rDNA Loci Research Articles

To Repeat or Not to Repeat: Repetitive Sequences Regulate Genome Stability in Candida albicans.

Genome instability often leads to cell death but can also give rise to innovative genotypic and phenotypic variation through mutation and structural rearrangements. Repetitive sequences and chromatin architecture in particular are critical modulators of recombination and mutability. In Candida albicans, four major classes of repeats exist in the genome: telomeres, subtelomeres, the major repeat sequence (MRS), and the ribosomal DNA (rDNA) locus. Characterization of these loci has revealed how their structure contributes to recombination and either promotes or restricts sequence evolution. The mechanisms of recombination that give rise to genome instability are known for some of these regions, whereas others are generally unexplored. More recent work has revealed additional repetitive elements, including expanded gene families and centromeric repeats that facilitate recombination and genetic innovation. Together, the repeats facilitate C. albicans evolution through construction of novel genotypes that underlie C. albicans adaptive potential and promote persistence across its human host.

Comparison of fish detections, community diversity, and relative abundance using environmental DNA metabarcoding and traditional gears

AbstractBackgroundDetecting species at low abundance, including aquatic invasive species (AIS), is critical for making informed management decisions. Environmental DNA (eDNA) methods have become a powerful tool for rare or cryptic species detection; however, many eDNA assays offer limited utility for community‐level analyses due to their use of species‐specific (presence/absence) ‘barcodes’. Metabarcoding methods provide information on entire communities based on sequencing of all taxon‐specific barcodes within an eDNA sample.AimsEvaluate measures of fish species detections, community diversity, and estimates of relative abundance based on eDNA metabarcoding and traditional fisheries sampling approaches in the context of fish community characterization and AIS survellience.Materials and MethodsIn 2016, eight limnologically diverse lakes (surface area range: 13 – 1,728 ha) in Michigan, USA were sampled using a variety of traditional fisheries gears to characterize fish community composition. Environmental DNAs from surface (33 ± 6, mean ± 1 SD) and benthic (14 ± 2) water samples from each lake were isolated and amplified for two metabarcoding markers (mitochondrial 12S and 16S rDNA loci) using fish‐specific primers. Fish species detected within each lake were determined by comparing the sequencing data to a database of sequences from native Michigan fish species and 19 AIS on the Michigan's Watch List.ResultsAnalysis of species accumulation curves indicated multi‐locus eDNA metabarcoding assays can enhance species detection capacities and characterize 95% of a fish community in fewer sampling efforts than traditional gear (range: 2 – 62, median: 14). In addition, all AIS detected in traditional gear samples were also detected by eDNA, while some AIS detected by eDNA assays were absent from traditional gear samples.DiscussionResults reported here are, in part, driven by the lack of species‐selectivity during eDNA sampling events. Given the efficacy of eDNA assays, we suggest multi‐locus eDNA metabarcoding assays be implemented in early detection efforts.

Karyotype evolution in cycads

Karyotype evolution in cycads has been described in 113 species covering all the 10 genera. Cycads are a conservative group in this regard as the genera with low basic numbers i.e. Dioon, Macrozamia, Lepidozamia, Encephalartos, Bowenia (2n = 18) and Ceratozamia and Stangeria (2n = 16) have relatively symmetrical karyotypes while those with higher numbers i.e. Cycas (2n = 22) and Microcycas (2n = 26) have more asymmetric karyotypes. The situation in Zamia is more complicated with different species showing a range of chromosome numbers of 2n = 16–28 while some species even show intraspecific variation. The increase or decrease in number as seen in some Zamia species has occurred mainly by chromosomal fission/fusion. Some variability is however, seen in chromosome banding as well as the position of rDNA loci. The phylogenetic relationships between the genera have been discussed on the basis of karyotype evolution.

IGS sequences in Cestrum present AT- and GC-rich conserved domains, with strong regulatory potential for 5S rDNA.

The 35S and 5S ribosomal DNA (rDNA) organized in thousands of copies in genomes, have been widely used in numerous comparative cytogenetic studies. Nevertheless, several questions related to the diversity and organization of regulatory motifs in 5S rDNA remain to be addressed. The 5S rDNA unit is composed of a conserved 120bp length coding region and an intergenic spacer (IGS) containing potential regulatory motifs (Poly-T, AT-rich and GC-rich) differing in number, redundancy and position along the IGS. The Cestrum species (Solanaceae) have large genomes (about 10pg/1C) and conserved 2n = 16 karyotypes. Strikingly, these genomes show high diversity of heterochromatin distribution, variability in 35S rDNA loci and the occurrence of B chromosomes. However, the 5S rDNA loci are highly conserved in the proximal region of chromosome 8. Comparison of seventy-one IGS sequences in plants revealed several conserved motifs with potential regulatory function. The AT- and GC-rich domains appeared highly conserved in Cestrum chromosomes. The 5S genic and the GC-rich IGS probe produced FISH signals in both A (pair 8) and B chromosomes. The GC-rich domain presented a strong potential for regulation because it may be associated with CpG islands organization, as well as to hairpin and loop organization. Another interesting aspect was the ability of AT- and GC-rich motifs to produce non-heterochromatic CMA/DAPI signals. While the length of the 5S rDNA IGS region varied in size between the Cestrum species, the individual sequence motifs seem to be conserved suggesting their regulatory function. The most striking feature was the conserved GC-rich domain in Cestrum, which is recognized as a signature trait of the proximal region of chromosome pair 8.

Multigene‐based phylogeny analyses of the controversial family Condylostomatidae (Ciliophora, Heterotrichea)

AbstractCondylostoma, the type genus of the family Condylostomatidae, is a well‐known and taxonomically confusing genus with more than 30 nominal species. The phylogenetic relationships among congeners and related genera remain obscure despite the continuous discovery of new species and detailed redescription of known species over the past decades. Based on a total of 24 new sequences obtained from nine Condylostoma isolates, we conducted phylogenetic analyses by combining multiple marker genes. The molecular phylogenetic trees indicate that: (a) the monophyly of the family Condylostomatidae is supported by the rDNA locus and SSU rDNA sequences with high support value but is no longer sustained by both the ITS1‐5.8S‐ITS2 and LSU rDNA sequences; (b) Condylostoma is very likely a paraphyletic genus, and the possibility that it is a polyphyletic group cannot be denied, and (c) the genera Condylostentor and Chattonidium are suggested to evolve from a radiation of Condylostoma species. Furthermore, in an effort to provide better resolutions for understanding the evolutionary relationships of condylostomatids, the secondary structures of the variable regions 2 and 4 (V2 and V4) of SSU rDNA and ITS2 transcripts are predicted. The results revealed that the V2 and V4 structures are rather conserved at the genus level, while the ITS2 structures are much more variable.

Accuracy of PCR and serological testing for the diagnosis of primary syphilis: Both tests are necessary.

Syphilis, caused by the spirochete Treponema pallidum subspecies pallidum, is a rising global public health concern and laboratory diagnostics remain challenging. Especially during early disease, rapid and accurate diagnosis is crucial to ensure patients and their contacts receive timely treatment to eradicate infection and prevent further transmission. In this prospective observational study, we evaluated the performance of polymerase chain reaction (PCR) and serological testing for the diagnosis of primary syphilis by evaluating anogenital swabs and sera from 178 Cuban patients presenting with ulcers. Three different PCR assays were evaluated targeting polA, tpp47 and 16S rDNA loci. Sera were evaluated with venereal disease research laboratory (VDRL) and T. pallidum hemagglutination (TPHA) assays. Assuming both methods were confirmatory, our data showed that PCR and serology did not correlate well (agreement = 52.3%, kappa 0.0512, 95% CI −0.0928–0.1951, p = 0.496). The sensitivities, specificities, positive and negative predictive values of the PCR assays were 76.1%, 100%, 100% and 57.9%, respectively, while the values for serology were 62.5%, 100%, 100% and 45.2%, respectively. The combination of PCR and serology can offer valuable information for the diagnosis of syphilis in patients presenting with anogenital ulceration avoiding further clinical complications and disease transmission.

LYAR potentiates rRNA synthesis by recruiting BRD2/4 and the MYST-type acetyltransferase KAT7 to rDNA.

Activation of ribosomal RNA (rRNA) synthesis is pivotal during cell growth and proliferation, but its aberrant upregulation may promote tumorigenesis. Here, we demonstrate that the candidate oncoprotein, LYAR, enhances ribosomal DNA (rDNA) transcription. Our data reveal that LYAR binds the histone-associated protein BRD2 without involvement of acetyl-lysine–binding bromodomains and recruits BRD2 to the rDNA promoter and transcribed regions via association with upstream binding factor. We show that BRD2 is required for the recruitment of the MYST-type acetyltransferase KAT7 to rDNA loci, resulting in enhanced local acetylation of histone H4. In addition, LYAR binds a complex of BRD4 and KAT7, which is then recruited to rDNA independently of the BRD2-KAT7 complex to accelerate the local acetylation of both H4 and H3. BRD2 also helps recruit BRD4 to rDNA. By contrast, LYAR has no effect on rDNA methylation or the binding of RNA polymerase I subunits to rDNA. These data suggest that LYAR promotes the association of the BRD2-KAT7 and BRD4-KAT7 complexes with transcription-competent rDNA loci but not to transcriptionally silent rDNA loci, thereby increasing rRNA synthesis by altering the local acetylation status of histone H3 and H4.

Phenotypic and Genotypic Consequences of CRISPR/Cas9 Editing of the Replication Origins in the rDNA of Saccharomyces cerevisiae.

The complex structure and repetitive nature of eukaryotic ribosomal DNA (rDNA) is a challenge for genome assembly, thus the consequences of sequence variation in rDNA remain unexplored. However, renewed interest in the role that rDNA variation may play in diverse cellular functions, aside from ribosome production, highlights the need for a method that would permit genetic manipulation of the rDNA. Here, we describe a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based strategy to edit the rDNA locus in the budding yeast Saccharomyces cerevisiae, developed independently but similar to one developed by others. Using this approach, we modified the endogenous rDNA origin of replication in each repeat by deleting or replacing its consensus sequence. We characterized the transformants that have successfully modified their rDNA locus and propose a mechanism for how CRISPR/Cas9-mediated editing of the rDNA occurs. In addition, we carried out extended growth and life span experiments to investigate the long-term consequences that altering the rDNA origin of replication have on cellular health. We find that long-term growth of the edited clones results in faster-growing suppressors that have acquired segmental aneusomy of the rDNA-containing region of chromosome XII or aneuploidy of chromosomes XII, II, or IV. Furthermore, we find that all edited isolates suffer a reduced life span, irrespective of their levels of extrachromosomal rDNA circles. Our work demonstrates that it is possible to quickly, efficiently, and homogeneously edit the rDNA origin via CRISPR/Cas9.

Genotyping Approach for Potential Common Source of Enterocytozoon bieneusi Infection in Hematology Unit

Microsporidiosis is a fungal infection that generally causes digestive disorders, especially in immunocompromised hosts. Over a 4-day period in January 2018, 3 patients with hematologic malignancies who were admitted to the hematology unit of a hospital in France received diagnoses of Enterocytozoon bieneusi microsporidiosis. This unusually high incidence was investigated by sequence analysis at the internal transcribed spacer rDNA locus and then by 3 microsatellites and 1 minisatellite for multilocus genotyping. The 3 isolates had many sequence similarities and belonged to a new genotype closely related to genotype C. In addition, multilocus genotyping showed high genetic distances with all the other strains collected from epidemiologically unrelated persons; none of these strains belonged to the new genotype. These data confirm the epidemiologic link among the 3 patients and support a common source of infection.

Karyotype structure and NOR activity in Brazilian Smilax Linnaeus, 1753 species (Smilacaceae).

The genus Smilax Linnaeus, 1753 (Smilacaceae) is a large genus of dioecious plants distributed in tropical, subtropical and temperate regions. Some Smilax species have medicinal importance and their identification is important for the control of raw material used in the manufacture of phytotherapeutical products. The karyotypes of seven Brazilian Smilax species were investigated. Mitotic metaphases of roots from young plants were analysed in Feulgen-stained preparations. The karyotypes were asymmetric and modal with 2n = 2x = 32 chromosomes gradually decreasing in size. In S. goyazana A De Candolle & C De Candolle, 1878, a polyploid species, 2n = 4x = 64. In all the species, the large and medium-sized chromosomes were subtelocentric and submetacentric and the small chromosomes were submetacentric or metacentric. Their karyotypes were quite similar, with differences in the arm ratio of some chromosomes. S. fluminensis Steudel, 1841 differed from the other species by having a large metacentric chromosome 1. These findings suggest that evolution occurred without drastic changes in the chromosomal structure in the species analyzed. Terminal secondary constrictions were visualized on the short arm of some chromosomes, but they were detected only in one homologue of each pair. Due to the terminal location and the degree of chromosome condensation, secondary constrictions were not visualized in some species. The nucleolus organizer regions (NORs) were mapped by silver-staining and fluorescent in situ hybridization (FISH) in S. rufescens Grisebach, 1842 and S. fluminensis. Silver-staining and FISH signals were colocalized on the short arms of six chromosomes in S. rufescens and four chromosomes in S. fluminensis. In FISH preparations, one of the largest chromosomes had the secondary constrictions highly decondensed in some cells. This finding and the heteromorphism observed in Feulgen-stained chromosomes suggest that differential rRNA gene expression between homologous rDNA loci can occur in some cells, resulting in different degrees of ribosomal chromatin decondensation. The presence of a heteromorphic chromosome pair in S. rufescens, S. polyantha Grisebach, 1842 and S. goyazana suggests a chromosomal sex determination in these dioecious species.

MODULATION OF RNA POLYMERASE I TRANSCRIPTION IN NORMAL AND MALIGNANT HAEMATOPOIESIS

RNA Polymerase I (PolI)-mediated transcription of ribosomal RNAs is the limiting factor of ribosomal biogenesis, is often dysregulated in cancer 1 and is a new target for cancer therapy 2 , 3 . Decreased levels of PolI transcription is also associated with cell differentiation 4 suggesting that high levels of PolI transcription are required for stemness. Mammalian cells contain over 200 copies of the ribosomal genes (rDNA) clustered in specific chromosomal regions that aggregate in the nucleolus, where only a proportion acquires an active chromatic configuration allowing PolI transcription to occur. Malignant transformation is associated with an increase in the portion of active rDNA loci in the nucleolus. We showed that during malignant progression of B-cell lymphoma, the rDNA chromatin acquires an open state and rDNA-genome contacts are reorganized, which correlate with the repression of genes involved in B-cell differentiation 5 . These data support the intriguing hypothesis that dynamic changes in rDNA chromatin and long-range rDNA-genomic interactions regulate distinct programs of gene expression required for stemness, differentiation and malignant transformation. To address this hypothesis we are; (i) examining the molecular mechanisms by which nucleolar-associated chromatin domains dynamically regulate spatial organisation of the genome; (ii) testing whether the concomitant changes in distinct patterns of gene expression actively drive developmental programs such as differentiation and contribute to malignant transformations; and (iii) examining whether the therapeutic efficacy of Pol I inhibitors is mediated in part through the disruption of long-range rDNA genomic interaction.

Identification of a novel species of Eimeria Schneider, 1875 from the woylie, Bettongia penicillata Gray (Diprotodontia: Potoroidae) and the genetic characterisation of three Eimeria spp. from other potoroid marsupials

Faecal samples (n = 1,093) collected from the woylie Bettongia penicillata Gray, in south-western Australia were examined for the presence of coccidian parasites. Eimeria sp. oöcysts were detected in 15.2% of samples. Faecal samples obtained from the eastern bettong Bettongia gaimardi (Desmarest) (n = 4) and long-nosed potoroo Potorous tridactylus (Kerr) (n = 12) in Tasmania, were also screened for the presence of Eimeria spp. (prevalence 50% and 41.7%, respectively). Morphological and genetic comparison with other known species of Eimeria indicates that the material identified in woylies is novel. This study aimed to (i) morphologically describe and genetically characterise Eimeria woyliei n. sp. found in woylies; and (ii) genetically characterise Eimeria gaimardi Barker, O’Callaghan & Beveridge, 1988, Eimeria potoroi Barker, O’Callaghan & Beveridge, 1988, and Eimeria mundayi Barker, O’Callaghan & Beveridge, 1988, from other potoroid marsupials. Molecular phylogenetic analyses conducted at the 18S rDNA and mitochondrial cytochrome c oxidase subunit 1 (cox1) loci revealed that E. woyliei n. sp. was most closely related to Eimeria setonicis Barker, O’Callaghan & Beveridge, 1988, at the 18S rDNA locus, and Eimeria trichosuri O’Callaghan & O’Donoghue, 2001, at the cox1 locus. Eimeria woyliei n. sp. is the sixth species of Eimeria to be formally described from potoroid marsupials.

Deciphering the Origin and Evolution of the X1X2Y System in Two Closely-Related Oplegnathus Species (Oplegnathidae and Centrarchiformes).

Oplegnathus fasciatus and O. punctatus (Teleostei: Centrarchiformes: Oplegnathidae), are commercially important rocky reef fishes, endemic to East Asia. Both species present an X1X2Y sex chromosome system. Here, we investigated the evolutionary forces behind the origin and differentiation of these sex chromosomes, with the aim to elucidate whether they had a single or convergent origin. To achieve this, conventional and molecular cytogenetic protocols, involving the mapping of repetitive DNA markers, comparative genomic hybridization (CGH), and whole chromosome painting (WCP) were applied. Both species presented similar 2n, karyotype structure and hybridization patterns of repetitive DNA classes. 5S rDNA loci, besides being placed on the autosomal pair 22, resided in the terminal region of the long arms of both X1 chromosomes in females, and on the X1 and Y chromosomes in males. Furthermore, WCP experiments with a probe derived from the Y chromosome of O. fasciatus (OFAS-Y) entirely painted the X1 and X2 chromosomes in females and the X1, X2, and Y chromosomes in males of both species. CGH failed to reveal any sign of sequence differentiation on the Y chromosome in both species, thereby suggesting the shared early stage of neo-Y chromosome differentiation. Altogether, the present findings confirmed the origin of the X1X2Y sex chromosomes via Y-autosome centric fusion and strongly suggested their common origin.

Prevalence, fecal egg counts, and species identification of gastrointestinal nematodes in replacement dairy heifers in Canada

Information is scarce regarding the epidemiology of gastrointestinal nematodes in Canadian dairy heifers. The objectives of this study were to estimate the prevalence and fecal egg counts of gastrointestinal nematodes in dairy heifers, and using a novel deep-amplicon sequencing approach, to identify the predominant gastrointestinal nematode species in Canadian dairy replacement heifers. Fresh environmental fecal samples (n = 2,369) were collected from replacement heifers on 306 dairy farms across western Canada, Ontario, Québec, and Atlantic Canada. Eggs per gram of feces (EPG) were determined using a modified Wisconsin double-centrifugation sugar flotation technique. Predominant nematode species at the farm level were identified by deep-amplicon nemabiome sequencing of the internal transcribed spacer-2 rDNA locus of nematode third-stage larvae. Generalized estimating equations were used to estimate predicted parasite prevalence and mean EPG in all heifers and by province, allowing for clustering within herds. Individual heifer egg counts ranged from 0 to 141 EPG (median: 0 EPG; interquartile range: 0 to 71 EPG). Gastrointestinal nematodes were detected in 20.9% (95% confidence interval: 17.2 to 24.6%) of heifers, and the predicted mean strongylid EPG accounting for clustering on farms was 1.1 (95% confidence interval: 0.6 to 1.6). The predominant parasite species were Cooperia oncophora and Ostertagia ostertagi. This is the first study in Canada to use a combination of deep-amplicon nemabiome sequencing and a traditional egg count method to describe the epidemiology of gastrointestinal nematodes in dairy heifers.

Superresolution microscopy reveals linkages between ribosomal DNA on heterologous chromosomes.

The spatial organization of the genome is enigmatic. Direct evidence of physical contacts between chromosomes and their visualization at nanoscale resolution has been limited. We used superresolution microscopy to demonstrate that ribosomal DNA (rDNA) can form linkages between chromosomes. We observed rDNA linkages in many different human cell types and demonstrated their resolution in anaphase. rDNA linkages are coated by the transcription factor UBF and their formation depends on UBF, indicating that they regularly occur between transcriptionally active loci. Overexpression of c-Myc increases rDNA transcription and the frequency of rDNA linkages, further suggesting that their formation depends on active transcription. Linkages persist in the absence of cohesion, but inhibition of topoisomerase II prevents their resolution in anaphase. We propose that linkages are topological intertwines occurring between transcriptionally active rDNA loci spatially colocated in the same nucleolar compartment. Our findings suggest that active DNA loci engage in physical interchromosomal connections that are an integral and pervasive feature of genome organization.

Molecular Cytogenetic Characterization of the Native Forage GrassTrichloris crinita

ABSTRACTTrichloris crinita(Lag.) Parodi is a perennial forage grass native to—and of wide distribution in—arid and semiarid regions of the American continent. In addition to its good forage quality, this species is particularly valued in these drylands due to its high tolerance to drought, salinity, trampling, and grazing. Genetic and cytogenetic information forT. crinitais very scarce, hindering progress in genetic research and breeding of the species. We describe chromosome numbers, karyotypes, heterochromatin distribution, physical mapping of ribosomal DNA (rDNA) genes (18–5.8–26S and 5S) by fluorescent in situ hybridization (FISH), and chromosome pairing in meiotic cells for the first time inT. crinitaaccessions. Karyotype based on chromosome morphology and cytogenetic landmarks are presented for eightT. crinitaaccessions. Physical mapping of rDNA loci revealed variation in the number, size, and intensity of the 18–5.8–26S FISH signals, whereas 5S rDNA produced two signals per accession. Considering a basic chromosome number of 10, as reported for most Chloridoideae species and the facts that all 20T. crinitaaccessions analyzed had 40 somatic chromosomes, that only bivalent chromosome pairings were observed in meiosis, and that chromosome morphology and sizes revealed high similarity between pairs of chromosomes, along with other reproductive features of the species, strongly suggest thatT. crinitais an allotetraploid (2n= 4x= 40). Our cytogenetic data represent a useful resource for advancing cytogenetic research and breeding inT. crinita.

Description of Xiphinema azarbaijanense n. sp. (Nematoda; Longidoridae) from West Azarbaijan province, northwestern Iran

A new species of the dagger nematode genus Xiphinema was recovered from three different points in West Azarbaijan province, northwestern Iran, from the rhizosphere of grasses and described based on both morphological and molecular data. Xiphinema azarbaijanense n. sp. belongs to the Xiphinema non-americanum group and is characterized by having 3.8-5.3 mm long females with long odontostyle (129-151 μm), two equally developed genital tracts with crystalloid bodies in the tubular part of uterus, short tail, four juvenile developmental stages and males absent. It is similar to 14 known species belonging to morphospecies group 6, and is almost indistinguishable from most of them using traditional taxonomy; thus, is part of a complex of cryptic species. Molecular phylogenetic data using two genomic structural (18S, 28S D2-D3) and one ITS1 ribosomal DNA (rDNA) loci were used to separate the new species from close taxa. The new species formed an independent lineage in three reconstructed trees based upon the resolved topologies, and was also morphologically compared with phylogenetically related species.

Detecting Brachypodium distachyon Chromosomes Bd4 and Bd5 in MH- and X-Ray-Induced Micronuclei Using mcFISH.

Micronuclei are biomarkers of genotoxic effects and chromosomal instability. They are formed when chromosome fragments or whole chromosomes fail to disjoin into daughter nuclei. We present qualitative and quantitative analyses of the involvement of specific chromosome regions of chromosomes Bd4 and Bd5 in the formation of micronuclei of Brachypodium distachyon root tip cells following maleic hydrazide (MH) treatment and X-radiation. This is visualised by cytomolecular approaches using bacterial artificial chromosome (BAC)-based multicolour fluorescence in situ hybridisation (mcFISH) in combination with 5S and 25S rDNA probes. The results showed that the long arm of submetacentric chromosome Bd4 forms micronuclei at twice the frequency of its short arm, suggesting that the former is more prone to double-strand breaks (DSBs). In contrast, no difference was observed in the frequency of micronuclei derived from the long and short arms of submetacentric chromosome Bd5. Interestingly, the proximal region of the short arm of Bd5 is more prone to DSBs than its distal part. This demonstrates that 5S rDNA and 35S rDNA loci are not “hot spots” for DNA breaks after the application of these mutagens.

The first survey of Cystobasidiomycete yeasts in the lichen genus Cladonia; with the description of Lichenozyma pisutiana gen. nov., sp. nov.

The view of lichens as a symbiosis only between a mycobiont and a photobiont has been challenged by discoveries of diverse associated organisms. Specific basidiomycete yeasts in the cortex of a range of macrolichens were hypothesized to influence the lichens' phenotype. The present study explores the occurrence and diversity of cystobasidiomycete yeasts in the lichen genus Cladonia. We obtained seven cultures and 56 additional sequences using specific primers from 27 Cladonia species from all over Europe and performed phylogenetic analyses based on ITS, LSU and SSU rDNA loci. We revealed yeast diversity distinct from any previously reported. Representatives of Cyphobasidiales, Microsporomycetaceae and of an unknown group related to Symmetrospora have been found. We present evidence that the Microsporomycetaceae contains mainly lichen-associated yeasts. Lichenozyma pisutiana is circumscribed here as a new genus and species. We report the first known associations between cystobasidiomycete yeasts and Cladonia (both corticate and ecorticate), and find that the association is geographically widespread in various habitats. Our results also suggest that a great diversity of lichen associated yeasts remains to be discovered.

How dynamic could be the 45S rDNA cistron? An intriguing variability in a grasshopper species revealed by integration of chromosomal and genomic data.

To better understand the structure and variability of the 45S rDNA cistron and its evolutionary dynamics in grasshoppers, we performed a detailed analysis combining classical and molecular cytogenetic data with whole-genome sequencing in Abracris flavolienata, which shows extraordinary variability in the chromosomal distribution for this element. We found astonishing variability in the number and size of rDNA clusters at intra- and inter-population levels. Interestingly, FISH using distinct parts of 45S rDNA cistron (18S rDNA, 28S rDNA, and ITS1) as probes revealed a distinct number of clusters, suggesting independent mobility and amplification of the 45S rDNA components. This hypothesis is consistent with the higher genomic coverage of almost the entire cistron of 45S rDNA observed in A. flavolineata compared to other grasshoppers, besides coverage variability along the 45S rDNA cistron in the species. In addition, these differences in coverage for distinct components of the 45S rDNA cistron indicate emergence of pseudogenes evidenced by existence of truncated sequences, demonstrating the rDNA dynamics in the species. Although the chromosomal distribution of 18S rDNA was highly variable, the chromosomes 1, 3, 6, and 9 harbored rDNA clusters in all individuals with the occurrence of NOR activity in pair 9, suggesting ancestry or selective pressures to prevent pseudogenization of rDNA sequences in this chromosome pair. Additionally, small NORs and cryptic rDNA loci were observed. Finally, there was no evidence of enrichment and association of transposable elements, at least, inside or nearby rDNA cistron. These findings broaden our knowledge of rDNA dynamics, revealing an independent movement and amplification of segments of 45S rDNA cistron, which in A. flavolineata could be attributed to ectopic recombination.