The genomic structure of a human chromosome 22 nucleolar organizer region determined by TAR cloning

The rDNA clusters and flanking sequences on human chromosomes 13, 14, 15, 21 and 22 represent large gaps in the current genomic assembly. The organization and the degree of divergence of the human rDNA units within an individual nucleolar organizer region (NOR) are only partially known. To address this lacuna, we previously applied transformation-associated recombination (TAR) cloning to isolate individual rDNA units from chromosome 21. That approach revealed an unexpectedly high level of heterogeneity in human rDNA, raising the possibility of corresponding variations in ribosome dynamics. We have now applied the same strategy to analyze an entire rDNA array end-to-end from a copy of chromosome 22. Sequencing of TAR isolates provided the entire NOR sequence, including proximal and distal junctions that may be involved in nucleolar function. Comparison of the newly sequenced rDNAs to reference sequence for chromosomes 22 and 21 revealed variants that are shared in human rDNA in individuals from different ethnic groups, many of them at high frequency. Analysis infers comparable intra- and inter-individual divergence of rDNA units on the same and different chromosomes, supporting the concerted evolution of rDNA units. The results provide a route to investigate further the role of rDNA variation in nucleolar formation and in the empirical associations of nucleoli with pathology.

The Black Sea Flexopecten species-complex (Mollusca: Bivalvia: Pectinidae): Shell morphology and 16S rDNA variation

The taxonomic status and population variability of a Black Sea scallop (Flexopecten: Mollusca, Pectinidae) was evaluated in a DNA barcoding study using the 16S ribosomal DNA gene (=16S rDNA), in conjunction with seven morphological features of the shell. We conclude that the Black Sea form represents an eastern extension of the Mediterranean scallop F. glaber (Linnaeus, 1758). It is characterized by a significant lack of genetic variability when compared to the Mediterranean form, although it does contain new haplotypes not found in that species. There is also an increase in the mismatch in the distribution of nucleotides for the Black Sea form when compared with pooled samples of Mediterranean scallop. Plausible reasons for the observed phenomena are hypothesized from the standpoint of the epigenetic theory of evolution and the time since the scallop penetrated into the Black Sea. The lack of genetic variability is potentially due to founder effect and genetic drift.

Low internal transcribed spacer rDNA variation in New Zealand Bonamia ostreae: evidence for a recent arrival

Bonamia ostreae is a haplosporidian parasite of oysters that was first reported to occur in the Southern Hemisphere in 2015 in the New Zealand flat oyster Ostrea chilensis. Until that report, B. ostreae had been restricted to populations of O. edulis within the Northern Hemisphere. This large range extension raised questions regarding B. ostreae dispersal, including whether B. ostreae is a recent introduction and from where it originated. The whole 18S rRNA gene of New Zealand B. ostreae revealed 99.9-100% sequence homology to other published B. ostreae 18S rDNA sequences. Internal transcribed spacer (ITS) rDNA sequences (n = 29) were generated from New Zealand B. ostreae and compared to published B. ostreae sequences from 3 Northern Hemisphere sites: California, USA (n = 18), Maine, USA (n = 7), and the Netherlands (n = 6) to investigate intraspecific variation. Low ITS rDNA variation was observed from New Zealand B. ostreae isolates, and high levels of variation were observed from Northern Hemisphere B. ostreae sequences. We hypothesise that the low ITS rDNA diversity found in New Zealand B. ostreae is the result of a founder effect resulting from a single introduction from a limited number of propagules. The high level of ITS rDNA variation from the Northern Hemisphere prevented inferences of dispersal origins. New Zealand B. ostreae were genetically differentiated from all sites, and additional genetic data are required to better determine the origin of B. ostreae in New Zealand.

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, and 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 CRISPR/Cas9 based strategy to edit the rDNA locus in the budding yeast Saccharomyces cerevisiae. 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 has 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 chr 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 locus via CRISPR/Cas9. It serves as a model for modifying other parts of the rDNA and, more generally, for editing other tandemly repeated sequences in higher eukaryotes.

An evaluation of rDNA variation in Lolium species (ryegrass)

Species of the genus Lolium are important fodder and turf grasses for agricultural and amenity use world-wide. Difficulties currently exist regarding the taxonomy of Lolium species and also in the demarcation between Lolium and the genus Festuca, with the debate focusing on the status of Festuca pratensis and its relations. The diversity in ribosomal DNA (rDNA) in Lolium was investigated. Cloned probes for rDNA, derived from the intergenic spacer (IGS-M) and transcribed regions (COD-M) of mustard (Sinapis alba) were used to investigate species relationships. Genomic DNA from seven Lolium taxa, F. pratensis, and xFestulolium braunii, were assessed for variation in rDNA by RFLP. Data support continued recognition of existing Lolium species' demarcations, and provided more taxonomic insight between allogamous Lolium taxa and F. pratensis. The COD-M and IGS-M probes grouped taxa by reproductive mode. The IGS-M probe distinguished all species as unique entities and established that hybrids, xFestulolium braunii and Lolium xboucheanum, had rDNA phenotypes that were composites of their parental species. The evolution of the ribosomal repeat unit is discussed and the potential for further rDNA taxonomic study in Lolium is considered.Key words: Lolium, variation, ribosomal, rDNA, RFLP.