Expansions and contractions of repetitive DNA elements reveal contrasting evolutionary responses to the polyploid genome shock hypothesis in Brachypodium model grasses.

Brachypodium grass species have been selected as model plants for functional genomics of grass crops, and to elucidate the origins of allopolyploidy and perenniality in monocots, due to their small genome sizes and feasibility of cultivation. However, genome sizes differ greatly between diploid or polyploid Brachypodium lineages. We have used genome skimming sequencing data to uncover the composition, abundance, and phylogenetic value of repetitive elements in 44 representatives of the major Brachypodium lineages and cytotypes. We also aimed to test the possible mechanisms and consequences of the "polyploid genome shock hypothesis" (PGSH) under three different evolutionary scenarios of variation in repeats and genome sizes of Brachypodium allopolyploids. Our data indicated that the proportion of the genome covered by the repeatome in the Brachypodium species showed a 3.3-fold difference between the highest content of B. mexicanum-4x (67.97%) and the lowest of B. stacei-2x (20.77%), and that changes in the sizes of their genomes were a consequence of gains or losses in their repeat elements. LTR-Retand and Tekay retrotransposons were the most frequent repeat elements in the Brachypodium genomes, while Ogre retrotransposons were found exclusively in B. mexicanum. The repeatome phylogenetic network showed a high topological congruence with plastome and nuclear rDNA and transcriptome trees, differentiating the ancestral outcore lineages from the recently evolved core-perennial lineages. The 5S rDNA graph topologies had a strong match with the ploidy levels and nature of the subgenomes of the Brachypodium polyploids. The core-perennial B. sylvaticum presents a large repeatome and characteristics of a potential post-polyploid diploidized origin. Our study evidenced that expansions and contractions in the repeatome were responsible for the three contrasting responses to the PGSH. The exacerbated genome expansion of the ancestral allotetraploid B. mexicanum was a consequence of chromosome-wide proliferation of TEs and not of WGD, the additive repeatome pattern of young allotetraploid B. hybridum of stabilized post-WGD genome evolution, and the genomecontraction of recent core-perennials polyploids (B. pinnatum, B. phoenicoides) of repeat losses through recombination of these highly hybridizing lineages. Our analyses have contributed to unraveling the evolution of the repeatome and the genome size variation in model Brachypodium grasses.

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.

Ancient hybridization and repetitive element proliferation in the evolutionary history of the monocot genus Amomum (Zingiberaceae).

Genome size variation is a crucial aspect of plant evolution, influenced by a complex interplay of factors. Repetitive elements, which are fundamental components of genomic architecture, often play a role in genome expansion by selectively amplifying specific repeat motifs. This study focuses on Amomum, a genus in the ginger family (Zingiberaceae), known for its 4.4-fold variation in genome size. Using a robust methodology involving PhyloNet reconstruction, RepeatExplorer clustering, and repeat similarity-based phylogenetic network construction, we investigated the repeatome composition, analyzed repeat dynamics, and identified potential hybridization events within the genus. Our analysis confirmed the presence of four major infrageneric clades (A-D) within Amomum, with clades A-C exclusively comprising diploid species (2n = 48) and clade D encompassing both diploid and tetraploid species (2n = 48 and 96). We observed an increase in the repeat content within the genus, ranging from 84% to 89%, compared to outgroup species with 75% of the repeatome. The SIRE lineage of the Ty1-Copia repeat superfamily was prevalent in most analyzed ingroup genomes. We identified significant difference in repeatome structure between the basal Amomum clades (A, B, C) and the most diverged clade D. Our investigation revealed evidence of ancient hybridization events within Amomum, coinciding with a substantial proliferation of multiple repeat groups. This finding supports the hypothesis that ancient hybridization is a driving force in the genomic evolution of Amomum. Furthermore, we contextualize our findings within the broader context of genome size variations and repeatome dynamics observed across major monocot lineages. This study enhances our understanding of evolutionary processes within monocots by highlighting the crucial roles of repetitive elements in shaping genome size and suggesting the mechanisms that drive these changes.

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.

Investigating the Origin and Evolution of Polyploid Trifolium medium L. Karyotype by Comparative Cytogenomic Methods.

Trifolium medium L. is a wild polyploid relative of the agriculturally important red clover that possesses traits promising for breeding purposes. To date, T. medium also remains the only clover species with which agriculturally important red clover has successfully been hybridized. Even though allopolyploid origin has previously been suggested, little has in fact been known about the T. medium karyotype and its origin. We researched T. medium and related karyotypes using comparative cytogenomic methods, such as fluorescent in situ hybridization (FISH) and RepeatExplorer cluster analysis. The results indicate an exceptional karyotype diversity regarding numbers and mutual positions of 5S and 26S rDNA loci and centromeric repeats in populations of T. medium ecotypes and varieties. The observed variability among T. medium ecotypes and varieties suggests current karyotype instability that can be attributed to ever-ongoing battle between satellite DNA together with genomic changes and rearrangements enhanced by post-hybridization events. Comparative cytogenomic analyses of a T. medium hexaploid variety and diploid relatives revealed stable karyotypes with a possible case of chromosomal rearrangement. Moreover, the results provided evidence of T. medium having autopolyploid origin.

Karyotypes and Physical Mapping of Ribosomal DNA with Oligo-Probes in Eranthis sect. Eranthis (Ranunculaceae).

A comparative karyotype analysis of four species of yellow-flowered Eranthis sect. Eranthis, i.e., E. bulgarica, E. cilicica, E. hyemalis, and E. longistipitata from different areas, has been carried out for the first time. All the studied specimens had somatic chromosome number 2n = 16 with basic chromosome number x = 8. Karyotypes of the investigated plants included five pairs of metacentric chromosomes and three pairs of submetacentric/subtelocentric chromosomes. The chromosome sets of the investigated species differ mainly in the ratio of submetacentric/subtelocentric chromosomes, their relative lengths, and arm ratios. A new oligonucleotide probe was developed and tested to detect 45S rDNA clusters. Using this probe and an oligonucleotide probe to 5S rDNA, 45S and 5S rDNA clusters were localized for the first time on chromosomes of E. cilicica, E. hyemalis, and E. longistipitata. Major 45S rDNA clusters were identified on satellite chromosomes in all the species; in E. cilicica, minor clusters were also identified in the terminal regions of one metacentric chromosome pair. The number and distribution of 5S rDNA clusters is more specific. In E. cilicica, two major clusters were identified in the pericentromeric region of a pair of metacentric chromosomes. Two major clusters in the pericentromeric region of a pair of submetacentric chromosomes and two major clusters in the interstitial region of a pair of metacentric chromosomes were observed in E. longistipitata. E. hyemalis has many clusters of different sizes, localized mainly in the pericentromeric regions. Summarizing new data on the karyotype structure of E. sect. Eranthis and previously obtained data on E. sect. Shibateranthis allowed conclusions to be formed about the clear interspecific karyological differences of the genus Eranthis.

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.

Integration of Repeatomic and Cytogenetic Data on Satellite DNA for the Genome Analysis in the Genus Salvia (Lamiaceae).

Within the complicated and controversial taxonomy of cosmopolitan genus Salvia L. (Lamiaceae) are valuable species Salvia officinalis L. and Salvia sclarea L., which are important for the pharmaceutical, ornamental horticulture, food, and perfume industries. Genome organization and chromosome structure of these essential oil species remain insufficiently studied. For the first time, the comparative repeatome analysis of S. officinalis and S. sclarea was performed using the obtained NGS data, RepeatExplorer/TAREAN pipelines and FISH-based chromosome mapping of the revealed satellite DNA families (satDNAs). In repeatomes of these species, LTR retrotransposons made up the majority of their repetitive DNA. Interspecific variations in genome abundance of Class I and Class II transposable elements, ribosomal DNA, and satellite DNA were revealed. Four (S. sclarea) and twelve (S. officinalis) putative satDNAs were identified. Based on patterns of chromosomal distribution of 45S rDNA; 5S rDNA and the revealed satDNAs, karyograms of S. officinalis and S. sclarea were constructed. Promising satDNAs which can be further used as chromosome markers to assess inter- and intraspecific chromosome variability in Salvia karyotypes were determined. The specific localization of homologous satDNA and 45S rDNA on chromosomes of the studied Salvia species confirmed their common origin, which is consistent with previously reported molecular phylogenetic data.

Integration of Genomic and Cytogenetic Data on Tandem DNAs for Analyzing the Genome Diversity Within the Genus Hedysarum L. (Fabaceae).

The section Multicaulia is the largest clade in the genus Hedysarum L. (Fabaceae). Representatives of the sect. Multicaulia are valuable plants used for medicinal and fodder purposes. The taxonomy and phylogeny of the sect. Multicaulia are still ambiguous. To clarify the species relationships within sect. Multicaulia, we, for the first time, explored repeatomes of H. grandiflorum Pall., H. zundukii Peschkova, and H. dahuricum Turcz. using next-generation sequencing technologies and a subsequent bioinformatic analysis by RepeatExplorer/TAREAN pipelines. The comparative repeatome analysis showed that mobile elements made up 20-24% (Class I) and about 2-2.5% (Class II) of their repetitive DNAs. The amount of ribosomal DNA varied from 1 to 2.6%, and the content of satellite DNA ranged from 2.7 to 5.1%. For each species, five high confident putative tandem DNA repeats and 5-10 low confident putative DNA repeats were identified. According to BLAST, these repeats demonstrated high sequence similarity within the studied species. FISH-based mapping of 35S rDNA, 5S rDNA, and satDNAs made it possible to detect new effective molecular chromosome markers for Hedysarum species and construct the species karyograms. Comparison of the patterns of satDNA localization on chromosomes of the studied species allowed us to assess genome diversity within the sect. Multicaulia. In all studied species, we revealed intra- and interspecific variabilities in patterns of the chromosomal distribution of molecular chromosome markers. In H. gmelinii Ledeb. and H. setigerum Turcz. ex Fisch. et Meyer, similar subgenomes were detected, which confirmed the polyploid status of their genomes. Our findings demonstrated a close genomic relationship among six studied species indicating their common origin and confirmed the taxonomic status of H. setigerum as a subspecies of H. gmelinii as well as the validity of combining the sect. Multicaulia and Subacaulia into one sect. Multicaulia.

Uneven Levels of 5S and 45S rDNA Site Number and Loci Variations across Wild Chrysanthemum Accessions.

Ribosomal DNA (rDNA) is an excellent cytogenetic marker owing to its tandem arrangement and high copy numbers. However, comparative studies have focused more on the number of rDNA site variations within the Chrysanthemum genus, and studies on the types of rDNA sites with the same experimental procedures at the species levels are lacking. To further explore the number and types of rDNA site variations, we combined related data to draw ideograms of the rDNA sites of Chrysanthemum accessions using oligonucleotide fluorescence in situ hybridization (Oligo-FISH). Latent variations (such as polymorphisms of 45S rDNA sites and co-localized 5S-45S rDNA) also occurred among the investigated accessions. Meanwhile, a significant correlation was observed between the number of 5S rDNA sites and chromosome number. Additionally, the clumped and concentrated geographical distribution of different ploidy Chrysanthemum accessions may significantly promote the karyotype evolution. Based on the results above, we identified the formation mechanism of rDNA variations. Furthermore, these findings may provide a reliable method to examine the sites and number of rDNA variations among Chrysanthemum and its related accessions and allow researchers to further understand the evolutionary and phylogenetic relationships of the Chrysanthemum genus.

5S Ribosomal DNA of Genus Solanum: Molecular Organization, Evolution, and Taxonomy.

The Solanum genus, being one of the largest among high plants, is distributed worldwide and comprises about 1,200 species. The genus includes numerous agronomically important species such as Solanum tuberosum (potato), Solanum lycopersicum (tomato), and Solanum melongena (eggplant) as well as medical and ornamental plants. The huge Solanum genus is a convenient model for research in the field of molecular evolution and structural and functional genomics. Clear knowledge of evolutionary relationships in the Solanum genus is required to increase the effectiveness of breeding programs, but the phylogeny of the genus is still not fully understood. The rapidly evolving intergenic spacer region (IGS) of 5S rDNA has been successfully used for inferring interspecific relationships in several groups of angiosperms. Here, combining cloning and sequencing with bioinformatic analysis of genomic data available in the SRA database, we evaluate the molecular organization and diversity of IGS for 184 accessions, representing 137 species of the Solanum genus. It was found that the main mechanisms of IGS molecular evolution was step-wise accumulation of single base substitution or short indels, and that long indels and multiple base substitutions, which arose repeatedly during evolution, were mostly not conserved and eliminated. The reason for this negative selection seems to be association between indels/multiple base substitutions and pseudogenization of 5S rDNA. Comparison of IGS sequences allowed us to reconstruct the phylogeny of the Solanum genus. The obtained dendrograms are mainly congruent with published data: same major and minor clades were found. However, relationships between these clades and position of some species (S. cochoae, S. clivorum, S. macrocarpon, and S. spirale) were different from those of previous results and require further clarification. Our results show that 5S IGS represents a convenient molecular marker for phylogenetic studies on the Solanum genus. In particular, the simultaneous presence of several structural variants of rDNA in the genome enables the detection of reticular evolution, especially in the largest and economically most important sect. Petota. The origin of several polyploid species should be reconsidered.

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.

Cytogenetic and Molecular Characterization of Oligosarcus pintoi (Characidae): A New Record of Supernumerary Chromosome in this Species.

The genus Oligosarcus currently comprises 24 valid species distributed in the major river basins of South America. In this group, nine species were cytogenetically investigated, and found to share a diploid number of 50 chromosomes. Despite the conservation of the diploid number, variations in the karyotypic formula, number and position of the nucleolar organizer regions, and longitudinal bands have been described between both species and populations. In this study, we present cytogenetic and molecular data from Oligosarcus pintoi specimens from the Keller River, a tributary of the Ivaí River (Upper Paraná basin), using DNA barcoding and cytogenetic markers (C-band, silver-stained nucleolar organizer regions, and fluorescence in situ hybridization of 18S and 5S rDNA). The genetic inferences reached after analyzing the cytochrome c oxidade subunit 1 gene allowed us to confirm the identity of the individuals with 2n = 50 chromosomes. However, one specimen contained a medium subtelocentric supernumerary chromosome (2n = 51). This is the second record of additional chromosomes in O. pintoi, thereby confirming the existence of a supernumerary chromosome in allopatric populations of this species, a fact that demonstrates an evolutionary path that is divergent from other populations and/or species of Oligosarcus analyzed so far, contributing to the karyotypic diversification of the group.

A set of sampling, preparation, and staining techniques for studying meiosis in cucumber.

The development of genetic and genomic resources for biological studies in cucumber has experienced an unprecedented boom in recent years. To investigate the function of putative meiotic genes and germplasm in breeding programs, an accurate cytogenetic characterization is required. Cytological methods and reference to investigate meiosis in cucumber are limited at present. Here we provide a set of cytological techniques that have been adapted for the study of meiosis in cucumber. The meiotic stages can be identified with high precision using hierarchical criteria from developing buds, undisturbed meiocytes, and freshly stained chromosomes. A meiotic cytological atlas of all stages is presented as a reference for identifying particular stages and for comparison of meiosis between normal and mutant plants. We performed a comparative analysis of the distribution of cytoplasmic organelles between cucumber and Arabidopsis, and we described a highly nonsynchronous condensation of chromosome parts during diplotene. A simplified fluorescence in situ hybridization (FISH) protocol, using robustly spread chromosomes, were developed. In addition, we designed a single oligonucleotide probe for 5S rDNA to use in karyotyping and monitoring of homologous chromosome pairing, which will make FISH analysis of 5S rDNA easier and more economical.

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.

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.

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.

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.

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

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

A New Type of Allodiploid Hybrids Derived From Female Megalobrama amblycephala × Male Gobiocypris rarus.

Distant hybridization can combine whole genomes from parent species and result in changes in the phenotypes and genotypes in hybrids. The characteristics of many hybrid fishes with even number of chromosomes have been reported, but the hybrids with odd number chromosomes are rarely reported. Blunt snout bream (Megalobrama amblycephala, BSB, 2n = 48) and rare gudgeon (Gobiocypris rarus, RG, 2n = 50) belong to two different subfamilies and have quite different biological characteristics. In this study, we obtain the hybrids (BR) derived from the inter-subfamily hybridization of female BSB and male RG. We investigate the fertilization rate, hatching rate, morphological traits, chromosomal numbers, DNA content, growth rates, and 5S rDNA in the BR. The results show that the BR is an allodiploid fish with 49 chromosomes, and all the measurable traits are significantly different (p < 0.05) among BR, BSB, and BR. Interestingly, the upper part of the BR body color is similar to BSB (gray), the lower part of the BR body color is similar to RG (light yellow), and the BR inherits a unique light yellow wide longitudinal band from the RG. Furthermore, the BR has a fast growth rate compared with RG. The 5S rDNA of the BR inherits the specific bands of its parental 5S rDNA respectively and has some mutations, which show obvious recombination, heredity, and variability in BR. This study will be of great significance in fish genetic breeding.