RepeatExplorer Pipeline Research Articles

In-Depth Satellitome Analyses of 37 Drosophila Species Illuminate Repetitive DNA Evolution in the Drosophila Genus.

Satellite DNAs (SatDNA) are ubiquitously present in eukaryotic genomes and have been recently associated with several biological roles. Understanding the evolution and significance of SatDNA requires an extensive comparison across multiple phylogenetic depths. We combined the RepeatExplorer pipeline and cytogenetic approaches to conduct a comprehensive identification and analysis of the satellitome in 37 species from the genus Drosophila. We identified 188 SatDNA-like families, 112 of them being characterized for the first time. Repeat analysis within a phylogenetic framework has revealed the deeply divergent nature of SatDNA sequences in the Drosophila genus. The SatDNA content varied from 0.54% of the D. arizonae genome to 38.8% of the D. albomicans genome, with the SatDNA content often following a phylogenetic signal. Monomer size and guanine–cytosine-content also showed extreme variation ranging 2–570 bp and 9.1–71.4%, respectively. SatDNA families are shared among closely related species, consistent with the SatDNA library hypothesis. However, we uncovered the emergence of species-specific SatDNA families through amplification of unique or low abundant sequences in a lineage. Finally, we found that genome sizes of the Sophophora subgenus are positively correlated with transposable element content, whereas genome size in the Drosophila subgenus is positively correlated with SatDNA. This finding indicates genome size could be driven by different categories of repetitive elements in each subgenus. Altogether, we conducted the most comprehensive satellitome analysis in Drosophila from a phylogenetic perspective and generated the largest catalog of SatDNA sequences to date, enabling future discoveries in SatDNA evolution and Drosophila genome architecture.

Identification and characterization of satellite DNAs in Poa L.

BackgroundPoa L. is a large genus of grass in Gramineae, among which P. pratensis is widely cultivated as turf and forage. Satellite DNA is the main components of the plant genome. Information of satellites will helpful for dissection the genome composition and definition of the phylogeny relationship of these species. However, the knowledge about the satellites in genus Poa is still limited.ResultsFour satellite DNAs were identified using the Repeat Explorer pipeline in HiSeq Illumina reads from diploid plants in P. malaca (2n = 26). Two satellites showed high similarity with the previously identified PpTr-1 and PpTr-3, whereas two others are newly identified with the monomer of 326 bp (Poa-326) and 353 bp (Poa-353) respectively. The clone DNAs of PpTr-1 and PpTr-3, and oligonucleotides designed representing satellites Poa-326 and Poa-353 were probed to test on chromosomes across 13 Poa speceis with different polyploidy level by fluorescent in situ hybridization (FISH). PpTr-1, PpTr-3, and Poa-362 were stably positioned in the subtelomeric regions in nearly all species with the variation of hybridization sites number. However, Poa-353 showed different FISH patterns of multiple regions with the variation of hybridization intensity and distribution sites across species. In addition, 5S rDNA and 45S rDNA were used to characterize the genome of the Poa species. Four rDNA FISH patterns were revealed in the tested species.ConclusionFour identified satellite were high conservable across Poa species. Genome distribution of these satellites can be characterized by FISH. The variation of satellite DNAs and rDNA chromosomal distributions between species provide useful information for phylogenetic analysis in genus Poa.

Comparative analyses of DNA repeats and identification of a novel Fesreba centromeric element in fescues and ryegrasses

BackgroundCultivated grasses are an important source of food for domestic animals worldwide. Increased knowledge of their genomes can speed up the development of new cultivars with better quality and greater resistance to biotic and abiotic stresses. The most widely grown grasses are tetraploid ryegrass species (Lolium) and diploid and hexaploid fescue species (Festuca). In this work, we characterized repetitive DNA sequences and their contribution to genome size in five fescue and two ryegrass species as well as one fescue and two ryegrass cultivars.ResultsPartial genome sequences produced by Illumina sequencing technology were used for genome-wide comparative analyses with the RepeatExplorer pipeline. Retrotransposons were the most abundant repeat type in all seven grass species. The Athila element of the Ty3/gypsy family showed the most striking differences in copy number between fescues and ryegrasses. The sequence data enabled the assembly of the long terminal repeat (LTR) element Fesreba, which is highly enriched in centromeric and (peri)centromeric regions in all species. A combination of fluorescence in situ hybridization (FISH) with a probe specific to the Fesreba element and immunostaining with centromeric histone H3 (CENH3) antibody showed their co-localization and indicated a possible role of Fesreba in centromere function.ConclusionsComparative repeatome analyses in a set of fescues and ryegrasses provided new insights into their genome organization and divergence, including the assembly of the LTR element Fesreba. A new LTR element Fesreba was identified and found in abundance in centromeric regions of the fescues and ryegrasses. It may play a role in the function of their centromeres.

Differential Genome Size and Repetitive DNA Evolution in Diploid Species of Melampodium sect. Melampodium (Asteraceae).

Plant genomes vary greatly in composition and size mainly due to the diversity of repetitive DNAs and the inherent propensity for their amplification and removal from the host genome. Most studies addressing repeatome dynamics focus on model organisms, whereas few provide comprehensive investigations across the genomes of related taxa. Herein, we analyze the evolution of repeats of the 13 species in Melampodium sect. Melampodium, representing all but two of its diploid taxa, in a phylogenetic context. The investigated genomes range in size from 0.49 to 2.27 pg/1C (ca. 4.5-fold variation), despite having the same base chromosome number (x = 10) and very strong phylogenetic affinities. Phylogenetic analysis performed in BEAST and ancestral genome size reconstruction revealed mixed patterns of genome size increases and decreases across the group. High-throughput genome skimming and the RepeatExplorer pipeline were utilized to determine the repeat families responsible for the differences in observed genome sizes. Patterns of repeat evolution were found to be highly correlated with phylogenetic position, namely taxonomic series circumscription. Major differences found were in the abundances of the SIRE (Ty1-copia), Athila (Ty3-gypsy), and CACTA (DNA transposon) lineages. Additionally, several satellite DNA families were found to be highly group-specific, although their overall contribution to genome size variation was relatively small. Evolutionary changes in repetitive DNA composition and genome size were complex, with independent patterns of genome up- and downsizing throughout the evolution of the analyzed diploids. A model-based analysis of genome size and repetitive DNA composition revealed evidence for strong phylogenetic signal and differential evolutionary rates of major lineages of repeats in the diploid genomes.

The Utility of Graph Clustering of 5S Ribosomal DNA Homoeologs in Plant Allopolyploids, Homoploid Hybrids, and Cryptic Introgressants.

IntroductionRibosomal DNA (rDNA) loci have been widely used for identification of allopolyploids and hybrids, although few of these studies employed high-throughput sequencing data. Here we use graph clustering implemented in the RepeatExplorer (RE) pipeline to analyze homoeologous 5S rDNA arrays at the genomic level searching for hybridogenic origin of species. Data were obtained from more than 80 plant species, including several well-defined allopolyploids and homoploid hybrids of different evolutionary ages and from widely dispersed taxonomic groups.Results(i) Diploids show simple circular-shaped graphs of their 5S rDNA clusters. In contrast, most allopolyploids and other interspecific hybrids exhibit more complex graphs composed of two or more interconnected loops representing intergenic spacers (IGS). (ii) There was a relationship between graph complexity and locus numbers. (iii) The sequences and lengths of the 5S rDNA units reconstituted in silico from k-mers were congruent with those experimentally determined. (iv) Three-genomic comparative cluster analysis of reads from allopolyploids and progenitor diploids allowed identification of homoeologous 5S rRNA gene families even in relatively ancient (c. 1 Myr) Gossypium and Brachypodium allopolyploids which already exhibit uniparental partial loss of rDNA repeats. (v) Finally, species harboring introgressed genomes exhibit exceptionally complex graph structures.ConclusionWe found that the cluster graph shapes and graph parameters (k-mer coverage scores and connected component index) well-reflect the organization and intragenomic homogeneity of 5S rDNA repeats. We propose that the analysis of 5S rDNA cluster graphs computed by the RE pipeline together with the cytogenetic analysis might be a reliable approach for the determination of the hybrid or allopolyploid plant species parentage and may also be useful for detecting historical introgression events.

In silico mining and FISH mapping of a chromosome-specific satellite DNA in Capsicum annuum L.

A large proportion of eukaryote nuclear genomes is composed of repetitive DNA. Tracing the dynamics of repetitive elements in the genomes of related taxa can reveal important information about their phylogenic relationships as well as traits that have become distinct to a lineage. Study the genomic abundance and chromosomal location of repetitive DNA in Capsicum annuum L. to understand the repeat dynamics. We quantified repeated DNA content in the C. annuum genome using the RepeatExplorer pipeline. About 42% of the C. annuum genome dataset comprised repetitive elements. Of these, 0.011, 0.98, 3.09, and 0.024% represented high and low confidence satellite repeats, putative long-terminal repeats (LTRs), and rDNA sequences, respectively. One novel high confidence 167-bp satellite repeat with a genomic proportion of 0.011%, Ca167TR, was identified. Furthermore, FISH with Ca167TR on metaphase chromosomes of C. annuum revealed signals in the subtelomeric regions of the short and long arms of chromosome 3 and 4, respectively. Further understanding of the origin and associated functions of Ca167TR and other repeats in Capsicum will give us insights into the genomic relationships and functions of the genome.

Low coverage sequencing for repetitive DNA analysis in Passiflora edulis Sims: citogenomic characterization of transposable elements and satellite DNA

BackgroundThe cytogenomic study of repetitive regions is fundamental for the understanding of morphofunctional mechanisms and genome evolution. Passiflora edulis a species of relevant agronomic value, this work had its genome sequenced by next generation sequencing and bioinformatics analysis performed by RepeatExplorer pipeline. The clusters allowed the identification and characterization of repetitive elements (predominant contributors to most plant genomes). The aim of this study was to identify, characterize and map the repetitive DNA of P. edulis, providing important cytogenomic markers, especially sequences associated with the centromere.ResultsThree clusters of satellite DNAs (69, 118 and 207) and seven clusters of Long Terminal Repeat (LTR) retrotransposons of the superfamilies Ty1/Copy and Ty3/Gypsy and families Angela, Athila, Chromovirus and Maximus-Sire (6, 11, 36, 43, 86, 94 and 135) were characterized and analyzed. The chromosome mapping of satellite DNAs showed two hybridization sites co-located in the 5S rDNA region (PeSat_1), subterminal hybridizations (PeSat_3) and hybridization in four sites, co-located in the 45S rDNA region (PeSat_2). Most of the retroelements hybridizations showed signals scattered in the chromosomes, diverging in abundance, and only the cluster 6 presented pericentromeric regions marking. No satellite DNAs and retroelement associated with centromere was observed.ConclusionP. edulis has a highly repetitive genome, with the predominance of Ty3/Gypsy LTR retrotransposon. The satellite DNAs and LTR retrotransposon characterized are promising markers for investigation of the evolutionary patterns and genetic distinction of species and hybrids of Passiflora.

Natural History of a Satellite DNA Family: From the Ancestral Genome Component to Species-Specific Sequences, Concerted and Non-Concerted Evolution.

Satellite DNA (satDNA) is the most variable fraction of the eukaryotic genome. Related species share a common ancestral satDNA library and changing of any library component in a particular lineage results in interspecific differences. Although the general developmental trend is clear, our knowledge of the origin and dynamics of satDNAs is still fragmentary. Here, we explore whole genome shotgun Illumina reads using the RepeatExplorer (RE) pipeline to infer satDNA family life stories in the genomes of Chenopodium species. The seven diploids studied represent separate lineages and provide an example of a species complex typical for angiosperms. Application of the RE pipeline allowed by similarity searches a determination of the satDNA family with a basic monomer of ~40 bp and to trace its transformation from the reconstructed ancestral to the species-specific sequences. As a result, three types of satDNA family evolutionary development were distinguished: (i) concerted evolution with mutation and recombination events; (ii) concerted evolution with a trend toward increased complexity and length of the satellite monomer; and (iii) non-concerted evolution, with low levels of homogenization and multidirectional trends. The third type is an example of entire repeatome transformation, thus producing a novel set of satDNA families, and genomes showing non-concerted evolution are proposed as a significant source for genomic diversity.

Satellite DNAs Unveil Clues about the Ancestry and Composition of B Chromosomes in Three Grasshopper Species.

Supernumerary (B) chromosomes are dispensable genomic elements occurring frequently among grasshoppers. Most B chromosomes are enriched with repetitive DNAs, including satellite DNAs (satDNAs) that could be implicated in their evolution. Although studied in some species, the specific ancestry of B chromosomes is difficult to ascertain and it was determined in only a few examples. Here we used bioinformatics and cytogenetics to characterize the composition and putative ancestry of B chromosomes in three grasshopper species, Rhammatocerus brasiliensis, Schistocerca rubiginosa, and Xyleus discoideus angulatus. Using the RepeatExplorer pipeline we searched for the most abundant satDNAs in Illumina sequenced reads, and then we generated probes used in fluorescent in situ hybridization (FISH) to determine chromosomal position. We used this information to infer ancestry and the events that likely occurred at the origin of B chromosomes. We found twelve, nine, and eighteen satDNA families in the genomes of R. brasiliensis, S. rubiginosa, and X. d. angulatus, respectively. Some satDNAs revealed clustered organization on A and B chromosomes varying in number of sites and position along chromosomes. We did not find specific satDNA occurring in the B chromosome. The satDNAs shared among A and B chromosomes support the idea of putative intraspecific ancestry from small autosomes in the three species, i.e., pair S11 in R. brasiliensis, pair S9 in S. rubiginosa, and pair S10 in X. d. angulatus. The possibility of involvement of other chromosomal pairs in B chromosome origin is also hypothesized. Finally, we discussed particular aspects in composition, origin, and evolution of the B chromosome for each species.

Comparative analysis of repetitive sequences among species from the potato and the tomato clades.

The genus Solanum includes important vegetable crops and their wild relatives. Introgression of their useful traits into elite cultivars requires effective recombination between hom(e)ologues, which is partially determined by genome sequence differentiation. In this study we compared the repetitive genome fractions of wild and cultivated species of the potato and tomato clades in a phylogenetic context. Genome skimming followed by a clustering approach was used as implemented in the RepeatExplorer pipeline. Repeat classes were annotated and the sequences of their main domains were compared. Repeat abundance and genome size were correlated and the larger genomes of species in the tomato clade were found to contain a higher proportion of unclassified elements. Families and lineages of repetitive elements were largely conserved between the clades, but their relative proportions differed. The most abundant repeats were Ty3/Gypsy elements. Striking differences in abundance were found in the highly dynamic Ty3/Gypsy Chromoviruses and Ty1/Copia Tork elements. Within the potato clade, early branching Solanum cardiophyllum showed a divergent repeat profile. There were also contrasts between cultivated and wild potatoes, mostly due to satellite amplification in the cultivated species. Interspersed repeat profiles were very similar among potatoes. The repeat profile of Solanum etuberosum was more similar to that of the potato clade. The repeat profiles in Solanum seem to be very similar despite genome differentiation at the level of collinearity. Removal of transposable elements by unequal recombination may have been responsible for structural rearrangements across the tomato clade. Sequence variability in the tomato clade is congruent with clade-specific amplification of repeats after its divergence from S. etuberosum and potatoes. The low differentiation among potato and its wild relatives at the level of interspersed repeats may explain the difficulty in discriminating their genomes by genomic in situ hybridization techniques.

Satellite DNA in Paphiopedilum subgenus Parvisepalum as revealed by high-throughput sequencing and fluorescent in situ hybridization

BackgroundSatellite DNA is a rapidly diverging, largely repetitive DNA component of many eukaryotic genomes. Here we analyse the evolutionary dynamics of a satellite DNA repeat in the genomes of a group of Asian subtropical lady slipper orchids (Paphiopedilum subgenus Parvisepalum and representative species in the other subgenera/sections across the genus). A new satellite repeat in Paphiopedilum subgenus Parvisepalum, SatA, was identified and characterized using the RepeatExplorer pipeline in HiSeq Illumina reads from P. armeniacum (2n = 26). Reconstructed monomers were used to design a satellite-specific fluorescent in situ hybridization (FISH) probe. The data were also analysed within a phylogenetic framework built using the internal transcribed spacer (ITS) sequences of 45S nuclear ribosomal DNA.ResultsSatA comprises c. 14.5% of the P. armeniacum genome and is specific to subgenus Parvisepalum. It is composed of four primary monomers that range from 230 to 359 bp and contains multiple inverted repeat regions with hairpin-loop motifs. A new karyotype of P. vietnamense (2n = 28) is presented and shows that the chromosome number in subgenus Parvisepalum is not conserved at 2n = 26, as previously reported. The physical locations of SatA sequences were visualised on the chromosomes of all seven Paphiopedilum species of subgenus Parvisepalum (2n = 26–28), together with the 5S and 45S rDNA loci using FISH. The SatA repeats were predominantly localisedin the centromeric, peri-centromeric and sub-telocentric chromosome regions, but the exact distribution pattern was species-specific.ConclusionsWe conclude that the newly discovered, highly abundant and rapidly evolving satellite sequence SatA is specific to Paphiopedilum subgenus Parvisepalum. SatA and rDNA chromosomal distributions are characteristic of species, and comparisons between species reveal that the distribution patterns generate a strong phylogenetic signal. We also conclude that the ancestral chromosome number of subgenus Parvisepalum and indeed of all Paphiopedilum could be either 2n = 26 or 28, if P. vietnamense is sister to all species in the subgenus as suggested by the ITS data.

Dating the Species Network: Allopolyploidy and Repetitive DNA Evolution in American Daisies (Melampodium sect. Melampodium, Asteraceae).

Allopolyploidy has played an important role in the evolution of the flowering plants. Genome mergers are often accompanied by significant and rapid alterations of genome size and structure via chromosomal rearrangements and altered dynamics of tandem and dispersed repetitive DNA families. Recent developments in sequencing technologies and bioinformatic methods allow for a comprehensive investigation of the repetitive component of plant genomes. Interpretation of evolutionary dynamics following allopolyploidization requires both the knowledge of parentage and the age of origin of an allopolyploid. Whereas parentage is typically inferred from cytogenetic and phylogenetic data, age inference is hampered by the reticulate nature of the phylogenetic relationships. Treating subgenomes of allopolyploids as if they belonged to different species (i.e., no recombination among subgenomes) and applying cross-bracing (i.e., putting a constraint on the age difference of nodes pertaining to the same event), we can infer the age of allopolyploids within the framework of the multispecies coalescent within BEAST2. Together with a comprehensive characterization of the repetitive DNA fraction using the RepeatExplorer pipeline, we apply the dating approach in a group of closely related allopolyploids and their progenitor species in the plant genus Melampodium (Asteraceae). We dated the origin of both the allotetraploid, Melampodium strigosum, and its two allohexaploid derivatives, Melampodium pringlei and Melampodium sericeum, which share both parentage and the direction of the cross, to the Pleistocene (n}{}<1.4 Ma). Thus, Pleistocene climatic fluctuations may have triggered formation of allopolyploids possibly in short intervals, contributing to difficulties in inferring the precise temporal order of allopolyploid species divergence of M. sericeum and M. pringlei. The relatively recent origin of the allopolyploids likely played a role in the near-absence of major changes in the repetitive fraction of the polyploids’ genomes. The repetitive elements most affected by the postpolyploidization changes represented retrotransposons of the Ty1-copia lineage Maximus and, to a lesser extent, also Athila elements of Ty3-gypsy family.

Satellite DNAs as potential taxonomic markers for Squirrel Monkeys, genus Saimiri

The number of squirrel monkey species (genus Saimiri, Platyrrhini) and their phylogenetic relationships are still uncertain. All squirrel monkeys analyzed to date presented a diploid number of 2n=44 with variable fundamental numbers. The repetitive DNA fraction of Saimiri genomes, as for most eukaryotes, is still poorly explored. Herein, we took advantage of the genome sequence of Saimiri boliviensis to explore the satellite DNAs (satDNAs) of the genus. After clustering Illumina reads of S. boliviensis (NCBI SRA access: SRR317821) by similarity using the Repeat Explorer pipeline, we identified two abundant satDNAs. The first represents nearly 1% of the S. boliviensis genome and is homologous to the alpha satDNA, known to have a centromeric location in primates. PCR and sequence analysis confirmed the ~340 bp monomer size in S. boliviensis, S. vanzolinii, S. sciureus and S. ustus. This result agrees with that found in most New World monkeys, which is a duplication-derivative of the 170 bp monomer found in Old World primates. In addition to the alpha satDNA, we identified a large satDNA (~1500 bp monomers) that comprises 2.2% of the genome and displays similarity with the CapA satDNA first described in Cebus appella. Using fluorescent in situ hybridization, we verified that CapA chromosome location varies among Saimiri species and is mainly located in subtelomeric regions of the short arms and in the interstitial heterochromatin of some chromosome pairs. In S. sciureus CapA was mapped to the interstitial heterochromatin of both arms of chromosome 2 and in the subtelomeric regions of chromosomes 4, 8, 10 and 14. In S. vanzolinii, CapA was located in the subtelomeric regions of chromosomes 4, 5, 8, 10 and 11. S. ustus is the species in which CapA is more ubiquitous, being present in the subtelomeric regions of chromosomes 4, 5, 7, 8, 10, 11 and 15. The CapA signal on pair 2 of S. vanzolinii and S. ustus displayed a much weaker intensity than that found in S. sciureus. The differences in CapA distribution and abundance within Saimiri species may provide valuable insights into the chromosome evolution and phylogenetic relationships within the genus.Financial support: CNPq, Fapemig, CAPES

Deciphering the repetitive DNA landscape in Phaseolus beans and Allied genera (Cajanus and Vigna) by a comparative cytogenomic approach

Several bean species (Fabaceae) have been undergoing intensive genomic research, which includes the generation of massive amount of DNA sequence data. We performed a comparative investigation of the repetitive DNA composition in some bean species (Phaseolus vulgaris - Pvu, P. acutifolius - Pac, P. coccineus - Pco, Cajanus cajan - Cca, Vigna angularis - Van and V. nakashimae - Vna). Genomic sequences were obtained from the NCBI public database Cca/SRX031567, Pac/SRX1182015), Pco/SRX1182014, Pvu-andean accession/SRX1182008 and mesoamerican accession/SRX1182013, Vna/SRX758247 and Van/SRX7575509) and run in the Repeat Explorer pipeline using an all-to-all comparison analysis. Physical mapping of the most abundant repetitive elements was performed by FISH using mitotic chromosomes. Overall, the repetitive DNA fraction makes up around 30% of the genomes, except for both Vigna species (9.7% and 14.6%, for Van and Vna, respectively). LTR-retrotransposons represented the major class, with Ogre-Tat elements showing a large variation, followed by LTR-Chromovirus, satellite DNA repeats, LINEs and DNA transposons. The composition of satellite repeats was variable and four new families were identified, together with five previously described. All satellites were genus- or species-specific. Chromosomal mapping of LTR sequences (Chromovirus and Ogre) showed a differential accumulation on the pericentromeres of most chromosomes, particularly on Phaseolus. Among the satellite repeats, five were used for FISH (four newly identified). The three satellite families from Phaseolus showed different species-specific distribution. PacSAT363 was detected in 12 pericentromeres. PcoSAT36 and PcoSAT336 were accumulated at pericentromes, but PcoSAT336 were also located in some chromosome termini co-localized with 45 rDNA, suggesting that this satellite have probably originated from ribosomal sequences. Cajanus cajan and Vigna showed only one satellite repeat each. CcaSAT100 was located in 18 centromeres, while VanSAT174 corresponded to the 45 rDNA in Vna, but appeared also as an independent centromeric satellite repeat in Van. Our analysis indicates a very dynamic landscape on the evolution of repetitive DNA among beans. Retrotransposons appeared as the main component of genomes and showed higher accumulation in the pericentromeric heterochromatin of Phaseolus species. Satellite repeats were mostly centromeric or pericentromeric and frequently species-specific, suggesting a fast evolution of this type of sequence in the bean genomes.

Next-generation sequencing reveals differentially amplified tandem repeats as a major genome component of Northern Europe’s oldest Camellia japonica

Northern Europe's oldest and largest Camellia japonica growing at the Pillnitz Castle (Germany) for over 200 years is of botanical and cultural importance and is a reference for C. japonica molecular scale analysis. In order to provide a fundament for genome analysis of the genus Camellia, we characterize the C. japonica tandem repeat fraction, constituting 12.5 % of the Pillnitz camellia's genome. A genomic library of the Pillnitz C. japonica was produced and Illumina sequenced to generate 36 Gb of paired-end reads. We performed graph-based read clustering implemented in the RepeatExplorer pipeline to estimate the C. japonica repeat fraction of 73 %. This enabled us to identify and characterize the most prominent satellite DNAs, Camellia japonica satellite 1-4 (CajaSat1-CajaSat4), and the 5S ribosomal DNA (rDNA) by bioinformatics, fluorescent in situ and Southern hybridization. Within the Camellia genus, satellite spreading, array expansion and formation of higher-order structures highlight different modes of repeat evolution. The CajaSat satellites localize at prominent chromosomal sites, including (peri)centromeres and subtelomeres of all chromosomes, thus serving as chromosomal landmarks for their identification. This work provides an insight into the C. japonica chromosome organization and significantly expands the Camellia genomic knowledge, also with respect to the tea plant Camellia sinensis.

Endogenous pararetrovirus sequences associated with 24 nt small RNAs at the centromeres of Fritillaria imperialis L. (Liliaceae), a species with a giant genome.

Endogenous pararetroviral sequences are the most commonly found virus sequences integrated into angiosperm genomes. We describe an endogenous pararetrovirus (EPRV) repeat in Fritillaria imperialis, a species that is under study as a result of its exceptionally large genome (1C = 42 096 Mbp, approximately 240 times bigger than Arabidopsis thaliana). The repeat (FriEPRV) was identified from Illumina reads using the RepeatExplorer pipeline, and exists in a complex genomic organization at the centromere of most, or all, chromosomes. The repeat was reconstructed into three consensus sequences that formed three interconnected loops, one of which carries sequence motifs expected of an EPRV (including the gag and pol domains). FriEPRV shows sequence similarity to members of the Caulimoviridae pararetrovirus family, with phylogenetic analysis indicating a close relationship to Petuvirus. It is possible that no complete EPRV sequence exists, although our data suggest an abundance that exceeds the genome size of Arabidopsis. Analysis of single nucleotide polymorphisms revealed elevated levels of C→T and G→A transitions, consistent with deamination of methylated cytosine. Bisulphite sequencing revealed high levels of methylation at CG and CHG motifs (up to 100%), and 15-20% methylation, on average, at CHH motifs. FriEPRV's centromeric location may suggest targeted insertion, perhaps associated with meiotic drive. We observed an abundance of 24 nt small RNAs that specifically target FriEPRV, potentially providing a signature of RNA-dependent DNA methylation. Such signatures of epigenetic regulation suggest that the huge genome of F. imperialis has not arisen as a consequence of a catastrophic breakdown in the regulation of repeat amplification.