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

Cultivated 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. Partial 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. Comparative 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.


Background
Grasses (Poaceae) are an important source of food for domestic animals worldwide and perform important ecological and environmental functions. The tribe Poeae is the largest tribe in family Poaceae, and species from its largest subtribe, Loliinae, grow in a range of habitats, including wetlands, dry areas, and regions with cold and temperate climates; some are even well adapted to the extreme conditions of mountain, arctic, and sub-Antarctic regions [1]. The subtribe Loliinae comprises a cosmopolitan genus Festuca and its satellite genera [2,3]. Festuca is the largest genus of the family Poaceae, containing more than 600 species, and Torrecilla and Catalán [4] discriminated its two main evolutionary lines: broad leaved and fine leaved (Fig. 1). Broad-leaved Festuca species (hereafter "fescues") include the subgenus Schedonorus, which gave rise to Lolium species (hereafter "ryegrasses"), a sister group of fescues ( Fig. 1) [1]. The evolution of grasses, including Loliinae, has been accompanied by frequent polyploidization and hybridization events, and about 70% of grass species are polyploid [6]. The species of Loliinae have large genomes ranging from 2. 6 Gbp/1C to 11.8 Gbp/1C [7,8].
This study focuses on species from the subgenus Schedonorus, a complex of species with various ploidy levels [7,9] that includes important species widely used for forage and turf. Although some Schedonorus species are diploid, such as Festuca pratensis Huds. (2n = 2x = 14) and Lolium multiflorum Lam. (2n = 2x = 14) and L.
Although fescues and ryegrasses have been intensively studied, their evolution and the origin of most polyploid representatives remain obscure [11,15,16]. Like in other species with large genomes, the nuclear genomes of fescues and ryegrasses include a large number and variety of repetitive DNA sequences [17,18]. Their amplification in the genome, accompanied by interspecific hybridization and polyploidization, has expanded the genome size [19][20][21][22][23][24]. However, these processes have likely been counterbalanced by recombination-based mechanisms that have removed substantial parts of nuclear genomes [25][26][27].
Repetitive DNA elements may play different roles in a nuclear genome. Tandem organized ribosomal RNA genes and telomeric sequences are the key components of nucleolar organizing regions and chromosome Fig. 1 Phylogenetic tree of Loliinae subtribe. Phylogeny of subtribe Loliinae with Brachypodium distachyon was used as an outgroup. The tree was constructed from ITS sequence regions of Loliinae species and B. distachyon with PhyML implemented in SeaView [5]. Detailed phylogeny of subgenus Schedonorus is depicted and shows the relationships of fescue and ryegrass species in this lineage (highlighted in light yellow) termini, respectively. Centromeric regions in Arabidopsis, Brachypodium, rice, and maize are partly formed from specific satellite DNAs with~130 bp long units [28][29][30][31], whereas in other plant species, including cereals, these regions are formed by large blocks of Ty3/gypsy retrotransposons containing chromodomain [29,[32][33][34]. In F. pratensis, a putative long terminal repeat (LTR) element localizing preferentially to centromeric regions has been identified [35]. In addition to elucidating the molecular organization of chromosome domains, characterization of repetitive parts of nuclear genomes helps in the development of cytogenetic markers [21,35,36]. Repetitive DNA sequences are also used extensively to study genetic diversity and processes of genome evolution and speciation [37][38][39][40].
The main goal of the present work was to elucidate the repetitive landscape and its impact on genome size and genome divergence in closely related land grasses, including natural polyploid species. We characterized repetitive DNA sequences in the nuclear genomes of 10 representatives of fescues and ryegrasses. We performed global analyses of repetitive DNA sequences and characterized their abundance and variability after partial Illumina sequencing. Moreover, we characterized and assembled the DNA sequence of an LTR element that was highly enriched in centromeric and (peri)centromeric chromosome regions in all 10 genotypes. Colocalization of the centromere-specific histone H3 variant CENH3 with the LTR element indicated its role in centromere function.

Genome size estimation
Flow cytometric analysis of propidium iodide-stained nuclei was conducted to estimate nuclear DNA content (Fig. 2). Because of the large differences in genome size between the species analyzed, two internal reference standards were used: Pisum sativum cv. Ctirad (2C = 9.09 pg DNA) [41] and Secale cereale cv. Dankovske (2C = 16.19 pg DNA) [41]. All histograms of relative DNA content represented two dominant peaks corresponding to G1 nuclei of the sample and the standard. The 2C nuclear DNA content thus determined ranged from 5.32 pg in L. multiflorum to 20.17 pg in F. gigantea. The monoploid genome (1Cx) ranged in size from 2.43 in F. mairei to 3.36 pg in F. gigantea (Table 1). The remaining representatives of fescues and ryegrasses had similar 1Cx sizes (~2.7 Gb).

Repeat composition and comparative analyses of repetitive DNA sequences
Interspecific comparisons, reconstruction, and quantification of major repeat families were performed with the RepeatExplorer pipeline [42]. The process, which involved grouping orthologous repeat families from all analyzed species in the same cluster, facilitated the assembly, identification, and quantification of individual repeat elements.
In all accessions, LTR retroelements were the most abundant component of the nuclear genome (Table 2, Fig. 3). Ty3/gypsy elements were more than 4 times more abundant than Ty1/copia retrotransposons ( Table  2). The biggest difference in copy number between fescues and ryegrasses was for an LTR element from the Athila clade. Whereas the nuclear genomes of both Lolium species were enriched for the element, which accounted for~25-30% of their genomes, the orthologous Athila element accounted for only~5-7% of the nuclear genomes of fescues (Table 2). A relatively large part of the genomes was represented by unclassified LTR sequences, which indicates a high frequency of unique LTR sequences. DNA transposons and long interspersed nuclear element (LINE) elements were found in low copy numbers, and tandem repeats accounted for 1.5% to more than 8% of the genome sequences (Table 2, Fig. 3).
Comparative analyses with RepeatExplorer showed that most clusters of orthologous repeat families contained reads from all accessions and that a large number of similar sequences were present in fescues and ryegrasses. Among the fescues, F. mairei and F. glaucescens showed the lowest similarity in DNA repeats. The composition as well as the abundance of DNA repeats in ryegrasses were highly conserved. Tandem organized repeats were the most diverged elements among the fescues and ryegrasses studied, and some of the repeats were species specific (Fig. 4, Additional file 1: Table S1). In addition to tandem repeats, some small sequence clusters contained reads from only a few species. Species-specific variants of the majority of repetitive elements within and between fescues and ryegrasses were identified only after detailed analyses of individual repeat clusters with SeqGrapheR ( Fig. 5a-c). Detailed analyses revealed the presence of species-specific DNA contigs, which may be used to develop molecular and cytogenetic markers.
To confirm the differences determined in silico, we analyzed selected repetitive DNA elements using Southern hybridization. We designed specific probes for those DNA repeats that seemed to have species-specific variants. A probe for the Ty3/gypsy Athila element that was reconstructed in cluster CL1 and showed the largest copy number variation between fescues and ryegrasses ( Table 2) gave strong hybridization signals on genomic DNA from ryegrasses but no or weak signals on DNA from fescues (Fig. 5d). Similarly, a probe for the Ty3/ gypsy Athila element that was reconstructed in cluster CL38 and contained mostly Festuca sequence reads ( Fig.  5b) provided strong visible signals only with fescue genomic DNA (Fig. 5e). Finally, Southern hybridization was performed with a probe for the Ty3/gypsy Ogre-Tat retrotransposon, identified in cluster CL20. The probe, which was designed from contigs representing fescues (Fig. 5c), provided strong hybridization signals on all fescues analyzed and low intensity signals on ryegrasses (Fig. 5f). In general, the signal intensities obtained after Southern hybridization corresponded to the copy numbers identified in silico.

Centromere composition
Partial genome sequence data obtained using Illumina sequencing technology made it possible to reconstruct nearly complete centromeric LTR elements in all 10 accessions of fescues and ryegrasses. Detailed characterization of the element called Fesreba confirmed that it belongs to the Ty3/gypsy Chromoviridae lineage. Phylogenetic analyses of its reverse transcriptase (RT) domain showed a close relationship with the Cereba element ( Fig. 6), which was identified earlier in barley (Hordeum vulgare) [43].
Southern hybridization with a probe for the RT domain of Fesreba and a probe for its LTR region [35] showed their presence in all fescues and ryegrasses included in this work (Additional file 2: Fig. S1). Similar hybridization patterns indicated sequence conservation between Fesreba repetitive DNA elements in these species. The results were supported by in silico data, which showed high similarity at the DNA sequence level (most abundant copies of Fesreba shared at least 92% similarity at the DNA level within and between fescues and ryegrasses) but lower abundance in ryegrasses. To confirm the differences in Fesreba copy number, we performed quantification for the RT domain and LTR sequence using droplet digital polymerase chain reaction (ddPCR). The results confirmed a two-fold higher copy number of Fesreba in fescues compared to ryegrasses (Additional file 3: Table S2). The assay also showed that the majority of genotypes analyzed contained 5 to 50 times more copies of the LTR region of Fesreba than its coding region (Additional file 3: Table S2).
To confirm preferential localization of Fesreba to centromeric chromosome regions, we conducted fluorescence in situ hybridization (FISH) on mitotic metaphase plates with probes derived from its RT domain and LTR region. In all fescues and ryegrasses, both probes localized preferentially to centromeric regions of all chromosomes (Fig. 7). Whereas the hybridization signals of the RT domain were observed almost exclusively in centromeric regions, a probe derived from the noncoding LTR region resulted in stronger signals in centromeric and/or pericentromeric regions and weak signals along the chromosomal arms, as shown previously in F. pratensis [35]. Weak signals of the LTR part of Fesreba , respectively, were used as internal reference standards. The ratio of relative G1 peak positions was used to calculate DNA amounts of the fescue and ryegrass accessions in distal parts of chromosomes indicate the presence of unique LTRs spread over the genome and correspond to a higher copy number of the LTR non-coding part of Fesreba compared to its coding sequence.
In addition to the fescues and ryegrasses included in this study, FISH was performed with the same probes on mitotic metaphase plates from related grass species, oat, barley, rye, bread wheat, and Aegilops tauschii. High homology of the RT coding domain resulted in successful in situ localization in all species. However, the probe specific to the LTR region of Fesreba provided visible signals only in A. sativa (Additional file 4: Fig. S2).
Finally, immunostaining with the centromere-specific histone H3 variant CENH3 [44] in combination with FISH with probes for the RT domain and LTR region of Fesreba resulted in overlapping signals in all fescues and ryegrasses studied (Fig. 8, Additional file 5: Fig. S3).

Discussion
Because of genome shock, the 1Cx size of polyploid species is often, but not always, lower than that of their progenitors [25,45]. In this study, we performed comparative analyses of repeatomes and analyzed the impact of DNA repeats on genome size in a set of Festuca and  Lolium species differing in ploidy. The set comprised hexaploids F. arundinacea subsp. arundinacea and F. gigantea; tetraploids F. glaucescens and F. mairei; and artificial autotetraploids F. pratensis cv. Westa, L. multiflorum cv. Mitos, and L. perenne cv. Neptun developed in breeding programs. We estimated nuclear DNA amounts using flow cytometry, and a test of normality confirmed that the data set had a normal distribution.
Our results suggest possible genome changes in hexaploid F. arundinacea and tetraploid ryegrasses compared to their probable progenitors. Although the differences in the 1Cx size of natural polyploid F. arundinacea and its probable parents (F. pratensis and F. glaucescens) are small, they are statistically significant (P < 0.01). The same is true for tetraploid ryegrass cultivars obtained after polyploidization. Genome downsizing was detected in the case of F. arundinacea (~2% difference between expected and estimated values) and tetraploid L. perenne (~1% decrease). In the tetraploid cultivar of L. multiflorum, a slight increase in genome size (~4%) was detected, corresponding with Kopecký et al. [8]. In the case of tetraploid fescue cultivars obtained after polyploidization, no statistically significant difference in 1Cx value was found (P > 0.01).
DNA retrotransposons are major contributors to the variation in nuclear genomes in plants [24,46,47]. Various approaches and tools have been developed to study these important parts of nuclear genomes, one of them being RepeatExplorer, which facilitates de novo repeat identification and characterization [42,48]. The pipeline uses graph-based clustering and analyzes nextgeneration sequencing data to reconstruct and characterize DNA repeats in a particular species or to compare DNA repeat composition in different genotypes [23,24,[49][50][51]. The pipeline has been frequently used to reconstruct DNA repeats in diversity studies, to create repeat databases for repeat masking [19,46,48], and to identify tandem organized repeats suitable as probes for molecular cytogenetics [35,[51][52][53].
Our work revealed that Ty3/gypsy elements had the highest impact on genome size in fescues and ryegrasses. Ty3/gypsy elements are also abundant in other Poaceae species, including wheat, rice, maize, and barley [8,[54][55][56]. In barley, about 50% of the genome is made up of 15 high-copy transposable element (TE) families, with elements of the Angela lineage (Ty1/copia family) being the most abundant and representing almost 14% of the genome [56]. The Ty3/gypsy superfamily is 1.5-fold more abundant than the Ty1/copia superfamily [56].
Festuca and Lolium genera comprise closely related complexes of species, and thus a high homology of DNA repeats was observed in this study. The main difference was the copy number. In Lolium species the Ty3/gypsy Athila LTR retroelement accounted for~25% of the nuclear genomes, whereas in fescues it accounted for0 .7% in tetraploids F. glaucescens and F. mairei and for 6% in other fescues analyzed. This indicates a burst of Athila LTR element linked with Lolium speciation.  6 Phylogenetic tree of Chromoviridae elements. The tree was constructed from a Jukes-Cantor distance matrix of the reverse transcriptase domains of Ty3/gypsy Chromoviridae elements described in Neumann et al. [34] and Fesreba elements identified in the present work with BioNJ implemented in Seaview [5]. The tree was rooted on the Ty3/gypsy/Tat element. The subclade of the Cereba element (in red) and other closely related elements identified in different plant species are labeled in blue. Fesreba elements identified in fescues and ryegrasses are also marked in blue Activation and integration of TEs (e.g., as a result of environment change) may lead to a rapid burst of the Athila element in a species-specific manner [46,47,57] and impact evolution and speciation [46,58]. In some species, a rapid increase in the number of lineagespecific retroelements can also result in significant genome upsizing [24,[58][59][60], which was not observed in the fescues and ryegrasses included in our study.
Species-specific DNA elements identified in this work were represented by tandem organized repeats (Additional file 1: Table S1). Unique tandem repeats are also found in other plant species, and thanks to their genus or species specificity they have been widely used in molecular cytogenetics (e.g., to identify chromosomes using FISH) [61][62][63][64]. Tandem repeats originally identified in F. pratensis chromosome 4F are useful as probes for FISH to identify individual chromosomes of the species [18,35] and in comparative karyotype analyses of its cultivars. The present work resulted in the identification of other putative tandem organized repeats, either genus or species specific (Additional file 1: Table S1). These observations expand the number of potential cytogenetic markers for comparative karyotyping and identification of chromosomes in other fescue and ryegrass species.
Although the sequencing of F. pratensis chromosome 4F revealed a relatively high number of tandem repeats, none of them localized to chromosome centromeric regions [18,35]. However, the mapping of other types of Fig. 7 Localization of the centromeric LTR retrotransposon Fesreba on mitotic metaphase chromosomes. Localization was performed in Festuca and Lolium species with fluorescence in situ hybridization (yellow-green or violet signals) with a probe for the reverse transcriptase domain of the Fesreba element. a F. arundinacea subsp. arundinacea (2n = 6x = 42). b F. gigantea (2n = 6x = 42). c F. mairei (2n = 4x = 28). d F. pratensis cv. Westa (2n = 4x = 28). (E) L. perenne GR 3320 (2n = 2x = 14). f L. multiflorum cv. Mitos (2n = 4x = 28). Chromosomes were counterstained with DAPI (blue). The bar corresponds to 10 μm DNA repeats on mitotic metaphase chromosomes showed preferential localization of one uncharacterized DNA element CL38 to centromeric regions of F. pratensis chromosomes [35]. In this work, the entire DNA element homologous to the CL38 repeat was reconstructed and its nature was clarified. Phylogenetic analyses of its coding domains (Fig. 6) confirmed close relationships with other plant centromeric elements of Ty3/gypsy Chromoviridae lineage, such as Cereba-like elements [43]. Preferential localization of the Cereba element to centromeric regions of barley chromosomes was shown by Hudakova et al. [33], and more complex study of centromere-specific elements belonginging to the lineage of Centromeric retrotransposons in maize (CRM) of the Ty3/gypsy family in larger set of plant species followed [20,34]. These studies imply a role for TEs at the structural level and their impact on centromere structure. Li et al. [65] showed that the Cereba element was strongly associated with the histone H3 variant CENH3, which plays a role in centromere function. Colocalization of the centromere-specific element Fesreba, reconstructed in this work with histone CENH3 (Fig. 8, Additional file 5: Fig. S3), indicates a role for this element in the function of fescue and ryegrass centromeres as well.

Conclusions
Partial sequencing of genomes of 10 fescues and ryegrasses revealed various types of retrotransposons as the most abundant repeat. These comparative repeatome analyses increase knowledge of genome organization in fescues and ryegrasses and confirm close relationships between Festuca and Lolium. The most striking difference was observed for the Athila element, which was~5 times more abundant in Lolium than Festuca. Highly diverged DNA repeats were represented by tandem organized repeats, which are candidates for species-specific cytogenetic markers. In addition to tandem repeats, other species-specific variants of the majority of repetitive DNA sequences within and between fescues and ryegrasses were identified. A nearly complete LTR element Fesreba was assembled and found to be highly enriched in centromeric and (peri)centromeric chromosome regions in all species. A combination of FISH with a probe for Fesreba and immunostaining with CENH3 antibody showed their co-

Estimation of nuclear genome size
Nuclear DNA amounts were determined according to Doležel et al. [66] following the two-step procedure of Otto [67] with modifications. Samples of isolated nuclei stained with propidium iodide were analyzed with a Sysmex CyFlow Space flow cytometer (Sysmex Partec, Münster, Germany) equipped with a 532 nm laser. Two reference standards were used to estimate DNA amounts in absolute units. Pea (Pisum sativum cv. Ctirad; 2C = 9.09 pg DNA) [41] served as an internal standard for estimating DNA content in all accessions except F. mairei, for which rye (Secale cereale cv. Dankovske; 2C = 16.19 pg DNA) [41] was used. Three plants were measured per accession, and each plant was analyzed three times on three different days. At least 5000 nuclei per sample were analyzed. Nuclear amounts were calculated from measurements of individual samples as follows: 2C nuclear DNA content (pg) = 2C nuclear DNA content of reference standard × sample G 1 peak mean / standard G 1 peak mean.
Mean nuclear DNA content (2C) was estimated for each plant, with 1 pg DNA equal to 0.978 × 10 9 bp [68]. The statistical significance of the differences between 1Cx sizes was determined with one-way ANOVA. Analyses were conducted with NCSS 97 (Statistical Solutions, Cork, Ireland). The significance level α = 0.01 was used.

Illumina sequencing and data analyses
Genomic DNA was isolated with the NucleoSpin PlantII kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's recommendations and used to prepare Illumina libraries with a Nextera® DNA Sample Preparation Kit (Illumina, San Diego, CA, USA). Briefly, 50 ng DNA was fragmented, purified, and amplified according to the protocol. The DNA concentration in individual libraries was measured with a Qubit fluorometer, adjusted to an equal molar concentration, and pooled prior to sequencing. DNA sequencing was done with an Illumina MiSeq with either single or paired-end sequencing to produce up to 500 bp reads. Sequence reads were deposited in the Sequence Read Archive (BioProject ID:  PRJNA601325, accessions SAMN13866227, SAMN1386  6228, SAMN13866229, SAMN13866230, SAMN138662  31, SAMN13866232, SAMN13866233, SAMN13866234,  SAMN13866235, SAMN13866236).
Illumina reads were trimmed for adapters and for quality with the FASTX-Toolkit (−q 20 -p 90; http://hannonlab. cshl.edu/fastx_toolkit/index.html). Detailed characterization of repeat families was performed with a stand-alone version of the RepeatExplorer pipeline [37] running on an IBM server with 16 processors, 100 Gb RAM, and 17 Tb disk space. In the first step, comparative analyses of repetitive parts of the genomes were performed with the RepeatExplorer pipeline according to Novák et al. [49]. Random data sets represented the same amount of reads (0.5× coverage of individual accessions) were used to reconstruct repetitive elements using a graph-based method according Novák et al. [48]. The RepeatExplorer pipeline led to the characterization of assembled sequences using different tools (e.g., BLASTN and BLASTX, phylogenetic analysis) [37,48]. Tandem organized repeats were identified with Dotter [72].
In the second step, the RepeatExplorer pipeline was applied to a merged data set containing all species marked by specific prefixes to perform comparative analyses [49]. The results of the clustering were then used to create repetitive databases. Databases of Illumina reads were deposited in the Sequence Read Archive (accessions: SRX7566047-SRX7566056). Assembled contigs from different types of repetitive DNA elements are publicly available online (https://olomouc.ueb.cas.cz/en/content/dna-repeats) and in the Dryad digital repository (doi:https://doi.org/10.5061/dryad.xksn02vch).

Southern hybridization
Genomic DNA corresponding to 3 × 10 6 copies of a 1Cx nuclear genome was digested by HaeIII enzyme (New England Biolabs, Ipswich, MA, USA). DNA fragments were size-fractionated by electrophoresis in 1.2% agarose gel and then transferred onto Hybond™ N+ nylon membranes (GE Healthcare, Chicago, IL, USA). Probes were prepared with F. pratensis genomic DNA as a template and polymerase chain reaction (PCR) with biotin-labeled dUTP (Roche, Mannheim, Germany) and specific primers (Additional file 6: Table S3, Additional file 7: Fig. S4). Southern hybridization was performed at 68°C overnight, and hybridization signals were detected with a Chemiluminescent Nucleic Acid Module (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's recommendations with 90% stringency. Hybridization signals were visualized with chemiluminiscent substrate on Medical X-Ray Film Blue (Agfa Healthcare, Mortsel, Belgium).
ddPCR Based on the assembled DNA contigs from the Fesreba retrotransposon, two restriction endonucleases with unique restriction sites in the retrotransposon (HpaI and HpaII) were identified and used for further analyses. Briefly, 3 μg genomic DNA was digested according to the manufacturer's recommendations (Bio-Rad Laboratories, Hercules, CA, USA) and then diluted 1000-fold to reach a starter concentration of 0.06 ng/μl. ddPCR was performed on a QX200 Droplet Digital PCR machine (Bio-Rad Laboratories) following the manufacturer's recommendations with EvaGreen Supermix (Bio-Rad Laboratories), template DNA, and specific primers for Fesreba (Additional file 6: Table S3). Three independent replicates were performed for every accession analyzed.

Cytogenetic mapping and immunostaining
Cytogenetic mapping of selected repeats was done by FISH on mitotic metaphase plates. Chromosome spreads were prepared according to Křivánková et al. [35], and immunostaining was performed according to Neumann et al. [73]. Root tips were collected in ice water for 28 h; washed in LB01 buffer [74]; fixed in 3.7% formaldehyde for 25 min; and digested using 2% cellulose, 2% pectinase, and 2% cytohelicase in 1× phosphate-buffered saline (PBS) for 90 min at 37°C. After the coverslip was removed, the preparations were washed in 1× PBS and then in PBS-Triton buffer (1× PBS, 0.5% Triton X-100, pH 7.4) for 25 min and then again in 1× PBS. For incubation with antigrass CENH3 primary antibody [75], the slides were washed in PBS-Tween buffer (1× PBS, 0.1% Tween 20, pH 7.4) for 25 min and then incubated with anti-grass CENH3 primary antibody (diluted 1:200 in PBS-Tween) overnight at 4°C. The next day the slides were washed in 1× PBS, CENH3 antibody was detected using the anti-Rabbit Alexa Fluor 546 secondary antibody (Thermo Fisher Scientific/Invitrogen) diluted 1:250 in PBS-Tween buffer for 1 h at room temperature, and washed 1× PBS. Before FISH, immunofluorescent signals were stabilized with ethanol:acetic acid (3:1) fixative and 3.7% formaldehyde for 10 min at room temperature. FISH was performed after three washes in 1× PBS.
Probes for FISH, derived from the RT and LTR regions of the Fesreba element, were labeled with digoxigenin-11-dUTP or biotin-16-dUTP (Roche Applied Science) using PCR with specific primers (Additional file 6: Table S3). FISH and detection of hybridization sites were performed according to Křivánková et al. [35]. The chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted in Vectashield (Vector Laboratories). The slides were examined with an Axio Imager.Z2 microscope (Carl Zeiss, Oberkochen, Germany) equipped with a Cool Cube 1 (Metasystems, Altlussheim, Germany) camera, and images were prepared with ISIS 5.4.7 (Metasystems). Final adjustments were made to figures in Adobe Photoshop 12.0.
Additional file 1: Table S1. List of clusters containing putative tandem repeats identified in Festuca and Lolium.