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Logo of gbeAboutAuthor GuidelinesEditorial BoardGenome Biology and Evolution
 
Genome Biol Evol. 2017 January; 9(1): 197–212.
Published online 2017 February 1. doi:  10.1093/gbe/evw303
PMCID: PMC5381607

Satellite DNA and Transposable Elements in Seabuckthorn (Hippophae rhamnoides), a Dioecious Plant with Small Y and Large X Chromosomes

Abstract

Seabuckthorn (Hippophae rhamnoides) is a dioecious shrub commonly used in the pharmaceutical, cosmetic, and environmental industry as a source of oil, minerals and vitamins. In this study, we analyzed the transposable elements and satellites in its genome. We carried out Illumina DNA sequencing and reconstructed the main repetitive DNA sequences. For data analysis, we developed a new bioinformatics approach for advanced satellite DNA analysis and showed that about 25% of the genome consists of satellite DNA and about 24% is formed of transposable elements, dominated by Ty3/Gypsy and Ty1/Copia LTR retrotransposons. FISH mapping revealed X chromosome-accumulated, Y chromosome-specific or both sex chromosomes-accumulated satellites but most satellites were found on autosomes. Transposable elements were located mostly in the subtelomeres of all chromosomes. The 5S rDNA and 45S rDNA were localized on one autosomal locus each. Although we demonstrated the small size of the Y chromosome of the seabuckthorn and accumulated satellite DNA there, we were unable to estimate the age and extent of the Y chromosome degeneration. Analysis of dioecious relatives such as Shepherdia would shed more light on the evolution of these sex chromosomes.

Keywords: sex chromosomes, genome composition, chromosomal localization, repetitive DNA

Introduction

Seabuckthorn (Hippophae rhamnoides) is a hardy, deciduous dioecious shrub belonging to the Elaeagnaceae family with a natural habitat extending widely across Europe and Asia. It is used in traditional Chinese, Tibetan and Siberian medicine and has special characteristics exploitable in biotechnology, pharmaceutical and cosmetic sciences, as a source of oil, minerals and vitamins. The size of seabuckthorn genome is ~2.55 Gbp/2C (Zhou et al. 2010) but there is a dearth of information on its composition. The ribosomal DNA ITS regions were compared among H . rhamnoides ssp chinensis from different geographical areas of China and showed distinct genetic variation (Chen et al 2010). RAPD markers (Sharma et al. 2010) were identified with the aim of determining the sex of individuals. Cytogenetic analysis is represented only by the older works of Shchapov (1979) and Rousi and Arohonka (1980) who both determined the diploid chromosome number 2n = 24. Shchapov (1979) revealed the small Y and large X chromosomes. Seabuckthorn transcriptome has been analyzed recently providing a resource for gene discovery and development of molecular markers (Ghangal et al. 2013).

Sex chromosomes have evolved repeatedly and independently in the plant kingdom with different age and degree of degeneration shown in various dioecious species (Ming et al. 2011; Hobza and Vyskot 2015; Charlesworth 2016). The evolution of the Y chromosomes is characterized by gene erosion/loss and accumulation of repetitive DNA (Kejnovsky et al. 2009). The most studied dioecious model species with heteromorphic sex chromosomes are white campion (Silene latifolia, Kejnovsky and Vyskot 2010), sorrel (Rumex acetosa, Steflova et al. 2013; R. hastatulus, Hough et al. 2014), ivy gourd (Coccinia grandis, Sousa et al. 2013), and members of the Cannabaceae family (Humulus lupulus, Divashuk et al. 2011; H. japonicus, Alexandrov et al. 2012; Cannabis sativa, Divashuk et al. 2014).

The majority of large plant genomes are formed of repetitive DNA, mostly by transposable elements and tandem repeats (satellite DNA). The processes of repetitive DNA amplification and elimination are only partially understood. Turnover of repeats is high and corresponds only to million of years (Lim et al. 2007). The localization of repetitive DNA on sex chromosomes is different from that of autosomes, reflecting different repeat dynamics, especially on the nonrecombining regions of the Y chromosomes (Kejnovsky et al. 2009). Satellite DNA has mostly discrete localization in the genome and some satellites are thus Y chromosome-specific (Mariotti et al. 2009). In contrast, transposable elements have more homogenous distribution and are only slightly enriched on the Y chromosome (Charlesworth 1991; Cermak et al. 2008) or alternatively absent on the Y chromosome as shown in Silene latifolia (Cermak et al. 2008; Kubat et al. 2014) and Rumex acetosa (Steflova et al. 2013) despite their presence in the rest of genome. The striking example is the large Y chromosome of the dioecious plant Coccinia grandis showing accumulation of transposable elements, satellites, and organellar DNA (Souza et al. 2016). One review published recently discusses the role of repetitive DNA in the evolution of sex chromosomes and includes a database of transposable elements of dioecious plants (Li et al. 2016a, 2016b).

In this study, we analyzed the transposable elements and satellites in the seabuckthorn genome and determined the chromosomal localization of these repeats. We showed that seabuckthorn has an XY system with large X and small Y chromosomes.

Materials and Methods

Illumina Sequencing

DNA isolation from male (Pollinator 1) and female (cv “Botanicheskaya lyubitelskaya”) plants was carried out according to Doyle and Doyle (1990). One Illumina MiSeq sequencing run was performed for each male and female genomic DNA. The voucher specimen of the plants used in the study was kept for record in the herbarium (AT) of Department of Botany and Breeding of Horticultural Crops of the Russian State Agrarian University – MTAA (Voucher No.5470). Sequencing reads were analyzed by quality control tool FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/; last accessed January 4, 2017) followed by quality filtering based on the sequence quality score, adaptors trimming, filtering out short or unpaired sequences and trimming all reads to lengths of 230 nucleotides using the Trimmomatic tool (Bolger et al. 2014), leading to 1,848,543 male and 1,863,670 female paired-end reads. Quality-filtered reads were randomly sampled to 415,650 paired-end reads for both male and female individuals and the reads were merged together (totally 1,662,600 reads). As the nuclear DNA content of H. rhamnoides reported in Zhou et al. (2010) was determined to be ~2.61/2C pg (without detailed specification of male or female) we converted it to genome size (in bp) using following formula (Doležel et al. 2003): g = DNA content (pg) × (0.978 × 109), resulting into ~2.55 Gbp/2C, our samples represent ~30% of haploid genome. Genome coverage was calculated as follow: cov = (r × l)/g, where r corresponds to number of reads used in our analysis, l to read length and g to haploid genome size of H. rhamnoides.

Repeat Identification and Annotation

In order to identify repetitive sequences in the H. rhamnoides genome we employed comparative graph-based clustering analysis of sequenced reads by RepeatExplorer pipeline (Novak et al. 2013). Only clusters containing at least 0.01% of all clustered reads were considered and they corresponded to 58.5% of the genome. These were further manually characterized based on the similarity search results from RepeatMasker (http://www.repeatmasker.org; last accessed January 4, 2017) against Viridiplantae database and blastn and blastx (Altschul et al. 1990) against GenBank nr (Benson et al. 2009), which are part of the RepeatExplorer output. Cluster shapes were also used for repeat identification as tandem repeats with monomer longer than read length have typical donut-shaped clusters (Novak et al. 2010). Additionally, advanced analysis of satellite sequences, described in the section Satellite DNA sequences analysis, was used in the manual annotation of clusters.

Structural Annotation of LTR Retrotransposons

We reconstructed several Ty3/Gypsy and Ty1/Copia retrotransposons. The reconstruction comprised several steps. First, clusters belonging to particular element were visualized inSeqGrapheR (https://cran.r-project.org/web/packages/SeqGrapheR/index.html; last accessed January 4, 2017) program and contigs which together covered the whole elements were selected. These contigs were searched for occurrences of protein domains (GAG, RT, RH, AP, INT) by querying them to CDD (Marchler-Bauer et al. 2015). We then did multiple sequence alignment to create a consensus sequence of these contigs using progressive pairwise alignment implemented in Geneious 8.1.7 (http://www.geneious.com; last accessed January 4, 2017, Kearse et al. 2012). If necessary, resulting alignments were manually modified with respect to the order of domains for particular type of transposable element. The consensus sequence of reconstructed elements was then searched for the structural motif characteristics (ORFs and LTRs). Possible ORFs were detected by ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/; last accessed January 4, 2017). LTRs were determined on the basis of shape of a cluster and the element’s coverage. Male and female coverage of reconstructed elements was determined by mapping reads which formed a current element to its consensus sequence using BowTie2 tool (Langmead and Salzberg 2012). Structural features and male and female coverage of reconstructed elements were visualized by custom R script and graph layouts of reconstructed elements were depicted by SeqGrapheR.

Phylogeny and Classification

Firstly, we created custom databases of plant LTR retrotransposon RT domains from sequences available in TREP (Wicker et al. 2002) and GyDB (Llorens et al. 2011) databases, independently for Ty3/Gypsy and Ty1/Copia retrotransposons. Contigs corresponding to retrotransposons were examined for the presence of a reverse transcriptase domain and Ty3/Gypsy and Ty1/Copia cores of RT domains were trimmed from these contigs based on the exact localization designated by CDD (Marchler-Bauer et al. 2015). Cores of RT domains were aligned by MUSCLE algorithm (Edgar 2004) together with our custom-made database of RT domains, and the resulting multiple sequence alignment was used as an input to create Neighbor-Joining tree (Saitou and Nei 1987) with Jukes-Cantor distance model using Geneious 8.1.7 (http://www.geneious.com; last accessed January 4, 2017, Kearse et al. 2012).

Preparation of Chromosomes and Probes and Fluorescence In S itu Hybridization

For chromosome preparations vegetatively propagated for commercial use, male (“Pollinator 1” and “Pollinator 3”) and female (cv “Lomonosovskaya” and cv “Botanicheskaya ljubitelskaya”) plants were used. Plant material was kindly provided by Dr G. Boyko, Lomonosov Moscow State University. The root tips were harvested separately from the individual male and female plants grown in pots. The harvested root tips were immediately pre-treated with a 2 mM aqueous solution of 8-hydroxyquinoline for 6 h at 20 °C. A 3:1 ethanol/glacial acetic acid (v/v) mix was used for fixation. Meristems 2 mm long were cut from the fixed root tips and digested in 10 μl enzyme solution [0.5% cellulase Onozuka R-10 (Serva, Germany) and 0.5% pectolyase Y-23 (Seishin Corp., Japan)] in 10 mM citrate buffer (pH = 4.9) for 2.5 h at 37 °C. The suspended cells were used for chromosome preparation as described by Kirov et al. (2014). The quality of spreads was assessed microscopically using phase-contrast and only preparations with at least 20 well-spread metaphases were used.

Probes for fluorescence in situ hybridization were generated using PCR-DIG Labeling Mix PLUS (Roche Diagnostics Gmbh) or by Biotin-11-dUTP 1/3 PCR labeling Mix (ZAO Sileks, Moscow). Primers for RT domain of selected transposable elements and determined monomer sequence of satellites were designed by Primer3 tool (Untergasser et al. 2012), were synthesized by ZAO “Syntol” (Moscow). These are available in supplementary table S1, Supplementary Material online. The pTa71 (45S rDNA) and pCT4.2 (5S rDNA) clones labeled by DIG-Nick translation kit were also used (Gerlach and Bedbrook 1979; Campell et al. 1992).

FISH experiments were performed as described in Alexandrov and Karlov (2016). For digoxigenin and biotin detection, slides were incubated with anti-DIG-FITC conjugate (Roche) and/or streptavidin-Cy3 conjugate (Sigma). The chromosomes were counterstained with DAPI (2 µg/ml) and mounted in Vectashield (Vector). An AxioImager M1 fluorescent microscope (Zeiss) was used to observe metaphase plates with fluorescent signals that were photographed with a monochrome AxioCam MRm CCD camera and visualized using Axiovision software (Zeiss).

Satellite DNA Sequences Analysis

As the seabuckthorn genome is abundant in satellite DNA and manual inspection would be exhaustive, we developed a custom bioinformatics approach which extended the basic analysis of RepeatExplorer tool. As an input the satellite clusters identified by RepeatExplorer are required. It is highly recommended to do manual inspection of these clusters and verify their structure and interaction with other clusters based on similarities among other clusters and pair-end reads connections. Our approach consisted of three basic steps.

  • (i)Detection of satellite monomers: First, assembled contigs of selected clusters were extracted from RepeatExplorer output and for each contig the monomer length was estimated from the distribution of distances between the same k-mers. The resulting monomer sequence was then extracted from the most covered part of the contig of previously determined length. Only the monomers with clearly distinguishable length, longer than 100 bp and reaching average coverage 50x and more were taken into account.
  • (ii)Estimation of satellite families composition in genome and their annotation: First, all to all monomer similarity was calculated. In order to do alignment of tandemly repeated monomers correctly (offsets between monomers are not known) we used one monomer as a subject and two copies in a row of the second monomer as a query. The similarity between monomers was then determined based on semiglobal alignment. To estimate the composition of satellite families in the genome, we clustered the monomer’s similarity matrix using UPGMA method. The resulting dendrogram was then inspected by the user and cut off at the level that best discriminated the individual families (usually 70-85% of monomer identity). Identified families were visualized by the algorithm described by Fruchterman and Reingold (1991) implemented in igraph library and only connections that exceeded specified cut-off were considered and depicted. Secondly, to annotate identified families, all monomers were searched for similarity hits with sequences in the public nucleotide database and PlantSat database (Macas et al. 2002) using blastn (Altschul et al. 1990) with word size set to 11. Only results with an e-value lower than 10  20 were considered as significant. Finally, to depict satellite diversity inside the family, we chose the most covered monomer as a reference and mapped all reads belonging to the family onto its reference using BWA-MEM mapping tool (Li 2013). Conservation of different parts of the monomer was depicted using sequence logo created by WebLogo (Crooks et al. 2004) tool.
  • (iii)Visualization of satellite families homogeneity : First, the relative abundance of male and female reads was calculated in each tandem repeat family. This enabled us to predict their presence in sex chromosomes. We visualized the satellite homogeneity using the following procedure: reads from each identified family were merged together and sampled randomly to limit the maximum number of reads to speed up the following analysis. Similarity of sampled reads from all families was calculated using the megablast tool (Camacho et al. 2009) that performed all against all sequence comparison. Pairs of reads that met specific similarity threshold (70% sequence identity over at least 55% of sequence length) were further used for graph construction and visualization. Male and female reads were distinguished by color (male—blue, female—red), tandem repeat families were highlighted by different colors and the algorithm by Fruchterman and Reingold (1991) was used to depict the results. Additionally, graphs for selected families were refined with similarity thresholds ranging from 70% to 95% sequence identity to show satellite composition more clearly. Each satellite falling within individual satellite family was marked by a different color.

Results

Genomic Composition

We performed one Illumina MiSeq platform sequencing run for each male and female genomic DNA followed by graph-based clustering of reads and characterization of repetitive sequences by RepeatExplorer (Novak et al. 2013). All 223 clusters (with more than 167 reads) contained 973,049 reads corresponding to 58.5% of genome (fig. 1) and their identification showed that dominant (first) clusters corresponded to satellite DNA followed by Ty3/Gypsy and Ty1/Copia LTR retrotransposons. One cluster (CL97) corresponded to 5S rDNA, two clusters (CL40, CL71) to 45S rDNA and 15 clusters to chloroplast DNA (cpDNA). Although the majority of chloroplast DNA reads probably originated from contaminating cpDNA, some proportion could come from nuclear cpDNA insertions (NUPTs).

Fig. 1.
Repeat composition of clusters and their genomic proportions. Each column corresponds to one cluster and repeat types are distinguished by colors. The height of columns represents number of reads in each cluster, the width of column indicate genomic proportion ...

We identified main types of repetitive DNA and their genome proportions in male and female individuals (table 1). All transposable elements represented together 24% of male and 23% of female genome. Ty1/Copia retrotransposons formed 12%, Ty3/Gypsy retrotransposons 11% and DNA transposons 1.5% of male genome. The most abundant among Ty1/Copia retrotransposons were Angela/Tork and Ale/Retrofit, among Ty3/Gypsy retrotransposons Athila and chromoviruses dominated. No LINE elements were found in the whole seabuckthorn genome. Satellites together comprised about 27% of male and 24% of female genomes. The 45S rDNA formed 0.7% of both male and female genomes and 5S rDNA represented 0.2% of both male and female genomes.

Table 1
Repeat Composition in Hippophae rhamnoides Genome

Transposable Elements

To determine the phylogenetic relationships of Ty1/Copia and Ty3/Gypsy retrotransposons, we aligned their reverse transcriptase (RT) domains from individual clusters and constructed the phylogenetic trees. Both Ty3/Gypsy (fig. 2A ) and Ty1/Copia (fig. 2B ) trees contained families identified in our clusters (in red) mixed with representatives of known subfamilies of Ty1/Copia or Ty3/Gypsy from other plant species (in black). Among Ty3/Gypsy retrotransposons, we identified five clusters containing Athila subfamilies, one CRM subfamily, one Galadriel, one Reina and one Tat/Ogre subfamily (fig. 2A ). Among Ty1/Copia retrotransposons, we found four subfamilies of Ale/Retrofit, four Angela/Tork subfamilies, one Maximus/SIRE subfamily, two TAR subfamilies and two Ivana/Oryco subfamilies (fig. 2B ). The Angela/Tork and Ale/Retrofit subfamilies showed higher variability while Athila subfamilies were homogenous. Highest homogeneity were shown by chromoviruses where all reads were assembled into a single cluster for CRM, Galadriel and Reina families (fig. 2A ).

Fig. 2.
Phylogenetic trees of Hippophae rhamnoides Ty3/Gypsy (A) and Ty1/Copia (B) retrotransposons based on reverse transcriptase sequences. RT domains of retrotransposons reconstructed from Illumina reads in this study are in red, representative RT domains ...

We reconstructed the structure of the main Ty3/Gypsy and Ty1/Copia subfamilies (fig. 3) and identified all main features such as gag and pol genes (with all domains) and long terminal repeats (LTRs). In some retrotransposons (CL6, CL16) LTR regions were assembled into one long terminal repeat while in other clusters (CL7, CL27) right and left LTR were distinguished. This may be a consequence of lower or higher mutual diversity of LTRs in one element, and could correspond to age differences of elements. Graph layouts (right part of fig. 3) show the variability of specific parts of elements as well as alternative variants of elements, e.g., potential spliced variant (Novak et al. 2010). The similar coverage of elements by male and female reads indicates that elements are present on all chromosomes without accumulation/absence on the X or Y chromosome. Some elements had uninterrupted ORF corresponding to gag and pol (CL7, CL27, and CL43) and hence they can be active. Interruption of ORFs in other elements may have been caused by assembling errors during reconstruction (CL6, CL16, and CL37).

Fig. 3.
Comparison of structure of selected retrotransposon families in Hippophae rhamnoides. Graphs of coverage by male (in blue) and female (in red) genomic reads are showed under the structure of Ty3/Gypsy (A, B) and Ty1/Copia (CF) elements shown ...

Satellite DNA

We developed a new bioinformatics approach for detailed analysis of satellite DNA in genomes. This method includes: (i) identification of satellite monomers based on distribution of distances of k-mers in assembled contigs, (ii) clustering of monomers allowing identification and annotation of satellite families in genome, and (iii) visualization of satellites homogeneity and male/female composition allowing better prediction of their localization with respect to sex chromosomes. Detailed description of the whole procedure is available in the section Materials and Methods and in supplementary figure S4, Supplementary Material online.

We utilized this approach for analysis of the seabuckthorn genome, but it is generally applicable in genomic studies of other species as well. As an input we used the 38 largest manually inspected satellite clusters from RepeatExplorer output extended by five smaller clusters with potentially interesting chromosomal localization (X, Y chromosomes). All clusters were grouped into 12 main superclusters that correspond to the 12 main families of satellite DNA in the seabuckthorn genome. Satellites were named HRTR1-HRTR12 (supplementary fig. S1, Supplementary Material online and table 2). Copy number of individual satellite families was determined based on following formula: cn = [(s x l)/m]/cov, where s represents number of reads of individual satellite family, l corresponds to read length, m represents estimated monomer length for satellite family and cov is genome coverage. Sequence logos show the monomer sequences of the main satellites and the sequence variability (supplementary fig. S2 AL, Supplementary Material online). Only HRTR1 and HRTR12 showed significant similarity hits with blast nucleotide (nr/nt) database (to previously deposited microsatellite markers of H. rhamnoides). There were no significant hits with PlantSat database for all satellite groups.

Table 2
Main Satellite Families in Hippophae rhamnoides Genome

Based on our detailed analysis of HRTR6 and HRTR7, sharing small part of monomers (supplementary fig. S3 C, Supplementary Material online), we decided to retain them as two separate tandem repeat families instead of one. These two families were very divergent and each showed variability in monomer’ length (HRTR6: 730–810 bp, HRTR7: 475–830 bp). Monomers in each family had a common sequence (HRTR6: 198 bp, HRTR7: 493 bp) while other parts of monomers were significantly different from each other. For this reason, we only created sequence logos for the shared part of monomers for each family (supplementary fig. S2 F and G, Supplementary Material online).

Male versus Female Comparison

To compare male and female genomes and to predict which repetitive DNA is specific for or accumulated on the X and Y chromosomes, we plotted the numbers of male versus female reads corresponding to individual clusters (fig. 4). This analysis involved all 223 clusters. The majority of clusters was located on the diagonal and these corresponded to transposable elements, rDNA and some satellites. However, some clusters containing satellites were enriched or even specific for males and represented potential Y-specific repeats. Other repeats, mostly satellites, were more abundant in females which could reflect their enrichment or specific localization on the X chromosome.

Fig. 4.
Comparison of repeats in male and female of Hippophae rhamnoides. Number of male versus female reads corresponding to individual clusters. Each circle in plot represents one cluster. Repeat types are marked by different color. Clusters in left upper part ...

The greatest differences in composition of male and female reads were observed in satellites (five clusters located in the left; fig. 4). Detailed analysis showed that one of these (CL123—HRTR12) formed an isolated family composed of male reads only which suggests its localization only on the Y chromosome (fig. 5). The other four male biased satellites represented either a variant of a specific widespread cluster with Y chromosome presence (CL99 and CL144—HRTR2) or a satellite with a minor presence on the Y chromosome (CL150—HRTR1 and CL132—HRTR3). Eight satellites contained more female than male reads (2:1) indicating its localization on the X chromosome (female has two X chromosomes, male only one). HRTR2 satellite also contained more female than male reads but the ratio was 1.3 to 1 which could be explained by the localization on both sex chromosomes with greater abundance on the X than on the Y chromosome (fig. 5). Most other satellites had similar abundance of male and female reads, suggesting their localization (at least mostly) on autosomes.

Fig. 5.
Visualization of male/female reads homogeneity in satellite families. Graph nodes correspond to sequenced reads and edges connect overlapping reads with more than 70% of sequence identity over at least 55% read length. Distances between reads are inversely ...

Chromosomal Localization of Transposable Elements and Satellites

For determination of the chromosomal localization of transposable elements and satellites in seabuckthorn, we prepared probes representing reverse transcriptase region of individual TE families or part of a satellite monomer (supplementary fig. S1, Supplementary Material online) and used them for fluorescence in situ hybridization (FISH). In all FISH experiments we used both male (Pollinator 1, Leningradskaya region) and female (cv “Botanicheskaya lyubitelskaya”) metaphases from plants that was used for sequencing. FISH experiments were also expanded to male (“Pollinator 3” Kaliningrad region) and female (cv “Lomonosovskaya”). In all ecotypes, we got the same results with X and Y.

FISH with satellite DNA showed various localization patterns on metaphase chromosomes of H. rhamnoides (fig. 6). The HRTR2, HRTR8 and HRTR12 show the sex specific or accumulation pattern of hybridization, while for HRTR3, HRTR4, HRTR5, HRTR6, HRTR7, HRTR9, HRTR10, and HRTR11 the hybridization patterns was the same for male as well as for female. The HRTR1 satellite hybridized mainly to heterochromatic arms of six pairs of small autosomes and weakly on one more pair of small autosomes (fig. 6A and B ). In addition, a weak signal was detected distal to centromere on one arm of one large chromosome (chromosome X) in male (fig. 6A ) and two large chromosomes in female (fig. 6B ). The HRTR2 satellite gave a strong FISH signal on one large chromosome (chromosome X) and on one small chromosome (chromosome Y) in male (fig. 6C ) and a strong FISH signal on two large chromosomes (chromosome X) in female (fig. 6D ). Also a weak signal on the centromeric region of a pair of large and a pair of small autosomes was detected in both sexes. The HRTR3 satellite was localized on two large autosomal pairs with the FISH signal dispersed along these chromosomes (fig. 6E ). The HRTR4 localized on one pair of large and on one pair of small autosomes (fig. 6F ). The HRTR5 signal was detected on one pair of small autosomes only (fig. 6G ). HRTR6 gave a strong signal on one autosomal pair and a weaker signals on two autosomal pairs (fig. 6H ). The HRTR7 showed two sites of hybridization on one arm of a pair of large autosomes and on the centromeric region of a pair of small autosomes (fig. 6I ). The HRTR8 hybridized mainly to the one large chromosome (chromosome X) in male (Fig. 6J ) and to the two large chromosomes (chromosomes X) in female (fig. 6K ). A weak signal was also detected on one pair of small autosomes. The HRTR9, HRTR10, and HRTR11 were localized on one pair of small autosomes each (fig. 6LN ). The HRTR12 hybridized specifically to the small chromosome (Y chromosome) (fig. 6O ) in male and no signal was detected in female (fig. 6D ). The FISH signal intensity from HRTRs on X chromosomes varied depending on genotype.

Fig. 6.
Localization of main satellite families on metaphase chromosomes of Hippophae rhamnoides using fluorescence in situ hybridization. The name of satellite family and sex of individual are indicated inside each figure. Blue are DAPI stained chromosomes, ...

Localization of the HRTR1 and the Y-specific (HRTR12), X-accumulated (HRTR8) and X and Y-accumulated (HRTR2) satellites on sex chromosomes was demonstrated by bicolor FISH using combinations of these probes and is summarized in a scheme (fig. 7). This together with specific or enriched representation of clusters in male and female (figs. 4 and 5), clearly demonstrates that H. rhamnoides has heteromorphic sex chromosomes (XY system) with large X and the small Y chromosomes.

Fig. 7.
FISH and scheme of four satellites on sex chromosomes. The HRTR1, Y-specific HRTR12, X-accumulated HRTR8, sex chromosome-accumulated HRTR2.

We also mapped ribosomal genes. 45S rDNA was localized on one pair of small autosomes (fig. 8A ) and 5S rDNA was localized on another pair of autosomes (fig. 8B ). FISH with probes derived from transposable elements showed that three of four studied groups of TEs are present mainly in subtelomeres of all chromosomes (fig. 8DF ) and only the CRM retroelements (CL4) that was localized in the centromeric region of all chromosomes (fig. 8C ).

Fig. 8.
Localization of transposable elements and rDNA on metaphase chromosomes of Hippophae rhamnoides using fluorescence in situ hybridization. The name of transposable element family (together with the number of corresponding cluster) or type of rDNA cluster ...

Discussion

We present the first comprehensive analysis of seabuckthorn (H . rhamnoides) genome. We found that about one quarter of the genome is composed of TEs and another quarter of satellite DNA which is comparable to other plant genomes. Nevertheless, the seabuckthorn genome contains an unusually large number of different satellites (table 2, 12 main tandem repeats) compared with most other plant genomes (Mehrotra and Goyal 2014). Moreover, some satellites evolve rapidly into new variants. In particular, HRTR2 and HRTR3 satellite superclusters are comprised of a number of smaller clusters where each cluster represents an individual satellite (supplementary fig. S3, Supplementary Material online). Thus, the number of different satellites may be even higher if more strict criteria were used for tandem repeat classification. Transposable elements are represented by all main families of both Ty3/Gypsy and Ty1/Copia retrotransposons (fig. 2) with chromoviruses (CRM and Galadriel) and TAR families dominating (table 1). Most transposable element families are represented by only one or two clusters indicating their long term presence without changes in sequence or structure. Only Athila, Angela, Tork and Ale/Retrofit retrotransposons are found in multiple clusters (data not shown) suggesting higher divergence. Well preserved long ORFs in some TEs indicate the recent amplification/younger age and low level of degeneration of these elements. All in all, high variability of some satellites and TE families indicate high tempo of their diversification in the seabuckthorn genome, while other repeats remain relatively conserved. Nevertheless, this conclusion should be verified by comparative analysis of at least two closely related species. Recent analysis by Macas et al. (2015) showed that it is not transposable elements but satellites that are the most variable repeats among closely related species of Fabae genus.

Comparison of numbers of male and female reads constituting satellite superclusters, enabled us to predict satellites localized on the Y chromosome, X chromosome, on both sex chromosomes or on autosomes as each specific ratio of abundance of male and female reads in a cluster corresponded to specific chromosomal distribution. Our FISH results showed that this prediction works well in most cases as verified by satellites accumulated on the X chromosome (HRTR8) and both X and Y chromosomes, and specific for the Y chromosome (HRTR12) and for autosomes (HRTR1, 3, 4, 5, 6, and 10). It is a question whether or not the higher number of different satellites in the seabuckthorn genome than in the majority of plant genomes (Mehrotra and Goyal 2014) somehow correlates with the presence of sex chromosomes representing a specific genomic context, each shaped by different evolutionary forces.

The localization of satellites is remarkable and shows that satellites are gathered not only on the nonrecombining region of the Y chromosome but some are specific for the X chromosome or for both sex chromosomes. They are gathered in heterochromatic parts of sex chromosomes what can reflect possible role of satellites in heterochromatinization. The list of chromosomal localization of satellites and TEs in dioecious plants was recently presented by Li et al. (2016a). Although Y chromosome divergence and specific repeat composition is a generally accepted feature, an accumulation of X-specific repeats during plant sex chromosome evolution has been suggested only by limited number of studies (Hobza et al. 2004). As satellites localized on either X or Y chromosomes are mutually different, we prefer the explanation that these satellites originated and expanded on the sex chromosomes long after the X–Y divergence. Therefore, it would be interesting to compare X and Y-linked variants of HRTR2 satellite and, if present, to assess the extent of X- and Y-linked satellite divergence.

The localization of transposable elements mainly in subtelomeres is a feature characteristic of the seabuckthorn genome. However, transposable elements are accumulated in subtelomeres in other plant species too (Zhang and Wessler 2004), and, among dioecious plants, subtelomeric localization was shown in Retand retrotransposon in Silene latifolia (Kejnovsky et al. 2006). Retrotransposons are found in or around centromeres as well (Miller et al. 1998; Neumann et al. 2011).

Our results clearly confirm the existence of the XY system in seabuckthorn found by Shchapov (1979) and they show that the Y chromosome is small and the X chromosome large. We mention in passing the work of Truta et al. (2011) who initially found a large Y chromosomes and small X chromosome in three Romanian seabuckthorn genotypes that later investigation of Romanian genotypes failed to confirm (Dr. Elena Truta, Institute of Biological Research Iasi, Romania, personal communication, June 15, 2016). Another cytogenetic study on seabuckthorn using C-banding that unfortunately showed only female karyotype without marking sex chromosomes (Rousi and Arohonka 1980).

Estimation of the age of sex chromosomes is not yet possible in this species because no X- and Y-linked genes are known. It remains a question whether the large size difference between X and Y chromosomes, the small size of the Y chromosome and accumulation of different satellites on both sex chromosomes indicates greater age of these sex chromosomes or not. It is remarkable that another genus of the Elaeagnaceae family—Shepherdia (Elaeagnaceae contains three genera—Elaeagnus, Hippophae, and Shepherdia) contains only three species that are all dioecious (Veldkamp 1986). Moreover, the Elaeagnaceae family belongs to the order of Rosales containing other plants with heteromorphic sex chromosomes like Humulus and Cannabis. Although karyotypes were described in Elaeagnus (2n = 28 in E. angustifolia) and Shepherdia (2n = 26 in S. argentea and 2n = 22 in S. canadensis), the sex chromosomes were not revealed (Rousi and Arohonka 1980). Therefore, it is not possible to draw conclusions about the formation or age of sex chromosomes during phylogeny.

The small Y chromosome containing several satellite DNA and a large X chromosome revealed in seabuckthorn resemble the mammalian sex chromosomal system. To the best of our knowledge, such a system is very rare among plants. Sex chromosomes in plants are mostly evolutionarily young—e.g., Silene latifolia (6 Ma, Kubat et al. 2014), Rumex acetosa (12–13 Ma, Navajas-Perez et al. 2005), or Coccinia grandis (3 Ma, Sousa et al. 2013)—and only sex chromosomes of Marchantia polymorpha are thought to be older (Yamato et al. 2007). A small Y chromosome and the large X chromosome were revealed in Humulus lupulus (Shephard et al. 2000; Karlov et al. 2003) and also in gymnosperm species Cycas revoluta (Segawa et al. 1971). The small size of the seabuckthorn Y chromosome may be caused by the loss of DNA which indicates that the Y chromosome could be in a shrinkage phase of evolution [reviewed in Hobza et al. (2015)] and thus could represent a rare example of an evolutionarily old plant sex chromosome. This assumption is supported by the FISH results which indicate that the large part of the Y chromosome arm that is homologous to the arm of the X chromosome, carrying HRTR8, was lost (fig. 7).

In this study, we developed and used a new bioinformatics approach for analysis of satellite DNA allowing prediction of satellite monomers, their grouping into clusters corresponding to main satellite families in the genome and visualization of their male/female homogeneity. This enabled prediction of satellite localization with respect to the sex determination system in species studied.

Supplementary Material

Supplementary data are available at Genome Biology and Evolution online.

Supplementary Material

Supplementary Data

Acknowledgments

This work was supported by the Czech Science Foundation (grant P501/12/G090). Access to computing and storage facilities owned by parties and projects contributing to the National Grid Infrastructure MetaCentrum, provided under the program “Projects of Large Infrastructure for Research, Development, and Innovations” (LM2010005), is greatly appreciated. The work of JP was supported by the Research and Application of Advanced Methods in ICT project (FIT-S-14-2299; http://www.fit.vutbr.cz/). The work of T.M. was supported from IT4Innovations excellence in science project (LQ1602). O.R., O.A., M.D., and G.K. were supported by Government Program supporting of the Leading Scientific Schools of Russian Federation (grant SS-8315.2016.11).

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