rDNA Evolution Research Articles

Recombination and incomplete concerted evolution of the ribosomal 18S (partial) -ITS1-5.8S-ITS2-28S (partial) rDNA in Cynoglossus trigrammus genome

Nuclear ribosomal RNA gene (nrDNA) has long been deemed as an archetypal example illustrating concerted evolution. However, nrDNA has been found to be polymorphic in an increasing number of species. The ribosomal internal transcribed spacer region (ITS1-5.8S-ITS2) is of great importance to molecular barcoding and phylogenetic studies, but it almost remains unknown in some large taxa of Pleuronectiformes (flatfish). In this study, the polymorphisms of the 18S (partial) -ITS1-5.8S-ITS2-28S (partial) rDNA sequence in two Cynoglossus trigrammus individuals were investigated using a PCR cloning approach. Four main types of rDNA sequences (Type A, R1, R2, and B) were found, indicating a consistent lack of homogeneity. This confirmed the existence of significant levels of variation within individuals in tonguefish (Cynoglossidae) genomes. Type A sequence was inferred to be a putative pseudogene because of its shorter length, lower minimum free energy, less stable secondary structure, and remarkable variations in the conserved regions. Type R1 and R2 sequences were detected as recombinants. The most likely turnover mechanism generating this variation pattern was gene conversion operating at the intra-chromosomal level. These results will not only provide useful information for polymorphism analysis of ribosomal RNA genes in flatfish, but also shed light on the complex dynamic evolution of rDNA in teleost genomes.

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.

The relevance of chromosome fissions for major ribosomal DNA dispersion in hymenopteran insects

Ribosomal DNA (rDNA) loci are essential for cellular metabolism due to their participation in ribosome biogenesis. Although these genes have been widely cytogenetically mapped, the evolutionary mechanisms behind their variability in number and chromosomal location remain elusive, even in well-known biological groups, such as ants, bees and wasps (Insecta: Hymenoptera). To address this question in Hymenoptera and therefore advance the understanding of rDNA evolution in insects in general, we integrated molecular cytogenetic data, a phylogenomic framework, model-based predictions and genome sequencing. Hence, we assessed the main evolutionary trends shaping the chromosomal distribution of rDNA loci in Hymenoptera. We noticed the conservation of one site of rDNA per haploid genome, suggesting that a single 45S rDNA locus is the putative ancestral pattern for aculeate Hymenoptera. Moreover, our results highlighted a nonrandom distribution of rDNA in Hymenoptera karyotypes, as well as a lineage-specific preferential location. The proximal location of rDNA is favoured in species with multiple loci and in the two families of Hymenoptera that show the highest range of chromosome numbers: Formicidae and Vespidae. We propose that chromosome fissions have played a crucial role in the distribution pattern of rDNA loci through the evolutionary diversification of Hymenoptera. Moreover, our genomic analysis of two species, one with a single locus of rDNA and one with multiple loci, supported that loci multiplication is followed by sequence divergence. Our results provide detailed information about the number and chromosomal position of rDNA in Hymenoptera and, therefore, broaden our knowledge regarding rDNA evolutionary dynamics in insects.

Intra-genomic variability and pseudogenes in ribosomal ITS regions of Paraplagusia blochii (Pleuronectiformes: Cynoglossidae)

Abstract Eukaryotic nuclear ribosomal DNA (rDNA) typically evolves in a concerted manner, in which hundreds of rDNA sequences within species show little or no variations, whereas the sequences of different species diverge. There are few studies of the internal transcribed spacer regions (ITS1-5.8S-ITS2) in teleostean fishes and only one report on flatfishes. Here, we reported the discovery of two types of highly divergent ITS1-5.8S-ITS2 rDNA sequences (Type A and B) in the genome of the Bloch’s tonguesole, Paraplagusia blochii. These sequences mainly differ in sequence length, secondary structure, and minimum free energy. According to the potential features of pseudogenes, Type B sequences are speculated to be putative pseudogenic ITS regions. Cluster analyses also supported two major clades that corresponded to the sequence type. To the best of our knowledge, this is the first report of the ITS regions of tonguefish, and may therefore provide useful information for future studies of the rDNA of flatfishes as well as the patterns of rDNA evolution in teleostean fishes.

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

BackgroundBerberis 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.ResultsBerberis 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.ConclusionsFISH 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.

Intra-individual variation and transcribed pseudogenes in the ribosomal ITS1-5.8S-ITS2 rDNA of Paraplagusia japonica (Pleuronectiformes: Cynoglossidae)

The eukaryotic ribosomal DNA (rDNA) cluster consists of multiple copies of three genes (18S, 5.8S, and 28S rDNA) and two internal transcribed spacers (ITS1 and ITS2). Typically, rDNA is thought to be evolved in a strict concerted evolution pattern, in which show little or even no variations within species, while sequence diversity between species or higher taxa. However, in the present study, we reported a high level of intra-individual polymorphism of ITS1-5.8S-ITS2 rDNA in the genome of Paraplagusia japonica (Pleuronectiformes: Cynoglossidae), suggesting it conformed a non-concerted evolutionary pattern. Three divergent type sequences (Type A, B, and C) differing in length, GC content, secondary structure stability and minimum free energy coexisted in the genome of each studied individual. According to the particular features of ribosomal pseudogenes, Type B and Type C sequences were postulated as pseudogenes. Expression analyses gave clear evidence that both the functional gene and pseudogenes were abundantly transcribed, indicating expression is not an applicable criterion for identifying ribosomal pseudogenes. These findings will enrich useful data for the studies on rDNA evolution in flatfishes, as well as provide fresh insight on the transcribed ribosomal pseudogenes and further consideration of the definition of ribosomal pseudogene.

An improved metagenomic strategy reveals an unprecedentedly high level of intragenomic polymorphism of ribosomal DNA in three species of Camellia

AbstractThe ribosomal DNA (rDNA) repeats of more and more species have been found to be polymorphic in the genome, and the understanding of this polymorphism is conducive not only to eliminating potential chaos in the metagenomic analysis (MGA), but also to providing rich information on rDNA evolution. In this study, the MGA previously used for the study of environmental microbial diversity was improved and extended to detect the intragenomic polymorphism (IGP) of rDNA in three species of Camellia L. The 3′‐end region of 26S rDNA of three individuals was amplified using degenerate primer pairs. Three annealing temperatures were applied to obtain as many ribotypes as possible, and equimolar amplicons from the three species were then pooled and sequenced using the Illumina MiSeq platform. An incredibly high level of IGP of rDNA was found in all three species. Nearly all of the ribotypes detected from this study were rDNA pseudogenes, and most of them have existed for a long time in the genome, with some generated from several rapid expansions. Our procedure provided an effective technique for detection of IGP of rDNA, and the particular evolutionary information of rDNAs in Camellia was also exploited.

Marked intra-genomic variation and pseudogenes in the ITS1-5.8S-ITS2 rDNA of Symphurus plagiusa (Pleuronectiformes: Cynoglossidae)

AbstractThe eukaryotic ribosomal DNA (rDNA) cluster consists of multiple copies of three genes (18S, 5.8S, and 28S rDNA) and two internal transcribed spacers (ITS1 and ITS2). In recent years, an increasing number of rDNA sequence polymorphisms have been identified in numerous species. In the present study, we provide 33 complete ITS (ITS1-5.8S-ITS2) sequences from twoSymphurus plagiusaindividuals. To the best of our knowledge, these sequences are the first detailed information on ITS sequences in Pleuronectiformes. Here, two divergent types (Type A and B) of the ITS1-5.8S-ITS2 rDNA sequence were found, which mainly differ in sequence length, GC content, nucleotide diversity (π), secondary structure and minimum free energy. The ITS1-5.8S-ITS2 rDNA sequence of Type B was speculated to be a putative pseudogene according to pseudogene identification criteria. Cluster analysis showed that sequences from the same type clustered into one group and two major groups were formed. The high degree of ITS1-5.8S-ITS2 sequence polymorphism at the intra-specific level indicated that theS. plagiusagenome has evolved in a non-concerted evolutionary manner. These results not only provide useful data for ribosomal pseudogene identification, but also further contribute to the study of rDNA evolution in teleostean genomes.

Variations in 5S rDNAs in diploid and tetraploid offspring of red crucian carp \xd7 common carp

BackgroundThe 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.ResultsThe 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).ConclusionsWe 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.

Contrasting Patterns of rDNA Homogenization within the Zygosaccharomyces rouxii Species Complex.

Arrays of repetitive ribosomal DNA (rDNA) sequences are generally expected to evolve as a coherent family, where repeats within such a family are more similar to each other than to orthologs in related species. The continuous homogenization of repeats within individual genomes is a recombination process termed concerted evolution. Here, we investigated the extent and the direction of concerted evolution in 43 yeast strains of the Zygosaccharomyces rouxii species complex (Z. rouxii, Z. sapae, Z. mellis), by analyzing two portions of the 35S rDNA cistron, namely the D1/D2 domains at the 5’ end of the 26S rRNA gene and the segment including the internal transcribed spacers (ITS) 1 and 2 (ITS regions). We demonstrate that intra-genomic rDNA sequence variation is unusually frequent in this clade and that rDNA arrays in single genomes consist of an intermixing of Z. rouxii, Z. sapae and Z. mellis-like sequences, putatively evolved by reticulate evolutionary events that involved repeated hybridization between lineages. The levels and distribution of sequence polymorphisms vary across rDNA repeats in different individuals, reflecting four patterns of rDNA evolution: I) rDNA repeats that are homogeneous within a genome but are chimeras derived from two parental lineages via recombination: Z. rouxii in the ITS region and Z. sapae in the D1/D2 region; II) intra-genomic rDNA repeats that retain polymorphisms only in ITS regions; III) rDNA repeats that vary only in their D1/D2 domains; IV) heterogeneous rDNA arrays that have both polymorphic ITS and D1/D2 regions. We argue that an ongoing process of homogenization following allodiplodization or incomplete lineage sorting gave rise to divergent evolutionary trajectories in different strains, depending upon temporal, structural and functional constraints. We discuss the consequences of these findings for Zygosaccharomyces species delineation and, more in general, for yeast barcoding.

Thinking beside the box: Should we care about the non-coding strand of the 16S rRNA gene?

The 16S rRNA gene (16S rDNA) codes for RNA that plays a fundamental role during translation in the ribosome and is used extensively as a marker gene to establish relationships among bacteria. However, the complementary non-coding 16S rDNA (nc16S rDNA) has been ignored. An idea emerged in the course of analyzing bacterial 16S rDNA sequences in search for nucleotide composition and substitution patterns: Does the nc16S rDNA code? If so, what does it code for? More importantly: Does 16S rDNA evolution reflect its own evolution or the evolution of its counterpart nc16S rDNA? The objective of this minireview is to discuss these thoughts. nc strands often encode small RNAs (sRNAs), ancient components of gene regulation. nc16S rDNA sequences from different bacterial groups were used to search for possible matches in the Bacterial Small Regulatory RNA Database. Intriguingly, the sequence of one published sRNA obtained from Legionella pneumophila (GenBank: AE0173541) showed high non-random similarity with nc16S rDNA corresponding in part to the V5 region especially from Legionella and relatives. While the target(s) of this sRNA is unclear at the moment, its mere existence might open up a new chapter in the use of the 16S rDNA to study relationships among bacteria.

The 5S rDNA in two Abracris grasshoppers (Ommatolampidinae: Acrididae): molecular and chromosomal organization.

The 5S ribosomal DNA (rDNA) sequences are subject of dynamic evolution at chromosomal and molecular levels, evolving through concerted and/or birth-and-death fashion. Among grasshoppers, the chromosomal location for this sequence was established for some species, but little molecular information was obtained to infer evolutionary patterns. Here, we integrated data from chromosomal and nucleotide sequence analysis for 5S rDNA in two Abracris species aiming to identify evolutionary dynamics. For both species, two arrays were identified, a larger sequence (named type-I) that consisted of the entire 5S rDNA gene plus NTS (non-transcribed spacer) and a smaller (named type-II) with truncated 5S rDNA gene plus short NTS that was considered a pseudogene. For type-I sequences, the gene corresponding region contained the internal control region and poly-T motif and the NTS presented partial transposable elements. Between the species, nucleotide differences for type-I were noticed, while type-II was identical, suggesting pseudogenization in a common ancestor. At chromosomal point to view, the type-II was placed in one bivalent, while type-I occurred in multiple copies in distinct chromosomes. In Abracris, the evolution of 5S rDNA was apparently influenced by the chromosomal distribution of clusters (single or multiple location), resulting in a mixed mechanism integrating concerted and birth-and-death evolution depending on the unit.

Polymorphism and evolution of ribosomal DNA in tea (Camellia sinensis, Theaceae)

Ribosomal DNA (rDNA) repeats often exhibit a high level of within-species homogeneity, but intra-individual polymorphism of rDNA has been found in Camellia species. In order to reveal the evolutionary pattern of rDNA repeats in tea [Camellia sinensis (L.) O. Kuntze], we identified the 45S rDNA loci, estimated their copy number, and cloned partial regions of them from different PCR products and from digested genomic DNA. The results show that there are 3 loci (6 sites) containing 45S rDNA in the tea genome; they are located at the ends of the short arms of 6 chromosomes and consist of a maximum of ∼6500 repeat units. On one hand, 164 sequences of the 26S rDNA cloned from PCR products contained 147 haplotypes, consisting of 62% pseudogenes, 24% putative functional genes, and 14% PCR-mediated recombinants, suggesting that the 45S rDNA of tea maintains an extremely high level of polymorphism and divergence; on the other hand, rDNA fragments cloned directly from genomic DNA exhibited a very high level of homogeneity: only one of 33 rDNA fragments was from a pseudogene. These results demonstrate that although over 60% of the 26S rDNA sequences identified in PCR products belong to pseudogenes, most 45S rDNA repeats are functional genes and have undergone concerted evolution. In this study, strong PCR bias and PCR-mediated recombination greatly increased the apparent proportion of pseudogenes in PCR products. Phylogenetic analysis and genetic divergence values for 26S rDNA sequences obtained in this study show that many pseudogenes have originated independently from functional genes at different times, and despite thus escaping from concerted evolution, they have failed to be eliminated from the tea genome over a long period, some of them having even produced addition copies by rapid expansion. Importantly, our study suggests that in order to determine the true pattern of evolution of rDNA it is necessary to combine data from more than one method rather than relying only on sequences from PCR products.

Structure of 5S rDNA sequence of the gibel carp (Carassius gibelio) with 2n=100 chromosomes

Event Abstract Back to Event Structure of 5S rDNA sequence of the gibel carp (Carassius gibelio) with 2n=100 chromosomes Aleksandra Szabelska1*, Lech Kirtiklis1 and Alicja Boron1 1 University of Warmia and Mazury in Olsztyn, Faculty of Biology and Biotechnology, Department of Zoology, Faculty of Biology and Biotechnology, Poland In vertebrate, the 5S ribosomal DNA (rDNA) consists of a conserved coding (transcriptional) unit of 120 bp (5S), which is separated from the next unit by a nontranscribed spacer (NTS). The coding sequence and the NTS together form a repeat unit which can be found in hundreds to thousands of copies tandemly repeated in the genomes. The coding region is highly conserved, even among nonrelated taxa, whereas the spacer region is variable, both in length and in sequence. Many studies have shown that birth-and-death processes can drive the evolution of 5S rDNA in distantly related taxa. The variations are due to insertions, internal subrepeats, deletions, base substitutions, pseudogenes, therefore the NTSs seem to be subject to rapid evolution. Thus, 5S rDNA sequences have been used as species – specific or population – specific genetic and cytogenetic markers in evolutionary studies: studying intraspecific variation, genome evolution, and phylogenetic reconstruction. Little is known about the nucleotide sequences and chromosomal location of 5S rDNA in teleosts in comparison with other vertebrate classes. Our preliminary research has related to diploid individuals of gibel carp (Carassius gibelio) with 2n=100 chromosomes. The PCR reaction has resulted in product 340 bp. Sequencing has yielded data on the exact nucleotide sequence and length of 5S rDNA fragment. Studies have shown no difference in length and nucleotide composition of the 5S sequence between males and females. Some study has revealed the influence of polyploidy on the organization and evolution of the 5S rDNA in teleosts fishes, therefore, then we will have studied the 5S rDNA sequences in triploid individuals. Acknowledgements The study was financed by a grant no. 12.610.006-300 of the UWM in Olsztyn Keywords: 5S rDNA, Carassius gibelio, Sequence Analysis, DNA, Sequence Alignment, Diploidy Conference: XV European Congress of Ichthyology, Porto, Portugal, 7 Sep - 11 Sep, 2015. Presentation Type: Poster Presentation Topic: Phylogeny, Systematics and Genetics Citation: Szabelska A, Kirtiklis L and Boron A (2015). Structure of 5S rDNA sequence of the gibel carp (Carassius gibelio) with 2n=100 chromosomes. Front. Mar. Sci. Conference Abstract: XV European Congress of Ichthyology. doi: 10.3389/conf.FMARS.2015.03.00187 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Nov 2015; Published Online: 01 Dec 2015. * Correspondence: Ms. Aleksandra Szabelska, University of Warmia and Mazury in Olsztyn, Faculty of Biology and Biotechnology, Department of Zoology, Faculty of Biology and Biotechnology, Olsztyn, 10 – 718, Poland, aleksandra.szabelska@uwm.edu.pl Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Aleksandra Szabelska Lech Kirtiklis Alicja Boron Google Aleksandra Szabelska Lech Kirtiklis Alicja Boron Google Scholar Aleksandra Szabelska Lech Kirtiklis Alicja Boron PubMed Aleksandra Szabelska Lech Kirtiklis Alicja Boron Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Gregarine site-heterogeneous 18S rDNA trees, revision of gregarine higher classification, and the evolutionary diversification of Sporozoa

Gregarine 18S ribosomal DNA trees are hard to resolve because they exhibit the most disparate rates of rDNA evolution of any eukaryote group. As site-heterogeneous tree-reconstruction algorithms can give more accurate trees, especially for technically unusually challenging groups, I present the first site-heterogeneous rDNA trees for 122 gregarines and an extensive set of 452 appropriate outgroups. While some features remain poorly resolved, these trees fit morphological diversity better than most previous, evolutionarily less realistic, maximum likelihood trees. Gregarines are probably polyphyletic, with some ‘eugregarines’ and all ‘neogregarines’ (both abandoned as taxa) being more closely related to Cryptosporidium and Rhytidocystidae than to archigregarines. I establish a new subclass Orthogregarinia (new orders Vermigregarida, Arthrogregarida) for gregarines most closely related to Cryptosporidium and group Orthogregarinia, Cryptosporidiidae, and Rhytidocystidae as revised class Gregarinomorphea. Archigregarines are excluded from Gregarinomorphea and grouped with new orders Velocida (Urosporoidea superfam. n. and Veloxidium) and Stenophorida as a new sporozoan class Paragregarea. Platyproteum and Filipodium never group with Orthogregarinia or Paragregarea and are sufficiently different morphologically to merit a new order Squirmida. I revise gregarine higher-level classification generally in the light of site-heterogeneous-model trees, discuss their evolution, and also sporozoan cell structure and life-history evolution, correcting widespread misinterpretations.

When fathers are instant losers: homogenization of rDNA loci in recently formed Cardamine×schulzii trigenomic allopolyploid.

Recently formed allopolyploids represent an excellent system to study the impacts of hybridization and genomic duplication on genome structure and evolution. Here we explored the 35SrRNA genes (rDNA) in the Cardamine×schulzii allohexaploid that was formed by two subsequent hybridization events within the past c. 150yr. The rDNA loci were analyzed by cloning, next generation sequencing (NGS), RT-PCR and FISH methods. The primary C.×insueta triploid hybrid derived from C.rivularis (♀) and C.amara (♂) had gene ratios highly skewed towards maternal sequences. Similarly, C.×schulzii, originating from the secondary hybridization event involving C.×insueta (♀) and C.pratensis (♂), showed a reduction in paternal rDNA homeologs despite an excess of chromosomes inherited from C.pratensis. We also identified novel rDNA loci in C.×schulzii, suggesting that lost loci might be slowly reinstalled by translocation (but not recombination) of genes from partner genomes. Prevalent clonal propagation of allopolyploids, C.×insueta and C.×schulzii, indicates that concerted evolution of rDNA may occur in the absence of extensive meiotic cycles. Adoption of NGS in rDNA variant analysis is highly informative for deciphering the evolutionary histories of allopolyploid species with ongoing homogenization processes.

(Cryptic) sex in the microsporidian Nosema granulosis – Evidence from parasite rDNA and host mitochondrial DNA

Microsporidia are single-celled, intracellular eukaryotes that parasitise a wide range of animals. The Nosema/Vairimorpha group includes some putative asexual species, and asexuality is proposed to have originated multiple times from sexual ancestors. Here, we studied the variation in the ribosomal DNA (rDNA) of 14 isolates of the presumed apomictic and vertically transmitted Nosema granulosis to evaluate its sexual status. The analysed DNA fragment contained a part of the small-subunit ribosomal gene (SSU) and the entire intergenic spacer (IGS). The mitochondrial cox1 gene of the host Gammarus duebeni (Crustacea) was analysed to temporally calibrate the system and to test the expectation of cophylogeny of host and parasite genealogies. Genetic variability of the SSU gene was very low within and between the isolates. In contrast, intraisolate (within a single host) variability of the IGS felt in two categories, because 12 isolates possess a very high IGS genetic diversity and two isolates were almost invariable in the IGS. This difference suggests variable models of rDNA evolution involving birth-and-death and unexpectedly concerted evolution. An alternative explanation could be a likewise unattended mixed infection of host individuals by more than one parasite strain. Despite considerable genetic divergence between associated host mitochondrial haplotypes, some N. granulosis ‘IGS populations’ seem not to belong to different gene pools; the relevant tests failed to show significant differences between populations. A set of recombinant IGS sequences made our data incompatible with the model of a solely maternally inherited, asexual species. In line with recent reports, our study supports the hypothesis that some assumed apomictic Microsporidia did not entirely abstain from the evolutionary advantages of sex. In addition, the presented data indicate that horizontal transmission may occur occasionally. This transmission mode could be a survival strategy of N. granulosis whose host often populates ephemeral habitats.

Contrasting Patterns of Evolution of 45S and 5S rDNA Families Uncover New Aspects in the Genome Constitution of the Agronomically Important Grass Thinopyrum intermedium (Triticeae)

We employed sequencing of clones and in situ hybridization (genomic and fluorescent in situ hybridization [GISH and rDNA-FISH]) to characterize both the sequence variation and genomic organization of 45S (herein ITS1-5.8S-ITS2 region) and 5S (5S gene + nontranscribed spacer) ribosomal DNA (rDNA) families in the allohexaploid grass Thinopyrum intermedium. Both rDNA families are organized within several rDNA loci within all three subgenomes of the allohexaploid species. Both families have undergone different patterns of evolution. The 45S rDNA family has evolved in a concerted manner: internal transcribed spacer (ITS) sequences residing within the arrays of two subgenomes out of three got homogenized toward one major ribotype, whereas the third subgenome contained a minor proportion of distinct unhomogenized copies. Homogenization mechanisms such as unequal crossover and/or gene conversion were coupled with the loss of certain 45S rDNA loci. Unlike in the 45S family, the data suggest that neither interlocus homogenization among homeologous chromosomes nor locus loss occurred in 5S rDNA. Consistently with other Triticeae, the 5S rDNA family in intermediate wheatgrass comprised two distinct array types-the long- and short-spacer unit classes. Within the long and short units, we distinguished five and three different types, respectively, likely representing homeologous unit classes donated by putative parental species. Although the major ITS ribotype corresponds in our phylogenetic analysis to the E-genome species, the minor ribotype corresponds to Dasypyrum. 5S sequences suggested the contributions from Pseudoroegneria, Dasypyrum, and Aegilops. The contribution from Aegilops to the intermediate wheatgrass' genome is a new finding with implications in wheat improvement. We discuss rDNA evolution and potential origin of intermediate wheatgrass.

Chromosomal Evolution in Tortricid Moths: Conserved Karyotypes with Diverged Features

Moths of the family Tortricidae constitute one of the major microlepidopteran groups in terms of species richness and economic importance. Yet, despite their overall significance, our knowledge of their genome organization is very limited. In order to understand karyotype evolution in the family Tortricidae, we performed detailed cytogenetic analysis of Grapholita molesta, G. funebrana, Lobesia botrana, and Eupoecilia ambiguella, representatives of two main tortricid subfamilies, Olethreutinae and Tortricinae. Besides standard cytogenetic methods, we used fluorescence in situ hybridization for mapping of major rRNA and histone gene clusters and comparative genomic hybridization to determine the level of molecular differentiation of the W and Z sex chromosomes. Our results in combination with available data in the codling moth, Cydia pomonella, and other tortricids allow us a comprehensive reconstruction of chromosomal evolution across the family Tortricidae. The emerging picture is that the karyotype of a common ancestor of Tortricinae and Olethreutinae differentiated from the ancestral lepidopteran chromosome print of n = 31 by a sex chromosome-autosome fusion. This rearrangement resulted in a large neo-sex chromosome pair and a karyotype with n = 30 conserved in most Tortricinae species, which was further reduced to n = 28 observed in Olethreutinae. Comparison of the tortricid neo-W chromosomes showed differences in their structure and composition presumably reflecting stochasticity of molecular degeneration of the autosomal part of the neo-W chromosome. Our analysis also revealed conservative pattern of the histone distribution, which is in contrast with high rDNA mobility. Despite the dynamic evolution of rDNA, we can infer a single NOR-chromosome pair as an ancestral state not only in tortricids but probably in all Lepidoptera. The results greatly expand our knowledge of the genome architecture in tortricids, but also contribute to the understanding of chromosomal evolution in Lepidoptera in general.

Chromosomal evolution of rDNA and H3 histone genes in representative Romaleidae grasshoppers from northeast Brazil.

BackgroundGrasshoppers from the Romaleidae family are well distributed in the Neotropical Region and represent a diversified and multicolored group in which the karyotype is conserved. Few studies have been conducted to understand the evolutionary dynamics of multigene families. Here, we report the chromosomal locations of the 18S and 5S rDNA and H3 histone multigene families in four grasshopper species from the Romaleidae family, revealed by fluorescent in situ hybridization (FISH).ResultsThe 5S rDNA gene was located in one or two chromosome pairs, depending on the species, and was found in a basal distribution pattern. Its chromosomal location was highly conserved among these species. The 18S rDNA was located in a single medium-sized chromosomal pair in all species analyzed. Its chromosomal location was near the centromere in the proximal or pericentromeric regions. The location of the H3 histone gene was highly conserved, with slight chromosomal location differences among some species. To our knowledge, this is the first report of a megameric chromosome carrying both the chromosomal markers 18S rDNA and the H3 histone genes, thereby expanding our understanding of such chromosomes.ConclusionsThe 5S and 18S rDNA genes and the H3 histone genes showed a conservative pattern in the species that we analyzed. A basal distribution pattern for 5S rDNA was observed with a location on the fourth chromosomal pair, and it was identified as the possible ancestral bearer. The 18S rDNA and H3 histone genes were restricted to a single pair of chromosomes, representing an ancestral pattern. Our results reinforce the known taxonomic relationships between Chromacris and Xestotrachelus, which are two close genera.