Plant material
L. perenne GR3320 (2n = 2x = 14), F. arundinacea subsp. arundinacea (2n = 6x = 42), F. gigantea GR11759 (2n = 6x = 42), F. mairei GR610941 (2n = 4x = 28) were obtained as seeds from IPK Gatersleben gene bank. Seeds of F. pratensis cv. Fure (2n = 2x = 14) were obtained from Dr. Arild Larson (Graminor, Norway), L. perenne cv. Neptun (2n = 4x = 28) and L. multiflorum cv. Kuri1 (2n = 2x = 14), from Dr. Vladimír Černoch (DLF Seeds, Czech Republic). Plants of L. multiflorum cv. Mitos (2n = 4x = 28), F. pratensis cv. Westa (2n = 4x = 28) and F. glaucescens (2n = 4x = 28) were obtained from Dr. David Kopecký (Institute of Experimental Botany, Czech Republic).
Seeds of barley (Hordeum vulgare) cv. Morex, rye (Secale cereale) cv. Diament, oats (Avena sativa) cv. Atego were obtained from IPK Gatersleben gene bank. Seeds of Triticum aestivum cv. Chinese Spring were obtained from Prof. Takashi R. Endo (Kyoto University, Japan) and seeds of Aegilops tauschii were provided by Dr. Valárik (Institute of Experimental Botany, Czech Republic). Seeds of pea (Pisum sativum cv. Ctirad) and rye (Secale cereale cv. Dankovske), which served as internal reference standards in flow cytometric analysis, were obtained from Semo breeding station (Smržice, Czech Republic) and OSEVA, AGRO Brno company (Brno, Czech Republic), respectively.
Estimation of genome size
Nuclear DNA amounts were determined according to Doležel et al. [57]. A two-step procedure (Otto, 1990) was used for sample preparation. Samples were analysed using Sysmex CyFlow Space flow cytometer (Sysmex Partec GmbH, Münster, Germany) equipped with a 532-nm laser. Two reference standards were used for flow cytometric analysis. Pea (Pisum sativum cv. Ctirad; 2C = 9.09 pg DNA, [35] served as an internal standard for DNA content estimation in all accessions with the exception of F. mairei, for which rye (Secale cereale cv. Dankovske; 2C = 16.19 pg DNA, [35] was used. Three plants were measured per accession and each plant was analysed three times on three different days. At least 5000 nuclei per sample were analysed. Nuclear DNA content was then calculated from individual measurements following the formula:
2C nuclear DNA content [pg] = 2C nuclear DNA content of reference standard × sample G1 peak mean / standard G1 peak mean
Mean nuclear DNA content (2C) was calculated for each plant. Genome size (1C value) was then determined considering 1 pg DNA equal to 0.978×109 bp [58].
Illumina sequencing and data analysis
Genomic DNA of ten accessions was isolated using the NucleoSpin PlantII kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) following the manufacturer's recommendations. Genomic DNA was then used for preparation of Illumina libraries using Nextera® DNA Sample Preparation Kit (Illumina, San Diego, USA). 50 ng of DNA was fragmented, purified and amplified according to the protocol. Concentrations of individual libraries were measured using a Qubit fluorometer. Library concentrations were adjusted to an equal molar concentration and pooled prior to sequencing. Data was collected with an Illumina MiSeq using either single or paired end sequencing to produce up to 500 base pair reads. Sequences were deposited in the Sequence Read Archive (BioProject ID: PRJNA601325, accessions SAMN13866227, SAMN13866228, SAMN13866229, SAMN13866230, SAMN13866231, SAMN13866232, SAMN13866233, SAMN13866234, SAMN13866235, SAMN13866236).
Illumina reads of all eight studied accessions were trimmed for adapters and for quality using FASTX-toolkit [-q 20 -p 90] (http://hannonlab.cshl.edu/fastx_toolkit/index.html). Detailed characterization of repeat families was performed using stand-alone version of RepeatExplorer pipeline [36] running on IBM server with 16 processors, 100Gb of RAM and 17Tb of disk space. In the first step, comparative analysis of repetitive parts of the genomes was performed using the RepeatExplorer pipeline according to Novák et al. [44]. Random data sets represented the same amount of reads 0.5× coverage of individual species and used to reconstruct repetitive elements using graph-based method according Novák et al. [43]. The assembled sequences within each individual cluster were characterized based on the homology searches and other tools useful for repeat characterization (e.g. BLASTN and BLASTX programs, phylogenetic analysis). Tandem organized repeats were identified using Dotter [59].
In the second step, RepeatExplorer pipeline was applied on the merged dataset containing all species marked by specific prefixes with the aim to perform comparative analysis [44]. The results of these clustering were then used for creation of repetitive databases. Databases of Illumina reads and assembled contigs specific to different types of repetitive DNA elements are publicly available on our web site (https://olomouc.ueb.cas.cz/en/content/dna-repeats).
Southern hybridization
For Southern hybridization, genomic DNA from six Festuca and four Lolium accessions corresponding to 3 × 106 of monoploid (1Cx) nuclear genomes was digested by HaeIII enzyme (New England Biolabs, Ipswick, Massachusetts, USA). DNA fragments were size-fractionated by electrophoresis in 1.2 % agarose gel and then transferred onto HybondTM N+ nylon membranes (GE Healthcare, Chicago, Illinois, USA). Probes were prepared from meadow fescue template by PCR with biotin-labelled dUTP (Roche, Mannheim, Germany) and specific primers (Table 3). Southern hybridization was performed at 68 °C overnight and signals were detected by Chemiluminescent Nucleic Acid Module (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to manufacturer's recommendations with 90 % stringency. Hybridization signals were visualized by chemiluminescent substrate on Medical X-Ray Film Blue (Agfa HealthCare NV, Mortsel, Belgium).
Droplet digital PCR
Based on the assembled contigs specific to centromeric retrotransposon Fesreba, two restriction endonucleases produced unique restriction site in whole LTR retrotransposon (HpaI and HpaII) were identified and used for further analysis. 3 µg of genomic DNA was digested according manufacturer’s recommendations (Bio-Rad Laboratories, Hercules, California, USA) and then diluted 1,000-fold into starter concentration 0,06 ng/µl. Droplet Digital PCR experiment was performed using QX200 Droplet Digital PCR machine (Bio-Rad Laboratories) under manufacturer’s recommendations with use EvaGreen Supermix (Bio-Rad Laboratories), template DNA and specific primers for Fesreba element (Table 3) as three independent replicates for every analyzed accession. Whole experiment was also repeated with template genomic DNA restricted by HpaI enzyme.
Cytogenetic mapping and immunostaining
Cytogenetic mapping of selected repeats was done by fluorescence in situ hybridization (FISH) on mitotic metaphase plates. Chromosome spreads were prepared according to Křivánková et al. [29]. Immunostaining was done according to Neumann et al. [60]. Root tips were collected into ice water for 28 h, washed in LB01 buffer [61], fixed in 3.7% formaldehyde for 25 min and digested using 2 % cellulose, 2 % pectinase and 2 % cytohelicase in 1× PBS for 90 min at 37 °C. After squashing the meristem and coverslip removal, the slides 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 primary antibody, the slides were washed in PBS-Tween buffer (1× PBS, 0.1 % Tween 20, pH 7.4) for 25 min and then incubated with primary antibody (diluted 1 : 200 in PBS-Tween) overnight at 4°C. Next day slides were washed 1× PBS, the antibodies were detected using the appropriate secondary antibodies (diluted 1 : 250 in PBS-Tween buffer) for 1 h at room temperature and washed 1× PBS. For following FISH procedure immunosignals were stabilized using ethanol : acetic acid (3 : 1) fixative and 3.7% formaldehyde for 10 min. After 3 times washes in 1× PBS FISH procedure was performed.
Probes for FISH were labelled by digoxigenin-11-dUTP or biotin-16-dUTP (Roche Applied Science) using PCR with specific primers (Table 3). Hybridization mixture consisting of 50% formamide, 10% dextran sulfate in 1× SSC and 1 μg/ml of each labelled probe was added onto slides and denatured at 80°C for 3 min. The hybridization was carried out at 37°C overnight. The sites of probe hybridization were detected using anti-digoxigenin-FITC (Roche Applied Science) and streptavidin-Cy3 (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and the chromosomes were counterstained with DAPI. The slides were examined with Axio Imager.Z2 microscope (Carl Zeiss, Oberkochen, Germany) equipped with Cool Cube 1 (Metasystems, Altlussheim, Germany) camera and appropriate filters. The capture of fluorescence signals and merging the layers were performed with ISIS software 5.4.7 (Metasystems), the final image adjustment was done in Adobe Photoshop 12.0.