The genus Silene is a model system for sex chromosome evolution, including about 700 species varying greatly in their mating system, ecology and sex determination (Bernasconi et al., 2009). Inside the genus two groups are considered as invaluable for the study of sex chromosome evolution and sex determination; section Melandrium and subsection Otites (reviewed in Vyskot and Hobza, 2015). Two dioecious plants S. latifolia (24, XY) and S. dioica (24, XY) from Melandrium have heteromorphic sex chromosomes and sex determination similar to mammals (Ming et al., 2007; Charlesworth, 2016). In contrast, related gynodioecious species S. vulgaris and S. maritima with the same number of autosomes (2n = 24), possess no sex chromosomes having a smaller genome compared to S. latifolia or S. dioica (Runyeon and Prentice, 1997; Charlesworth and Laporte, 1998; Široký et al., 2001; Stone et al., 2017).

It is generally accepted that the sex chromosomes are derived from an ordinary pair of autosomes (reviewed in Bachtrog, 2006). As a result of suppressed recombination and accumulation of deleterious mutations, the sex chromosomes differ in their structure, function and gene density. The X chromosome becomes hemizygous and X hemizygosity in males leads to special regulatory mechanisms to equalize the transcription ratio between the X chromosomes and autosomes (Charlesworth and Charlesworth, 2005; Muyle et al., 2017; Darolti et al., 2019). As a result of accumulation of deleterious mutations, the Y chromosome is degenerated and the sex chromosomes may differ even within closely related species as demonstrated in human and chimpanzee (Hughes et al., 2010). Interestingly, newly formed sex chromosomes show the same signs of sex chromosome evolutionary pathways, as described in Drosophila (Bachtrog et al., 2009) or stickleback species (Yoshida et al., 2014) in which the ancestral Y chromosome fused with an autosome.

In S. latifolia and S. dioica, sequence data showed that the sex chromosomes evolved from one pair of autosomes with the divergence of X and Y homologous sequences <10 million years ago (Filatov, 2005), estimating the age of older and younger strata (non-recombining part of the sex chromosomes that differ from each other in level of divergence) around 11 and 6.32 mya (Krasovec et al., 2018). The sex chromosomes in S. latifolia and S. dioica, vary greatly in size having Y chromosome 1.4 larger than X (heteromorphism) (Vyskot and Hobza, 2015). The Y chromosome has a large non-recombining region and the size of the PAR (pseudo-autosomal region) is less than 10% of its chromosome length (Filatov et al., 2009). Both sex chromosomes accumulated various transposable elements (Bergero et al., 2008b; Kubat et al., 2014) and satellites (Cermak et al., 2008; Kejnovský et al., 2013), and differ in histone modifications and DNA methylation (Rodríguez Lorenzo et al., 2018; Bačovský et al., 2019).

Previous studies suggested that the sex chromosomes in S. latifolia, especially the Y chromosome, were derived through multiple rearrangements (Bergero et al., 2008a). Deletion mapping revealed that at least one larger inversion occurred after recombination suppression on the Y chromosome (Zluvova et al., 2005), supported also by findings of four genetically mapped genes between S. latifolia and S. dioica (Filatov, 2005). Later, Hobza et al. (2007) used physical mapping and confirmed two large inversions on the Y chromosome. These findings were further verified by Y deletion mapping showing that at least one inversion had to have occurred during the formation of the Y chromosome (Kazama et al., 2016), accelerating the recombination suppression (Bergero and Charlesworth, 2009). In contrast, comparative mapping between S. latifolia and S. vulgaris revealed the existence of one large (SvLG12) and two relatively small (SvLG9, Sv small LG) linkage groups that accompanied the sex chromosomes formation in S. latifolia (Bergero et al., 2013; Campos et al., 2017). Yet, it is still not clear what pair(s) of autosomes were included in such translocation and if such linkage groups also exist in other gynodioecious species. Thus, this raises two important questions: if S. vulgaris possesses three putative parts of three linkage groups corresponding to the X chromosome in S. latifolia, what is the origin of these linkage groups and how many chromosomes were involved in sex chromosome formation?

Recent advances in fluorescence in situ hybridization (FISH) experiments have provided a variety of techniques which can be used to study the structure, dynamics and origin of certain loci, chromosomal arms and/or specific chromosomes (reviewed in Cui et al., 2016; Bačovský et al., 2018; Huber et al., 2018). Previous cytogenetic studies in Silene species were based mainly on physical mapping of satellite rDNA (Široký et al., 2001), repeats and transposable elements (Cermak et al., 2008; Kejnovsky et al., 2009). Although Lengerova et al. (2004) managed to produce discrete signals using various DNA repeats (satellites, rDNA) and specific BAC clones, this approach proved to be cost ineffective due to the large screening of BACs containing only a low amount of repetitive DNA. As an another option, Hobza et al. (2004) designed a protocol using microdissected X and Y chromosomes from S. latifolia for whole chromosome painting. These probes produced relatively discrete signals on both sex chromosomes, but high amount of suppressive unlabeled DNA with very strict hybridization conditions made the use of such method very problematic in other Silene species (Hobza and Vyskot, 2007). The recent development of oligo painting probes in plants has proved to be useful in the detection of chromosomal aberrations and in comparative cytogenetics (reviewed in Jiang, 2019). In principal, oligo painting probe can be used to label particular regions containing enough short unique oligo sequences to be computationally isolated, synthesized, pooled and labeled (Han et al., 2015). Such probes, designed from conserved sequences of one species were used e.g. for developing karyotype among genetically related Solanum species (Braz et al., 2018), for differentiating of A, B, and D genomes in wheat (Tang et al., 2018), in comparative physical mapping of sex chromosomes in Populus (Xin et al., 2018) and in examination of meiotic pairing in polyploid Solanum species (He et al., 2018).

In this work, we designed an X chromosome-specific oligo probe enriched by X-linked scaffolds based on the S. latifolia female genome. We show that such technique is useful for the detection of discrete signals in sex chromosomes in S. latifolia and closely related S. dioica. In addition, the probe works well in the related gynodioecious species of S. vulgaris and S. maritima. Based on our results, we discuss the origin of sex chromosomes from one autosomal pair and the possible application of oligo painting probe in further studies. Our findings support the general hypothesis that multiple chromosomal changes took place during the formation of X and Y chromosomes.

The oligo painting probe specifically hybridized to pseudo-autosomal region (PAR) located on the Y q-arm and interstitial Y p-arm region, and to both sub-telomeric regions on the X chromosome in S. latifolia and S. dioica, enriched on the X p-arm sub-telomere. This distribution of the oligo painting probe signal correlates with the pattern of specific histone modifications for active chromatin, namely H3K4me1, H3K4me2, H3K4me3, H3K9ac and gene repressive mark H3K27me3 (Bačovský et al., 2019). Since the Y chromosome still contains many active genes (Bergero and Charlesworth, 2011), the additional band on the X p-arm together with the interstitial Y p-arm region probably represent unique/important gene clusters (Hobza et al., 2018). In related S. vulgaris and S. maritima, the oligo painting probe clearly differentiates three pairs of autosomes. In these gynodioecious species, the oligo painting probe labels three autosomal p-arms as shown on early metaphase/prophase chromosomes (Supplementary Figure S3). These chromosomal patterns support previous evidence that parts of three linkage groups in S. vulgaris were translocated to the pseudo-autosomal region in S. latifolia through a double translocation event (Bergero et al., 2013). Our results support previous studies showing expansion of the PAR region in S. latifolia (Bergero et al., 2013; Campos et al., 2017) and newly in S. dioica (Figure 3). Alternatively, rearrangements could be accompanied by whole chromosome fusion(s) as documented in other species. In Japan Sea stickleback fish, an ancestral Y chromosome fused with the autosome, forming a neo-Y chromosome and the X₁X₂Y sex determination system (compared to closely related Pacific Ocean stickleback with XY system) (Yoshida et al., 2014). In Rumex hastatulus, North Carolina male race possesses XY₁Y₂, having the older Y (ancestral) chromosome heterochromatinised, and the younger Y with translocated autosomal part (Grabowska-Joachimiak et al., 2015). In Silene species “fusion” scenario is unlikely since it usually influences basic karyotype chromosome number (all studied species have n = 12). Moreover, existing genetic maps do not suggest large scale fusion events during karyotype evolution in studied species (Bergero et al., 2013). The existence of telomere-like sequences in S. latifolia Y chromosome (Uchida et al., 2002) can be explained as remnant of chromosomal inversion as was demonstrated in some Silene species (Filatov, 2005; Zluvova et al., 2005). It was reported that such chromosomal rearrangements included at least one paracentric and one pericentric inversion on the Y (Hobza et al., 2007; Bergero and Charlesworth, 2009). On the other hand, additional rearrangements that would suggest alternative scenarios cannot be excluded. Based on deletion mapping one inversion also occurred during the formation of the Y (Kazama et al., 2016). Bergero et al. (2013) and Campos et al. (2017) showed that PAR expanded through two step translocations. Our data support such translocation by the existence of three pair of autosomes (in gynodioecious species) corresponding to sex chromosomes in S. latifolia and S. dioica. Since sex chromosomes in studied dioecious species originated from one of these three chromosomal pairs, we assume that two additional loci were translocated on proto sex chromosomes in S. latifolia and S. dioica during sex chromosome evolution (Figure 3).

We have also tested the robustness of oligo painting method to study the dynamics of sex chromosomes and PAR in early metaphase/prophase (Supplementary Figure S3). In prophase/early metaphase in which the chromosomal (spatial) resolution limit is 10 times higher than in the metaphase and chromosomes are 10 times more extended (reviewed in Figueroa and Bass, 2010), the strength of the signal is relatively low. Therefore, it will be necessary to use more cytogenetic markers (together with oligo sequences) for discrimination of relaxed chromosomes such as e.g. specific antibodies against synaptonemal complexes in meiosis as described in Hurel et al. (2018).

Our X chromosome-specific oligo probe serves as useful tool to study the evolution of sex chromosomes in S. latifolia, S. dioica and their relatives. Since the genome of S. latifolia is still not fully sequenced we would like to leave the possibility that some sequences targeted by the probe might occur more than once in the genome open (e.g. low repetitive or duplicated). Nevertheless, compared to previous attempts and labeling of the sex chromosomes in Silene species using e.g. unique BAC clones (Lengerova et al., 2004) or dissected chromosomal probes (Hobza et al., 2004), the oligo painting probe provides an unique signal on both sex chromosomes and is also suitable to study other related species. In addition, the probe simplifies future analysis of chromosome pairing and facilitates the study of the dynamics of the PAR region in interphase or during cell division.

All datasets generated for this study are included in the article/Supplementary Material.

VB, RČ, and RH conceived and designed the research. VB, RČ, EH, and DŠ conducted the experiments. DŠ and EH contributed the reagents. VB analyzed the data. VB wrote the manuscript. All the authors read and approved the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.