rDNA Variation Research Articles

Transcriptional Silencing of 35S rDNA in Tragopogon porrifolius Correlates with Cytosine Methylation in Sequence-Specific Manner.

Despite the widely accepted involvement of DNA methylation in the regulation of rDNA transcription, the relative participation of different cytosine methylation pathways is currently described only for a few model plants. Using PacBio, Bisulfite, and RNA sequencing; PCR; Southern hybridizations; and FISH, the epigenetic consequences of rDNA copy number variation were estimated in two T. porrifolius lineages, por1 and por2, the latter with more than twice the rDNA copy numbers distributed approximately equally between NORs on chromosomes A and D. The lower rDNA content in por1 correlated with significantly reduced (>90%) sizes of both D-NORs. Moreover, two (L and S) prominent rDNA variants, differing in the repetitive organization of intergenic spacers, were detected in por2, while only the S-rDNA variant was detected in por1. Transcriptional activity of S-rDNA in por1 was associated with secondary constriction of both A-NORs. In contrast, silencing of S-rDNA in por2 was accompanied by condensation of A-NORs, secondary constriction on D-NORs, and L-rDNA transcriptional activity, suggesting (i) bidirectional nucleolar dominance and (ii) association of S-rDNAs with A-NORs and L-rDNAs with D-NORs in T. porrifolius. Each S- and L-rDNA array was formed of several sub-variants differentiating both genetically (specific SNPs) and epigenetically (transcriptional efficiency and cytosine methylation). The most significant correlations between rDNA silencing and methylation were detected for symmetric CWG motifs followed by CG motifs. No correlations were detected for external cytosine in CCGs or asymmetric CHHs, where methylation was rather position-dependent, particularly for AT-rich variants. We conclude that variations in rDNA copy numbers in plant diploids can be accompanied by prompt epigenetic responses to maintain an appropriate number of active rDNAs. The methylation dynamics of CWGs are likely to be the most responsible for regulating silent and active rDNA states.

Copy number and sequence variation in rDNA of Daphnia pulex from natural populations: insights from whole-genome sequencing.

Ribosomal DNA (rDNA) has a vital role in ribosome biogenesis as it contains the genes that encode ribosomal RNA (rRNA) separated by intergenic spacers (IGSs). The rRNA genes occur in hundreds to tens of thousands of copies per haploid genome in eukaryotes and are generally highly conserved with low variation within species. Due to the repetitive nature and large size of rDNA arrays, detecting intraindividual variation can be difficult. In this study, we use whole-genome sequences of 169 Daphnia pulex individuals from 10 natural populations to measure the copy number and sequence variation in rDNA. This revealed that variation in rDNA copy number between individuals spans an order of magnitude. We further observed a substantial level of sequence variation within individual genomes. As expected, single-nucleotide polymorphisms occurred in regions of lower functional constraint such as the IGS and expansion segments of the rRNA genes. The presence of strong linkage disequilibrium among variants facilitated identification of haplotypes within each population. Although there was evidence of recombination among haplotypes from different populations, it is insufficient to eliminate linkage disequilibrium within populations. Estimating copy number and haplotype diversity within individuals revealed that the level of intraindividual sequence variation is not strongly correlated with copy number. The observed patterns of variation highlight a complex evolutionary history of rDNA in D. pulex. Future research should explore the functional implications of rDNA copy number and sequence variation on organismal phenotypes.

Contrasting patterns of 5S rDNA repeats in European and Asian ecotypes of greater duckweed, Spirodela polyrhiza (Lemnaceae).

Ribosomal DNA (rDNA) contains highly conserved, specifically organized sequences encoding ribosomal RNAs (rRNAs) separated by variable non-transcribed intergenic spacers (NTSs) and is abundant in eukaryotic genomes. These characteristics make the rDNA an informative molecular target to study genome organization, molecular evolution, and phylogenetics. In this study, we characterized the 5S rDNA repeats in the greater duckweed Spiroldela polyrhiza, a species known for its small size, rapid growth, highly conserved genome organization, and low mutation rate. Sequence analysis of at least 12 individually cloned PCR fragments containing the 5S rDNA units for each of six ecotypes that originated from Europe (Ukraine) and Asia (China) revealed two distinct types of 5S rDNA repeats containing NTSs of different lengths and nucleotide compositions. The shorter 5S rDNA repeat units had a highly homogeneous 400-bp NTS, with few ecotype- or region-specific single-nucleotide polymorphisms (SNPs). The longer 5S rDNA units had NTSs of 1056-1084 bp with characteristic intra- and inter-genomic variants due to specific SNPs and insertions/deletions of 4-15-bp DNA elements. We also detected significant variability in the ratio of short/long 5S rDNA variants between ecotypes of S. polyrhiza. The contrasting dynamics of the two types of 5S rDNA units, combined with the unusually low repeat copy number (for plants) in S. polyrhiza (46-220 copies per genome), shows that this species could serve as an excellent model for examining the mechanisms of concerted evolution and functional significance of rDNA variability.

Comparative analysis and classification of highly divergent mouse rDNA units based on their intergenic spacer (IGS) variability.

Ribosomal DNA (rDNA) repeat units are organized into tandem clusters in eukaryotic cells. In mice, these clusters are located on at least eight chromosomes and show extensive variation in the number of repeats between mouse genomes. To analyze intra- and inter-genomic variation of mouse rDNA repeats, we selectively isolated 25 individual rDNA units using Transformation-Associated Recombination (TAR) cloning. Long-read sequencing and subsequent comparative sequence analysis revealed that each full-length unit comprises an intergenic spacer (IGS) and a ∼13.4 kb long transcribed region encoding the three rRNAs, but with substantial variability in rDNA unit size, ranging from ∼35to ∼46 kb. Within the transcribed regions of rDNA units, we found 209 variants, 70 of which are in external transcribed spacers (ETSs); but the rDNA size differences are driven primarily by IGS size heterogeneity, due to indels containing repetitive elements and some functional signals such as enhancers. Further evolutionary analysis categorized rDNA units into distinct clusters with characteristic IGS lengths; numbers of enhancers; and presence/absence of two common SNPs in promoter regions, one of which is located within promoter (p)RNA and may influence pRNA folding stability. These characteristic features of IGSs also correlated significantly with 5'ETS variant patterns described previously and associated with differential expression of rDNA units. Our results suggest that variant rDNA units are differentially regulated and open a route to investigate the role of rDNA variation on nucleolar formation and possible associations with pathology.

Evolving Together: Cassandra Retrotransposons Gradually Mirror Promoter Mutations of the 5S rRNA Genes.

The 5S rRNA genes are among the most conserved nucleotide sequences across all species. Similar to the 5S preservation we observe the occurrence of 5S-related nonautonomous retrotransposons, so-called Cassandras. Cassandras harbor highly conserved 5S rDNA-related sequences within their long terminal repeats, advantageously providing them with the 5S internal promoter. However, the dynamics of Cassandra retrotransposon evolution in the context of 5S rRNA gene sequence information and structural arrangement are still unclear, especially: (1) do we observe repeated or gradual domestication of the highly conserved 5S promoter by Cassandras and (2) do changes in 5S organization such as in the linked 35S-5S rDNA arrangements impact Cassandra evolution? Here, we show evidence for gradual co-evolution of Cassandra sequences with their corresponding 5S rDNAs. To follow the impact of 5S rDNA variability on Cassandra TEs, we investigate the Asteraceae family where highly variable 5S rDNAs, including 5S promoter shifts and both linked and separated 35S-5S rDNA arrangements have been reported. Cassandras within the Asteraceae mirror 5S rDNA promoter mutations of their host genome, likely as an adaptation to the host's specific 5S transcription factors and hence compensating for evolutionary changes in the 5S rDNA sequence. Changes in the 5S rDNA sequence and in Cassandras seem uncorrelated with linked/separated rDNA arrangements. We place all these observations into the context of angiosperm 5S rDNA-Cassandra evolution, discuss Cassandra's origin hypotheses (single or multiple) and Cassandra's possible impact on rDNA and plant genome organization, giving new insights into the interplay of ribosomal genes and transposable elements.

Therapeutic effects of curcumin on constipation-predominant irritable bowel syndrome is associated with modulating gut microbiota and neurotransmitters.

Constipation-predominant irritable bowel syndrome (IBS-C) is a functional bowel disease that affects 10-20% of the population worldwide. Curcumin (CUR) is widely used in traditional Chinese medicine to treat IBS, but its mechanism of action needs further investigation. In this study, we used mosapride (MOS) as a positive control to evaluate the changes in gut microbiota in IBS-C rat models after treatment with CUR or MOS by analyzing 16S rDNA variation. In addition, we used enzyme immunoassay kits and immunohistochemical analysis to investigate whether CUR or MOS influenced serotonin (5-HT), substance P (SP), and vasoactive intestinal peptide (VIP) levels in the serum and colon of IBS-C rats. The study showed that rats supplemented with CUR showed significantly increased fecal weight, fecal water content, small intestine transit rate and significantly decreased serum levels of 5-HT, VIP and SP compared to the IBS group (p < 0.05). In addition, treatment with CUR changed the relative abundance of Blautia, Sutterella, Acetanaerobacterium and Ruminococcus2 in the gut microbiota. This study showed that the efficacy of CUR on IBS-C was possibly by modulating the microbiota and lowering the serum levels of HT, SP, and VIP.

Intragenomic rDNA variation - the product of concerted evolution, mutation, or something in between?

The classical model of concerted evolution states that hundreds to thousands of ribosomal DNA (rDNA) units undergo homogenization, making the multiple copies of the individual units more uniform across the genome than would be expected given mutation frequencies and gene redundancy. While the universality of this over 50-year-old model has been confirmed in a range of organisms, advanced high throughput sequencing techniques have also revealed that rDNA homogenization in many organisms is partial and, in rare cases, even apparently failing. The potential underpinning processes leading to unexpected intragenomic variation have been discussed in a number of studies, but a comprehensive understanding remains to be determined. In this work, we summarize information on variation or polymorphisms in rDNAs across a wide range of taxa amongst animals, fungi, plants, and protists. We discuss the definition and description of concerted evolution and describe whether incomplete concerted evolution of rDNAs predominantly affects coding or non-coding regions of rDNA units and if it leads to the formation of pseudogenes or not. We also discuss the factors contributing to rDNA variation, such as interspecific hybridization, meiotic cycles, rDNA expression status, genome size, and the activity of effector genes involved in genetic recombination, epigenetic modifications, and DNA editing. Finally, we argue that a combination of approaches is needed to target genetic and epigenetic phenomena influencing incomplete concerted evolution, to give a comprehensive understanding of the evolution and functional consequences of intragenomic variation in rDNA.

RDNA Transcription in Developmental Diseases and Stem Cells.

As the first and rate-limiting step in ribosome biogenesis, rDNA transcription undergoes significant dynamic changes during cell pluripotency alteration. Over the past decades, rDNA activity has demonstrated dynamic changes, but most people view it as passive compliance with cellular needs. The evidence for rDNA transcriptional activity determining stem cell pluripotency is growing as research advances, resulting in the arrest of embryonic development and impairment of stem cell lines stemness by rDNA transcription inhibition. The exact mechanism by which rDNA activation influences pluripotency remains unknown. The first objective of this opinion article is to describe rDNA changes in the pathological and physiological course of life, including developmental diseases, tumor genesis, and stem cell differentiation. After that, we propose three hypotheses regarding rDNA regulation of pluripotency: 1) Specialized ribosomes synthesized from rDNA variant, 2) Nucleolar stress induced by the drop of rDNA transcription, 3) Interchromosomal interactions between rDNA and other genes. The pluripotency regulatory center is expected to focus strongly on rDNA. A small molecule inhibitor of rDNA is used to treat tumors caused by abnormal pluripotency activation. By understanding how rDNA regulates pluripotency, we hope to treat developmental diseases and safely apply somatic cell reprogramming in clinical settings.

First molecular evidence of hybridization in endosymbiotic ciliates (Protista, Ciliophora).

Hybridization is an important evolutionary process that can fuel diversification via formation of hybrid species or can lead to fusion of previously separated lineages by forming highly diverse species complexes. We provide here the first molecular evidence of hybridization in wild populations of ciliates, a highly diverse group of free-living and symbiotic eukaryotic microbes. The impact of hybridization was studied on the model of Plagiotoma, an obligate endosymbiont of the digestive tube of earthworms, using split decomposition analyses and species networks, 2D modeling of the nuclear rRNA molecules and compensatory base change analyses as well as multidimensional morphometrics. Gene flow slowed down and eventually hampered the diversification of Lumbricus-dwelling plagiotomids, which collapsed into a single highly variable biological entity, the P. lumbrici complex. Disruption of the species boundaries was suggested also by the continuum of morphological variability in the phenotypic space. On the other hand, hybridization conspicuously increased diversity in the nuclear rDNA cistron and somewhat weakened the host structural specificity of the P. lumbrici complex, whose members colonize a variety of phylogenetically closely related anecic and epigeic earthworms. By contrast, another recorded species, P. aporrectodeae sp. n., showed no signs of introgression, no variability in the rDNA cistron, and very high host specificity. These contrasting eco-evolutionary patterns indicate that hybridization might decrease the alpha-diversity by dissolving species boundaries, weaken the structural host specificity by broadening ecological amplitudes, and increase the nuclear rDNA variability by overcoming concerted evolution within the P. lumbrici species complex.

Production of a diploid hybrid with fast growth performance derived from the distant hybridization of Hypophthalmichthys nobilis (female) × Megalobrama amblycephala (male)

Based on the distant hybridization derived from Hypophthalmichthys nobilis (bighead carp, BIC, 2n ​= ​48) (♀) ​× ​Megalobrama amblycephala (blunt snout bream, BSB, 2n ​= ​48) (♂), we obtained a novel hybrid (BB) in this study. We comparatively investigated the morphological traits, chromosome numbers, DNA contents, microsatellite patterns, and DNA variations in 5S rDNA and Hox genes. The chromosome and flow cytometer results indicated that BB was a diploid hybrid with 48 chromosomes. We confirmed successful hybridization with microsatellite DNA markers and analyzed the Hox gene family in the parental and hybrid lines. The sequences of 5S rDNA in BB mapped to those in BIC and BSB, indicating that the genetic materials of BB were derived from BIC and BSB. The breeding experiment showed that BB had the characteristics of herbivore and fast growth. This diploid hybrid is of great significance for fish genetic breeding and applied research.

Comparative FISH mapping of 45S and 5S rDNA in the genus Gambierdiscus advances understanding of the cytogenetic diversity and mitosis of dinoflagellates

ABSTRACT Dinoflagellates are protists with exceptionally large genomes and chromosomes that lack nucleosomes. To date, information on their genome organization is scarce. Studies investigating the distribution of ribosomal RNA genes (rDNA) in plants and animals by fluorescence in situ hybridization (FISH) have provided important information to elucidate the genome organization and evolution of many species. In the present study, FISH was used to analyse the chromosomal organization of 45S and 5S rDNA and the extent of rDNA variation among five species of Gambierdiscus (G. australes, G. excentricus, G. silvae, G. carolinianus and G. caribaeus). The results, the intensity and location of the FISH signals, are summarized as follows: (1) All Gambierdiscus species have extremely high copy number of 45S rDNA repeat units arranged in a large nucleolus situated in the concave region of the typical interphasic bean-shaped nucleus. In addition, outside the large nucleolus, G. silvae and G. carolinianus have minor clusters of 45S rDNA repeats. (2) Many copies of 5S rDNA organized in one or a few clusters, depending on the species, are physically separated from the 45S rDNA but located close, peripherally, to the nucleolus. This nuclear organization is preserved during cell division. (3) Gambierdiscus australes was the species with the lowest 5S rDNA FISH signals clustered into a single site. In contrast, one major and several minor 5S rDNA clusters of FISH signals were observed in G. excentricus. We find that the species analysed can be differentiated by their species-specific FISH rDNA phenotypes demonstrating the usefulness of FISH in comparative cytogenomic studies of Gambierdiscus. In addition, using G. australes as a model, we demonstrate the usefulness of 45S and 5S rDNA as chromosomal markers to study dinomitosis, the atypical nuclear division that solely occurs in the class Dinophyceae.

Variation in Ribosomal DNA in the Genus Trifolium (Fabaceae).

The genus Trifolium L. is characterized by basic chromosome numbers 8, 7, 6, and 5. We conducted a genus-wide study of ribosomal DNA (rDNA) structure variability in diploids and polyploids to gain insight into evolutionary history. We used fluorescent in situ hybridization to newly investigate rDNA variation by number and position in 30 Trifolium species. Evolutionary history among species was examined using 85 available sequences of internal transcribed spacer 1 (ITS1) of 35S rDNA. In diploid species with ancestral basic chromosome number (x = 8), one pair of 5S and 26S rDNA in separate or adjacent positions on a pair of chromosomes was prevalent. Genomes of species with reduced basic chromosome numbers were characterized by increased number of signals determined on one pair of chromosomes or all chromosomes. Increased number of signals was observed also in diploids Trifolium alpestre and Trifolium microcephalum and in polyploids. Sequence alignment revealed ITS1 sequences with mostly single nucleotide polymorphisms, and ITS1 diversity was greater in diploids with reduced basic chromosome numbers compared to diploids with ancestral basic chromosome number (x = 8) and polyploids. Our results suggest the presence of one 5S rDNA site and one 26S rDNA site as an ancestral state.

Intragenomic sequence variations in the second internal transcribed spacer (ITS2) ribosomal DNA of the malaria vector Anopheles stephensi.

Second Internal Transcribed Spacer (ITS2) ribosomal DNA (rDNA) sequence is a widely used molecular marker for species-identification or -delimitation due to observed concerted evolution which is believed to homogenize rDNA copies in an interbreeding population. However, intra-specific differences in ITS2 of Anopheles stephensi have been reported. This study reports the presence of intragenomic sequence variation in the ITS2-rDNA of An. stephensi and hypothesizes that observed intra-specific differences in this species may have resulted due to ambiguous DNA sequence-chromatogram resulting from intragenomic heterogeneity. Anopheles stephensi collected from different parts of India were sequenced for complete ITS2 and the variable region of 28S-rDNA (d1-d3 domains). Intragenomic variations were found in ITS2 region of all An. stephensi sequenced, but no such variation was observed in d1 to d3 domains of 28S-rDNA. Cloning and sequencing of ITS2 through the d3 domain of the 28S region of rDNA from representative samples from northern, central, and southern India confirmed the presence of intragenomic variation in ITS2 due to transitions at three loci and two bp indel in a di-nucleotide microsatellite locus. Multiple haplotypes were observed in ITS2 raised from such variations. Due to the absence of detectable intragenomic sequence variation in the d1 to d3 domain of 28S rDNA of An. stephensi, this region can serve as an ideal reference sequence for taxonomic and phylogenetic studies. The presence of intragenomic variation in rDNA should be carefully examined before using this as a molecular marker for species delimitation or phylogenetic analyses.

Tremellomycetes Yeasts in Kernel Ecological Niche: Early Indicators of Enhanced Competitiveness of Endophytic and Mycoparasitic Symbionts against Wheat Pathobiota.

Tremellomycetes rDNA sequences previously detected in wheat kernels by MiSeq were not reliably assigned to a genus or clade. From comparisons of ribosomal internal transcribed spacer region (ITS) and subsequent phylogenetic analyses, the following three basidiomycetous yeasts were resolved and identified: Vishniacozyma victoriae, V. tephrensis, and an undescribed Vishniacozyma rDNA variant. The Vishniacozyma variant’s clade is evolutionarily close to, but phylogenetically distinct from, the V. carnescens clade. These three yeasts were discovered in wheat kernel samples from the Canadian prairies. Variations in relative Vishniacozyma species abundances coincided with altered wheat kernel weight, as well as host resistance to chemibiotrophic Tilletia (Common bunt—CB) and necrotrophic Fusarium (Fusarium head blight—FHB) pathogens. Wheat kernel weight was influenced by the coexistence of Vishniacozyma with endophytic plant growth-promoting and mycoparasitic biocontrol fungi that were acquired by plants. Kernels were coated with beneficial Penicillium endophyte and Sphaerodes mycoparasite, each of which had different influences on the wild yeast population. Its integral role in the kernel microbiome renders Vishniacozyma a measurable indicator of the microbiome–plant interaction. The ability of NGS technology to detect specific endophytic DNA variants and early changes in dynamics among symbionts within the kernel ecological niche enables the prediction of crop disease emergence, suggesting that advanced microbiological testing may be a potentially useful tool for both phytoprotection and more efficient wheat breeding programs.

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.

Single Strain High-Depth NGS Reveals High rDNA (ITS-LSU) Variability in the Four Prevalent Pathogenic Species of the Genus Candida.

Ribosomal RNA in fungi is encoded by a series of genes and spacers included in a large operon present in 100 tandem repeats, normally in a single locus. The multigene nature of this locus was somehow masked by Sanger sequencing, which produces a single sequence reporting the prevalent nucleotide of each site. The introduction of next generation sequencing led to deeper knowledge of the individual sequences (reads) and therefore of the variants between the same DNA sequences located in different tandem repeats. In this framework, NGS sequencing of the rDNA region was used to elucidate the extent of intra- and inter-genomic variation at both the strain and species level. Specifically, the use of an innovative NGS technique allowed the high-throughput high-depth sequencing of the ITS1-LSU D1/D2 amplicons of 252 strains belonging to four opportunistic yeast species of the genus Candida. Results showed the presence of a large extent of variability among strains and species. These variants were differently distributed throughout the analyzed regions with a higher concentration within the Internally Transcribed Spacer (ITS) region, suggesting that concerted evolution was not able to totally homogenize these sequences. Both the internal variability and the SNPs between strain can be used for a deep typing of the strains and to study their ecology.

Comparative Phylogeography and Integrative Taxonomy of Ochlerotatus caspius (Dipera: Culicidae) and Ochlerotatus dorsalis

Given that accurately identifying pathogen vectors is vital for designing efficient mosquito control programs based on the proper surveillance of the epidemiologically important species, it has been suggested the complementary use of independently evolving genes and morphometric traits as a reliable approach for the characterization and delimitation of related species. Hence, we examined the spatial distribution of COI mtDNA and ITS2 rDNA variation from the historical perspective of Ochlerotatus caspius (Pallas, 1771) and O. dorsalis (Meigen, 1830), while simultaneously testing the utility of the two markers in integrative species delimitation when combined with phenotypic character analyses of larvae and adults. Despite the striking difference in haplotype diversity (high in COI mtDNA, low in ITS2 rDNA), no evident phylogeographic structure was apparent in the Palearctic O. caspius. The Holarctic O. dorsalis species was subdivided into two highly distinctive COI mtDNA phylogroups which corresponded to the Nearctic and Palearctic regions. Strong support for the independence of the two allopatric evolutionary lineages suggested that geographical barrier and climatic changes during Pleistocene caused vicariance of the ancestral range. COI mtDNA reliably distinguished O. caspius and O. dorsalis, while ITS2 rDNA yet again lacked the proper resolution for solving this problem. An integrative approach based on the larval and adult morphological traits have varying taxonomic applications due to their differential diagnostic values. Thus, by the implementation of an integrative taxonomic approach, we successfully detected species borders between the two epidemiologically relevant species and uncovered the presence of cryptic diversity within O. dorsalis.

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.

Two new types of triploid hybrids derived from Cyprinus carpio (♀) × Megalobrama amblycephala (♂)

Based on the formation of an autotetraploid fish lineage (4nNC, 4n = 200, F1-F4) derived from common carp (Cyprinus carpio, 2nCOC, 2n = 100) (♀) × blunt snout bream (Megalobrama amblycephala, 2nBSB, 2n = 48) (♂), we produced two new types of triploid hybrids. One type of triploid hybrid (3nNCRC) was produced by crossing females of a crucian carp-like homodiploid fish (2nNCRC, 2n = 100) with males of 4nNC; the other type of triploid hybrid (3nMCC) was generated by crossing females of an improved diploid scattered mirror carp (2nIDMC) with males of 4nNC. The 2nNCRC and 2nIDMC were derived from the first generation of 2nCOC (♀) × 2nBSB (♂). In this study, we comparatively investigated the morphological traits, chromosomal numbers, DNA contents, erythrocyte traits, and DNA variations in 5S rDNA and Hox genes in 3nNCRC and 3nMCC. The results at the chromosome and DNA content levels indicated that 3nNCRC and 3nMCC had 150 chromosomes originating from common carp and were triploid hybrids. The proportion of erythrocytes with a dumbbell-shaped nucleus in 3nNCRC (1.74 ± 0.44%) was not significantly different (P > .05) from that in 3nMCC (2.86 ± 0.59%). Regarding the cluster organization of six Hox genes, 3nNCRC and 3nMCC had greater genetic variations; they not only inherited some Hox gene cluster types (including partial recombinant clusters) from each of the parents but also presented some new gene cluster types (including a richer variety of recombinant clusters) or lost some gene cluster types, suggesting a clear source of hybridization for 3nNCRC and 3nMCC and instability in the newly established triploid hybrid genomes. Moreover, 3nNCRC and 3nMCC had derived the same type of HoxB5b gene cluster, or some derived gene clusters from one fish were identical to those of the other fish with the same gene clusters inherited from their parents. Meanwhile, compared with 3nMCC, 3nNCRC exhibited greater variation in 5S rDNA and Hox gene structures, which revealed that the genome composition of 3nNCRC was more complex and less stable. The successful development of these two new types of triploid hybrids is of great significance for fish genetic breeding.

Distribution of 18s rDNA variants of Diderma hemisphaericum (Myxomycota) specimens obtained from moist chambers of materials collected in the Philippines and Vietnam

Since there are only limited biogeographic studies of myxomycetes in Southeast Asia, this study was conducted to assess the molecular diversity of Diderma hemisphaericum, a cosmopolitan myxomycetes species that abundantly occurs in most moist chamber collections in tropical Southeast Asia. Specimens that contain D. hemisphaericum were gathered from various parts of the Philippines: (i) Los Banos, (ii) Calauan, (iii) Basud, (iv) Isarog and (v) Malilipot; and Vietnam: (i) Thai Nguyen (ii) Ha Noi and (iii) Da Nang. Using modern molecular techniques, the common barcode nuclear marker 18S rDNA for myxomycetes was targeted for the 50 specimen used in this study. A total of 27 ribotypes (11 from the Philippines and 16 from Vietnam) were obtained. The Mantel test showed that genetic differentiation among ribotypes is not correlated with geographic distances. The FST index (0.083) indicates high genetic intermixing between the Philippines and Vietnam. Hence, it seems that the variation across regional scale gene pool is not directly influenced by geographic isolation but rather by environmental selection.