rDNA Repeats Research Articles

Stalled RNAP‐II molecules bound to non‐coding rDNA spacers are required for normal nucleolus architecture

The correct distribution of nuclear domains is critical for the maintenance of normal cellular processes such as transcription and replication, which are regulated depending on their location and surroundings. The most well-characterized nuclear domain, the nucleolus, is essential for cell survival and metabolism. Alterations in nucleolar structure affect nuclear dynamics; however, how the nucleolus and the rest of the nuclear domains are interconnected is largely unknown. In this report, we demonstrate that RNAP-II is vital for the maintenance of the typical crescent-shaped structure of the nucleolar rDNA repeats and rRNA transcription. When stalled RNAP-II molecules are not bound to the chromatin, the nucleolus loses its typical crescent-shaped structure. However, the RNAP-II interaction with Seh1p, or cryptic transcription by RNAP-II, is not critical for morphological changes.

The million mutation project: A new approach to genetics in Caenorhabditis elegans

We have created a library of 2007 mutagenized Caenorhabditis elegans strains, each sequenced to a target depth of 15-fold coverage, to provide the research community with mutant alleles for each of the worm's more than 20,000 genes. The library contains over 800,000 unique single nucleotide variants (SNVs) with an average of eight nonsynonymous changes per gene and more than 16,000 insertion/deletion (indel) and copy number changes, providing an unprecedented genetic resource for this multicellular organism. To supplement this collection, we also sequenced 40 wild isolates, identifying more than 630,000 unique SNVs and 220,000 indels. Comparison of the two sets demonstrates that the mutant collection has a much richer array of both nonsense and missense mutations than the wild isolate set. We also find a wide range of rDNA and telomere repeat copy number in both sets. Scanning the mutant collection for molecular phenotypes reveals a nonsense suppressor as well as strains with higher levels of indels that harbor mutations in DNA repair genes and strains with abundant males associated with him mutations. All the strains are available through the Caenorhabditis Genetics Center and all the sequence changes have been deposited in WormBase and are available through an interactive website.

Molecular characterization of major and minor rDNA repeats and genetic variability assessment in different species of mahseer found in North India

Relationship among the mahseer species (Family: Cyprinidae) has long been debated in fish systematics. Present study concentrates on the nature of the phylogenetic relationship among the five mahseer species using the sequence of major ribosomal DNA (45S rDNA). We have covered rDNA sequence of approximately 5.2kb per individual, 26.0kb per species and 130.0kb as a whole. We also characterized the 45S and 5S rDNA regions with respect to their nucleotide composition. For phylogenetic analyses, nucleotide sequences were divided into four datasets. First and second datasets contained 18S rDNA and ITS1 sequence, whereas third and fourth datasets consisted of ITS2 and complete 18S-ITS1-5.8S-ITS2-28S, respectively. The NJ tree was constructed for all the datasets. The mahseer species under study formed a monophyletic group well separated from the outgroup species. Similarly, the individuals of Neolissochilus hexagonolepis form monophyletic group with Tor species, indicating Neolissochilus as a sister genus of Tor. The findings from the present study provide greater insights into taxonomic status of mahseer, and set the stage for future investigations dealing with phylo-geography, taxonomy, conservation and co-evolution within this interesting and important group of fish.

Metschnikowia Species Share a Pool of Diverse rRNA Genes Differing in Regions That Determine Hairpin-Loop Structures and Evolve by Reticulation.

Modern taxonomy of yeasts is mainly based on phylogenetic analysis of conserved DNA and protein sequences. By far the most frequently used sequences are those of the repeats of the chromosomal rDNA array. It is generally accepted that the rDNA repeats of a genome have identical sequences due to the phenomenon of sequence homogenisation and can thus be used for identification and barcoding of species. Here we show that the rDNA arrays of the type strains of Metschnikowia andauensis and M. fructicola are not homogenised. Both have arrays consisting of diverse repeats that differ from each other in the D1/D2 domains by up to 18 and 25 substitutions. The variable sites are concentrated in two regions that correspond to back-folding stretches of hairpin loops in the predicted secondary structure of the RNA molecules. The substitutions do not alter significantly the overall hairpin-loop structure due to wobble base pairing at sites of C-T transitions and compensatory mutations in the complementary strand of the hairpin stem. The phylogenetic and network analyses of the cloned sequences revealed that the repeats had not evolved in a vertical tree-like way but reticulation might have shaped the rDNA arrays of both strains. The neighbour-net analysis of all cloned sequences of the type strains and the database sequences of different strains further showed that these species share a continuous pool of diverse repeats that appear to evolve by reticulate evolution.

Intragenomic Profiling Using Multicopy Genes: The rDNA Internal Transcribed Spacer Sequences of the Freshwater Sponge Ephydatia fluviatilis

Multicopy genes, like ribosomal RNA genes (rDNA), are widely used to describe and distinguish individuals. Despite concerted evolution that homogenizes a large number of rDNA gene copies, the presence of different gene variants within a genome has been reported. Characterization of an organism by defining every single variant of tens to thousands of rDNA repeat units present in a eukaryotic genome would be quite unreasonable. Here we provide an alternative approach for the characterization of a set of internal transcribed spacer sequences found within every rDNA repeat unit by implementing direct sequencing methodology. The prominent allelic variants and their relative amounts characterizing an individual can be described by a single sequencing electropherogram of the mixed amplicon containing the variants present within the genome. We propose a method for rational analysis of heterogeneity of multicopy genes by compiling a profile based on quantification of different sequence variants of the internal transcribed spacers of the freshwater sponge Ephydatia fluviatilis as an example. In addition to using conventional substitution analysis, we have developed a mathematical method, the proportion model method, to quantify the relative amounts of allelic variants of different length using data from direct sequencing of the heterogeneous amplicon. This method is based on determining the expected signal intensity values (corresponding to peak heights from the sequencing electropherogram) by sequencing clones from the same or highly similar amplicon and comparing hypothesized combinations against the values obtained by direct sequencing of the heterogeneous amplicon. This method allowed to differentiate between all specimens analysed.

Comparative cytogenetic study onTrifolium subterraneum(2n= 16) andTrifolium israeliticum(2n= 12)

Changes in chromosome number have played an important role in the evolution of the genus Trifolium. Along with a few species of polyploid origin there are several cases of dysploidy as evidenced by the presence of four basic chromosome numbers (x = 8, 7, 6, 5). Trifolium subterraneum and Trifolium israeliticum are related species with chromosome complements 2n = 16 and 2n = 12, respectively. Although they represent an interesting case of speciation based on chromosome number reduction, no attempts to demonstrate their cytogenetic affinity have been carried out to date. With this study we performed a comparative cytogenetic study with the purpose of clarifying the evolutionary relationship between these species and to verify whether genomic rearrangements, other than modification of the chromosome number, are associated with the speciation process. Although karyomorphological analysis supports the hypothesis that chromosome rearrangements had a role in the reduction of the chromosome number, the physical mapping of the rDNA sequences revealed a significant remodelling of the 45S and 5S rDNA sites that greatly contributed to the differentiation of the 2n = 16 and 2n = 12 karyotypes. The nucleotide analysis of 5S rDNA repeats confirmed that the two species are related but constitute distinct entities. The observed genomic changes lead to the hypothesis that the 2n = 12 species is the result of an evolutionary pathway that passed through intermediate forms. It cannot be excluded that the most direct ancestor of T. israeliticum is a species with 2n = 14.

Cytogenetic characterization by in situ hybridization techniques and molecular analysis of 5S rRNA genes of the European hazelnut (Corylus avellana)

The European hazelnut (Corylus avellana L.) is widespread in Europe, where it has been cultivated for centuries. Despite progress in genetics, most of the cytogenetic aspects of this species have been overlooked. The aim of this study was to fill in this gap and obtain basic information on the chromosome structure of this species. Karyomorphological analysis confirmed the chromosome number 2n = 22 and showed that, despite their apparent uniformity, the chromosomes could be separated into three groups of different size: large (L), medium (M), and small (S). As a first step towards the physical mapping of the hazelnut chromosomes, we applied FISH to localize the position of rRNA genes (rDNA). The sites of 45S and 5S rDNA enabled us to identify two chromosome pairs belonging, respectively, to the L and S groups. The self-GISH procedure revealed that repetitive DNA is concentrated in the pericentromeric regions of the chromosomes, as with other species with rather small genomes. The analysis of 5S rDNA repeats offered additional information on the hazelnut genome by obtaining the whole sequence of the transcribed region so far unpublished. The overall results constitute a substantial advance in hazelnut cytogenetics. Further investigation of other species of Corylus could be an effective approach to understanding the phylogenesis of the genus and resolving taxonomic problems.

The SWI/SNF Chromatin Remodeling Complex Influences Transcription by RNA Polymerase I in Saccharomyces cerevisiae

SWI/SNF is a chromatin remodeling complex that affects transcription initiation and elongation by RNA polymerase II. Here we report that SWI/SNF also plays a role in transcription by RNA polymerase I (Pol I) in Saccharomyces cerevisiae. Deletion of the genes encoding the Snf6p or Snf5p subunits of SWI/SNF was lethal in combination with mutations that impair Pol I transcription initiation and elongation. SWI/SNF physically associated with ribosomal DNA (rDNA) within the coding region, with an apparent peak near the 5′ end of the gene. In snf6Δ cells there was a ∼2.5-fold reduction in rRNA synthesis rate compared to WT, but there was no change in average polymerase occupancy per gene, the number of rDNA gene repeats, or the percentage of transcriptionally active rDNA genes. However, both ChIP and EM analyses showed a small but reproducible increase in Pol I density in a region near the 5′ end of the gene. Based on these data, we conclude that SWI/SNF plays a positive role in Pol I transcription, potentially by modifying chromatin structure in the rDNA repeats. Our findings demonstrate that SWI/SNF influences the most robust transcription machinery in proliferating cells.

Heterochromatin dynamics during developmental transitions in Arabidopsis — a focus on ribosomal DNA loci

The Arabidopsis chromosomes contain conspicuous heterochromatin domains comprising the repetitive 45S and 5S ribosomal DNA loci as well as centromeric and pericentromeric repeats that organize into chromocenters during interphase. During developmental phase transitions such as seed maturation, germination, seedling growth and flowering that require large-scale reprogramming of gene expression patterns, the organization of repetitive sequences into chromocenters dynamically changes. Here we illustrate recent studies that shed light on the heterochromatin dynamics in cotyledons, the first aerial tissues preformed in the embryo, and in true leaves. We will summarize available data for the 5S rDNA repeat loci, in particular their chromatin organization and expression dynamics during the first days of post-germination development, and discuss how the plant accommodates 5S rRNA transcription during large-scale chromatin reorganization events.

Centromeric motion facilitates the mobility of interphase genomic regions in fission yeast

Dispersed genetic elements, such as retrotransposons and Pol-III-transcribed genes, including tRNA and 5S rRNA, cluster and associate with centromeres in fission yeast through the function of condensin. However, the dynamics of these condensin-mediated genomic associations remains unknown. We have examined the 3D motions of genomic loci including the centromere, telomere, rDNA repeat locus, and the loci carrying Pol-III-transcribed genes or long-terminal repeat (LTR) retrotransposons in live cells at as short as 1.5-second intervals. Treatment with carbendazim (CBZ), a microtubule-destabilizing agent, not only prevents centromeric motion, but also reduces the mobility of the other genomic loci during interphase. Further analyses demonstrate that condensin-mediated associations between centromeres and the genomic loci are clonal, infrequent and transient. However, when associated, centromeres and the genomic loci migrate together in a coordinated fashion. In addition, a condensin mutation that disrupts associations between centromeres and the genomic loci results in a concomitant decrease in the mobility of the loci. Our study suggests that highly mobile centromeres pulled by microtubules in cytoplasm serve as 'genome mobility elements' by facilitating physical relocations of associating genomic regions.

Genetic variability in the ITS and IGS regions of the ribosomal DNA of Acremonium cavaraeanum exhibiting antimicrobial activity

Endophytic microorganisms represent promising alternatives for obtaining new drugs of biotechnological importance. In this study, the endophytic species Acremonium cavaraeanum (A1a) isolated from Cocos nucifera was cultivated for the production of secondary metabolites, and its extracts and fractions were evaluated by the dilution method (MIC). The EtOAc extracts and MeOH fractions were tested against Gram-positive and -negative bacteria, and had an MIC of 125 µg/mL when evaluated in the EtOAc extract (EBI). The EtOAc extract (EBII) had an MIC of 62.25 µg/mL for Staphylococcus aureus and an MIC between 125 and 250 µg/mL for Gram-negative bacteria. The methanolic fractions showed activity with MIC between 125 and 250 µg/mL for all bacteria tested. The IGS region of the rDNA repeat unit of genomic DNA was analyzed by PCR/RFLPs, including endonucleases PstI, BamHII, HinfI, and EcoRI. The physical maps showed different restriction sites for the 6 Acremonium sp isolates, and revealed 5 RFLP patterns. The results showed that isolates of the same Acremonium species exhibited variation in this specific region. The sequences of ITS1-5.8S-ITS2 regions were aligned by Clustal W using the neighbor joining method, which grouped the isolates into 5 distinct clusters. This study aimed to evaluate the genetic diversity of A. cavaraeanum crops exhibiting antibacterial activity. The results of this study indicate that different fungal genetic isolates have biotechnological potential for the production of active bio-compounds against several human pathogens.

Phylogenetic Analysis of Common Millet (Panicum miliaceum L.) using NTS of 5S Ribosomal DNA

Twenty two common millet (Panicum miliaceum L.) varieties collected from Korea, China and Russia were investigated for their phylogenetic relationship using 5S ribosomal DNA sequences with a hope to provide the basic information on their exact origin. Sequences of 5S rDNA were isolated by PCR. The primers, 5s-rRNA1 and 5s-rRNA2, were designed to isolate the complete NTS. Genomic DNA amplification produced two fragments with different length, 900 bp and 400 bp fragments, confirming the presence of two types of 5S rDNA repeats that differed from each other in the length of the NTS region. Amplified DNAs of 400 bp fragment were subcloned and used for further investigation. The obtained NTS sequences ranged from 200 to 300 bp and homology of sequences among plant materials was much higher than long repeat. CLUSTALW multiple aligment of 5S rDNA sequences from 22 different common millets revealed the clear difference by their origin. And critically different areas with insert or deletion were also confirmed. Those sequence difference seems to be used for discrimination of cultivars from different origin and use as molecular markers for origin identification. In phylogenic tree construction, the clear classification was shown where the genotypes from China and Russia is positioned together and stay away from domestic genotypes.

Collaborating functions of BLM and DNA topoisomerase I in regulating human rDNA transcription

Bloom's syndrome (BS) is an inherited disorder caused by loss of function of the recQ-like BLM helicase. It is characterized clinically by severe growth retardation and cancer predisposition. BLM localizes to PML nuclear bodies and to the nucleolus; its deficiency results in increased intra- and inter-chromosomal recombination, including hyper-recombination of rDNA repeats. Our previous work has shown that BLM facilitates RNA polymerase I-mediated rRNA transcription in the nucleolus (Grierson et al., 2012 [18]). This study uses protein co-immunoprecipitation and in vitro transcription/translation (IVTT) to identify a direct interaction of DNA topoisomerase I with the C-terminus of BLM in the nucleolus. In vitro helicase assays demonstrate that DNA topoisomerase I stimulates BLM helicase activity on a nucleolar-relevant RNA:DNA hybrid, but has an insignificant effect on BLM helicase activity on a control DNA:DNA duplex substrate. Reciprocally, BLM enhances the DNA relaxation activity of DNA topoisomerase I on supercoiled DNA substrates. Our study suggests that BLM and DNA topoisomerase I function coordinately to modulate RNA:DNA hybrid formation as well as relaxation of DNA supercoils in the context of nucleolar transcription.

Lsd1 and Lsd2 Control Programmed Replication Fork Pauses and Imprinting in Fission Yeast

In the fission yeast Schizosaccharomyces pombe, a chromosomal imprinting event controls the asymmetric pattern of mating-type switching. The orientation of DNA replication at the mating-type locus is instrumental in this process. However, the factors leading to imprinting are not fully identified and the mechanism is poorly understood. Here, we show that the replication fork pause at the mat1 locus (MPS1), essential for imprint formation, depends on the lysine-specific demethylase Lsd1. We demonstrate that either Lsd1 or Lsd2 amine oxidase activity is required for these processes, working upstream of the imprinting factors Swi1 and Swi3 (homologs of mammalian Timeless and Tipin, respectively). We also show that the Lsd1/2 complex controls the replication fork terminators, within the rDNA repeats. These findings reveal a role for the Lsd1/2 demethylases in controlling polar replication fork progression, imprint formation, and subsequent asymmetric cell divisions.

Enforcement of a lifespan‐sustaining distribution of Sir2 between telomeres, mating‐type loci, and rDNA repeats by Rif1

Telomere dysfunction is linked with genome instability and premature aging. Roles for sirtuin proteins at telomeres are thought to promote lifespan in yeast and mammals. However, replicative lifespan of the budding yeast Saccharomyces cerevisiae shortens upon deletion of Rif1, a protein that limits the recruitment of the sirtuin histone deacetylase Sir2 to telomeres. Here we show that Rif1 maintains replicative lifespan by ultimately stabilizing another age-related chromosomal domain harboring the ribosomal DNA (rDNA) repeats. Deletion of Rif1 increases Sir2 localization to telomeres and the silent mating-type loci, while releasing a pool of the histone deacetylase from the intergenic spacer 1 (IGS1) of rDNA. This is accompanied by a disruption of IGS1 silent chromatin assembly and increases in aberrant recombination within rDNA repeats. Lifespan defects linked with Rif1 deletion are abolished if rDNA repeats are forcibly stabilized via deletion of the replication fork-blocking protein Fob1. In addition, Sir2 overexpression prevents Rif1 deletion from disrupting Sir2 at IGS1 and shortening lifespan. Moreover, subjecting cells lacking Rif1 to caloric restriction increases IGS1 histone deacetylation and lifespan, while uncovering novel genetic interactions between RIF1 and SIR2. Our data indicate that Rif1 maintains lifespan-sustaining levels of Sir2 at rDNA by preventing excessive recruitment of the histone deacetylase to telomeric and silent mating-type loci. As sirtuin histone deacetylases, such as Sir2 or mammalian SIRT6, each operate at multiple age-related loci, we propose that factors limiting the localization of sirtuins to certain age-related loci can promote lifespan-sustaining roles of these sirtuins elsewhere in the genome.

AAV Vectors for the Nucleolus

During the replication of wild-type adeno-associated virus (AAV), viral capsids are assembled in the nucleolus,1 and the capsid proteins interact with the nucleolar proteins nucleolin (NCL) and nucleophosmin (NPM1), possibly promoting their transport from the cytoplasm into the nucleolus.2,3 In the case of replication-incompetent AAV vectors, the nucleolus is also important because vector virions can be found inside the nucleolus soon after entering the cell, and both NCL and NPM1 are involved in this trafficking.4 The nucleolar localization of vector virions is probably responsible for the unusual preference of AAV vectors for integration within the ribosomal DNA (rDNA) repeats, which accounts for 3–8% of all nonhomologous integration events,5,6 or 10- to 30-fold more than expected based on the amount of rDNA per cell. In this issue of Molecular Therapy, two reports from the labs of Markus Grompe and Mark Kay exploit this tendency of AAV virions to localize to the nucleolus by incorporating rDNA sequences into the vector genome and transducing mouse hepatocytes with these “rDNA vectors.”7,8 This simple trick produced dramatic effects, resulting in a 30-fold increase in chromosomal integration, about one third of which occurred within the rDNA repeats.8

AAV Vectors Containing rDNA Homology Display Increased Chromosomal Integration and Transgene Persistence

Although recombinant adeno-associated viral (rAAV) vectors are promising tools for gene therapy of genetic disorders, they remain mostly episomal and hence are lost during cell replication. For this reason, rAAV vectors capable of chromosomal integration would be desirable. Ribosomal DNA (rDNA) repeat sequences are overrepresented during random integration of rAAV. We therefore sought to enhance AAV integration frequency by including 28S rDNA homology arms into our vector design. A vector containing ~1 kb of homology on each side of a cDNA expression cassette for human fumarylacetoacetate hydrolase (FAH) was constructed. rAAV of serotypes 2 and 8 were injected into Fah-deficient mice, a model for human tyrosinemia type 1. Integrated FAH transgenes are positively selected in this model and rDNA-containing AAV vectors had a ~30× higher integration frequency than controls. Integration by homologous recombination (HR) into the 28S rDNA locus was seen in multiple tissues. Furthermore, rDNA-containing AAV vectors for human factor IX (hFIX) demonstrated increased transgene persistence after liver regeneration. We conclude that rDNA containing AAV vectors may be superior to conventional vector design for the treatment of genetic diseases, especially those associated with increased hepatocyte replication.

The evolution and utility of ribosomal ITS sequences in Bambusinae and related species: divergence, pseudogenes, and implications for phylogeny

Ribosomal internal transcribed spacer (ITS) sequences are commonly used for phylogenetic reconstruction because they are highly reiterated as components of rDNA repeats, and hence are often subject to rapid homogenization through concerted evolution. Concerted evolution leads to intragenomic uniformity of repeats even between loci on nonhomologous chromosomes. However, a number of studies have shown that the ITS polymorphism within individuals is quite common. The molecular systematics of Bambusinae and related species were recently assessed by different teams using independently generated ITS sequences, and the results disagreed in some remarkable features. Here we compared the ITS sequences of the members of Bambusa s. l., the genera Dendrocalamus, Dinochloa, Gigantochloa, Guadua, Melocalamus, Monocladus, Oxytenanthera, Thyrsostachys, Pleioblastus, Pseudosasa and Schizostachyum.We have reanalysed the ITS sequences used by different research teams to reveal the underlying patterns of their different results. After excluding the sequences suspected to represent paralogous loci, a phylogenetic analysis of the subtribe Bambusinae species were performed using maximum parsimony and maximum-likelihood methods. The implications of the findings are discussed. The risk of incorporating ITS paralogues in plant evolutionary studies that can distort the phylogenetic signal should caution molecular systematists.

Karyotype variation is indicative of subgenomic and ecotypic differentiation in switchgrass

BackgroundKaryotypes can provide information about taxonomic relationships, genetic aberrations, and the evolutionary origins of species. However, differentiation of the tiny chromosomes of switchgrass (Panicum virgatum L.) and creation of a standard karyotype for this bioenergy crop has not been accomplished due to lack of distinguishing features and polyploidy.ResultsA cytogenetic study was conducted on a dihaploid individual (2n = 2X = 18) of switchgrass to establish a chromosome karyotype. Size differences, condensation patterns, and arm-length ratios were used as identifying features and fluorescence in-situ hybridization (FISH) assigned 5S and 45S rDNA loci to chromosomes 7 and 2 respectively. Both a maize CentC and a native switchgrass centromeric repeat (PviCentC) that shared 73% sequence identity demonstrated a strong signal on chromosome 3. However, only the PviCentC probe labeled the centromeres of all chromosomes. Unexpected PviCentC and 5S rDNA hybidization patterns were consistent with severe reduction or total deletion of these repeats in one subgenome. These patterns were maintained in tetraploid and octoploid individuals. The 45S rDNA repeat produced the expected number of loci in dihaploid, tetraploid and octoploid individuals. Differences observed at the 5S rDNA loci between the upland and lowland ecotypes of switchgrass provided a basis for distinguishing these subpopulations.ConclusionCollectively, these results provide a quantitative karyotype of switchgrass chromosomes. FISH analyses indicate genetic divergence between subgenomes and allow for the classification of switchgrass plants belonging to divergent genetic pools. Furthermore, the karyotype structure and cytogenetic analysis of switchgrass provides a framework for future genetic and genomic studies.

Karyotype polymorphism and chromosomal rearrangement in populations of the phytopathogenic fungus, Ascochyta rabiei

The fungus Ascochyta rabiei is the causal agent of Ascochyta blight of chickpea and the most serious threat to chickpea production. Little is currently known about the genome size or organization of A. rabiei. Given recent genome sequencing efforts, characterization of the genome at a population scale will provide a framework for genome interpretation and direction of future resequencing efforts. Electrophoretic karyotype profiles of 112 isolates from 21 countries revealed 12–16 chromosomes between 0.9 Mb and 4.6 Mb with an estimated genome size of 23 Mb–34 Mb. Three general karyotype profiles A, B, and C were defined by the arrangement of the largest chromosomes. Approximately one-third of isolates (group A) possessed a chromosome larger than 4.0 Mb that was absent from group B and C isolates. The ribosomal RNA gene (rDNA) cluster was assigned to the largest chromosome in all except four isolates (group C) whose rDNA cluster was located on the second largest chromosome (3.2 Mb). Analysis of progeny from an in vitro sexual cross between two group B isolates revealed one of 16 progeny with an rDNA-encoding chromosome larger than 4.0 Mb similar to group A isolates, even though a chromosome of this size was not present in either parent. No expansion of the rDNA cluster was detected in the progeny, indicating the increase in chromosome size was not due to an expansion in number of rDNA repeats. The karyotype of A. rabiei is relatively conserved when compared with published examples of asexual ascomycetes, but labile with the potential for large scale chromosomal rearrangements during meiosis. The results of this study will allow for the targeted sequencing of specific isolates to determine the molecular mechanisms of karyotype variation within this species.