Uniparental silencing of 5S rRNA genes in plant allopolyploids - insights from Cardamine (Brassicaceae).

While the phenomenon of uniparental silencing of 35S rDNA in interspecific hybrids and allopolyploids is well documented, there is a notable absence of information regarding whether such silencing extends to the 5S RNA component of ribosomes. To address this gap in knowledge, we analyzed the 5S and 35S rDNA expression in Cardamine (Brassicaceae) allopolyploids, namely C. × insueta (2n = 3x = 24, genome composition RRA), C. flexuosa (2n = 4x = 32, AAHH), and C. scutata (2n = 4x = 32, PPAA) which share a common diploid ancestor (AA). We employed high-throughput sequencing of transcriptomes and genomes and phylogenetic analyses of 5S rRNA variants. The genomic organization of rDNA was further scrutinized through clustering and fluorescence in situ hybridization. In the C. × insueta allotriploid, we observed uniparental dominant expression of 5S and 35S rDNA loci. In the C. flexuosa and C. scutata allotetraploids, the expression pattern differed, with the 35S rDNA being expressed from the A subgenome, whereas the 5S rDNA was expressed from the partner subgenome. Both C. flexuosa and C. scutata but not C. × insueta showed copy and locus number changes. We conclude that in stabilized allopolyploids, transcription of ribosomal RNA components occurs from different subgenomes. This phenomenon appears to result in the formation of chimeric ribosomes comprising rRNA molecules derived from distinct parental origins. We speculate that the interplay of epigenetic silencing and rDNA rearrangements introduces an additional layer of variation in multimolecule ribosomal complexes, potentially contributing to the evolutionary success of allopolyploids.

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.

5S-IGS rDNA in wind-pollinated trees (Fagus L.) encapsulates 55 million years of reticulate evolution and hybrid origins of modern species.

Standard models of plant speciation assume strictly dichotomous genealogies in which a species, the ancestor, is replaced by two offspring species. The reality in wind-pollinated trees with long evolutionary histories is more complex: species evolve from other species through isolation when genetic drift exceeds gene flow; lineage mixing can give rise to new species (hybrid taxa such as nothospecies and allopolyploids). The multi-copy, potentially multi-locus 5S rDNA is one of few gene regions conserving signal from dichotomous and reticulate evolutionary processes down to the level of intra-genomic recombination. Therefore, it can provide unique insights into the dynamic speciation processes of lineages that diversified tens of millions of years ago. Here, we provide the first high-throughput sequencing (HTS) of the 5S intergenic spacers (5S-IGS) for a lineage of wind-pollinated subtropical to temperate trees, the Fagus crenata - F. sylvatica s.l. lineage, and its distant relative F. japonica. The observed 4963 unique 5S-IGS variants reflect a complex history of hybrid origins, lineage sorting, mixing via secondary gene flow, and intra-genomic competition between two or more paralogous-homoeologous 5S rDNA lineages. We show that modern species are genetic mosaics and represent a striking case of ongoing reticulate evolution during the past 55 million years.

Genomic architecture of 5S rDNA cluster and its variations within and between species.

BACKGROUND: Ribosomal DNAs (rDNAs) are arranged in purely tandem repeats, preventing them from being reliably assembled onto chromosomes during generation of genome assembly. The uncertainty of rDNA genomic structure presents a significant barrier for studying their function and evolution. RESULTS: Here we generate ultra-long Oxford Nanopore Technologies (ONT) and short NGS reads to delineate the architecture and variation of the 5S rDNA cluster in the different strains of C. elegans and C. briggsae. We classify the individual rDNA's repeating units into 25 types based on the unique sequence variations in each unit of C. elegans (N2). We next perform assembly of the cluster by taking advantage of the long reads that carry these units, which led to an assembly of 5S rDNA cluster consisting of up to 167 consecutive 5S rDNA units in the N2 strain. The ordering and copy number of various rDNA units are consistent with the separation time between strains. Surprisingly, we observed a drastically reduced level of variation in the unit composition in the 5S rDNA cluster in the C. elegans CB4856 and C. briggsae AF16 strains than in the C. elegans N2 strain, suggesting that N2, a widely used reference strain, is likely to be defective in maintaining the 5S rDNA cluster stability compared with other wild isolates of C. elegans or C. briggsae. CONCLUSIONS: The results demonstrate that Nanopore DNA sequencing reads are capable of generating assembly of highly repetitive sequences, and rDNA units are highly dynamic both within and between population(s) of the same species in terms of sequence and copy number. The detailed structure and variation of the 5S rDNA units within the rDNA cluster pave the way for functional and evolutionary studies.

Molecular and Cytogenetic Analysis of rDNA Evolution in Crepis Sensu Lato.

Although Crepis was the first model plant group in which chromosomal changes were considered to play an important role in speciation, their chromosome structure and evolution have been barely investigated using molecular cytogenetic methods. The aim of the study was to provide a better understanding of the patterns and directions of Crepis chromosome evolution, using comparative analyses of rDNA loci number and localisation. The chromosome base number and chromosomal organisation of 5S and 35S rDNA loci were analysed in the phylogenetic background for 39 species of Crepis, which represent the evolutionary lineages of Crepis sensu stricto and Lagoseris, including Lapsana communis. The phylogenetic relationships among all the species were inferred from nrITS and newly obtained 5S rDNA NTS sequences. Despite high variations in rDNA loci chromosomal organisation, most species had a chromosome with both rDNA loci within the same (usually short) chromosomal arm. The comparative analyses revealed several independent rDNA loci number gains and loci repositioning that accompanied diversification and speciation in Crepis. Some of the changes in rDNA loci patterns were reconstructed for the same evolutionary lineages as descending dysploidy.

Karyotype Analysis, Genomic and Fluorescence In Situ Hybridization (GISH and FISH) Reveal the Ploidy and Parental Origin of Chromosomes in Paeonia Itoh Hybrids.

Itoh hybrids are intersectional hybrids in Paeonia L. with sect. Moutan and sect. Paeonia as paternal and maternal parents, respectively. Therefore, these hybrids have herbaceous stems with improved ornamental value introduced by the paternal parent. Although both of their parents are diploids, Itoh hybrids are triploids. Moreover, the parental origin of their chromosomes has not been extensively studied. This study systematically analyzed the genome size, ploidy, and karyotype of Itoh hybrids and compared them with their parental taxa. Although the monoploid genome size of Itoh hybrids was different, it was not significantly different from that of the parents. However, the size of varieties in the two parental taxa was significantly different from the wild species, probably due to genome rearrangements caused by artificial selection. Further karyotype analysis, correlation analysis, and hierarchical clustering could not identify the parental origin of chromosomes in Itoh hybrids. Verification through genomic and fluorescence in situ hybridization (GISH and FISH) suggested that for the three sets of chromosomes in Itoh hybrids, two were from the paternal parent, and one was from the maternal parent. One of the first two sets was from wild species, and the other from a cultivated variety. GISH could not label the chromosomes of cultivated peonies from the sect. Moutan, probably due to the huge and complex genomes compared with the wild species. Meanwhile, 5S rDNA-based FISH was first applied in Paeonia, which may be used for ploidy assessment. This work may give insights into the utilization of Itoh hybrid resources.

Satellite DNA landscapes after allotetraploidization of quinoa (Chenopodium quinoa) reveal unique A and B subgenomes.

If two related plant species hybridize, their genomes may be combined and duplicated within a single nucleus, thereby forming an allotetraploid. How the emerging plant balances two co-evolved genomes is still a matter of ongoing research. Here, we focus on satellite DNA (satDNA), the fastest turn-over sequence class in eukaryotes, aiming to trace its emergence, amplification, and loss during plant speciation and allopolyploidization. As a model, we used Chenopodium quinoa Willd. (quinoa), an allopolyploid crop with 2n = 4x = 36 chromosomes. Quinoa originated by hybridization of an unknown female American Chenopodium diploid (AA genome) with an unknown male Old World diploid species (BB genome), dating back 3.3-6.3 million years. Applying short read clustering to quinoa (AABB), C. pallidicaule (AA), and C. suecicum (BB) whole genome shotgun sequences, we classified their repetitive fractions, and identified and characterized seven satDNA families, together with the 5S rDNA model repeat. We show unequal satDNA amplification (two families) and exclusive occurrence (four families) in the AA and BB diploids by read mapping as well as Southern, genomic, and fluorescent in situ hybridization. Whereas the satDNA distributions support C. suecicum as possible parental species, we were able to exclude C. pallidicaule as progenitor due to unique repeat profiles. Using quinoa long reads and scaffolds, we detected only limited evidence of intergenomic homogenization of satDNA after allopolyploidization, but were able to exclude dispersal of 5S rRNA genes between subgenomes. Our results exemplify the complex route of tandem repeat evolution through Chenopodium speciation and allopolyploidization, and may provide sequence targets for the identification of quinoa's progenitors.

Identification of 5S and 45S rDNA sites in Chrysanthemum species by using oligonucleotide fluorescence in situ hybridization (Oligo-FISH).

Fluorescence in situ hybridization (FISH) is a conventional method used to visualize the distribution of DNA elements within a genome. To examine the relationships within the Chrysanthemum genus, ribosomal DNA (rDNA), a popular cytogenetic marker, was utilized as a probe for FISH within this genus. Based on the genome data of Chrysanthemum nankingense, C. seticuspe and its allied genera in the Compositae(Asteraceae), we explored rDNA sequences to design oligonucleotide probes and perform oligonucleotide fluorescence in situ hybridization (Oligo-FISH) in eight Chrysanthemum accessions. The results showed that the majority of 5S rDNA signals were located in subterminal chromosome regions and that the number of 5S rDNA sites might be tightly associated with ploidy. For 45S rDNA sites, the number and intensity of signals differed from those of previously investigated Chrysanthemum resources. These findings may provide an optimally reliable method of examining the chromosome composition and structural variation of Chrysanthemum and its related species and allow researchers to understand the evolutionary history and phylogenetic relationships of Chrysanthemum.

[Genomic Markers Associated with Cold-Hardiness in Brassica rapa L.]

Brassica rapa L. is a valuable and widespread species, but its cultivation in risk farming areas requires high-quality cold-hardy varieties to be developed. Mechanisms of the cold stress response in plants involve expression of numerous genes, including ribosomal ones, and are related to plant chromosome variability. FISH- and PCR-based methods were used to study intraspecific chromosome variability in the number and localization of 45S and 5S rDNA clusters and also to examine a set of molecular markers associated with cold-hardiness in winter B. rapa cultivars from high-risk farming areas. Several SSR (Na10-CO3 and BrgMS5339-1) and SCAR (BoCCA1-F/BoCCA1-1R1 and BoCCA1-F/BoCCA1-2R1) markers were identified as suitable for diagnosing cold-resistant and cold-susceptible genotypes in B. rapa. Compared with fodder cultivars, oilseed and leaf cultivars were shown to have more molecular markers associated with cold-hardiness and a higher level of polymorphism for the chromosomal distribution of 45S and 5S rDNAs, including chromosome heteromorphism. Thus, the least cold-resistant genotypes were found to display the lowest level of chromosome variability in the distribution of the 45S and 5S rDNA clusters and vice versa. The findings could be useful for the development of new cold-tolerant B. rapa varieties.

Evolutionary trends in animal ribosomal DNA loci: introduction to a new online database.

Ribosomal DNA (rDNA) loci encoding 5S and 45S (18S-5.8S-28S) rRNAs are important components of eukaryotic chromosomes. Here, we set up the animal rDNA database containing cytogenetic information about these loci in 1343 animal species (264 families) collected from 542 publications. The data are based on in situ hybridisation studies (both radioactive and fluorescent) carried out in major groups of vertebrates (fish, reptiles, amphibians, birds, and mammals) and invertebrates (mostly insects and mollusks). The database is accessible online at www.animalrdnadatabase.com . The median number of 45S and 5S sites was close to two per diploid chromosome set for both rDNAs despite large variation (1-74 for 5S and 1-54 for 45S sites). No significant correlation between the number of 5S and 45S rDNA loci was observed, suggesting that their distribution and amplification across the chromosomes follow independent evolutionary trajectories. Each group, irrespective of taxonomic classification, contained rDNA sites at any chromosome location. However, the distal and pericentromeric positions were the most prevalent (> 75% karyotypes) for 45S loci, while the position of 5S loci was more variable. We also examined potential relationships between molecular attributes of rDNA (homogenisation and expression) and cytogenetic parameters such as rDNA positions, chromosome number, and morphology.

Chromosomal Evolution in the Amolops mantzorum Species Group (Ranidae; Anura) Narrated by Repetitive DNAs.

In an attempt to analyze the organization of repetitive DNAs in the amphibian genome, 7 microsatellite motifs and a 5S rDNA sequence were synthesized and mapped in the karyotypes of 5 Amolops species. The results revealed nonrandom distribution of the microsatellite repeats, usually in the heterochromatic regions, as found in other organisms. These microsatellite repeats showed rapid changes among Amolops species, documenting the recent evolutionary history within this lineage. In contrast, 5S rDNA was localized in chromosomes 5 of all species, suggesting that these chromosomes are homologous within the monophyletic clade. Furthermore, the heteromorphic X and Y sex chromosomes (chromosomes 5) of A.mantzorum, had identical patterns of 5S rDNA, indicating that the subtelocentric Y resulted from a pericentric inversion. Several microsatellite repeats were found in the heteromorphic sex chromosomes, verifying the association of repetitive DNAs with sex chromosome differentiation in A. mantzorum.

First report of bicolour FISH of Berberis diaphana and B. soulieana reveals interspecific differences and co-localization of (AGGGTTT)3 and rDNA 5S in B. diaphana.

BACKGROUND: Berberis consists of approximately 500 species and is the largest genus in Berberidaceae. Most Berberis species lack cytological data, and bicolour fluorescence in situ hybridization (FISH) has never been performed on Berberis. In this work, a karyotype of Berberis diaphana, an alpine Berberis species obtained from an altitude of 3600 m in Wolong National Nature Reserve, China, was analysed and compared with Berberis soulieana Schneid. via FISH using oligonucleotide telomere probes for (AGGGTTT)3 and 5S rDNA (41 bp) for the first time. RESULTS: Berberis diaphana belonged to cytotype 2A and had the karyotype formula 2n = 2x = 28 = 26 m + 2 sm (2SAT). The mitotic metaphase chromosome lengths ranged from 1.82 ± 0.04 µm to 2.75 ± 0.00 µm. Clear (AGGGTTT)3 signals were detected at two telomeres in every chromosome and were co-localized with 5S rDNA at the terminal regions of the long arms in the 6th pair of chromosomes. One pair of (AGGGTTT)3 sites was localized in the satellites of the 7th pair of chromosomes, which are the only submetacentric chromosomes in this species. Totally 28 chromosomes with one pair of satellited chromosomes were observed in B. soulieana. This species had four 5S rDNA signals with two weak signals at the end of long arms in the 5th pair of chromosomes and another two strong signals detected in the interstitial region close to the end of short arms in the 6th pair of chromosomes. Each large signal consisted of two smaller signals with secondary constrictions around them. CONCLUSIONS: FISH physical mapping of B. diaphana suggested that (AGGGTTT)3 and rDNA 5S co-localize at the 6th pair of chromosomes. The density, location and number difference of 5S rDNA loci indicated structural differences among the chromosomes between B. diaphana and B. soulieana. Our results provide information that may contribute to future studies on the physical assembly of the Berberis genome and the evolution of rDNA and telomere FISH patterns in Berberis.

Retroelements and DNA Methylation Could Contribute to Diversity of 5S rDNA in Agave L.

Agave L. is a genus of economic importance, and many of the 166 species in the American plant genus Agave L. inhabit high-stress environments, which makes the genus promising for facing global climate change. However, sustainable use of economically important species without interfering with their ecology and evolution requires generating knowledge about the factors responsible for their genetic variation and diversity and, on this basis, their adaptation and speciation. Few genetic studies exploring the evolutionary relationships, speciation processes, genetic variability and diversity within species of Agave are currently available. Analyses of rDNA loci have been performed with the purpose of determining the genetic variability and diversity of the genus Agave, and these loci have been used as genetic markers of ploidy. However, the factors involved in the diversity of 5S rDNA regions in Agave have not yet been studied in depth. Our study explored the possible mechanisms of genetic (retroelements) and epigenetic (DNA methylation) diversity in 5S rDNA regions in Agave. We characterized the 5S rDNA gene tandem in species of the genus with different ploidy numbers and determined the levels of methylation in 13 haplotypes of 5S rDNA and in four non-transcribed spacers (NTS). Our results showed highly dynamic methylation with a high percentage in haplotypes and NTS of 5S rDNA regions in Agave. The characterization of the 5S rDNA tandem array in Agave revealed vestigial remains of the Cassandra terminal-repeat retrotransposon in miniature (TRIM). Our analysis supported previous results suggesting that in species of Agave L., regulation and diversity of 5S rDNA regions are controlled by coordinated genetic and epigenetic events, which will vary according to the species and the level of ploidy. The artificial pressure to which some agave crops are subjected may affect the mechanisms of evolution of gene 5S rDNA.

Evolutionary Dynamics of Copy Number and Meiotic Recombination in Murine 5S rDNA: Possible Involvement of Natural Selection.

We investigated evolutionary trends of the 5S ribosomal RNA gene in the house mouse, Mus musculus. First, we assessed the 5S cluster and copy numbers in eight laboratory strains by pulsed-field gel electrophoresis. The copy numbers in seven lines were estimated to be around 130-170 copies per cluster, with 63 copies in the remaining strain, implying that the copy number can change drastically and has been maintained under certain evolutionary constraints at ~ 140 copies. Second, we addressed the frequency of meiotic recombination mediated by the 5S cluster by performing a mating experiment with laboratory strains, and found that the 5S cluster did not accelerate recombination events. Third, we surveyed recombination events of the 5S-containing chromosome region in wild mice from the Japanese Islands, where the two subspecies lineages, M. m. castaneus and M. m. musculus, are historically mingled, and found that the influence of the 5S cluster on meiotic recombination was limited. Finally, we examined the nucleotide diversity of six genes in the neighboring regions of the 5S cluster and found reduced genetic diversity in the regions on both sides of the cluster, suggesting the involvement of either positive or background selection in the population-level sequence similarity of the 5S clusters. Therefore, the mouse 5S genes are considered to be evolving toward sequence similarity within a given cluster by certain intrachromosomal mechanisms and toward sharing of a specific 5S cluster within a population by certain selective processes.

Fine organization of genomic regions tagged to the 5S rDNA locus of the bread wheat 5B chromosome.

BACKGROUND: The multigene family encoding the 5S rRNA, one of the most important structurally-functional part of the large ribosomal subunit, is an obligate component of all eukaryotic genomes. 5S rDNA has long been a favored target for cytological and phylogenetic studies due to the inherent peculiarities of its structural organization, such as the tandem arrays of repetitive units and their high interspecific divergence. The complex polyploid nature of the genome of bread wheat, Triticum aestivum, and the technically difficult task of sequencing clusters of tandem repeats mean that the detailed organization of extended genomic regions containing 5S rRNA genes remains unclear. This is despite the recent progress made in wheat genomic sequencing. Using pyrosequencing of BAC clones, in this work we studied the organization of two distinct 5S rDNA-tagged regions of the 5BS chromosome of bread wheat. RESULTS: Three BAC-clones containing 5S rDNA were identified in the 5BS chromosome-specific BAC-library of Triticum aestivum. Using the results of pyrosequencing and assembling, we obtained six 5S rDNA- containing contigs with a total length of 140,417 bp, and two sets (pools) of individual 5S rDNA sequences belonging to separate, but closely located genomic regions on the 5BS chromosome. Both regions are characterized by the presence of approximately 70-80 copies of 5S rDNA, however, they are completely different in their structural organization. The first region contained highly diverged short-type 5S rDNA units that were disrupted by multiple insertions of transposable elements. The second region contained the more conserved long-type 5S rDNA, organized as a single tandem array. FISH using probes specific to both 5S rDNA unit types showed differences in the distribution and intensity of signals on the chromosomes of polyploid wheat species and their diploid progenitors. CONCLUSION: A detailed structural organization of two closely located 5S rDNA-tagged genomic regions on the 5BS chromosome of bread wheat has been established. These two regions differ in the organization of both 5S rDNA and the neighboring sequences comprised of transposable elements, implying different modes of evolution for these regions.

A Tandemly Arranged Pattern of Two 5S rDNA Arrays in Amolops mantzorum (Anura, Ranidae).

In an attempt to extend the knowledge of the 5S rDNA organization in anurans, the 5S rDNA sequences of Amolops mantzorum were isolated, characterized, and mapped by FISH. Two forms of 5S rDNA, type I (209 bp) and type II (about 870 bp), were found in specimens investigated from various populations. Both of them contained a 118-bp coding sequence, readily differentiated by their non-transcribed spacer (NTS) sizes and compositions. Four probes (the 5S rDNA coding sequences, the type I NTS, the type II NTS, and the entire type II 5S rDNA sequences) were respectively labeled with TAMRA or digoxigenin to hybridize with mitotic chromosomes for samples of all localities. It turned out that all probes showed the same signals that appeared in every centromeric region and in the telomeric regions of chromosome 5, without differences within or between populations. Obviously, both type I and type II of the 5S rDNA arrays arranged in tandem, which was contrasting with other frogs or fishes recorded to date. More interestingly, all the probes detected centromeric regions in all karyotypes, suggesting the presence of a satellite DNA family derived from 5S rDNA.

Variations in 5S rDNAs in diploid and tetraploid offspring of red crucian carp × common carp.

BACKGROUND: The allotetraploid hybrid fish (4nAT) that was created in a previous study through an intergeneric cross between red crucian carp (Carassius auratus red var., ♀) and common carp (Cyprinus carpio L., ♂) provided an excellent platform to investigate the effect of hybridization and polyploidization on the evolution of 5S rDNA. The 5S rDNAs of paternal common carp were made up of a coding sequence (CDS) and a non-transcribed spacer (NTS) unit, and while the 5S rDNAs of maternal red crucian carp contained a CDS and a NTS unit, they also contained a variable number of interposed regions (IPRs). The CDSs of the 5S rDNAs in both parental fishes were conserved, while their NTS units seemed to have been subjected to rapid evolution. RESULTS: The diploid hybrid 2nF1 inherited all the types of 5S rDNAs in both progenitors and there were no signs of homeologous recombination in the 5S rDNAs of 2nF1 by sequencing of PCR products. We obtained two segments of 5S rDNA with a total length of 16,457 bp from allotetraploid offspring 4nAT through bacterial artificial chromosome (BAC) sequencing. Using this sequence together with the 5S rDNA sequences amplified from the genomic DNA of 4nAT, we deduced that the 5S rDNAs of 4nAT might be inherited from the maternal progenitor red crucian carp. Additionally, the IPRs in the 5S rDNAs of 4nAT contained A-repeats and TA-repeats, which was not the case for the IPRs in the 5S rDNAs of 2nF1. We also detected two signals of a 200-bp fragment of 5S rDNA in the chromosomes of parental progenitors and hybrid progenies by fluorescence in situ hybridization (FISH). CONCLUSIONS: We deduced that during the evolution of 5S rDNAs in different ploidy hybrid fishes, interlocus gene conversion events and tandem repeat insertion events might occurred in the process of polyploidization. This study provided new insights into the relationship among the evolution of 5S rDNAs, hybridization and polyploidization, which were significant in clarifying the genome evolution of polyploid fish.

First case of Trichinella nativa infection in wild boar in Central Europe-molecular characterization of the parasite.

The examination of wild boars gained in Poland shows for the first time occurrence of Trichinella nativa, freeze-resistant species of Trichinella in this host from the central Europe region. This finding is not only one of several cases of T. nativa invasion in wild boars all over the world but also one of the very few cases of T. nativa detected so far beyond the known boundary of occurrence of this species. The molecular characterization of discovered larvae based on analysis of partial genes: 5s rDNA-ISR and CO1 confirm the findings. Moreover, the analyzed DNA sequences of both genes present new haplotypes of T. nativa in comparison to that described previously.

A new multiple sex chromosome system X1X1X2X2/X1Y1X2Y2 in Siluriformes: cytogenetic characterization of Bunocephalus coracoideus (Aspredinidae).

We analyzed one Bunocephalus coracoideus population from the Negro River basin using cytogenetic techniques. The results showed a diploid number of 42 chromosomes in both sexes, with the karyotypic formula 4m + 14sm + 24a and fundamental number (FN) = 60 for females and the formula 5m + 14sm + 23a and FN = 61 for males, constituting an X1X1X2X2/X1Y1X2Y2 multiple sex chromosome system. The constitutive heterochromatin is distributed in the pericentromeric regions of most of the chromosomes, except for the sex chromosomes, of which the X1, X2, and Y1 chromosomes were euchromatic and the Y2 chromosome was partially heterochromatic. 18S rDNA mapping confirmed the presence of nucleolar organizer regions on the short arms of the fifth chromosomal pair for both sexes. The 5S rDNA is present in the terminal regions of the short arms on the 2nd, 10th, and 12th pairs and on the X2 chromosome of both sexes; however, we observed variations in the presence of these ribosomal cistrons on the Y1 chromosome, on which the cistrons are pericentromeric, and on the Y2 chromosome, on which these cistrons are present in the terminal portions of the short and long arms. Telomeric sequences are located in the terminal regions of all of the chromosomes, particularly conspicuous blocks on the 10th and 12th pairs and internal telomeric sequences in the centromeric regions of the 1st, 6th, and 9th pairs for both sexes. This work describes an new sex chromosomes system for the Siluriformes and increases our genetic knowledge of the Aspredinidae family.

Evidence of birth-and-death evolution of 5S rRNA gene in Channa species (Teleostei, Perciformes).

In higher eukaryotes, minor rDNA family codes for 5S rRNA that is arranged in tandem arrays and comprises of a highly conserved 120 bp long coding sequence with a variable non-transcribed spacer (NTS). Initially the 5S rDNA repeats are considered to be evolved by the process of concerted evolution. But some recent reports, including teleost fishes suggested that evolution of 5S rDNA repeat does not fit into the concerted evolution model and evolution of 5S rDNA family may be explained by a birth-and-death evolution model. In order to study the mode of evolution of 5S rDNA repeats in Perciformes fish species, nucleotide sequence and molecular organization of five species of genus Channa were analyzed in the present study. Molecular analyses revealed several variants of 5S rDNA repeats (four types of NTS) and networks created by a neighbor net algorithm for each type of sequences (I, II, III and IV) did not show a clear clustering in species specific manner. The stable secondary structure is predicted and upstream and downstream conserved regulatory elements were characterized. Sequence analyses also shown the presence of two putative pseudogenes in Channa marulius. Present study supported that 5S rDNA repeats in genus Channa were evolved under the process of birth-and-death.