PCR products of SSU-rDNA gene were purified using a SV gel and PCR clean up system purification kit (Pro- mega; Wi, USA). Sequencing of PCR products was per- formed at the Biotechnology Institute (UNAM, Mexico) using an ABI 377 automated DNA sequencer and appro- priate primers to each amplicon. Phylogenetic and mo- lecular evolutionary analyses of sequences were con- ducted using MEGA version 5 .
Phylogenetic analyses using the two 18S SSU rDNA mole- cules indicate that L. loboi is a distinct and novel species phylogenetically close to but fundamentally different from P. brasiliensis. Of interest to this study were findings that placed the dimorphic fungal pathogens within the Onygenales (4–6, 14, 21) and also a recent study that connected P. brasiliensis with the same order (22). These studies and the sequences that they provided made it possible for us to place L. loboi within the classical dimorphic fungal pathogens. Clustal alignment (Sequence Navigator, version 1.0.1; Perkin-Elmer) of the three P. brasiliensis 18S SSU rDNA sequences available in GenBank (one of them sequenced by us during this analysis [AF238302]) FIG. 3. Neighbor-joining tree of aligned CHS2 nucleotide sequences of one L. loboi strain, other dimorphic human pathogens, and two Eurotiales and two Chaetothyriales species as outgroup taxa. The L. loboi sequence is the sister taxon to P. brasiliensis and a member of the strongly supported clade of all dimorphic human pathogens except C. immitis. Changes in the parameters of maximum-likelihood multiple-hit correction affected the placement of C. immitis but not of the other taxa. Parsimony analysis (branch and bound) gave one tree with the same topology as the neighbor-joining tree. Numbers above and below the branches are percentages of bootstrap-resampled data sets supporting the branch as obtained by neighbor-joining and parsimony analyses, respectively. The scale bar represents 0.1 nucleotide substitution per nucleotide. The organisms used in this tree and the GenBank accession numbers of the CHS nucleotide sequences are as follows: B. dermatitidis M82943, C. immitis U60213, Emericella nidulans M82941, Exophiala jeanselmei M82945, H. capsulatum M82949, L. loboi AF238303, P. brasiliensis Y09231, Penicillium marneffei U60516, Phaeococcus exophiale M82953.
PCR-RFLP analysis. Genotypic characterization of Blastocystis from humans and animals was determined by an RFLP analysis of partial ssu rDNA (4). Although a few different primer pairs were used for the PCR-RFLP analysis of this gene in recent reports (3, 7, 12, 19), we chose a primer pair specific to partial ssu rDNA of B. hominis (3) since mixed infection with intestinal protozoa might be found in some specimens. Genomic DNA and a primer pair (forward, 5⬘-G GAGGTAGTGACAATAAATC-3⬘; reverse, 5⬘-CGTTCATGATGAACAATT- 3⬘) were used in PCR with conditions as described by Bo¨hm-Gloning et al. (3). PCR amplification was performed using a Perkin-Elmer 480 thermal cycler. After an initial 4-min denaturing step at 94°C, 35 PCR cycles were carried out, each consisting of a 30-s annealing at 54°C, 30-s extension at 72°C, 30-s dena- turing at 94°C, and an additional cycle with a 5-min chain elongation at 72°C. The PCR products and molecular markers were electrophoresed in 2% agarose gel (FMC Bioproducts, Philadelphia, Pa.) with 1.5% Tris-borate-EDTA (TBE) buffer using a Mupid gel electrophoresis device (Cosmo Bio, Tokyo, Japan). Bands were visualized under UV light after being stained with ethidium bromide and documented on Polaroid film using a FoTo Prep (Fotodyne). Digestions of the PCR products were performed using three restriction enzymes, HinfI, RsaI, and AluI (Gibco-BRL, Gaithersburg, Md.), and separated by 2% agarose gel electrophoresis.
X-cell disease in fish typically develops either as epider- mal pseudotumours, gill filament lesions or pseudobran- chial swellings in various marine species . X-cells associated with epidermal pseudotumours in the flat- head flounder, Hippoglossoides dubius Schmidt, 1904 and the yellowfin goby Acanthogobius flavimanus (Tem- minck et Schlegel, 1845) from northern Japan, have been shown, using small subunit ribosomal DNA (SSU rDNA) sequence data, to be related protozoan parasites that have an unresolved taxonomic identity . Freeman  further demonstrated that the X-cell parasite causing gill filament lesions in the European dab, Limanda limanda (L., 1758), is related to the two Japanese X-cell parasites, and suggested they belong in the alveolate group and that they are basal members of the Myzozoa. Pseudobranchial X-cell pseudotumours occur in gadoid fish from the Pacific and Atlantic Oceans , but thus far have not been studied phylogenetically.
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sequence data available that can be used to infer molecu- lar phylogenetic relationships. It is not possible to provide a comprehensive molecular phylogeny for the family until more DNA data for the various genera become available. However, available molecular data largely come from two of the most specious genera, Caligus and Lepeophtheirus, and can be used to identify important molecular phylogen- etic relationships between these related taxa. Morphologic- ally, Lepeophtheirus differ from Caligus as they lack lunules. Lunules are paired sucker-like structures on the frontal plates that are used for attachment to the fish host, and are unique to nearly half of the genera within the Cali- gidae, including Caligus and Pseudocaligus [2,26]. Lu- nules are thought to have evolved only once in the Caligidae and character based phylogenetic analysis of the family suggests that several genera, such as Lepeophtheirus, have secondarily lost their lunules . In accordance with this theory, we consistently retrieve the majority of Lepeophtheirus spp. as a separate well-supported clade in our analyses, with the lunule bearing Caligus and Pseudocaligus, grouping together as a sister taxon. How- ever, within the lunule-bearing group, the three sequences available for Pseudocaligus are consistently placed apart from each other, and form parts of recoverable clades, that appear to reflect the geographical location and/or the fish host taxon. In the SSU rDNA analyses, P. fugu groups with Caligus quadratus also infecting fish from Japan and forms part of the C. elongatus clade; P. uniartus groups with an unidentified Caligus sp. that also infects siganid fish from Indo-Pacific region, and P. brevipedis, found on gadoid fish in the Atlantic, consistently groups with two species of Caligus from the Atlantic, one is the type species of the genus, C. curtus, which is also found on
The morphological species belonging to the P. pulmo- narius complex seem to comprise more closely related taxa, because all members in this biological species do not show any variation in the 5 ⬘ portion of the mt SSU rDNA and are placed in a common clade. On the other hand, the high homology of the sequences of the 5⬘ portion of the mt SSU rDNA (⬎99%) in the P. ostreatus complex shows that the 5⬘ portion of the mt SSU rDNA is a relatively highly con- served region that has hardly evolved since the biological speciation event.
Conclusions from the multigene phylogenetic analysis. When the ITS1-5.8S-ITS2 region was first applied to the study of oligotrichs and choreotrichs, it was suggested that the ITS and 5.8S regions could provide adequate polymorphism data to assess ge- netic variation at the genus/population level within these groups (11). In the meantime, SSU rDNA sequences have been widely used to infer evolutionary relationships among spirotrichs, and phylogenetic trees based on such data are generally concordant with many morphological hypotheses, albeit with some discrep- ancies (17, 35). The value of using a single gene marker in order to elucidate evolutionary relationships among ciliates has been ques- tioned (10). Therefore, we have used multigenes, i.e., SSU rRNA gene and ITS-5.8S region sequences, to increase the robustness of our analyses of phylogenetic relationships among oligotrichs. In the present study, the overall mean distances in oligotrichs are 0.104 in the SSU rRNA gene and 0.159 in the ITS-5.8S region, while in choreotrichs, they are 0.067 in the SSU rRNA gene and 0.090 in the ITS-5.8S region, confirming that oligotrichs are more genetically variable than choreotrichs in both the SSU rRNA gene and the ITS-5.8S region (11). Our findings support the removal of Laboea from the Strombidiidae to the Tontoniidae, thus render- ing the family Strombidiidae monophyletic. Furthermore, the monophyly of Novistrombidium was doubted in the topologies of trees and morphological features. However, we are currently un- able to resolve a number of phylogenetic relationships due to (i) differences between the two genes in length and variation rate, (ii) the lack of available ITS-5.8S region sequence data for oligotrichs, and (iii) undersampling of certain key taxa, such as Varistrom- bidium, Parallelostrombidium, and Omegastrombidium. There- fore, further studies are required to increase the resolution of the oligotrich systematics.
Molecular phylogenetic analysis of naturally occurring archaeal communities in deep-sea hydrothermal vent environments was carried out by PCR-mediated small subunit rRNA gene (SSU rDNA) sequencing. As determined through partial sequencing of rDNA clones amplified with archaea-specific primers, the archaeal populations in deep-sea hydrothermal vent environments showed a great genetic diversity, and most members of these populations appeared to be uncultivated and unidentified organisms. In the phylogenetic analysis, a number of rDNA sequences obtained from deep-sea hydrothermal vents were placed in deep lineages of the crenarchaeotic phylum prior to the divergence of cultivated thermophilic members of the crenarchaeota or between thermophilic members of the euryarchaeota and members of the methanogen-halophile clade. Whole cell in situ hybridization analysis suggested that some microorgan- isms of novel phylotypes predicted by molecular phylogenetic analysis were likely present in deep-sea hydrothermal vent environments. These findings expand our view of the genetic diversity of archaea in deep-sea hydrothermal vent environments and of the phylogenetic organization of archaea.
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SSU rRNA gene was the most widely accepted mo- lecular marker in identifying different malaria parasites infecting humans with PCR method. The common PCR systems based on SSU rDNA sequences were NP-1993 . Latterly, NP-2002  and NP-2005  were devel- oped as new variant SSU rDNA sequences were discov- ered. At first, the P. ovale wallikeri made the NP-1993 system unreliable [7,36] and the system of NP-2002 seemed to resolve this problem, in which not only the PCR product size of first round extended from 1,100 bp to 1,700 bp along the forward direction of SSU rDNA sequences, but the reverse ovale-specific primer was designed at conserved region. However, another PCR system of NP-2005 in which a pair of new P. ovale species-specific primers was applied, proved to be more accurate than both NP-1993 and NP-2002 in detecting P. ovale. For the present, the problem is that one of the species-specific primers used in these three systems is located at the variable region R2 or B6. In this region, variant sequences obtained in this study showed low similarity (<60%) with normal ones. But high similarity (>90%) presented between variant and normal ones where genus-specific primers usually matched. So, the- oretically, all the SSU rDNA types could be amplified synchronously with same genus-specific primers. If most of the PCR products obtained in the first round reaction did not match the species-specific primer (as shown in this study), all of the PCR results in the three systems Table 2 The occurred percentage of eight variant SSU rDNA sequences among the positive sequence clones
Owing to the difficulties in DNA isolation and amplification of phylogenetically informative genes, the molecular phylogenetic relationships within this group have been understudied. Weir & Hughes (2002) constructed a partial SSU rDNA phylogeny of ten species of Laboulbeniales, representing three subfamilies (Ceratomycetoideae, Laboulbenioideae, Peyritschielloideae). A combined dataset of the partial SSU and ITS rDNA regions was used to study the phenomenon of position specificity in 13 species of Chitonomyces on Laccophilus maculosus (Coleoptera: Dytiscidae; Goldmann & Weir 2012). Goldmann et al. (2013) described two position specific species of Hesperomyces on Coleomegilla maculata (Coleoptera: Coccinellidae), again based on partial SSU+ITS rDNA. All these studies used the extraction methodology of Weir & Blackwell (2001b).
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FIG. 1. Phylogenetic comparison of 18S SSU rDNA from R. seeberi and 23 other organisms showing that this human and animal pathogen is a member of the Mesomycetozoa clade. (a) Parsimony tree (length ⫽ 1,688 steps; consistency index ⫽ ⫹0.53; retention index ⫽ 0.51; homoplasy index ⫽ 0.47) based on a heuristic search with 1,000 random taxon input orders. The numbers on the internal branches are percentages of trees based on 1,000 bootstrapped data sets possessing the branch. The branch lengths reflect the length in steps. An italicized scale is given above the Ochromonas danica branch. (b) Neighbor-joining distance tree with Kimura’s 3-parameter correction for multiple hits. The numbers on the internal branches are percentages of trees based on 1,000 bootstrapped data sets possessing the branch. The branch lengths reflect corrected distances. An italicized scale is given above the O. danica branch. The GenBank accession numbers of the organisms are as follows: Acanthocoepsis unguiculata, L10823; Diaphanoeca grandis, L10824; Dermocystidium sp., U21336; Dermocystidium salmonis, U21337; I. hoferi, U43712; Psorospermium haeckelli, U33180; the rosette agent, L29455; R. seeberi, AF118851; Microciona prolifera, L10825; Mnemiopsis leidyi, L10826; Blastocladiella emersonii, X54264; Chytridium confervae, M59758; Glomus intraradices, X58725; Pneumocystis carinii, X12708; Saccharomyces cerevisiae, J01353; Schizosaccharomyces pombe, X58056; Chlorella lobophora, X63504; Chlamydomonas reinhardtii, M32703; Gracilaria lemaneiformis, M54986; Perkinsus sp., L07375; Perkinsus marinus, X75762; Achlya bisexualis, M32705; O. danica, M32704; and Prorocentrum micans, M14649.
Abstract Ammonia and Elphidium collected in the Kiel Fjord for the present study were first identified on mor- phological bases as Ammonia beccarii (Linne´, 1758) and Elphidium excavatum (Terquem, 1876). Phylogenetic analyses based on partial SSU rDNA and LSU rDNA sequences show that Ammonia specimens sampled in the Kiel Fjord belong to the phylotype T6, which has a disjunct distribution (Wadden and Baltic Seas/China and Japan) and has been identified as Ammonia aomoriensis (Asano, 1951). Partial SSU rDNA sequence analyses indicate that Elphidium specimens from the Kiel Fjord belong to the clade E. excavatum, confirming the morphological identi- fication. This clade can be further divided in three subc- lades. Kiel Fjord Elphidium belong to two of these subclades and were identified morphologically as the
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Our objective was to find a hypervariable rDNA stretch, flanked by strongly conserved regions, which can be used for molecular species identification of species within the Neisseri- aceae and Moraxellaceae families. The longest coherent vari- able region in the 16S rDNA fulfilling these criteria spans the region from E. coli positions 54 to 510 and in the 23S rDNA spans the region from positions 1400 to 1600 (16). This is well illustrated by the quantitative map of nucleotide substitution rates in bacterial rDNAs published by Van de Peer et al. (37). The inter- and intrageneric relationships of members of the Neisseriaceae and Moraxellaceae were therefore investigated by carrying out comparative sequence analysis of PCR-amplified partial 16S and 23S rDNAs of these regions in a total of 94 strains. An ideal region should show a low intraspecies and a high interspecies variability. When the DNA sequences of the 16S and 23S rRNA genes were used as a measure of the intraspecies and interspecies distances within a genus, no great differences between the two regions could be observed (Fig. 1). Only in the case of the 23S rDNA in Neisseria was the inter- species variation significantly lower than that of the 16S rDNA. The interspecies distances between genera for the 23S rDNA sequences on the other hand, were consistently higher than those for the 16S rDNA. A comparison of an absolute measure (i.e., polymorphic positions), however, showed that 16S rDNA always had significantly more variable positions and this was because the relevant region in the 16S rDNA was more than twice as long as that in the 23S rDNA (Table 2). We therefore concluded that the selected region of the 16S rDNA is more suitable than that of the 23S rDNA for identification purposes because of its greater length.
ABSTRACT 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 ﬁ nd 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 ﬁ nd 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.
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In terms of the evolution of the Legionellaceae, the results of this analysis suggest that the degree of genetic drift required before differences in the intergenic spacers can be observed is greater than that required for the recognition of different spe- cies by DNA homologies. This is demonstrated by the absence of intraspecies PCR product polymorphism. In addition, some groups of species, including the closely related red autofluo- rescent legionellae (18), shared 16S-23S rDNA profiles (Fig. 1).
The discovery of mUltiple rDNA loci in these endophytes suggests that several intact rDNA arrays should be able to be resolved on CHEF gels with the appropriate conditions. Therefore intact chromosomal preparations from Lp l AO were digested with BamHI and HindIII, two restriction endonucleases that had been previously shown not to cleave within the rDNA (Ganley, 1 993 and A.R.D. Ganley, unpublished results). These were separated by CHEF gel electrophoresis and the gel was Southern blotted and hybridised to the 5.6 kb Sal! coding region probe. A variety of separation conditions were trjaIled, and those that gave the best separation of the rDNA arrays are shown in Figure 3 .20. There are clearly two high molecular weight rDNA arrays present in the BamHl digest, and it is possible that the largest band contains more than one rDNA array that migrate together, although no separation conditions were found that could resolve this into more than one band. This large band is at least 1 .6 Mb in size, as determined by comparison to the S. cerevisiae chromosome standard (Bio-Rad). Also present is a very diffuse smear of hybridisation of lower molecular weight than the well-resolved arrays (Figure 3 . 20B). These diffuse "bands" are located above the smear of ethidium bromide-stained DNA seen in the HindIII digest that corresponds to the bulk of the digested DNA but not in the BamHI digest (Figure 3 .20A). The reason for the highly diffuse nature of these lower molecular weight bands is not clear, as the S. cerevisiae chromosomes are well-resolved in the same region of the gel. The size range covered by this diffuse smear is from less than 200 kb up to 1 . 1 Mb, as determined by comparison to the S. cerevisiae chromosome standard (Bio-Rad). Interestingly, the two high molecular weight rDNA arrays that are resolved in the BamHI digest are not seen at all in the HindUI digest. This was the case in all the separations that were trialled (results not shown). The reason for this is not clear.
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The Prussian carp may occur as diploid (2n=100) and/or triploid (3n=150) individuals, co-existing in many natural populations. The simultaneous occurrence of individuals with different ploidy makes the taxonomy of this species unclear. Additionally, the taxonomic status of C. gibelio has become even more enigmatic due to its hybridizing with other non- indigenous cyprinids. Since the variation within 5S rDNA can serve as a suitable marker for molecular identification of the fish ploidy, the main aim of present study was to compare this rDNA sequence between Prussian carp individuals with different ploidy levels: diploids (2n =100) and triploids (3n=150-160). PCR amplification of 5S rDNA generated two bands of approximately 340 and 470 bp in length (in both diploids and triploids) and band 200 bp visible in some individuals. These results indicate the presence of at least two different classes of 5S rRNA gene. Analysis of their nucleotide composition revealed no differences within the class of 340 bp and several nucleotide differences within the class of 470 bp between diploid and triploid individuals. The 5S rDNA variability detected in this study indicates the potential usefulness of this sequence for the identification of diploid and triploid individuals of the Prussian carp.
In previous studies we reported the presence of self-spli- cing group I introns in the rRNA small subunit (SSU) genes of Cenococcum geophilum , as well as the loca- tion and self-splicing ability of a small (67 nucleotides, nt) group I intron in Phialophora americana Wang 1046 [13,14]. This small intron may have originated from an unequal crossover in an ancestor that was amplified in the genome. Gene conversion, which is active in the rRNA gene locus, may have increased copy number of the mutant. Strong selective pressure is in effect in the rRNA locus to retain the production of the large number of rRNAs required in each cell. Therefore, the small introns must have retained their ability to splice in order to allow the production of functional small subunit rRNA. In this study we compared eight small putative group I introns from different isolates of Phialophora species (anamorphic dematiaceous fungi) in order to study the structure and evolution of these introns after their establishment in the rRNA genes. We show that the small introns differ in sequence, but retain similar structural features.
Objective: Cyanobacteria are an ancient phylum of prokaryotes that contain the class Oxyphotobacteria. This group has been extensively studied by phylogenomics notably because it is widely accepted that Cyanobacteria were responsible for the spread of photosynthesis to the eukaryotic domain. The aim of this study was to evaluate the fraction of the oxyphotobacterial diversity for which sequenced genomes are available for genomic studies. For this, we built a phylogenomic-constrained SSU rRNA (16S) tree to pinpoint unexploited clusters of Oxyphotobacteria that should be targeted for future genome sequencing, so as to improve our understanding of Oxyphotobacteria evolution.
To overcome the challenges of amplifying a diverse set of genes within a large number of taxa, with which they had not been tested previously (or tested with no success), an array of PCR protocols was used (25 different protocols with 43 different primers). PCR protocol data is summarized in Table 2.1, and primers that were used are summarized in Table 2.2. Four different PCR reagent kits were used: Finnzymes Phusion Hot Start II (Thermo Fisher Scientific, Wilmington, Deleware), Go-Taq Green Master Mix, Go-Taq Green Hot Master Mix (Promega, Madison, Wisconsin), and TaKaRa LA PCR (TaKaRa Bio, Otsa, Shiga, Japan). Reaction chemistry was kept consistent within a given PCR kit with two exceptions: protocols designed to amplify protein-coding genes were run with a higher primer concentration than protocols designed to amplify rDNA, and some protocols included either betaine, DMSO, or BSA (see Table 2.1).
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