Repeatome Analyses and Satellite DNA Chromosome Patterns in Deschampsia sukatschewii, D. cespitosa, and D. antarctica (Poaceae)

Subpolar and polar ecotypes of Deschampsia sukatschewii (Popl.) Roshev, D. cespitosa (L.) P. Beauv, and D. antarctica E. Desv. are well adapted to stressful environmental conditions, which make them useful model plants for genetic research and breeding. For the first time, the comparative repeatome analyses of subpolar and polar D. sukatschewii, D. cespitosa, and D. antarctica was performed using RepeatExplorer/TAREAN pipelines and FISH-based chromosomal mapping of the identified satellite DNA families (satDNAs). In the studied species, mobile genetic elements of class 1 made up the majority of their repetitive DNA; interspecific variations in the total amount of Ty3/Gypsy and Ty1/Copia retroelements, DNA transposons, ribosomal, and satellite DNA were revealed; 12–18 high confident and 7–9 low confident putative satDNAs were identified. According to BLAST, most D. sukatschewii satDNAs demonstrated sequence similarity with satDNAs of D. antarctica and D. cespitosa indicating their common origin. Chromosomal mapping of 45S rDNA, 5S rDNA, and satDNAs of D. sukatschewii allowed us to construct the species karyograms and detect new molecular chromosome markers important for Deschampsia species. Our findings confirmed that genomes of D. sukatschewii and D. cespitosa were more closely related compared to D. antarctica according to repeatome composition and patterns of satDNA chromosomal distribution.


Introduction
Several species of the cosmopolitan grass genus Deschampsia P. Beauv. (Poaceae) are well adapted to stressful environmental conditions including extreme polar habitats [1][2][3]. In particular, polar and subpolar ecotypes of D. sukatschewii (Popl.) Roshev and D. cespitosa (L.) P. Beauv. are widespread in the Arctic and sub-Arctic regions of Canada, Europe, Siberia, Chukotka Peninsula, and the Altai mountains [1,[4][5][6]. D. antarctica E. Desv. is one of two native angiosperms adapted to extreme Antarctic environments, which can be found in diverse Antarctic habitats, including the west coast of the Antarctic Peninsula, the Maritime Antarctic, sub-Antarctic Islands, and northern Patagonia [3,[7][8][9]. Such native cold-hardy ecotypes of the Deschampsia species are resources of genes associated with environmental stress tolerance and can also serve as models in crop breeding strategies [10,11].
Plant responses to environmental stresses might include some genetic changes (e.g., alternation in metabolic pathways and transcriptional regulation of genes) and cytological alterations [12]. Currently, genome diversity and comparative chromosomal phylogeny of cold-hardy ecotypes of Deschampsia are being intensively studied. Transcriptome sequencing of D. antarctica has been performed under various abiotic stress conditions and its expression profile has been examined [10]. For D. antarctica populations from the Maritime

Genomic DNA Extraction and Sequencing
Genomic DNA of D. sukatschewii and D. cespitosa were isolated from young leaves of the studied accessions using the GeneJet Plant Genomic DNA Purification Kit (Thermo Fisher Scientific, Vilnius, Lithuania). The quality of the DNA samples was checked with the Implen Nano Photometer N50 (Implen, Munich, Germany). The concentration and purification of the extracted DNAs were assessed with the Qubit 4.0 fluorometer and Qubit 1X dsDNA HS Assay Kit (Thermo Fisher Scientific, Eugene, OR, USA).

Sequence Analysis and Identification of DNA Repeats
For genome-wide comparative analyses, genome sequences of D. sukatschewii and D. cespitosa and also the publicly available D. antarctica sequencing data (https://www. ebi.ac.uk/ena/browser/view/PRJNA237267?show=reads, (accessed on 28 January 2018) PRJNA237267 gDNA-Seq for Antarctic hairgrass, Korea Polar Research Institute), were used. Interspecific comparisons, reconstruction, and quantification of major repeat families were performed with the use of RepeatExplorer 2 and TAREAN pipelines [27,38]. For each studied species, the genomic reads were filtered by quality, and 1 million high-quality reads were randomly selected for further analyses, which corresponds to 0.0147-0.0350× of a coverage of the D. cespitosa genome (1C = 4283.64-5105. 16 Mbp, Eurasian region) [36,37], and is within the limits recommended by the developers of these programs (genome coverage of 0.01-0.50× is recommended) [38]. RepeatExplorer/TAREAN was launched with the preset settings based on Galaxy platform (https://repeatexplorer-elixir.cerit-sc.cz/ galaxy/, 25 March 2022). Initially, the preprocessing of the genomic reads was performed. The reads were filtered in terms of quality using a cut-off of 10, trimmed, and filtered by size to obtain high-quality reads. Default threshold was explicitly set to 90% sequence similarity spanning at least 55% of the read length (in the case of reads differing in length it applies to the longer one). The sequence homology of the identified satDNAs of D. sukatschewii with repeats of D. cespitosa and D. antarctica was estimated by BLAST (NCBI, Bethesda, MD, USA

Chromosome Spread Preparation
Root tips (0.5-1 cm long) were kept in ice water for 24 h for accumulation of mitotic divisions and then fixed in the ethanol and glacial acetic acid fixative (3:1) for 2 days at room temperature. The fixed roots were incubated in 1% acetocarmine solution (in 45% acetic acid) for 30-40 min. Then, the root meristem was cut from the tip cap, macerated in 45% acetic acid, and a squashed preparation was made with the use of a cover slip. After freezing in liquid nitrogen, the cover slip was removed; the obtained preparation was dehydrated in 96% ethanol for 3 min and air dried for 15 min.
Several sequential FISH procedures were performed with various combinations of these labeled DNA probes as described previously [6,42]. Before the first FISH procedure, chromosome slides were pretreated with 1 mg/mL RNase A (Roche Diagnostics, Mannheim, Germany) in 2 × SSC at 37 • C for 1 h. Then, the slides were washed three times for 10 min in 2 × SSC, dehydrated through a graded ethanol series (70%, 85%, and 96%) for 3 min each and air dried for 15 min. A total of 15 µL of hybridization mixture containing 40 ng of each labeled probe was added to each slide. The slides with DNA probes were covered with coverslips, sealed with rubber cement, denatured at 74 • C for 5 min, chilled on ice and placed in a moisture chamber at 37 • C. After overnight hybridization, the slides were washed in 0.1 × SSC (10 min, 44 • C), twice in 2 × SSC for 10 min at 44 • C, followed by a 5-min wash in 2 × SSC and three 3-min washes in PBS at room temperature. Then, the slides were dehydrated through the graded ethanol series for 3 min each, air dried for 15 min, and stained with DAPI (4 ,6-diamidino-2-phenylindole) dissolved (0.1 µg/mL) in Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). After documenting FISH results, the chromosome slides were washed twice in 2 × SSC for 10 min. Then, sequential FISH procedures were conducted on the same slides.

Chromosome Analysis
The chromosome slides were inspected using the epifluorescence Olympus BX61 microscope with the standard narrow band pass filter set and UPlanSApo 100×/1.40 oil UIS2 objective (Olympus, Tokyo, Japan). Chromosome images were captured with a monochrome CCD (charge-coupled device) camera (Snap, Roper Scientific, Tucson, AZ, USA) in grayscale channels, pseudo-colored, and processed with Adobe Photoshop 10.0 (Adobe Systems, Birmingham, AL, USA) and VideoTesT-FISH 2.1 (IstaVideoTesT, St. Petersburg, Russia) software. At least five plants and 15 metaphase plates were examined in each sample. Chromosome pairs in karyotypes were identified according to the chromosome size and morphology, localization of chromosome markers, and also the cytological nomenclature proposed previously [16].

Comparative Analyses of the Repetitive DNA Sequences
The comparative repeatome analysis of D. antarctica, D. cespitosa, and D. sukatschewii showed that mobile genetic elements made up the majority of their repetitive DNAs ( Table 2). Retrotransposon elements, including Ty3-Gypsy and Ty1-Copia superfamilies (transposable elements of Class I), were highly abundant and represented 41.21-43.41% of their genomes. Within the Ty1-Copia superfamily, SIRE and Angela were most abundant, and Ty3-Gypsy retroelements were dominated by the Tat-Retand and Athila nonchromoviruses and chromovirus Tekay. In D. cespitosa and D. sukatschewii, Ty3-Gypsy elements significantly exceeded Ty1-Copia retrotransposons. In D. antarctica, however, Ty1-Copia retroelements were roughly twice abundant than Ty3-Gypsy elements. The genome of D. antarctica contained the largest proportion of unclassified LTR retroelements (14.03%) if compared with D. cespitosa (1.31%) and D. sukatschewii (4.27%). DNA transposons (Class II) were found in lower amount (2.48-2.89%) compared to retrotransposons, and the least quantity was revealed in D. sukatschewii. The total amount of satellite DNA ranged from 1.61% (D. sukatschewii) to 2.85% (D. cespitosa). The content of ribosomal DNA was notably less in D. antarctica (0.06%) if compared with D. sukatschewii (0.29%) and D. cespitosa (0.6%). In the studied accessions, 12-18 high confident and 7-9 low confident putative satellites were revealed by TAREAN ( Figure 1, Table 2).  Repeat Name Genome Proportion (%) D. antarctica D. cespitosa D. sukatschewii
In karyotypes of D. sukatschewii and D. cespitosa, similar patterns of chromosome distribution of 45S and 5S rDNA clusters were observed. Six bright 45S rDNA signals were detected in the short arms of chromosome pairs 5, 6, and 9 with satellites of different sizes and secondary constrictions. Ten hybridization signals of 5S rDNA were observed on chromosome pairs 1 (in the proximal regions of both arms), 3 (in the terminal regions of the long arms), and also in the proximal regions of the long arms of chromosome pairs 7 and 10 ( Figures 4 and 5).
In the karyotype of D. antarctica, four hybridization signals of 45S rDNA were revealed in the short arms of two chromosome pairs 5 and 9 with satellites of different sizes and secondary constrictions (Figures 4 and 5). Ten loci of 5S rDNA were localized on chromosome pairs 1 (in the proximal regions of the short arm), 3 (in the terminal regions of the long arms), 6 (in the distal regions of the short arms), and also in the proximal regions of the long arms of chromosome pairs 7 and 10 ( Figures 4 and 5).

Discussion
Most eukaryotic genomes contain large numbers of repetitive DNA sequences [43,44]. Transposable elements (TEs) as well as tandem repeats (satellite DNA) are highly abundant and diverse parts of genomes [45,46]. In plants, TEs can constitute up to 90% of their genomes [47][48][49]. Due to the fact that TEs are capable of changing their location and/or copy numbers, they can influence the genome organization and evolution [50,51]. Currently, TEs are separated into two major classes, class I (retrotransposons, including LTR retrotransposons) and class II (DNA transposons), based on TEs structural characteristics and mode of replication [50,52]. In plant genomes, LTR retrotransposons include the Ty1-Copia and Ty3-Gypsy superfamilies, which are further divided into a number of families mostly specific to a single or a group of closely related species [53]. In plant genomes, LTR retrotransposons are highly abundant, making up to 75% of nuclear DNA [54,55]. In our study, a comparative repeatome analysis of D. sukatschewii, D. cespitosa, and D. antarctica also showed that LTR retrotransposons made up the majority of their genomes. LTR retrotransposons are considered to be main contributors to the variations of nuclear genomes within angiosperms [33,[56][57][58][59]. These retroelements are able to replicate using the copy and paste mechanism and, thus, generate new copies of the elements and increase the size of the genome [45]. However, the LTR copies can also be efficiently eliminated from the genome, through both solo LTR formation and accumulation of deletions, which reduces the genome size [54]. Genome size is often treated as an intrinsic property of a species, and intra-and interspecific variations in genome size might reflect different evolutionary processes during speciation [60].
D. cespitosa is a variable and widespread species with many subspecies and closely related species (including D. sukatschewii) [61], and the genome size of D. cespitosa accessions highly depends on their geographical location and habitat [37]. Nevertheless, the average genome size of diploid D. cespitosa (1C = 4.38-5.22 pg, Eurasian region) roughly corresponds to that of diploid D. antarctica (1C = 4.98-5.31 pg) [36,37,62,63]. These data are consistent with our results showing about the same content of retrotransposons in genomes of the studied Deschampsia species, which constituted an essential portion of their repeatomes (41-43%). At the same time, we revealed interspecific differences in content of Ty3-Gypsy and Ty1-Copia and also in genome proportions of SIRE, Angela, non-chromovirus Retand, and chromovirus Tekay. For instance, a ratio of Ty3-Gypsy/Ty1-Copia retrotransposons revealed in genomes of closely related D. cespitosa and D. sukatschewii differed greatly from that detected in D. antarctica. In genomes of D. cespitosa and D. sukatschewii, the Ty3-Gypsy elements were about 1.5 times more abundant than Ty1-Copia. More content of Ty3-Gypsy retroelements in the genome compared to Ty1-Copia is typical for many taxa of Poaceae. For example, in Avena genomes, Ty3-Gypsy elements were nearly three times more abundant than Ty1-Copia [64]; in genomes of Lolium and Festuca species, Ty3-Cypsy retrotransposons were four times more abundant compared to Ty1-Copia elements [33]. Moreover, among the studied species, some interspecific variations in the total amount of DNA transposons were detected. The observed interspecific differences might be related to the processes occurred in genomes of these Deschampsia species during speciation, which is supported by some previous research. In particular, it was shown that some evolutionary changes in genomes of diploid species of Melampodium correlated with differences in the abundance of the SIRE (Ty1-Copia), Athila (Ty3-Gypsy), and CACTA (DNA transposon) lineages [58].
We also found that in D. antarctica, the genome proportion of unclassified LTR retroelements significantly exceeded that revealed in the other two Deschampsia species, which highlights the need for more research on these TEs in D. antarctica. These differences could be related to specific attributes of the D. antarctica genome or environmentally induced genetic peculiarities of the studied accessions. Environmentally induced retrotransposonbased genetic diversity was previously described in populations of D. antarctica from the Maritime Antarctic [13]. Intense stress might induce rapid changes in the structure, organization, and function of plant genomes especially in populations with low genetic diversity [65], which is typical for D. antarctica [66][67][68]. Moreover, in many plant species, which grew under various abiotic and biotic stresses, transcriptional activation of TEs was revealed [69][70][71], and it was regarded as a mechanism responsible for genome plasticity under changing environmental conditions [72].
It was reported for different Poaceae species that satDNAs sequences can vary in a number of features, including nucleotide composition, abundance, and distribution in genomes [73,74]. The comparative analysis of the studied accessions detected interspecific variations in the content of ribosomal DNA, which was notably lower in D. antarctica compared to D. cespitosa and D. sukatschewii. These data are consistent with the different number of satellite chromosomes bearing nucleolar organizer regions (NORs) identified in karyotypes of D. antarctica (two pairs) and the other two species (three pairs) [15,16] since it is known that NORs contain tandemly repeated rDNA sequences [75]. Moreover, our results showed that genomes of the studied Deschampsia accessions contained substantial portions of satellite DNA sequences, and interspecific variations in their abundance were also revealed. D. cespitosa has the highest amount of satellite DNA among the studied species, which is consistent with earlier reported data [34]. Tandem repeats, such as rDNA and other satDNAs, are generally found to be a fast-evolving fraction of the repeatome, showing divergence in both copy number and sequence between closely related species [60]. SatDNAs are known to have a variable length of the repeat unit (monomer) and usually form tandem arrays up to 100 Mb [20,76]. Although they are considered to be non-coding sequences, the satellite monomers mostly exhibit lengths of 160 to 180 bp or 320 to 370 bp though other lengths are also found in plants [77], which correspond to the length of monoand dinucleosomes [78,79]. The sequences of satellite monomers evolve concertedly via the process of molecular drive; and mutations are homogenized in a genome and become fixed in the populations [80]. The sequence identity inside an array evolves according to the process called 'concerted evolution', which results to the maintenance of homogeneity of satDNA monomers within a species during evolution [81]. The abundance of satDNA can vary within the plant genomes even between generations resulting in high polymorphism in the length of satellite arrays [80]. At the same time, some satDNA sequences demonstrate sequence conservation for long evolutionary periods [82]. Since many satellite DNAs exist in a genome, the evolution of species-specific satDNA might be the result of copy number changes within a library of satellite sequences common for a group of species [79,80,82].
The high-throughput DNA sequencing and subsequent genome-wide bioinformatic analysis provide important data on the structural diversity of satDNA [21,83,84]. In the studied accessions of D. antarctica, D. cespitosa, and D. sukatschewii, more satDNA families (20, 27, and 21, correspondingly) were identified by genomic analyses with TAREAN if compared with reported earlier data on South American accessions of D. antarctica and D. cespitosa (34 satDNAs in total) [34], which indicated a high level of satDNA diversity in Deschampsia genomes. Moreover, a relatively large number of the satDNAs were identified in Deschampsia genomes compared to several other Poaceae species including Festuca pratensis (eight satDNAs), Agropyron cristatum (fourteen satDNAs), and Poa species (four satDNAs) [31,32,85], which might be related to some features of Deschampsia genomes.
Despite satDNAs are considered to be fast-evolving genome fractions, some of them remain preserved for long evolutionary periods and have a highly conserved monomer sequence, which might be related to their interaction with specific proteins necessary for heterochromatin formation and also to their putative regulatory role in gene expression [80,86]. SatDNAs are known to contribute to the essential processes of formation of crucial chromosome structures, e.g., DNA packaging and chromatin condensation [19,79,87,88]. In the present study, three Ds repeats (Ds 56, Ds 83, and Ds 124) showed high sequence similarity with CON1, CON2, and COM2 sequences. CON/COM satDNAs were originally isolated from the Helictotrichon genome [22,89] and then revealed in several taxa of the Aveneae/Poeae tribe complex including Deschampsia [23,25,90]. In different taxa, the nucleotide sequences in monomers of CON/COM satDNAs demonstrated a high degree of identity, which suggested their ancient origin, though they could change slightly and independently in different species of Deschampsia and related genera [22,25,89] SatDNAs are often associated with heterochromatin regions and are localized in the certain chromosome regions (centromeric, terminal, and/or intercalary), which allow them to be explored with cytogenetic techniques, including FISH. The patterns of chromosomal distribution of satDNAs facilitate the recognition of homologous chromosome pairs and recombination as well as differences between lineages and species [19,20]. High sequence homology of certain satDNAs allowed us to use the oligonucleotide FISH probes, developed based on the most abundant Ds satDNAs, in the comparative karyotype analysis of the studied Deschampsia species. However, despite the large number of common repeats, different patterns of chromosomal distribution of these Ds were observed, and depending on the species, localization of most examined Ds satDNAs could be clustered and/or dispersed, which was probably related to different amount and organization of these homologous repeats in genomes of the related species. Large Ds clusters were predominantly localized in the pericentromeric and/or terminal regions of chromosomes of the studied species. Moreover, other patterns of Ds chromosomal distribution were observed including bright clusters combined with dispersed signals or small satDNA clusters in the intercalary chromosome regions, which is typical for plants [19][20][21]. Several Ds satDNAs exhibited only specific clustered localization on chromosomes of all studied species, which allowed us to explore interspecific variations in their distribution on chromosomes. These results were consistent with earlier reported data on patterns of chromosomal distribution of CON/COM and Da satDNAs in several Deschampsia species [25,34,35].
According to BLAST, any satDNAs, which would be homologous to Ds 81/Dc 135, were not identified in the D. antarctica genome. Moreover, BLAST did not detect any satDNAs homologous to Ds 146 within Deschampsia or other taxa. However, the performed FISH-based chromosome mapping of both Ds 81 and Ds 146 revealed bright hybridization signals in karyotypes of all studied Deschampsia species. This could be related to some peculiarities of the used sequencing technique, subsequent bioinformatic processing, and also the satDNA abundancy in the genome. Thus, our results demonstrate that the cytogenetic studies can increase the possibilities for satellite DNA analysis as they provide valuable additional data on genomic relationships among related species.
Among the examined Ds tandem repeats, four satDNAs (Ds 52, Ds 81, Ds 65, and Ds 146) demonstrated species-specific patterns of their chromosomal distribution in all studied Deschampsia species, which is important for comparative karyotype studies and also analyze the genome differentiation within Deschampsia. Specifically, hybridization signals of Ds 52 and also Ds 81 partially overlapped with sites of CON1 satDNA studied previously [25]. Both Ds 65 and Ds 146 demonstrated unique clustered species-specific patterns of chromosomal distribution indicating that they could be used as new promising chromosomal markers for Deschampsia species.
SatDNA repeats was shown to represent recombination "hotspots" of genome reorganization, and the occurrence of satDNA in interstitial and telomeric heterochromatin reduces genetic recombination in the adjacent regions [91]. In our study, the comparison of patterns of chromosomal distribution of Ds 65 and Ds 146 made it possible to identify different chromosomal rearrangements in some karyotypes of D. sukatschewii, D. cespitosa, and D. antarctica and detect the breakpoints on chromosomes.
The comparison of patterns of chromosomal distribution of Ds 52, Ds 81, Ds 65, and Ds 146 indicated predominant similarity between karyotypes of D. sukatschewii and D. cespitosa compared to D. antarctica, which was consistent with our previously reported data on other chromosomal markers [16,25]. Notably, the chromosomes bearing 45S and 5S rDNA clusters had the most similar patterns in all three species indicating that structures of these chromosomes were rather conserved. Satellite DNA-based chromosomal markers are particularly useful for chromosome identification, the analysis of chromosome rearrangements, as well as evolution of genomes within Poaceae [24,64,92]. This is especially important for Deschampsia due to the lack of effective molecular cytogenetic markers suitable for karyotype analyses within this genus [15]. At the same time, comprehensive genomic studies to assess the variability of satDNA arrays are still required to provide valuable data for investigating the functional and structural features of Deschampsia genomes, and also the paths of chromosomal reorganization of genomes during speciation.

Conclusions
For the first time, the comparative repeatome analyses among valuable subpolar and polar accessions of D. sukatschewii, D. cespitosa, and D. antarctica was performed with the use of the modern effective approach (combining high-throughput DNA sequencing, genomewide bioinformatic analyses, and FISH-based chromosome mapping of the identified specific satDNAs). Analyses of chromosome patterns of distribution of twelve abundant D. sukatschewii satDNAs allowed us to detect four new effective molecular chromosome markers. Due to the shortage of such markers in Deschampsia, this is especially important for comparative karyotypic studies within the genus to analyze the changes occurring in their genomes during speciation. For the first time, the unique species karyograms were constructed, which made it possible to compare the localization of these markers on homologous chromosomes of the studied species. Our results confirmed that genomes of the subarctic D. sukatschewii and D. cespitosa accessions were more closely related if compared with the D. antarctica accession according to repeatome composition and patterns of satDNA chromosomal distribution. Our findings demonstrated that cytogenetic studies might expand the possibilities of repeatome analyses as they provide important additional data on genomic relationships within Deschampsia as well as increase knowledge on genome organization in these species.