Non-transcribed Spacer Research Articles

Description of the complete rDNA repeat unit structure of Coturnixjaponica Temminck et Schlegel, 1849 (Aves).

Ribosomal RNA (18S, 5.8S, 28S) gene clusters in genomes form regions that consist of multiple tandem repeats. They are located on a single or several pairs of chromosomes and play an important role in the formation of the nucleolus responsible for the assembly of ribosome subunits. The rRNA gene cluster sequences are widely used for taxonomic studies, however at present, complete information on the avian rDNA repeat unit structure including intergenic spacer sequence is available only for the chicken (Gallusgallusdomesticus Linnaeus, 1758). The GC enrichment and high-order repeats peculiarities within the intergenic spacer described for the chicken rDNA cluster may be responsible for these failures. The karyotype of the Japanese quail (Coturnixjaponica Temminck et Schlegel, 1849) deserves close attention because, unlike most birds, it has three pairs of nucleolar organizer bearing chromosomes, two of which are microchromosomes enriched in repeating elements and heterochromatin that carry translocated terminal nucleolar organizers. Here we assembled and annotated the complete Japanese quail ribosomal gene cluster sequence of 21166 base pairs (GenBank under the registration tag BankIt2509210 CoturnixOK523374). This is the second deciphered avian rDNA cluster after the chicken. Despite the revealed high similarity with the chicken corresponding sequence, it has a number of specific features, which include a slightly lower degree of GC content and the presence of bendable elements in the content of both the transcribed spacer I and the non-transcribed intergenic spacer.

Molecular organisation and chromosomal localisation of 5S rDNA tandem arrays in evolutionary polyploid cyprinids from the genera Carassius and Barbus

Some evolutionary polyploid Carassius and Barbus species are poorly differentiated morphologically and hybridise to form viable intrageneric hybrids. 5S rDNA arrays and their chromosomal localisation were studied in C. carassius, C. gibelio, B. barbus, B. carpathicus and B. waleckii distributed throughout Poland, in order to contribute to the knowledge of their genetic and cytogenetic differences. Two 5S rDNA array classes in C. carassius (203 bp, 380 bp) and in three Barbus species (196 bp, 215 bp) were characterised by a highly-conserved coding region. The adjacent non-transcribed spacer (NTS) of C. Carassius exhibited a relatively high level of variation due to base substitutions and insertions/deletions, whereas the NTS of the Barbus species were highly conserved. Fluorescence in situ hybridisation with 5S rDNA array classes as probes indicated their syntenic chromosomal location and the utility of these sites as suitable marker of the analysed Carassius taxa ploidy. 5S rDNAs in two chromosomes of the Barbus species was discovered. The observed low variability of 5S rDNA arrays makes them an inappropriate tool for an investigation of the Barbus species, whereas the suitable for investigation within karyologically varied in the Carassius taxa.

The Genome Organization of 5S rRNA Genes in the Model Organism Tribolium castaneum and Its Sibling Species Tribolium freemani.

5S ribosomal DNAs (rDNAs) are arranged in tandem and are often under-represented in genome assemblies. In the present study, we performed a global and in-depth analysis of the 5S rDNAs in the model insect Tribolium castaneum and its closely related species Tribolium freemani. To accomplish this goal, we used our recently published genome assemblies based on Nanopore and PacBio long-read sequencing. Although these closely related species share the 5S rRNA gene sequence with high homology, they show a different organization of the 5S rDNA locus. Analysis of 5S rDNA arrays in T. castaneum revealed a typical tandemly repeated organization characterized by repeat units consisting of the 121 bp long 5S rRNA gene and the 71 bp long nontranscribed spacer (NTS). In contrast, T. freemani showed a much more complex organization of 5S rDNA arrays characterized by two patterns. The first is based on the association of 5S rRNA gene with arrays of a satellite DNA, representing the NTS sequence of the 5S rDNA genes in T. freemani. The second, more complex type is characterized by a somewhat less frequent occurrence of the 5S rRNA gene and its association with longer satellite DNA arrays that are regularly interrupted by Jockey-like retrotransposons. This organization, in which the ribosomal gene is associated with two completely different repetitive elements such as satellite DNAs and retrotransposons, suggests that the 5S rRNA gene, regardless of its crucial function in the genome, could be a subject of extremely dynamic genomic rearrangements.

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.

The improvement of the CAPS-marker for St, J and V subgenome identification in Triticeae tribe plants using the 5S non-transcribed spacer polymorphism

In the Triticeae tribe, several subgenomes (St, J, V and others) were described, and a subgenome-specific molecular marker development is an actual branch of studies. Non-transcribed spacers (NTS) of 5S rDNA are perspective to create species-specific or genome-specific molecular markers in closely related organisms. Early, the 5S rDNA NTS based CAPS marker (StJV-f/StJV-r primers with the SmiM1 enzyme digestion) was designed for identification of the St, J and V subgenomes. The V subgenome amplicons were differed from the St and J amplicons in length. The St amplicons were distinguishable from the J amplicons after the SmiM1 enzyme digestion (St fragments were digested, J fragments were not digested, which is a disadvantage, because a failure with the digestion of St amplicons can be mistaken for J amplicons). This article presents the results of the StJV marker improvement. Comparative analysis of restriction enzyme maps revealed that the Hpy166 II enzyme cuts the StJV amplicons of all studied subgenomes in such a way that different product patterns are obtained. Experimental testing confirmed this finding. Thus, the improved CAPS marker can serve as a promising tool for verifying samples in Triticeae plant collections, screening herbarium materials, as well as in the breeding process aimed at improving wheat by involving its wild relatives in traditional breeding programs efficiently.

Molecular typing of a collection of Iranian clinical Trichophyton tonsurans isolates based on the non-transcribed spacer region of rDNA and antifungal susceptibility testing of the species.

Wrestling, considered the national sport of Iran, has gained immense popularity among Iranians. Wrestlers frequently encounter skin conditions, with dermatophyte fungal infections, particularly tinea gladiatorum (TG), being a common issue. TG, caused by the Trichophyton genus, has emerged as a major health concern for wrestlers and other contact sport athletes worldwide. This study aimed to assess the genotypic diversity and antifungal susceptibility of Trichophyton tonsurans isolates responsible for TG in Iranian wrestlers from Mazandaran province, northern Iran. A total of 60 clinical T. tonsurans isolates collected from various cities in Mazandaran, were included in the study. The isolates were identified through PCR-restriction fragment length polymorphism and sequencing methods. Genomic DNA was extracted from these isolates, and the non-transcribed spacer (NTS) region of ribosomal RNA (rRNA) was targeted for genotyping using newly designed primers. Haplotype analysis was performed to explore genetic diversity, and antifungal susceptibility to terbinafine (TRB) and itraconazole (ITC) was assessed. The results revealed five distinct NTS types: NTS-I, NTS-II, NTS-III, NTS-IV and NTS-V, with NTS-IV being the most prevalent. The distribution of NTS types varied across different cities, suggesting potential transmission patterns among wrestlers. Antifungal susceptibility testing showed that all isolates were susceptible to TRB, while one isolate demonstrated resistance to ITC. Genotypic diversity was not correlated with antifungal susceptibility, emphasising the importance of monitoring susceptibility to ensure effective treatment. Haplotype analysis highlighted significant genetic diversity among the T. tonsurans isolates. This diversity may be attributed to factors such as human-to-human transmission, geographic location and lifestyle changes. The study's findings underscore the need for comprehensive genotypic analysis to understand the epidemiology and evolution of T. tonsurans infections in athletes. In conclusion, this study provides valuable insights into the genotypic diversity and antifungal susceptibility of T. tonsurans isolates causing TG in Iranian wrestlers. The presence of multiple NTS types and varying susceptibility patterns highlights the complexity of T. tonsurans infections in this population. Further research is warranted to track the transmission routes and genetic evolution of T. tonsurans strains among wrestlers and develop effective control measures.

Genetic variation between a hybrid abalone and its parents (Haliotis discus hannai ♀ and H. fulgens ♂) based on 5S rDNA gene and genomic resequencing

Abalones are a group of economically important species of mollusks that represent a source of high-quality protein. China accounts for 90% of the world's abalone production, and make a significant contribution to the aquaculture industry's economic value. With the increasing scale of abalone farming, problems such as the degradation of germplasm resources and a reduced ability to resist environmental changes and stresses have become increasingly prominent. The hybrid breeding technique has significantly ameliorated this situation. The distant hybrid species H. discus hannai ♀ × H. fulgens ♂ (the Lvpan abalone) shows significant heterosis in terms of growth and stress resistance. However, our knowledge of the cytogenetics and molecular mechanisms of heterosis in the hybrid abalone remains limited. In this study, we found significant non-transcribed spacer (NTS) variation of 5S rDNA gene among H. discus hannai, H. fulgens, and the Lvpan abalone, with five, two, and four types of NTSs identified, respectively. These NTSs exhibited genomic polymorphisms within and between species, making them reliable markers for species identification. Furthermore, the sequence alignment of NTSs and genomic resequencing analysis indicated that the hybrid abalone was biased toward H. discus hannai, suggesting a maternal effect. Moreover, numerous mutation sites that were identified only in the hybrid abalone potentially contributed to heterosis. This study provides a valuable molecular data reference for future research on distant hybridization and contributes to a better understanding of the molecular mechanisms underlying heterosis in mollusks.

Analysis and evaluation of morphological and molecular polymorphism in the hybridization of <i>Elaeagnus ×maritima</i> and <i>E. ×submacrophylla</i>

The taxonomic identity of <i>Elaeagnus ×maritima</i> and <i>E. ×submacrophylla</i> (Elaeagnaceae) in Korea is unclear, yet they are presumed to be hybrid taxa based on their morphology. To determine their hybrid origins, a morphological analysis (field surveys and specimen examinations) and a molecular analysis involving two nuclear ribosomal DNA (nrDNA) regions (internal transcribed spacer and 5S non-transcribed spacer) and one chloroplast DNA (cpDNA) region (<i>matK</i>) were conducted. The morphological analysis revealed that <i>E. ×maritima</i> showed certain morphological similarities to <i>E. glabra</i>, whereas <i>E. ×submacrophylla</i> showed certain morphological similarities to <i>E. pungens</i>. However, the molecular analysis indicated that <i>E. ×maritima</i> exhibited additive species-specific sites of <i>E. glabra</i> and <i>E. macrophylla</i> in the nrDNA regions. Notably, <i>E. ×submacrophylla</i> showed various aspects, with some individuals exhibiting additive species-specific sites of <i>E. pungens</i> and <i>E. macrophylla</i> in the nrDNA and <i>E. macrophylla</i> sequences in the cpDNA regions, some individuals exhibiting <i>E. macrophylla</i> sequences in the nrDNA and <i>E. pungens</i> sequences in the cpDNA regions, and some individuals displaying <i>E. macrophylla</i> sequences in both the nrDNA and cpDNA regions, despite an intermediate morphology between <i>E. pungens</i> and <i>E. macrophylla</i>. These results indicate that these two species are of hybrid origin and frequently cross between parental and hybrid individuals.

A pseudo-outbreak of Cyberlindnera fabianii funguria: Implication from whole genome sequencing assay.

Although the yeast Cyberlindnera fabianii (C. fabianii) has been rarely reported in human infections, nosocomial outbreaks caused by this organism have been documented. Here we report a pseudo-outbreak of C. fabianii in a urology department of a Chinese hospital over a two-week period. Three patients were admitted to the urology department of a tertiary teaching hospital in Beijing, China, from Nov to Dec 2018, for different medical intervention demands. During the period Nov 28 to Dec 5, funguria occurred in these three patients, and two of them had positive urine cultures multiple times. Sequencing of rDNA internal transcribed spacer (ITS) region and MALDI-TOF MS were applied for strain identification. Further, sequencing of rDNA non-transcribed spacer (NTS) region and whole genome sequencing approaches were used for outbreak investigation purpose. All the cultured yeast strains were identified as C. fabianii by sequencing of ITS region, and were 100% identical to the C. fabianii type strain CBS 5640T. However, the MALDI-TOF MS system failed to correctly identify this yeast pathogen. Moreover, isolates from these three clustered cases shared 99.91%-100% identical NTS region sequences, which could not rule out the possibility of an outbreak. However, whole genome sequencing results revealed that only two of the C. fabianii cases were genetically-related with a pairwise SNP of 192 nt, whilst the third case had over 26,000 SNPs on its genome, suggesting a different origin. Furthermore, the genomes of the first three case strains were phylogenetically even more diverged when compared to a C. fabianii strain identified from another patient, who was admitted to a general surgical department of the same hospital 7 months later. One of the first three patients eventually passed away due to poor general conditions, one was asymptomatic, and other clinically improved. In conclusion, nosocomial outbreaks caused by emerging and uncommon fungal species are increasingly being reported, hence awareness must be raised. Genotyping with commonly used universal gene targets may have limited discriminatory power in tracing the sources of infection for these organisms, requiring use of whole genome sequencing to confirm outbreak events.

A Fast and Simple DNA Mini-barcoding and RPA Assay Coupled with Lateral Flow Assay for Fresh and Canned Mackerel Authentication.

The online version contains supplementary material available at 10.1007/s12161-022-02429-6.

High diversity of 5S ribosomal DNA and evidence of recombination with the satellite DNA PcP190 in frogs

Distinct types of 5S rDNA repeats, differing by size and composition of its non-transcribed spacer (NTS), have been found in diverse taxa. Both concerted evolution and birth-and-death evolution have proven to play important roles in the evolution of the 5S rDNA family. In anurans, however, this subject has been underexplored as only a few anuran species had their 5S rDNA characterized and evolutionary analyzed to date. In the present study, we characterized the 5S rDNA sequences from species of two anuran families using classical molecular biology techniques and bioinformatic approaches. Based on the NTS, more than one type of 5S rDNA was identified in each analyzed species, which suggests that birth-and-death processes take part in the evolution of these sequences along the Anura tree of life. In addition, closely related species shared the same types of sequences, in accordance with the model of concerted evolution. We also found evidence of recombination between 5S rDNA and PcP190 satellite DNA, a repetitive sequence that is derived from the 5S rDNA. The interplay between 5S rDNA and PcP190 satellite DNA might favor the maintenance of the latter in the genome and respond to its presence in several species of frogs. The analysis of 5S rRNA transcripts confirmed the type I 5S rDNA of leiuperine as a functional 5S rRNA gene. Finally, the chromosome mapping of 5S rDNA sequences allowed some inferences of chromosome homology in Leiuperinae. In conclusion, our study provides additional information about the organization, differentiation and functionality of 5S rDNA in anuran species, revealing the potential participation of satellite DNA in the evolution of this family of rDNA.

P309 Clonal outbreak of Trichophyton tonsurans causing tinea capitis among wrestlers in Beijing, China

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectives Trichophyton tonsurans, an anthropophilic dermatophyte, mostly causes tinea capitis and tinea corporis and is often associated with outbreaks among athletes involved in combat sports, such as wrestlers and judo athletes. Here, we report an outbreak of tinea capitis caused by Trichophyton tonsurans among five juvenile athletes aged ten to fourteen in a wrestling team in Beijing, China.MethodsA total of 5 wrestlers aged 10-14 years presented with alopecia, erythema, scales, and pustule on the scalp (Fig. 1a). Scrapings from the lesions were performed by direct microscopic examination using 10% potassium hydroxide (KOH) with calcofluor white and fungal culture. The fungal pathogens were identified by morphology and sequencing of the internal transcribed spacer (ITS) regions. Multilocus genotyping analysis was performed by sequencing of the five gene loci including the ITS and non-transcribed spacer (NTS) of the ribosomal RNA (rRNA) locus, alkalineprotease-1 (ALP1), metalloprotease-5 (MEP5), carboxypeptidases Y (CarbY), which show intraspecies diversity and can be applied to epidemiological investigations and determining the route of infection transmission. Antifungal susceptibility of terbinafine (TBF), itraconazole (ITC), fluconazole (FLC), ketoconazole (KTC), and amphotericin B (AMB) against the causative fungal isolates was determined by broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) M38-A3 document.ResultsThe five patients were diagnosed with tinea capitis because of the scalp lesions, spores, and hyphae observed by direct microscopic examination (Fig. 1b), and positive fungal culture. The causative isolates were all identified as T. tonsurans by macroscopic and microscopic characteristics (Fig. 2) and sequencing of ITS regions. The sequences of ITS, NTS, ALP1, MEP5, and CarbY were identical among the five isolates, revealing a single genotype. The abovementioned sequences have been deposited to Genbank. The minimum inhibitory concentrations (MICs) of TBF, ITC, FLC, KTC, and AMB against five T. tonsurans isolates were 0.015 μg/ml, 0.015 μg/ml, 8 μg/ml, 0.06 μg/ml, and 0.5 μg/ml, respectively.ConclusionWe report an outbreak of tinea capitis caused by T. tonsurans among wrestlers in Beijing, China. Multilocus genotyping analysis revealed that all isolates consisted of a single genotype, suggesting the outbreak may be caused by a single strain of T. tonsurans. The isolates were all susceptible to the common antifungal drugs treating tinea capitis.

Interrelationship amongst varieties of edible mushroom through Molecular marker Study

The information regarding the DNA sequences of plant is very limited. When knowledge of the DNA sequence of the targeted genome is unavailable several PCR based techniques (RAPD, ITS, AFLP, SSR markers, SNP markers) are utilized to accumulate information about the genetic adaptability. Random Amplification of Polymorphic DNA is a technique by which the random segments of DNA are amplified. Short nucleotide primers (8–12 nucleotides) are used to proceed with the PCR using genomic DNA, for fragments to amplify. The primers bind somewhere in the sequence. By comparing the banding matrix the DNA patterns can be ascertained. RAPD has been utilized to characterize, trace the phylogeny of diverse plant species. Internal transcribed spacer (ITS) is to a non-functional RNA situated between structural ribosomal RNAs (rRNA) on a transcript. The sequence of rRNA precursor transcript is the 5' external transcribed sequence (5' ETS), 18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA and the 3'ETS read from 5' to 3' direction. The complete sequence repeats themselves in tandem array for thousands of copies separated by regions of non-transcribed DNA termed non-transcribed spacer (NTS) or intergenic spacer (IGS). There is the presence of two ITS regions in eukaryotes. The first one located between 18S and 5.8S rRNA genes is called ITS1, while the next in the sequence is ITS2 that lies between 5.8S and 26S rRNA genes in plants. Each eukaryotic ribosomal cluster follows the sequence of the 5' external transcribed sequence (5' ETS), trailed by the 18S rRNA gene, the ITS1 and the 5.8S rRNA gene, the ITS2, the 28S rRNA gene, and finally the 3' ETS. ETS and ITS pieces are excised and rapidly degraded during rRNA maturation. In our study four different gilled mushrooms were analyzed for RAPD and ITS study.

The Ribosomal DNA Loci of the Ancient Monocot Pistia stratiotes L. (Araceae) Contain Different Variants of the 35S and 5S Ribosomal RNA Gene Units.

The freshwater plant water lettuce (Pistia stratiotes L.) grows in warm climatic zones and is used for phytoremediation and biomass production. P. stratiotes belongs to the Araceae, an ecologically and structurally diverse early monocot family, but the phylogenetic relationships among Araceae members are poorly understood. Ribosomal DNAs (rDNAs), including the 35S and 5S rDNA, encode the RNA components of ribosomes and are widely used in phylogenetic and evolutionary studies of various plant taxa. Here, we comprehensively characterized the chromosomal locations and molecular organization of 35S and 5S rDNA genes in water lettuce using karyological and molecular methods. Fluorescence in situ hybridization revealed a single location for the 35S and 5S rDNA loci, each on a different pair of the species’ 28 chromosomes. Molecular cloning and nucleotide sequencing of 35S rDNA of P. stratiotes, the first representative Araceae sensu stricto in which such a study was performed, displayed typical structural characteristics. The full-length repeat showed high sequence conservation of the regions producing the 18S, 5.8S, and 25S rRNAs and divergence of the internal transcribed spacers ITS1 and ITS2 as well as the large intergenic spacer (IGS). Alignments of the deduced sequence of 18S rDNA with the sequences available for other Araceae and representatives of other clades were used for phylogenetic analysis. Examination of 11 IGS sequences revealed significant intra-genomic length variability due to variation in subrepeat number, with four types of units detected within the 35S rDNA locus of the P. stratiotes genome (estimated size 407 Mb/1C). Similarly, the 5S rDNA locus harbors gene units comprising a conserved 119-bp sequence encoding 5S rRNA and two types of non-transcribed spacer (NTS) sequences. Type I was classified into four subtypes, which apparently originated via progressive loss of subrepeats within the duplicated NTS region containing the 3’ part of the 5S rRNA gene. The minor Type II NTS is shorter than Type I and differs in nucleotide composition. Some DNA clones containing two or three consecutive 5S rDNA repeats harbored 5S rDNA genes with different types of NTSs, confirming the mosaic composition of the 5S rDNA locus.

Partial Ribosomal Nontranscribed Spacer Sequences DistinguishRhagoletis zephyria(Diptera: Tephritidae) From the Apple Maggot,R. pomonella

The apple maggot, Rhagoletis pomonella (Walsh), was introduced into the apple-growing regions of the Pacific Northwest in the U.S.A. during the past 60–100 yr. Apple maggot (larvae, puparia, and adults) is difficult to distinguish from its morphologically similar sister species, Rhagoletis zephyria Snow, which is native and abundant in the Pacific Northwest. While morphological identifications are common practice, a simple, inexpensive assay based on genetic differences would be very useful when morphological traits are unclear. Here we report nucleotide substitution and insertion–deletion mutations in the nontranscribed spacer (NTS) of the ribosomal RNA gene cistron of R. pomonella and R. zephyria that appear to be diagnostic for these two fly species. Insertion–deletion variation is substantial and results in a 49 base-pair difference in PCR amplicon size between R. zephyria and R. pomonella that can be scored using agarose gel electrophoresis. PCR amplification and DNA sequencing of 766 bp of the NTS region from 38 R. pomonella individuals and 35 R. zephyria individuals from across their geographic ranges led to the expected PCR fragments of approx. 840 bp and 790 bp, respectively, as did amplification and sequencing of a smaller set of 26 R. pomonella and 16 R. zephyria flies from a sympatric site in Washington State. Conversely, 633 bp mitochondrial COI barcode sequences from this set of flies were polyphyletic with respect to R. pomonella and R. zephyria. Thus, differences in NTS PCR products on agarose gels potentially provide a simple way to distinguish between R. pomonella and R. zephyria.

The Development of New Species-Specific Molecular Markers Based on 5S rDNA in Elaeagnus L. Species.

The Elaeagnus L. species are trees and bushes that mainly grow in temperate zones of Western Europe; Minor, Central, and Southeast Asia; the Far East; and North America. Some species are used as fruit or ornamental plants and have economic value. Problems with the identification of species in the Elaeagnus genus by molecular genetical methods arise in the study of populations, systematics, breeding, and other areas of plant science and practice. Recently, the polymorphism of 5S ribosomal DNA non-transcribed spacers (5S rDNA NTSs) in Elaeagnaceae Adans. has been described. The results were used in our study as a basis for development of new species-specific molecular markers for some members of the Elaeagnus genus. The author’s method was applied for finding regions that were potentially applicable for species-specific primer design. As a result, some species-specific molecular markers were developed for Elaeagnus angustifolia L., E. commutata Bernh., E. pungens Thunb., and E. multiflora Thunb. These markers were tested in a range of samples and showed the presence of amplified fragments in lanes of the marked species only. Samples of other species showed no amplifications. Thus, the developed markers may be useful for the species identification of the studied Elaeagnus plants in botanical, dendrological, and genetic research (especially in a leafless period of year), as well as in breeding and hybridization experiments.

Molecular and FISH analysis of 45S rDNA on BAC molecule of Saccharina japonica

IGS is abundant in polymorphism, which is widely used in the analysis of intraspecific genetic diversity and phylogenetic relationships among geographical populations. In this study, the 45S rDNA repeat unit of Saccharina japonica was obtained for the first time by BAC clone sequencing. The total length of the 45S rDNA repeat unit of S. japonica was 8 995 bp, including 5 420 bp of 18S-5.8S–25S rDNA, and 3 575 bp of IGS (Intergenic Spacer), with the GC content of 51.4 %. The IGS was composed of a 465 bp of 3′-outer transcribed spacer (ETS), an 874 bp 5′-ETS, and a 2 236 bp non-transcribed spacer (NTS), with the GC content of 50.1 %. Fiber-FISH (fiber-fluorescence in situ hybridization) analysis of the distribution of 45S rDNA repeat units on the bacterial artificial chromosome illustrated that each fiber had at least five continuously moniliform hybridization signal points. This study provided a new candidate molecular marker for detecting intraspecific polymorphisms of S. japonica. In addition, the successful fiber-FISH analysis of the 45S rDNA on BAC molecule would contribute to the construction of the physical map and map-based cloning of this kelp.

Comparative Analysis of the nrDNA Repeat Unit of Manila Clam Ruditapes philippinarum and Quahog Mercenaria mercenaria

Ruditapes philippinarum and Mercenaria mercenaria are economically important bivalve species. The complete ribosomal DNA (rDNA) unit sequences of R. philippinarum and M. mercenaria, with as-sembled rDNA unit lengths of 12,910 and 12,100 bp, respectively, were obtained in this study for the first time. The rDNA unit structural organisation was similar to that in other eukaryotes, in-cluding the following elements in order: 18S rRNA-internal transcribed spacer 1 (ITS1); 5.8S rRNA-ITS2-28S rRNA-intergenic spacer (IGS) (3′ external transcribed spacer (ETS); non-transcribed spacer (NTS)-5′ ETS). The genetic differences between R. philippinarum and M. mercenaria were mainly attributable to non-coding regions (ITS1, ITS2 and IGS), especially the IGS region. The boundaries of putative 3′ ETS, NTS and 5′ ETS were confirmed. Seven and three sub-repeat fragments were found in R. philippinarum and M. mercenaria, respectively. These frag-ments ranged from 4 to 154 bp in length, and were located at the NTS and 5′ ETS regions. Five and six cytosine–guanine (CpG) islands were detected in R. philippinarum and M. mercenaria, respec-tively, and these covered 85.58% and 79.29% of the entire IGS sequence, respectively. The phylo-genetic tree was constructed based on Veneridae ITS and 18S rRNA sequences using the maxi-mum likelihood (ML) method. The ML tree based on ITS revealed that species within the same genus clearly clustered together with relatively high supporting values, and all the genera were recovered as monophyletic. The phylogenetic analyses using 18S rRNA provided a weaker phy-logenetic signal than ITS.

Generation of large mitochondrial and nuclear nucleotide sequences and phylogenetic analyses using high-throughput short-read datasets for endangered Placostylinae snails of the southwest Pacific

ABSTRACT Placostylinae are a sub-family of terrestrial land snails endemic to the southwest Pacific. Some species are harvested for food, and others are critically endangered. Here we assemble and characterise complete mitochondrial genomes, as well as three nuclear markers (partial 45S ribosomal cassettes and the histone genes H3 and H4) of five snail species from three geographical regions (New Zealand, New Caledonia and the Solomon Islands). Mitogenomes of Placostylinae snails ranged between 14,544 bp and 14,711 bp, with minor variation in the position of tRNA tyrosine (Y) and tRNA tryptophane (W). The 45S ribosomal cassette contained intra-genomic nucleotide variation in ITS2. Cassettes containing histone genes H3 and H4 and their non-transcribed spacer region were assembled for three species, with the two genes coded in the same direction. Phylogenetic analysis on this large dataset (mitochondrial genome + nuclear markers) supported geographical clustering of species but could not confidently infer monophyly of the four Placostylus species with respect to Eumecostylus cleryi. Analysis based on shorter cytochrome c oxidase sequences with a wider taxon sampling found species representing the genera Eumecostylus and Placocharis were nested within the phylogenetic diversity of Placostylus. Multi-locus phylogenetic analysis containing mitochondrial and nuclear sequences did not support monophyly of Placostylinae.

Mosaic Arrangement of the 5S rDNA in the Aquatic Plant Landoltia punctata (Lemnaceae).

Duckweeds are a group of monocotyledonous aquatic plants in the Araceae superfamily, represented by 37 species divided into five genera. Duckweeds are the fastest growing flowering plants and are distributed around the globe; moreover, these plants have multiple applications, including biomass production, wastewater remediation, and making pharmaceutical proteins. Dotted duckweed (Landoltia punctata), the sole species in genus Landoltia, is one of the most resilient duckweed species. The ribosomal DNA (rDNA) encodes the RNA components of ribosomes and represents a significant part of plant genomes but has not been comprehensively studied in duckweeds. Here, we characterized the 5S rDNA genes in L. punctata by cloning and sequencing 25 PCR fragments containing the 5S rDNA repeats. No length variation was detected in the 5S rDNA gene sequence, whereas the nontranscribed spacer (NTS) varied from 151 to 524 bp. The NTS variants were grouped into two major classes, which differed both in nucleotide sequence and the type and arrangement of the spacer subrepeats. The dominant class I NTS, with a characteristic 12-bp TC-rich sequence present in 3–18 copies, was classified into four subclasses, whereas the minor class II NTS, with shorter, 9-bp nucleotide repeats, was represented by two identical sequences. In addition to these diverse subrepeats, class I and class II NTSs differed in their representation of cis-elements and the patterns of predicted G-quadruplex structures, which may influence the transcription of the 5S rDNA. Similar to related duckweed species in the genus Spirodela, L. punctata has a relatively low rDNA copy number, but in contrast to Spirodela and the majority of other plants, the arrangement of the 5S rDNA units demonstrated an unusual, heterogeneous pattern in L. punctata, as revealed by analyzing clones containing double 5S rDNA neighboring units. Our findings may further stimulate the research on the evolution of the plant rDNA and discussion of the molecular forces driving homogenization of rDNA repeats in concerted evolution.