The oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), known as oxi-mCs, garners significant interest in plants as potential epigenetic marks. While research in mammals has established a role in cell reprogramming, carcinogenesis, and gene regulation, their functions in plants remain unclear. In rice, 5hmC has been associated with transposable elements (TEs) and heterochromatin. This study utilizes Silene latifolia, a dioecious plant with heteromorphic sex chromosomes and a genome with a large proportion of TEs, which provides a favourable environment for the study of oxi-mCs in individual sexes. Notably, we detected surprisingly high levels of oxi-mCs in S. latifolia comparable with mammals. Nuclei showed enrichment in heterochromatic regions, except for 5hmC whose signal was homogeneously distributed. Intriguingly, the same X chromosome in females displayed overall enrichment of 5hmC and 5fC compared with its counterpart. This fact is shared with 5mC, resembling dosage compensation. Co-localization showed higher correlation between 5mC and 5fC than with 5hmC, indicating no potential relationship between 5hmC and 5fC. Additionally, the promoter of several sex-linked genes and sex-biased TEs clustered in a clear sex-dependent way. Together, these findings unveil a hypothetical role for oxi-mCs in S. latifolia sex chromosome development, warranting further exploration.

• Keywords
• Silene latifolia, Cytosine modifications, dosage compensation, oxi-mCs, sex chromosomes, transposable elements,
• MeSH
• 5-Methylcytosine metabolism   analogs & derivatives   MeSH
• Chromosomes, Plant * genetics   MeSH
• Epigenesis, Genetic MeSH
• Sex Chromosomes * genetics   MeSH
• Silene * genetics   MeSH
• DNA Transposable Elements genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 5-Methylcytosine MeSH
• DNA Transposable Elements MeSH

The genus Silene includes a plethora of dioecious and gynodioecious species. Two species, Silene latifolia (white campion) and Silene dioica (red campion), are dioecious plants, having heteromorphic sex chromosomes with an XX/XY sex determination system. The X and Y chromosomes differ mainly in size, DNA content and posttranslational histone modifications. Although it is generally assumed that the sex chromosomes evolved from a single pair of autosomes, it is difficult to distinguish the ancestral pair of chromosomes in related gynodioecious and hermaphroditic plants. We designed an oligo painting probe enriched for X-linked scaffolds from currently available genomic data and used this probe on metaphase chromosomes of S. latifolia (2n = 24, XY), S. dioica (2n = 24, XY), and two gynodioecious species, S. vulgaris (2n = 24) and S. maritima (2n = 24). The X chromosome-specific oligo probe produces a signal specifically on the X and Y chromosomes in S. latifolia and S. dioica, mainly in the subtelomeric regions. Surprisingly, in S. vulgaris and S. maritima, the probe hybridized to three pairs of autosomes labeling their p-arms. This distribution suggests that sex chromosome evolution was accompanied by extensive chromosomal rearrangements in studied dioecious plants.

• Keywords
• Silene, Y chromosome, chromosome painting, double-translocation, pseudo-autosomal region,
• Publication type
• Journal Article MeSH

Contrasting patterns of histone modifications between the X and Y chromosome in Silene latifolia show euchromatic histone mark depletion on the Y chromosome and indicate hyperactivation of one X chromosome in females. Silene latifolia (white campion) is a dioecious plant with heteromorphic sex chromosomes (24, XX in females and 24, XY in males), and a genetically degenerated Y chromosome that is 1.4 times larger than the X chromosome. Although the two sex chromosomes differ in their DNA content, information about epigenetic histone marks and evidence of their function are scarce. We performed immunolabeling experiments using antibodies specific for active and suppressive histone modifications as well as pericentromere-specific histone modifications. We show that the Y chromosome is partially depleted of histone modifications important for transcriptionally active chromatin, and carries these marks only in the pseudo-autosomal region, but that it is not enriched for suppressive and pericentromere histone marks. We also show that two of the active marks are specifically enriched in one of the X chromosomes in females and in the X chromosome in males. Our data support recent findings that genetic imprinting mediates dosage compensation of sex chromosomes in S. latifolia.

• Keywords
• Posttranslational histone modifications, Pseudo-autosomal region, Sex chromosomes,
• MeSH
• Chromosomes, Plant genetics   MeSH
• Epigenesis, Genetic * MeSH
• Histone Code genetics   MeSH
• Silene genetics   MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: The rise and fall of the Y chromosome was demonstrated in animals but plants often possess the large evolutionarily young Y chromosome that is thought has expanded recently. Break-even points dividing expansion and shrinkage phase of plant Y chromosome evolution are still to be determined. To assess the size dynamics of the Y chromosome, we studied intraspecific genome size variation and genome composition of male and female individuals in a dioecious plant Silene latifolia, a well-established model for sex-chromosomes evolution. RESULTS: Our genome size data are the first to demonstrate that regardless of intraspecific genome size variation, Y chromosome has retained its size in S. latifolia. Bioinformatics study of genome composition showed that constancy of Y chromosome size was caused by Y chromosome DNA loss and the female-specific proliferation of recently active dominant retrotransposons. We show that several families of retrotransposons have contributed to genome size variation but not to Y chromosome size change. CONCLUSIONS: Our results suggest that the large Y chromosome of S. latifolia has slowed down or stopped its expansion. Female-specific proliferation of retrotransposons, enlarging the genome with exception of the Y chromosome, was probably caused by silencing of highly active retrotransposons in males and represents an adaptive mechanism to suppress degenerative processes in the haploid stage. Sex specific silencing of transposons might be widespread in plants but hidden in traditional hermaphroditic model plants.

• Keywords
• Epigenetics, Genome size, Silene latifolia, Transposable elements, Y chromosome,
• MeSH
• Chromosomes, Plant * MeSH
• Genome Size MeSH
• DNA, Plant * MeSH
• Genome, Plant MeSH
• In Situ Hybridization, Fluorescence MeSH
• Terminal Repeat Sequences MeSH
• Chromosome Mapping MeSH
• Evolution, Molecular * MeSH
• Repetitive Sequences, Nucleic Acid MeSH
• Retroelements * MeSH
• Sequence Deletion * MeSH
• Silene classification   genetics   MeSH
• Gene Silencing * MeSH
• DNA Copy Number Variations MeSH
• Base Composition MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant * MeSH
• Retroelements * MeSH

BACKGROUND: Genome size evolution is a complex process influenced by polyploidization, satellite DNA accumulation, and expansion of retroelements. How this process could be affected by different reproductive strategies is still poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed differences in the number and distribution of major repetitive DNA elements in two closely related species, Silene latifolia and S. vulgaris. Both species are diploid and possess the same chromosome number (2n = 24), but differ in their genome size and mode of reproduction. The dioecious S. latifolia (1C = 2.70 pg DNA) possesses sex chromosomes and its genome is 2.5× larger than that of the gynodioecious S. vulgaris (1C = 1.13 pg DNA), which does not possess sex chromosomes. We discovered that the genome of S. latifolia is larger mainly due to the expansion of Ogre retrotransposons. Surprisingly, the centromeric STAR-C and TR1 tandem repeats were found to be more abundant in S. vulgaris, the species with the smaller genome. We further examined the distribution of major repetitive sequences in related species in the Caryophyllaceae family. The results of FISH (fluorescence in situ hybridization) on mitotic chromosomes with the Retand element indicate that large rearrangements occurred during the evolution of the Caryophyllaceae family. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate that the evolution of genome size in the genus Silene is accompanied by the expansion of different repetitive elements with specific patterns in the dioecious species possessing the sex chromosomes.

• MeSH
• Chromosomes, Plant MeSH
• Genome Size * MeSH
• Genetic Variation MeSH
• Genome, Plant MeSH
• Genomics MeSH
• In Situ Hybridization, Fluorescence MeSH
• Nucleic Acid Hybridization MeSH
• Magnoliopsida genetics   MeSH
• Microsatellite Repeats genetics   MeSH
• Models, Genetic MeSH
• Evolution, Molecular MeSH
• Polyploidy MeSH
• Repetitive Sequences, Nucleic Acid genetics   MeSH
• Genes, Plant MeSH
• Plant Proteins genetics   MeSH
• DNA, Satellite genetics   MeSH
• Silene classification   genetics   MeSH
• Computational Biology methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Plant Proteins MeSH
• DNA, Satellite MeSH

BACKGROUND: Silene latifolia is a dioecious [corrected] plant with well distinguished X and Y chromosomes that is used as a model to study sex determination and sex chromosome evolution in plants. However, efficient utilization of this species has been hampered by the lack of large-scale sequencing resources and detailed analysis of its genome composition, especially with respect to repetitive DNA, which makes up the majority of the genome. METHODOLOGY/PRINCIPAL FINDINGS: We performed low-pass 454 sequencing followed by similarity-based clustering of 454 reads in order to identify and characterize sequences of all major groups of S. latifolia repeats. Illumina sequencing data from male and female genomes were also generated and employed to quantify the genomic proportions of individual repeat families. The majority of identified repeats belonged to LTR-retrotransposons, constituting about 50% of genomic DNA, with Ty3/gypsy elements being more frequent than Ty1/copia. While there were differences between the male and female genome in the abundance of several repeat families, their overall repeat composition was highly similar. Specific localization patterns on sex chromosomes were found for several satellite repeats using in situ hybridization with probes based on k-mer frequency analysis of Illumina sequencing data. CONCLUSIONS/SIGNIFICANCE: This study provides comprehensive information about the sequence composition and abundance of repeats representing over 60% of the S. latifolia genome. The results revealed generally low divergence in repeat composition between the sex chromosomes, which is consistent with their relatively recent origin. In addition, the study generated various data resources that are available for future exploration of the S. latifolia genome.

• MeSH
• DNA, Plant genetics   MeSH
• Repetitive Sequences, Nucleic Acid genetics   MeSH
• Silene genetics   MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH

BACKGROUND: The evolution of sex chromosomes is often accompanied by gene or chromosome rearrangements. Recently, the gene AP3 was characterized in the dioecious plant species Silene latifolia. It was suggested that this gene had been transferred from an autosome to the Y chromosome. RESULTS: In the present study we provide evidence for the existence of an X linked copy of the AP3 gene. We further show that the Y copy is probably located in a chromosomal region where recombination restriction occurred during the first steps of sex chromosome evolution. A comparison of X and Y copies did not reveal any clear signs of degenerative processes in exon regions. Instead, both X and Y copies show evidence for relaxed selection compared to the autosomal orthologues in S. vulgaris and S. conica. We further found that promoter sequences differ significantly. Comparison of the genic region of AP3 between the X and Y alleles and the corresponding autosomal copies in the gynodioecious species S. vulgaris revealed a massive accumulation of retrotransposons within one intron of the Y copy of AP3. Analysis of the genomic distribution of these repetitive elements does not indicate that these elements played an important role in the size increase characteristic of the Y chromosome. However, in silico expression analysis shows biased expression of individual domains of the identified retroelements in male plants. CONCLUSIONS: We characterized the structure and evolution of AP3, a sex linked gene with copies on the X and Y chromosomes in the dioecious plant S. latifolia. These copies showed complementary expression patterns and relaxed evolution at protein level compared to autosomal orthologues, which suggests subfunctionalization. One intron of the Y-linked allele was invaded by retrotransposons that display sex-specific expression patterns that are similar to the expression pattern of the corresponding allele, which suggests that these transposable elements may have influenced evolution of expression patterns of the Y copy. These data could help researchers decipher the role of transposable elements in degenerative processes during sex chromosome evolution.

• MeSH
• Alleles MeSH
• Chromosomes, Plant genetics   MeSH
• DNA, Plant genetics   MeSH
• Exons MeSH
• Introns MeSH
• Evolution, Molecular * MeSH
• Promoter Regions, Genetic MeSH
• Gene Expression Regulation, Plant MeSH
• Repetitive Sequences, Nucleic Acid MeSH
• Retroelements MeSH
• Genes, Plant MeSH
• Plant Proteins genetics   MeSH
• Sequence Analysis, DNA MeSH
• Silene genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH
• Retroelements MeSH
• Plant Proteins MeSH

We carried out a global survey of all major types of transposable elements in Silene latifolia, a model species with sex chromosomes that are in the early stages of their evolution. A shotgun genomic library was screened with genomic DNA to isolate and characterize the most abundant elements. We found that the most common types of elements were the subtelomeric tandem repeat X-43.1 and Gypsy retrotransposons, followed by Copia retrotransposons and LINE non-LTR elements. SINE elements and DNA transposons were less abundant. We also amplified transposable elements with degenerate primers and used them to screen the library. The localization of elements by FISH revealed that most of the Copia elements were accumulated on the Y chromosome. Surprisingly, one type of Gypsy element, which was similar to Ogre elements known from legumes, was almost absent on the Y chromosome but otherwise uniformly distributed on all chromosomes. Other types of elements were ubiquitous on all chromosomes. Moreover, we isolated and characterized two new tandem repeats. One of them, STAR-C, was localized at the centromeres of all chromosomes except the Y chromosome, where it was present on the p-arm. Its variant, STAR-Y, carrying a small deletion, was specifically localized on the q-arm of the Y chromosome. The second tandem repeat, TR1, co-localized with the 45S rDNA cluster in the subtelomeres of five pairs of autosomes. FISH analysis of other Silene species revealed that some elements (e.g., Ogre-like elements) are confined to the section Elisanthe while others (e.g. Copia or Athila-like elements) are present also in more distant species. Similarly, the centromeric satellite STAR-C was conserved in the genus Silene whereas the subtelomeric satellite X-43.1 was specific for Elisanthe section. Altogether, our data provide an overview of the repetitive sequences in Silene latifolia and revealed that genomic distribution and evolutionary dynamics differ among various repetitive elements. The unique pattern of repeat distribution is found on the Y chromosome, where some elements are accumulated while other elements are conspicuously absent, which probably reflects different forces shaping the Y chromosome.

• MeSH
• Chromosomes, Plant genetics   MeSH
• DNA, Plant genetics   MeSH
• Species Specificity MeSH
• In Situ Hybridization, Fluorescence MeSH
• Repetitive Sequences, Nucleic Acid genetics   MeSH
• Silene classification   genetics   MeSH
• Tandem Repeat Sequences genetics   MeSH
• DNA Transposable Elements genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH
• DNA Transposable Elements MeSH

Here we introduce a new model species, Silene colpophylla, that could facilitate research of sex chromosome evolution and sex-determining systems. This species is related to the well-established dioecious plant model Silene latifolia. Our results show that S. colpophylla is, similarly to S. latifolia, a male heterogametic species, but its sex chromosomes have evolved from a different pair of autosomes than in S. latifolia. The results of our phylogenetic study and mapping of homologs of S. latifolia X-linked genes indicate that the sex determination system in S. colpophylla evolved independently from that in S. latifolia. We assert that this model species pair will make it possible to study two independent patterns of sex chromosome evolution in related species.

• MeSH
• Amplified Fragment Length Polymorphism Analysis MeSH
• Biological Evolution MeSH
• Chromosomes, Plant genetics   MeSH
• Species Specificity MeSH
• Phylogeny MeSH
• Models, Genetic MeSH
• Genes, Plant MeSH
• Silene classification   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH

We established a new auxiliary phylogenetic approach based on genomic in situ hybridization technique (GISH). We used an interspecific hybrid Silene latifolia x Silene viscosa to compare two different genomes simultaneously on one slide. By using GISH with genomic DNA from another closely related species as a probe, we directly compared the level of relatedness between the genomes of the studied species and parental species. This experimental design enabled us to approximately estimate evolutionary relationships between the genome of tested plant species and genomes of both parental species of the hybrid by using the ratio of intensities of fluorescence signals. We tested this technique in various Silene species and the results were in accordance with the topology of the phylogenetic tree we constructed based on rDNA sequences. The results were also well correlated with phylogenetic distances between species that we estimated from an rDNA-based phylogenetic tree. Our experimental approach could help to improve tree topology and serve as a useful complementary tool in molecular phylogenetic studies in related species.

• MeSH
• DNA, Plant MeSH
• Phylogeny * MeSH
• Genome, Plant MeSH
• In Situ Hybridization, Fluorescence * MeSH
• In Situ Hybridization * MeSH
• DNA, Ribosomal genetics   MeSH
• Silene classification   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH
• DNA, Ribosomal MeSH