DISTINCTIVE SIRE1 RETROTRANSPOSON PATTERNS ON BARLEY CHROMOSOMES?

: SIRE1 is an active and relatively high copy-number retroelement belongs to the Tyl/Copia long terminal repeat (LTR) retrotransposon superfamily. Distinctive SIRE1 elements ( ENV and GAG ) distributions in barley genome were observed by using fluorescent in situ hybridization (FISH). We performed PCR to obtain tetramethylrhodamine-dUTP (TRITC)-labelled probes. Localizations of SIRE1 ENV and GAG domains were demonstrated under confocal microscope on Hordeum vulgare L. cv. Hasat root preparations. Our results revealed the distributions of SIRE1 elements ENV and GAG in barley genome. These results may provide to uncover the organization of SIRE retrotransposon pattern in barley genome. Özet: SIRE1 , Tyl/Copia Uzun Uç Tekrarlı (Long Terminal Repeats- LTR) retrotranspozon üst ailesine ait olan aktif, nispeten yüksek kopyalı bir retroementtir. Arpa genomundaki ayırt edici SIRE1 elementlerinin ( ENV ve GAG ) dağılımları floresan in situ hibridizasyonu (FISH) kullanılarak gözlemlendi. Tetramethylrhodamine-dUTP (TRITC)-işaretli probların elde edilmesinde PCR gerçekleştirildi. SIRE1 ENV ve GAG domainlerinin yerleşimleri, Hordeum vulgare L. cv. Hasat kök preparatlarında konfokal mikroskobu altında gösterildi. Sonuçlarımız, arpa genomundaki SIRE1 elementlerinin ENV ve GAG dağılımlarını göstermektedir. Bu sonuçlar, SIRE1 elementlerinin arpa genomunun organizasyonunun ortaya çıkarılmasına katkı sağlayacaktır.


Introduction
Transposable elements (TEs) are mobile genetic elements and are distributed throughout the plant genome, over the 14% of Arabidopsis thaliana (L.) Heynh. genome, over the 80% maize genome and 80.8% of barley genome consist of TEs (AGI 2000, Schnable et al. 2009, Mascher et al. 2017. TEs constitute DNA fragments which can move through the genome using a DNA intermediate (DNA transposons) or an RNA intermediate (retrotransposons). Retrotransposons encode their own proteins for replication and integration back into the genome can be subdivided into two main subclasses as LTR retrotransposons and non-LTR retrotransposons. Although both LTR and non-LTR retrotransposons are located on plant genomes, LTR retrotransposons are more abundant than non-LTR retrotransposons (Voytas & Boeke 2002, Sandmeyer et al. 2002, Schulman & Wicker 2013 and are divided into two main superfamilies known as Tyl/copia and Ty3/gypsy groups (Wicker et al. 2007).
Tyl/copia (Pseudoviridae) elements are prevalent in plants and grouped into three genera: Pseudovirus (Ty1), Hemivirus (Copia) and the Sirevirus which are closely related to the retroviruses, but have a different polyprotein (pol) gene order (Kumar & Bennetzen 1999, Llorens et al. 2009). Sireviruses are plant specific unique retrotransposons among Tyl/copia elements (Gao et al. 2003, Bousios et al. 2010. Sequence studies revealed that highly conserved sequence motifs of Sireviruses have been found within the extremely divergent non-coding part of the genome (Bousios et al. 2010 Mascher et al. (2017) revealed that 3.7 Gb (80.8%) of barley genome consists of transposable elements and that only 10% of them are intact and potentially active. They distinguished the seven barley chromosomes into three fractions as fractions 1, 2 and 3. In the proximal fraction 3 -where older transposable elements are diverged, unique and predominated-contains mostly repetitive 20-mers. Moreover, fraction 3 is favoured by Gypsy retrotransposons, while fractions 1 and 2 are populated by Copia elements. These differences in the relative abundance of retrotransposon families are considered as distinct distributions of functional domains. Additionally, Mariner transposons preferentially occupy within 1 kb up-or downstream of the coding regions of genes. However, Harbinger and long interspersed elements are observed to be located further away from genes. Selective pressures are considered to underlie the distribution of different types of transposable elements around genes. As expected, the smallest transposable elements such as Mariner may be more tolerated in regions close to the genes. However, intriguingly, the Harbinger superfamily elements possess a clear preference for promoter regions, while long interspersed elements preferentially possess for downstream regions of genes. Moreover, the large transposable elements such as LTR retrotransposons and CACTA elements are located at greater distances from genes (Mascher et al. 2017).
Fluorescence in situ hybridization (FISH) using target-specific DNA probes have become a routine technique in modern molecular biology, cell research and medical diagnosis (Hausmann & Cremer 2003). However, application of FISH has been shown to be more difficult, because of the cell wall, the cytoplasm, which prevents chromosome spreading, low metaphase indices and often similar chromosome morphology (Salvo-Garrido et al. used FISH analysis and YAC library to isolate Ror1 gene, which encodes Required for mlo-specified resistance genes and important for basal defence, due to fine mapping and gene synteny strategies. Additionally, BAC clones have also been used to demonstrate BARE1 patterns on barley chromosomes by FISH (Vicient et al. 1999).
The aim of this study was to present the localization of SIRE1 patterns in Hordeum vulgare L. chromosomes using labelled-PCR products via FISH. SIRE1 patterns were observed under confocal microscope on barley root preparations. Our results indicated the SIRE1 localizations in barley genome.

Plant materials
Hordeum vulgare L.cv. Hasat was provided from the Directorate of Trakya Agricultural Research Institute. The plants were grown in a growth chamber under 16 h light/8 h dark and 25°C ± 2°C conditions. Relative humidity was kept at 60-75%. After 72 hours, plants were harvested, directly treated with liquid nitrogen and then stored at -80°C until DNA extraction. The mixture was centrifugated at 16,000 g for 15 min. afterwards, the supernatant was extracted twice with chloroform. The aqueous phase was incubated with 2 volumes of CTAB precipitation solution, then the CTAB protocol was followed as previously described (Mafra et al. 2008). DNA yield and purity were measured by UV spectrophotometry at 230, 260 & 280 nm using a NanoDrop 2000c instrument (Thermo Scientific, Wilmington, DE, USA). DNA integrity was evaluated by agarose gel electrophoresis with which samples were separated on 1% agarose gels in 1X TAE buffer.

Chromosome preparation for FISH analysis
Seeds were placed randomly in Petri dishes containing filter paper soaked in only water to germinate in an incubator at 18-25°C in the dark for 3 days. Root tips were harvested, directly fixed in Carnoy fixative (3:1 ethanol:acetic acid solution) without any chemical pretreatment and stored at 4ºC. Chromosome preparations and FISH analysis were performed according to Jenkins & Hasterok (2001 with modifications. The slides were checked under the light microscope (Olympus U-TVO.5XC-3) and kept in a freezer at -20ºC.

Development of probes and labelling
The FISH probes used in this study were generated from two sets of data which are the ENV (envelope-like) and GAG (encoding a structural protein) genes of SIRE1.
To investigate the distribution of SIRE1, we amplified ENV and GAG domains of SIRE1 using designed specific primer sets ( Table 1). The probes for ENV and GAG domains were designated by using IDT's PrimerQuest © Tool (2012). GC% and Tm values of the probes were around 62 and between 47°and 55°C, respectively. The sequences of SIRE1 ENV and GAG were obtained from barley (KP420209 for GAG and KP420210 for ENV).
Probe synthesis was carried out individually by using SIRE1 ENV and GAG primers. The reactions were performed in a total volume of 50 μl including 18.25 μl nuclease-free dH2O, 25 μl of HotStart PCR Master Mix (Bio-Rad), 1.5 μl of each primer (10 μM/μl), 1.75 μl of tetramethylrhodamine-dUTP (TRITC) (1 mM), and 2 μl template DNA (40 ng/μl). PCR conditions were as follows: 94°C for 5 min followed by 40 cycles of 94°C for 25 s, annealing 50°C for 25 s and 72°C for 30 s. The reaction was completed by a final extension step at 72°C for 5 min.

Fluorescence in situ hybridization (FISH) analysis
The FISH analysis protocol was adopted from Jenkins and Hasterok (2001, 2007) protocol with modifications. Chromosome spreads were scanned under light microscope with ×40 objective to determine the number and quality of well-spread metaphase plates, and then they were treated with 100 μg/ml of RNase at 37°C for 1 h. The hybridization mixture contains 20 μl of deionised formamide (50%), 8 μl of dextran sulphate (10%), 4 μl of 20X SSC (2X SSC), 2 μl of 10% SDS (0.5%), 10 μl of probe (75-200ng/slide), 1 μl of blocking DNA (sonicated salmon sperm DNA) (25-100X probe) and added sterile dH2O to bring final volume 40 μl. Final concentrations were indicated in parenthesis. The mixture was denatured at 85°C for 10 min and kept on ice for 10 min. A 38 μl aliquot of the hybridization mixture was applied onto each slide, covered with a coverslip and sealed with paper bond. Both chromosomal DNA and probe DNA on the slides were denatured together in a thermal cycler at 70°C for 6 min and hybridized with each other at 37°C overnight in a humid dark box. Afterwards, hybridized the chromosome spreads were washed three times in 2X SSC: once 2X SSC to float coverslips off, once in 15% formamide/0.1X SSC and again once in 15% formamide/0.1X SSC, each for 10 min at 42°C. Then, the slides were washed in 2X SSC for 3 min at 42°C. This step was repeated twice with fresh 2X SSC at 42°C. Ultimately, the slides were washed three times in 2X SSC for 3 min at RT. Then, the slides were dehydrated in alcohol series (70, 90 and 100%), each for 1 min at RT and waited in the dark for 15-20 min. Vectashield-DAPI mounting-staining medium (7-10 μL) was dropped onto the chromosome spreads, which were then stored at 4°C until used.

Image acquisition
To image the slides, 551-575 nm wavelengths for probes labelled with TRITC and 420-480 nm wavelengths for DAPI were used for fluorescence detection in the Leica DM5500 confocal microscope. The different fluorescent images were acquired separately. Afterwards, they were merged into single composite images. The signal images were analysed by Adobe Photoshop CC 2014.

Results
To characterize the abundance and localization patterns of soybean SIRE1 ENV and GAG domains in barley, we used root tip cells and SIRE1 ENV and GAG probes were labelled with TRITC. Specifically, we observed TRITC labelled SIRE1 ENV exhibited characteristic patterns at prophase in nucleus (Fig. 1). TRITC labelled SIRE1 GAG probes also demonstrated distinctive patterns in prophase phase in barley chromosomes (Fig. 2).    Lee et al. 2013). In the current study, localizations of SIRE1 ENV and GAG domains were indicated on barley chromosomes (see Fig. 1 and 2). The results were obtained from Hordeum vulgare L. cv. Hasat root tips preparations using FISH analysis. Due to the large genome size of barley, researchers mostly prefer to use single-copy probes derived from BAC or YAC contigs (Vicient et al. 1999, Acevedo-Garcia et al. 2013, Bustamante et al. 2017). In our study, we used direct PCR products derived from barley DNA to produce singlestranded probes were small, appx. 233 bp for ENV and 120 bp for GAG. These small PCR probes were highly specific and stable for hybridization thus, it leads to very good amplification of the signals. By using short size of probes, we were able to visualize the first time SIRE1 ENV and GAG distribution patterns on barley chromosomes, indicating they intend to demonstrate different patterns. Additionally, it is possible to observe localization of lncRNAs on chromosomes and in the cells using direct labelled-PCR products via FISH techniques (Karlik et al. 2018).
In plant genomes, most of the TEs are amplified to thousands of copies and present as low-or moderate- The distribution of SIRE1 ENV and GAG barley were consistent with each other that they were mostly characterized close to the centromere regions (see Fig. 1 and 2). Presting et al. (1998) demonstrated the centromere of barley is enriched with cereba is a member of the Ty3/Gypsy class, that ~200 copies of cereba have been determined in each barley centromere (Hudakova et al. 2001, Houben & Schubert 2003. Two conserved centromere-specific repeats [Cereal Centromeric Sequence1 (CCS1) and Sau3A9] were reported, later is revealed that they are related to Ty3/Gypsy-like retrotransposons. In barley, parts of the LTRs of cereba element and parts of integrase region of its polygene correspond to CCS1 and Sau3A9, respectively (Hudakova et al. 2001). Our results also indicated that presence of SIRE1 is favoured mostly in regions close to centre locations that as we know SIRE1 is active and belongs to the Tyl/Copia retrotransposon superfamily (Lin et al. 2005). However, SIRE1 may be related to the centromeric repeats BCS1/CCS1 (barley cereal centromeric sequence), thus these elements may also contribute to SIRE1 elements movements. Lin et al. (2005) reported the presence of a 102-bp tandem repeat (STR102) which was a SIRE1 element and a SIRE1 solo LTR in soybean. However, the flanking sequences of 3 of 10 SIRE1 insertions were found to be repetitive, belonging to either Ty3/gypsy or other repetitive families (Laten et al. 1998). Moreover, these fragments have been demonstrated for maize and rice (Ananiev et al. 1998, Cheng et al. 2002 suggesting that they are common motifs of higher plants of centromeric/pericentromeric regions (Martinez-Zapater et al. 1986, Ananiev et al. 1998, Cheng et al. 2002, Lin et al. 2005, which is also consistent with our results. In soybean, Lin et al. (2005) also considered that SIRE1 may preferentially get in heterochromatic and/or pericentromeric regions which may alternatively be selected against the gene-rich euchromatic regions. In barley, we also observed that SIRE1 elements were mostly concentrated in centred regions. FISH analysis of barley SIRE1 elements provided us to allow cursory insight into the barley genome organization.
In conclusion, we were able to observe the distinctive distributions of the SIRE1 ENV and GAG elements via FISH analysis by using labelled-PCR products as probes in barley root preparations. How these elements function to organize genome is still a mystery, however the applications of FISH analysis of TEs may have an important potential to uncover the organization of higher plant genomes.