugal. A total of 756 samples were collected from 288, 242, and 226 wild carnivores, wild ungulates, and domestic animals, respectively. Overall, eight specimens were E. bieneusi-positive (1.1%, 8/756) obtained from five wild (Iberian lynx, Iberian wolf, red fox, stone marten, and wild boar) and one domestic (sheep) host. Nucleotide sequence analysis identified four genotypes of E. bieneusi, Type IV, Wildboar3, BEB6, and PtEbIX. Three of those genotypes belong to Groups 1 (Type IV and Wildboar3) and 2 (BEB6), which are known to contain genotypes capable of infecting a variety of hosts, including humans, highlighting their public health importance. PtEbIX belongs to the dog-specific Group 11. This study represents the first, largest, and most comprehensive molecular-based epidemiology survey carried out in Portugal in wild and domestic animals to date and the first worldwide identification of E. bieneusi in wolf species. Our study showed that wild carnivores and ungulates may act as reservoirs of zoonotic genotypes of E. bieneusi, establishing their role in maintaining the sylvatic cycle of this parasite while representing a potential source of infection for humans and domestic animals.

Figueiredo, Ana M. (ORCID:0000000226236340)↗

Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III‐secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the Acidovorax genus

Summary The cucurbit pathogenic bacterium Acidovorax citrulli requires a functional type III secretion system (T3SS) for pathogenicity. In this bacterium, as with Xanthomonas and Ralstonia spp . , an AraC‐type transcriptional regulator, HrpX, regulates expression of genes encoding T3SS components and type III‐secreted effectors (T3Es) . The annotation of a sequenced A. citrulli strain revealed 11 T3E genes. Assuming that this could be an underestimation, we aimed to uncover the T3E arsenal of the A. citrulli model strain, M6. Thorough sequence analysis revealed 51 M6 genes whose products are similar to known T3Es. Furthermore, we combined machine learning and transcriptomics to identify novel T3Es. The machine‐learning approach ranked all A. citrulli M6 genes according to their propensity to encode T3Es. RNA‐Seq revealed differential gene expression between wild‐type M6 and a mutant defective in HrpX: 159 and 28 genes showed significantly reduced and increased expression in the mutant relative to wild‐type M6, respectively. Data combined from these approaches led to the identification of seven novel T3E candidates that were further validated using a T3SS‐dependent translocation assay. These T3E genes encode hypothetical proteins that seem to be restricted to plant pathogenic Acidovorax species. Transient expression in Nicotiana benthamiana revealed that two of these T3Es localize to the cell nucleus and one interacts with the endoplasmic reticulum. This study places A. citrulli among the ‘richest’ bacterial pathogens in terms of T3E cargo. It also revealed novel T3Es that appear to be involved in the pathoadaptive evolution of plant pathogenic Acidovorax species.

Jiménez‐Guerrero, Irene↗

Multi-species outbreak of SARS-CoV-2 Delta variant in a zoological institution, with the detection in two new families of carnivores

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a worldwide distribution in humans and many other mammalian species. In late September 2021, 12 animals maintained by the Chicago Zoological Society's Brookfield Zoo were observed with variable clinical signs. The Delta variant of SARS-CoV-2 was detected in faeces and nasal swabs by qRT-PCR, including the first detection in animals from the families Procyonidae and Viverridae. Test positivity rate was 12.5% for 35 animals tested. All animals had been vaccinated with at least one dose of a recombinant vaccine designed for animals and all recovered with variable supportive treatment. Sequence analysis showed that six zoo animal strains were closely correlated with 18 human SARS-CoV-2 strains, suggestive of potential human-to-animal transmission events. This report documents the expanding host range of COVID-19 during the ongoing pandemic.

59 BASIC BIOLOGICAL SCIENCES↗

The fate of 35S rRNA genes in the allotetraploid grass Brachypodium hybridum

Nucleolar dominance (ND) consists of the reversible silencing of 35S/45S rDNA loci inherited from one of the ancestors of an allopolyploid. The molecular mechanisms by which one ancestral rDNA set is selected for silencing remain unclear. We applied a combination of molecular (Southern blot hybridization and reverse-transcription cleaved amplified polymorphic sequence analysis), genomic (analysis of variants) and cytogenetic (fluorescence in situ hybridization) approaches to study the structure, expression and epigenetic landscape of 35S rDNA in an allotetraploid grass that exhibits ND, Brachypodium hybridum (genome composition DDSS), and its putative progenitors, Brachypodium distachyon (DD) and Brachypodium stacei (SS). In progenitor genomes, B. stacei showed a higher intragenomic heterogeneity of rDNA compared with B. distachyon. In all studied accessions of B. hybridum, there was a reduction in the copy number of S homoeologues, which was accompanied by their inactive transcriptional status. The involvement of DNA methylation in CG and CHG contexts in the silencing of the S-genome rDNA loci was revealed. In the B. hybridum allotetraploid, ND is stabilized towards the D-genome units, irrespective of the polyphyletic origin of the species, and does not seem to be influenced by homoeologous 35S rDNA ratios and developmental stage.

35S rDNA evolution↗