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The genus Silene brings many opportunities for the study of various processes involved in the evolution of dioecy and young sex chromosomes. Here we focus on a dioecious clade in Silene subgenus Silene and closely related species. This study provides improved support for monophyly of this clade (based on inclusion of further dioecious species) and a new estimate of its age ( ca 2.3 million years). We observed a rise in adaptive evolution in the autosomal and pseudoautosomal parts of the genome on the branch where dioecy originated. This increase is not a result of the accumulation of sexually antagonistic genes in the pseudoautosomal region. It is also not caused by the coevolution of genes acting in mitochondria (despite the possibility that dioecy along this branch could have evolved from a nucleo-cytoplasmic male sterility-based system). After considering other possibilities, the most parsimonious explanation for the increase seen in the number of positively selected codons is the adaptive evolution of genes involved in the adaptation of the autosomal part of the genome to dioecy, as described in Charnov's sex-allocation theory. As the observed coincidence cannot prove causality, studies in other dioecious clades are necessary to allow the formation of general conclusions. This article is part of the theme issue ‘Sex determination and sex chromosome evolution in land plants’.

We have attempted to answer the question of whether the presence of sex chromosomes in the genome can affect the evolution of the autosomal part of the genome. As a model, we used dioecious plants from the section Otites of the genus Silene. We have observed a rise in adaptive evolution in the autosomal and pseudoautosomal parts of the genome, which are associated with the evolution of dioecy. This rise is caused neither by the accumulation of sexually antagonistic genes in the pseudoautosomal region nor by the co-evolution of genes acting in mitochondria (in spite of the fact that the dioecy evolved in this case most likely from cytoplasmic male sterility). Thus, this rise in the amount of positively selected codons is most likely caused by the adaptive evolution of genes involved in the specialization of the autosomal part of the genome on the dioecy as described in sex-allocation theory.

Switches in heterogamety are known to occur in both animals and plants. Although plant sex determination systems probably often evolved more recently than those in several well-studied animals, including mammals, and have had less time for switches to occur, we previously detected a switch in heterogamety in the plant genus Silene: section Otites has both female and male heterogamety, whereas S. latifolia and its close relatives, in a different section of the genus, Melandrium (subgenus Behenantha), all have male heterogamety. Here we analyse the evolution of sex chromosomes in section Otites, which is estimated to have evolved only about 0.55 MYA. Our study confirms female heterogamety in S. otites and newly reveals female heterogamety in S. borysthenica. Sequence analyses and genetic mapping show that the sex-linked regions of these two species are the same, but the region in S. colpophylla, a close relative with male heterogamety, is different. The sex chromosome pairs of S. colpophylla and S. otites each correspond to an autosome of the other species, and both differ from the XY pair in S. latifolia. Silene section Otites species are suitable for detailed studies of the events involved in such changes, and our phylogenetic analysis suggests a possible change from female to male heterogamety within this section. Our analyses suggest a possibility that has so far not been considered, change in heterogamety through hybridization, in which a male-determining chromosome from one species is introgressed into another one, and over-rides its previous sex-determining system.

Some transposable elements (TEs) show extraordinary variance in abundance along sex chromosomes but the mechanisms responsible for this variance are unknown. Here, we studied Ogre long terminal repeat (LTR) retrotransposons in Silene latifolia, a dioecious plant with evolutionarily young heteromorphic sex chromosomes. Ogre elements are ubiquitous in the S. latifolia genome but surprisingly absent on the Y chromosome. Bacterial artificial chromosome (BAC) library analysis and fluorescence in situ hybridization (FISH) were used to determine Ogre structure and chromosomal localization. Next generation sequencing (NGS) data were analysed to assess the transcription level and abundance of small RNAs. Methylation of Ogres was determined by bisulphite sequencing. Phylogenetic analysis was used to determine mobilization time and selection forces acting on Ogre elements. We characterized three Ogre families ubiquitous in the S. latifolia genome. One family is nearly absent on the Y chromosome despite all the families having similar structures and spreading mechanisms. We showed that Ogre retrotransposons evolved before sex chromosomes appeared but were mobilized after formation of the Y chromosome. Our data suggest that the absence of one Ogre family on the Y chromosome may be caused by 24-nucleotide (24-nt) small RNA-mediated silencing leading to female-specific spreading. Our findings highlight epigenetic silencing mechanisms as potentially crucial factors in sex-specific spreading of some TEs, but other possible mechanisms are also discussed.

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.

The aim of this work was to isolate new DNA markers linked to the Silene latifolia Y chromosome. To do this we created a chromosome-specific plasmid library after DOP-PCR amplification of laser-microdissected Y-chromosomes. The library screening led to the isolation of several clones yielding mostly to exclusive male specific hybridization signals. Subsequent PCR confirmed the Y-unique linkage for one of the sequences. This DNA sequence called MK17 has no homology to any known DNA sequence and it is not expressed. Based on PCR and Southern analyses, MK17 is present only in dioecious species of the Elisanthe section of the genus Silene (S. latifolia, S. dioica, and S. diclinis) and it is absent in related gynodioecious and hermaphroditic species. The mapping analysis using a panel of deletion mutants showed that MK17 is closely linked to the region controlling suppression of gynoecium development. Hence MK17 represents a valuable marker to isolate genes controlling the gynoecium development suppression on the Y chromosome of S. latifolia.

The genus Silene is a good model for studying evolution of the sex chromosomes, since it includes species that are hermaphroditic and dioecious, while maintain a basic chromosome number of 2n = 24. For some combinations of Silene species it is possible to construct interspecific hybrids. Here, we present a detailed karyological analysis of a hybrid between the dioecious Silene latifolia as the maternal plant and a related species, hermaphroditic Silene viscosa, used as a pollen partner. Using genomic probes (the genomic in situ hybridization (GISH) technique), we were able to clearly discriminate parental genomes and to show that they are largely separated in distinct nuclear domains. Molecular GISH and fluorescence in situ hybridization (FISH) markers document that the hybrid genome of somatic cells was strictly additive and stable, and that it had 12 chromosomes originating from each parent, including the only X chromosome of S. latifolia. Meiotic analysis revealed that, although related, respective parental chromosomes did not pair or paired only partially, which resulted in frequent chromosome abnormalities such as bridges and irregular non-disjunctions. GISH and FISH markers clearly document that the larger genome of S. latifolia and its largest chromosome component, the X chromosome, were mostly employed in chromosome lagging and misdivision.Key words: sex chromosome, Silene, interspecific hybrid, meiotic pairing, misdivision.

Here we compare gene orders on the Silene latifolia sex chromosomes. On the basis of the deletion mapping results (11 markers and 23 independent Y chromosome deletion lines used), we conclude that a part of the Y chromosome (covering a region corresponding to at least 23.9 cM on the X chromosome) has been inverted. The gradient in silent-site divergence suggests that this inversion took place after the recombination arrest in this region. Because recombination arrest events followed by Y chromosome rearrangements also have been found in the human Y chromosome, this process seems to be a general evolutionary pathway.

The dioecious plant species Silene latifolia has a sex determination mechanism based on an active Y chromosome. Here, we used inter-specific hybrids in the genus Silene to study the effects of gene complexes on the Y chromosome. If the function of Y-linked genes has been maintained in the same state as in the hermaphrodite progenitor species, it should be possible to substitute such genes by genes coming from a related hermaphrodite species. In the inter-specific hybrid, S. latifolia x S. viscosa, anthers indeed develop far beyond the early bilobal stage characteristic of XX S. latifolia female plants. The S. viscosa genome can thus replace the key sex determination gene whose absence abolishes early stamen development in females (loss of the stamen-promoting function, SPF), so that hybrid plants are morphologically hermaphrodite. However, the hybrids have two anther development defects, loss of adhesion of the tapetum to the endothecium, and precocious endothecium maturation. Both these defects were also found in independent Y-chromosome deletion mutants of S. latifolia. The data support the hypothesis that the evolution of complete gender dimorphism from hermaphroditism involved a major largely recessive male-sterility factor that created females, and the appearance of new, dominant genes on the Y chromosome, including both the well-documented gynoecium-suppressing factor, and two other Y specific genes promoting anther development.

Chromosomal aberrations and the nuclear topography of retinoblastoma tumour cells as well as lymphocytes of patients suffering from the familiar or sporadic form of retinoblastoma were studied. Fluorescence in situ hybridisation (FISH) on fresh, paraffin-embedded tumour tissues and on peripheral blood leukocytes was used for cytogenetic analysis. The cell cycle profile and induction of apoptosis was studied by flow cytometry and gene expression changes were detected by RT-PCR. Using the repeated FISH technique, the average distances between the nuclear membrane and the fluorescence gravity centre (FGC) of seven selected chromosomes were determined in the same tumour population and three other cell types. Chromosome order in positioning from the nuclear membrane was similar in all cell populations investigated. Our experimental studies were focused on specific genetic loci relevant for retinoblastoma tumour pathogenesis. We revealed a certain heterogeneity in the copy number of the Rb1, N-myc, and TP53 gene loci in tumour cells. In addition, in lymphocytes isolated from peripheral blood of the patients, a high degree of copy number heterogeneity was also detected. In 60% of analysed retinoblastomas we observed numerical aberration involving the centromeric region of chromosome 6. In these tumours, apoptotic bodies were found irrespective of clinical therapy. Chromosome instability seems to be a typical feature of primary retinoblastomas as well as of the human pseudodiploid cell line Y79. These cells, of a hereditary form of retinoblastoma (Y79), were irradiated by gamma rays and exposed to anti-tumour drugs such as etoposide, vincristine, and cisplatin. These treatments induced apoptosis, changes in the cell cycle profile, and specific modifications in the nuclear topography of selected loci. Treatment with a non-lethal concentration of hydroxyurea was shown to induce the loss of the amplified N-myc gene involved in the homogenously staining region (HSR) that was found to be associated with the nuclear membrane of retinoblastoma Y79 cells. We assume that not only cytological and cytogenetic parameters but also aberrant chromatin structures and their nuclear topography can be useful tools for optimal tumour marker specification.

The first pollen mitosis results in generative and vegetative cells which are characterised by a striking difference in their chromatin structure. In this study, histone H4 acetylation and DNA methylation have been analysed during pollen development inLilium longiflorum. Indirect immunofluorescence procedures followed by epifluorescence and laser scanning microscopy enabled a relative quantification of H4 acetylation and DNA methylation in microspores, immature binucleate pollen, mature pollen, and pollen tubes. The results show that histone H4 of the vegetative nucleus, in spite of its decondensed chromatin structure, is strongly hypoacetylated at lysine positions 5 and 8 in comparison with both the original microspore nucleus and the generative-cell nucleus. These H4 terminal lysines in the vegetative nucleus are, however, progressively acetylated during the following pollen tube growth. The DNA methylation analysis inversely correlates with the histone acetylation data. The vegetative nucleus in mature pollen grains is heavily methylated, but a dramatic nonreplicative demethylation occurs during the pollen tube development. Changes neither in H4 acetylation nor in DNA methylation have been found during development of the generative nucleus. The results obtained indicate that the vegetative nucleus enters the quiescent state (accompanied by DNA hypermethylation and H4 underacetylation) during the maturation of pollen grain which enables pollen grains a long-term survival without external source of nutrients until they reach the stigma.

Few cases of spontaneously horizontally transferred bacterial genes into plant genomes have been described to date. The occurrence of horizontally transferred genes from the T-DNA of Agrobacterium rhizogenes into the plant genome has been reported in the genus Nicotiana and in the species Linaria vulgaris. Here we compare patterns of evolution in one of these genes (a gene encoding mikimopine synthase, mis) following three different events of horizontal gene transfer (HGT). As this gene plays an important role in Agrobacterium, and there are known cases showing that genes from pathogens can acquire plant protection function, we hypothesised that in at least some of the studied species we will find signs of selective pressures influencing mis sequence. The mikimopine synthase (mis) gene evolved in a different manner in the branch leading to Nicotiana tabacum and N. tomentosiformis, in the branch leading to N. glauca and in the genus Linaria. Our analyses of the genus Linaria suggest that the mis gene began to degenerate soon after the HGT. In contrast, in the case of N. glauca, the mis gene evolved under significant selective pressures. This suggests a possible role of mikimopine synthase in current N. glauca and its ancestor(s). In N. tabacum and N. tomentosiformis, the mis gene has a common frameshift mutation that disrupted its open reading frame. Interestingly, our results suggest that in spite of the frameshift, the mis gene could evolve under selective pressures. This sequence may still have some regulatory role at the RNA level as suggested by coverage of this sequence by small RNAs in N. tabacum.

Understanding the origin and evolution of sex chromosomes requires studying recently evolved X–Y chromosome systems such as those in some flowering plants. We describe Y chromosome deletion mutants of Silene latifolia, a dioecious plant with heteromorphic sex chromosomes. The combination of results from new and previously described deletions with histological descriptions of their stamen development defects indicates the presence of two distinct Y regions containing loci with indispensable roles in male reproduction. We determined their positions relative to the two main sex determination functions (female suppressing and the other male promoting). A region proximal to the centromere on the Y p arm containing the putative stamen promoting sex determination locus includes additional early stamen developmental factors. A medial region of the Y q arm carries late pollen fertility factors. Cytological analysis of meiotic X–Y pairing in one of the male-sterile mutants indicates that the Y carries sequences or functions specifically affecting sex chromosome pairing.

Most dioecious plant species are believed to derive from hermaphrodite ancestors. The regulatory pathways that have been modified during evolution of the hermaphrodite ancestors and led to the emergence of dioecious species still remain unknown. Silene latifolia is a dioecious plant species harboring XY sex chromosomes. To identify the molecular mechanisms involved in female organ suppression in male flowers of S. latifolia , we looked for genes potentially involved in the establishment of floral organ and whorl boundaries. We identified homologs of Arabidopsis thaliana SHOOTMERISTEMLESS ( STM ) and CUP SHAPED COTYLEDON ( CUC ) 1 and CUC2 genes in S. latifolia . Our phylogenetic analyses suggest that we identified true orthologs for both types of genes. Detailed expression analyses showed a conserved expression pattern for these genes between S. latifolia and A. thaliana , suggesting a conserved function of the corresponding proteins. Comparative in situ hybridization experiments between male, female, and hermaphrodite individuals reveal that these genes show a male-specific pattern of expression before any morphological difference become apparent. Our results make SlSTM and SlCUC strong candidates for being involved in sex determination in S. latifolia .

The ends of eukaryotic chromosomes are capped with special nucleoprotein structures called telomeres. Telomere shortening due to telomerase inactivation may result in fusion of homologous or heterologous chromosomes, leading to their successive breakage during anaphase movement, followed by fusion of broken ends in the next cell cycle, i.e. the breakage-fusion-bridge (BFB) cycle. Using fluorescence in situ hybridization (FISH) with 25S rDNA and specific bacterial artificial chromosome (BAC) probes we demonstrate participation of chromosomes 2 and 4 of Arabidopsis thaliana AtTERT null plants in the formation of anaphase bridges. Both homologous and non-homologous chromosomes formed transient anaphase bridges whose breakage and unequal separation led to genome rearrangement, including non-reciprocal translocations and aneuploidy. The 45S rDNA regions located at the ends of chromosomes 2 and 4 were observed in chromosome bridges at a frequency approximately ten times higher than expected in the case of random fusion events. This outcome could result from a functional association of rDNA repeats at nucleoli. We also describe increased variation in the number of nucleoli in some interphase cells with supernumerary rDNA FISH signals. These data indicate that dysfunctional telomeres in Arabidopsis lead to massive genome instability, which is induced by multiple rounds of the BFB mechanism.

Pollen grains of angiosperm plants represent a good model system for studies of chromatin structure and remodelling factors, but very little is known about the DNA methylation status of particular genes in pollen. In this study, we present an analysis of the DNA methylation patterns of the MROS1 gene, which is expressed in the late phases of pollen development in Silene latifolia (syn. Meladrium album). The genomic sequencing technique revealed similar DNA methylation patterns in leaves, binucleate pollen, and trinucleate pollen. Extremely high DNA methylation levels occurred in the CG dinucleotides of the upstream region (99%), whereas only a low level of CG methylation was observed in the transcribed sequence (7%). Low levels of methylation were also observed in asymmetric sequences (in both regions; 2% methylated). The results obtained in the MROS1 gene are discussed in consequence with the immunohistochemical data showing a hypermethylation of DNA in the vegetative nucleus.Key words: DNA methylation, genomic sequencing, immunocytology, pollen, Silene latifolia.

DNA methylation represents one of the key processes that play an important role in the transcriptional control of gene expression. The role of cytosine methylation in plant development has been demonstrated by at least three different kinds of evidence: parent-specific expression of some genes in developing seeds, control of flowering time and floral morphogenesis, and correlation with silencing of intrusive DNA sequences (mobile genetic elements and transgenes). In this work global changes in DNA methylation during seed germination and shoot apical meristem development in Silene latifolia have been studied using an indirect immunohistochemical approach. The data presented show that a rapid decrease in global DNA methylation during seed germination occurs first in endosperm tissue and subsequently in the hypocotyl. Using 5-bromo-2'-deoxyuridine pulses, it has been demonstrated that these demethylation events occurred before cell division had begun. In the early post-germination period, a decrease in DNA methylation was detected in cotyledons, also before cell division was observed. Taken together, these results indicate that DNA demethylation takes place in a non-replicative way, probably by an active mechanism. The central zone of the shoot apical meristem remains highly methylated during the whole period of vegetative growth and in this region, only a low cell division activity was found. However, upon the transition of the shoot apical meristem to the floral bud, the meristem both decreased its high methylation status and its cells started to divide. These data indicate that the central zone of the shoot apical meristem can represent a relatively quiescent 'germ-line' which is activated upon flowering to form spores and gametes.

Background Prior to this study, no differences in gene expression between male and female dioecious plants in the vegetative state had been detected. Among dioecious plants displaying sexual dimorphism, Silene latifolia is one of the most studied species. Although many sexually dimorphic traits have been described in S. latifolia, all of them are quantitative, and they usually become apparent only after the initiation of flowering. Results We present RT-PCR-based evidence that in S. latifolia, sexual dimorphism in gene expression is present long before the initiation of flowering. We describe three ESTs that show sex-specific (two male specific and one female specific) transcription at the rosette stage before the first flowering season. Conclusions To our knowledge, this study provides the first molecular evidence of early pre-flowering sexual dimorphism in angiosperms.