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1 DNA replication and chromosome positioning throughout the interphase in three- 2 dimensional space of plant nuclei 3 4 5 Němečková Alžběta, Veronika Koláčková, Vrána Jan, Doležel Jaroslav, Hřibová Eva* 6 7 8 Institute of Experimental Botany of the Czech Academy of Sciences, Centre of the Region 9 Hana for Biotechnological and Agricultural Research, Olomouc, Czech Republic 10 11 Short Title: DNA replication and interphase chromosome positioning 12 13 *Corresponding Author: 14 Eva Hřibová 15 Institute of Experimental Botany 16 Šlechtitelů 31 17 779 00 Olomouc 18 Czech Republic 19 Tel: +420 585 238 713 20 E-mail:[email protected] 21 22 E-mail addresses: 23 Němečková A. - [email protected] 24 Koláčková V. - [email protected] 25 Vrána J. - [email protected] 26 Doležel J. - [email protected] 27 28 29 Highlight 30 Telomere and centromere replication timing and interphase chromosome positioning in seven 31 grass species differing in genome size indicates a more complex relation between genome size 32 and the chromosome positioning. 33 34

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35 Abstract 36 Despite the recent progress, our understanding of the principles of plant genome organization 37 and its dynamics in three-dimensional space of interphase nuclei remains limited. In this 38 study, DNA replication timing and interphase chromosome positioning was analyzed in seven 39 Poaceae species differing in genome size. A multidisciplinary approach combining newly 40 replicated DNA labelling by EdU, nuclei sorting by flow cytometry, three-dimensional 41 immuno-FISH, and confocal microscopy revealed similar replication timing order for 42 telomeres and centromeres as well as for euchromatin and heterochromatin in all seven 43 species. The Rabl configuration of chromosomes that lay parallel to each other and their 44 centromeres and telomeres are localized at opposite nuclear poles, was observed in wheat, oat, 45 rye and barley with large genomes, as well as in Brachypodium with a small genome. On the 46 other hand, chromosomes of rice with a small genome and maize with relatively large genome 47 did not assume proper Rabl configuration. In all species, the interphase chromosome 48 positioning inferred from the location of centromeres and telomeres was stable throughout the 49 interphase. These observations extend earlier studies indicating a more complex relation 50 between genome size and interphase chromosome positioning, which is controlled by factors 51 currently not known. 52 53 54 Introduction 55 One of the exciting features of eukaryotic genomes is their organization in three- 56 dimensional space of cell nuclei and changes during cell cycle. The chromatin organization 57 and positioning of individual chromosomes in the nucleus was a great enigma until recently. 58 Nevertheless, based on microscopic observations of dividing cells of Salamandra maculata 59 and Proteus anguinus, Carl Rabl predicted already in 1885 that the positioning of 60 chromosomes in interphase nuclei follows their orientation in the preceding mitosis 61 (Reviewed by Cremer et al., 2006). The hypothesis was confirmed later by cytogenetic 62 studies in human, animals as well as in plants with larger chromosomes, which demonstrated 63 that centromeric and telomeric regions cluster at opposite poles of interphase nuclei (Cremer 64 et al., 1982; Schwarzacher and Heslop-Harrison 1991; Werner et al., 1992). This arrangement 65 of chromosomes in interphase was dubbed the Rabl configuration. 66 Interestingly, a different arrangement of chromosomes in interphase nuclei was 67 observed in Arabidopsis thaliana, a model plant with small genome (1C~157 Mbp). Here, the 68 centromeres are located at the nuclear periphery, whereas the telomeres congregate around

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69 nucleolus (Armstrong et al., 2001; Fransz et al., 2002). Centromeric heterochromatin forms 70 dense bodies called chromocenters, while euchromatin domains form 0.2 - 2 Mb loops that 71 are organized into Rosette-like structures (Fransz et al., 2002; Pecinka et al., 2004; Tiang et 72 al., 2012; Schubert et al., 2014). 73 Some earlier studies suggested that chromatin arrangement in interphase nuclei of 74 wheat, oat and rice, and in particular the centromere - telomere orientation, may be tissue- 75 specific and cell cycle-dependent (Dong and Jiang 1998; Prieto et al., 2004; Santos and Show, 76 2004). While a majority of nuclei in somatic cells of rice (Oryza sativa), which has a small 77 genome (1C~490 Mbp, Bennett et al., 1976) lack Rabl configuration, chromosomes in the 78 nuclei of some rice tissues, e.g., pre-meiotic cells in anthers or xylem - vessel precursor cells 79 seem to assume the configuration (Prieto et al., 2004; Santos and Show 2004). Such 80 chromosome arrangement and orientation in somatic cell nuclei was also observed in other 81 plants with small genomes, including Brachypodium distachyon (1C~ 355 Mbp) (Idziak et al., 82 2015). Although the majority of cell types of small plant genomes of B. stacei (1C~276 Mbp, 83 Catalán et al., 2012) and B. hybridum (1C~619 Mbp, Catalán et al., 2012) did not show Rabl 84 configuration. On the other hand, recent study of Idziak et al. (2015) showed that Rabl 85 configuration is present in small proportion (13 and 17%) of root meristematic cells of both 86 these plant species. Rabl configuration was not observed also in plants with large genomes 87 such as Allium cepa (~ 1.5 Gb), Vicia faba (~ 15Gb), Solanum tuberosum (~ 900 Mbp) and 88 Pisum sativum (~ 3.9 - 4.4 Gb) (Rawlins et al., 1991; Fussell, 1992; Kamm et al., 1995; 89 Harrison and Heslop-Harrison, 1995). 90 Development of a three-dimensional fluorescence in situ hybridization (3D-FISH) 91 method provided an opportunity to map telomere positions at meiotic prophase in maize (Bass 92 et al., 1997), Arabidopsis and oat (Howe et al., 2013). In order to characterize changes in 93 chromosome positioning in 3D nuclear space throughout the interphase, cell nuclei at 94 particular cell cycle phase can be isolated using flow cytometry. Embedding flow-sorted 95 nuclei in polyacrylamide gel stabilizes their structure during the 3D-FISH procedure and 3D 96 images can be captured by confocal microscopy (Kotogány et al., 2010; Hayashi et al., 2013; 97 Bass et al., 2014; Koláčková et al., 2019). 98 Chromosome conformation capture (3C) technique (Dekker 2002) and its variants 99 offer an alternative approach to study spatial organization of chromatin in cell nuclei. So 100 called Hi-C method (Lieberman-Aiden et al., 2009) analyses contacts between DNA loci 101 across the whole genome. The contact maps thus obtained enable the analysis of chromosome 102 contact patterns, genome packing and 3D chromatin architecture (Dong et al., 2018; Kempfer

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103 and Pombo, 2019). It should be noted, however, that Hi-C identifies genome loci that are 104 associated in 3D space, but it does not provide the information on their physical position in 105 the nuclei. 106 In a majority of cases, spatial organization of chromatin in interphase nuclei revealed 107 by FISH corresponded to the results obtained by Hi-C (Sexton et al., 2012; Dong et al., 2017; 108 Mascher et al., 2017; Liu et al., 2017). The only exception was Arabidopsis thaliana where 109 Hi-C analysis did not confirm the Rosette-like organization of chromosomal domains. 110 According to Hi-C, telomeres of different chromosomes should cluster, but FISH studies 111 show telomeres located around nucleolus (Feng et al., 2014; Liu and Weigel et al., 2015). 112 Several investigations focused on organization of chromatin, its structure and changes 113 during cell cycle. In mammals, DNA sequences located at interior regions of nuclei are 114 replicated earlier, while late replication occurred mostly in nuclear periphery (Gilbert et al., 115 2010; Bryant and Aves 2011). Cell cycle kinetics and the progression of cells through S phase 116 can be followed in detail after labelling newly synthesized DNA by a thymidine analogue 5- 117 ethynyl-2´-deoxyuridine (EdU) and subsequent bivariate flow cytometric analysis of nuclear 118 DNA content and the amount of incorporated EdU (Mickelson-Young et al., 2016). EdU- 119 labelled nuclei can be sorted by flow cytometry and used as templates for FISH to analyze 120 replication timing of particular DNA sequences and their positioning in 3D nuclear space 121 (Hayashi et al., 2013; Bass et al., 2014; Bass et al., 2015; Dvořáčková et al., 2018). 122 Using 3D microscopy to analyze DNA replication dynamics in maize root meristem 123 cells, Bass et al. (2015) observed distinct patterns of EdU signal distribution during early and 124 middle S phase. In early S phase, DNA replication primarily occurred in regions characterized 125 by weak DAPI fluorescence, while replication in middle S phase correlate with strong DAPI 126 signals. Authors also revealed that knob regions and centromeric regions associated with 127 heterochromatin were replicated during late S phase. Based on their findings they proposed 128 “mini-domain model”, when gene islands are replicated in early S phase, and blocks of 129 repetitive DNA in middle S phase (Bass et al., 2015). 130 DNA replication dynamics was analyzed in detail by microscopy in several plant 131 species, including monocot species maize and barley, and dicots Arabidopsis and field bean 132 (Jasencakova et al., 2001; Bass et al., 2014; Jacob et al., 2014; Robledillo et al., 2018). In 133 general, different stages of S phase contrasted in DNA replication pattern. The early S phase 134 was characterized by weak dispersed signals of EdU, speckled signals of EdU were typical for 135 late S phase, while the nuclei in the middle S phase were all covered with EdU signals, except

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136 of nucleolar area (Jasencakova et al., 2001; Kotogány et al., 2010; Bass et al., 2014; Bass et 137 al., 2015;). 138 Interestingly, the dynamics of interphase chromosome positioning during cell cycle in 139 plants was not studied to date. In order to fill this gap, we characterized chromosome 140 positioning in 3D nuclear space during cell cycle and identified replication timing of DNA in 141 centromeric and telomeric regions. To do this, we labelled newly synthesized DNA by EdU, 142 followed cell cycle kinetics by flow cytometry and performed 3D immuno-FISH on flow- 143 sorted nuclei. Chromosome positioning was analyzed in interphase nuclei of root meristem 144 cells in seven Poaceae species differing in nuclear genome size. Confocal microscopy of 145 nuclei embedded in polyacrylamide gel allowed us to locate centromeres and telomeres in 3D 146 space and to analyze replication timing of these chromosome regions. The results provided 147 new information on chromosome positioning and spatio-temporal pattern of DNA replication 148 of the important chromosome domains. 149 150 Keywords DNA replication, EdU labelling, flow cytometry, Poaceae, Rabl configuration, S phase, 151three- dimensional fluorescence in situ hybridization (3D-FISH) 152 153 154 Abbreviations

155 3D – three-dimensional 156 CenH3 – centromere-specific variant of histone H3 157 BrdU – 5-bromo-2'-deoxyuridine 158 EdU – 5-ethynyl-2'-deoxyuridine 159 FISH – fluorescence in situ hybridization 160 PAA – polyacrylamide 161 rDNA – ribosomal deoxyribonucleic acid 162 ROI – region of interest 163 RT – room temperature 164 165 Material and methods 166 Plant material and seeds germination 167 Plants used in the present study included wheat (Triticum aestivum L.) cultivar Chinese 168 Spring (2n=2x=42), oat (Avena sativa L.) cultivar Atego (2x=2x=42), barley (Hordeum

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169 vulgare L.) cultivar Morex (2n=2x=14), rye (Secale cereale L.) cultivar Dánkowskie Diament 170 (2n=2x=14), rice (Oryza sativa L.) cultivar Nipponbare (2n=2x=24), maize (Zea mays L.) line 171 B73 (2n=2x=20) and Brachypodium distachyon L. cultivar Bd21 (2n=2x=10). All seeds were 172 obtained from IPK Genebank (Gaterleben, Germany) except for rice, which was kindly 173 provided by prof. Takashi Ryu Endo (Kyoto University, Kyoto, Japan) and wheat, which was 174 obtained from Wheat Genetics & Genomic Resources Center (Kansas state university, 175 Manhattan, KS 66506). All seeds were germinated in a biological incubator at 24 °C in glass 176 Petri dishes on moistened filter paper until the primary roots were 2.5 – 4 cm long. 177 178 EdU labelling of replicating DNA 179 Young seedlings were incubated in 20 µM 5-ethynyl-2′-deoxyuridine (EdU) (Click-iT™ EdU 180 Alexa Fluor™ 488 Flow Cytometry Assay Kit, ThermoFisher Scientific/Invitrogen, Waltham,

181 Massachusetts, USA) made in ddH2O for 30 min at 24 °C. Root tips were excised and fixed in 182 2% (v/v) formaldehyde in Tris buffer for 20 min at 4°C, and washed 3 times in Tris buffer at 183 4°C (Doležel et al., 1992). About ten root tips with incorporated EdU were treated in 0.5 ml 184 Click-iT reaction cocktail (Click-iT™ EdU Alexa Fluor™ 488 Flow Cytometry Assay Kit) 185 prepared according to the manufacturer instructions, incubated for 10 min under vacuum, 186 followed by incubation in the dark for 45 min at room temperature (RT). After the labelling, 187 roots were washed 3 times for 5 min in phosphate buffered saline (PBS; 10 mM Na HPO , 2 4 188 2mM KH PO , 137mM NaCl, 2.7mM 2 4 189 KCl, pH 7.4) on a rotating shaker (160 rpm) at RT. 190 The root tips were mounted in 0.1 % agarose made in re-distilled water onto cavity 191 microscopic slides (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) and 192 mounted in Vectashield with DAPI (Vector Laboratories, Ltd., Peterborough, UK) to 193 counterstain the chromosomes. The preparations were imaged using a Leica TCS SP8 STED 194 3X confocal microscope (Leica Microsystems, Wetzlar, Germany) with 10x/0.4 NA Plan- 195 Apochromat objective (z-stacks, pinhole Airy). Image stacks were captured separately for 196 DAPI using 405 nm laser and for EdU labelled by Alexa Fluor 488 using 488 nm laser, and 197 appropriate emission filters. Typically, image stacks of 36 slides in average with 138 µm 198 spacing were acquired. Finally, maximum intensity projections were done using Leica LAS-X 199 software and final image processing was done using Adobe Photoshop 6 (Adobe Systems). 200 201 EdU labelling of replicating DNA and nuclei preparation for flow cytometry

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202 Roots of young seedlings were incubated in 20 µM EdU made in ddH2O (Click-iT™ EdU 203 Alexa Fluor™ 488 Flow Cytometry Assay Kit) for 30 min at 24 °C. Suspensions of intact 204 nuclei were prepared according to Doležel et al. (1992). Briefly, ~1 cm-long root tips were

205 cut and fixed with 2% (v/v) formaldehyde in Tris buffer (10 mM Tris, 10 mM Na2EDTA, 100 206 mM NaCl, 0.1% Triton X-100, 2 % formaldehyde, pH 7.5) at 4°C and washed three times 207 with Tris buffer at 4°C. Meristematic parts of root tips (~1 mm-long) were excised from 70 208 roots per sample in barley, oats, wheat, rye, and from 100 roots in rice and Brachypodium. 209 Root meristems were homogenized in 500 µl LB01 buffer (Doležel et al., 1989) by Polytron 210 PT 1200 homogenizer (Kinematica AG, Littua, Switzeland) for 13 s at 10 000 – 24 000 rpm 211 depending on a species. For maize, 50 root meristems were chopped using a razor blade 212 according to Doležel et al. (1989). The crude homogenates were passed through 50 µm nylon 213 mesh and nuclei were pelleted at 500 g, at 4 °C for 10 min. The pellet was resuspended in 0.5 214 ml Click-iT reaction cocktail prepared according to the manufacturer instructions and the 215 nuclei were incubated in the dark at 24 °C for 30 min. Then the nuclei were pelleted at 500 g 216 for 10 min, resuspended in 500 µl of LB01 buffer and stained by DAPI (0.2 µg/ ml final 217 concentration). Finally, the suspensions were filtered through a 20 µm nylon mesh and 218 analyzed using FASCAria II SORP flow cytometer and sorter (BD Bioscience, San Jose, 219 USA) equipped with a UV (355 nm) and blue (488 nm) lasers. Nuclei representing different 220 phases of cell cycle were sorted into 1x meiocyte buffer A (1x buffer A salts, 0.32M sorbitol, 221 1x DTT, 1x polyamines) (Bass et al., 1997; Howe et al., 2013).

222 223 Nuclei mounting in polyacrylamide gel 224 Flow sorted nuclei were mounted in 5% polyacrylamide (PAA) gel as described by Howe et 225 al. (2013) and Bass et al. (1997) with minor modifications. Briefly, 500 µl PAA mix 226 containing 15 % (w/v) acrylamide/bisacrylamide (akrylamid/bisakrylamid 30% NF 227 ROTIPHORESE (29 : 1) Roche Applied Science, Penzberg, Germany), 1x Buffer A salts (10 228 x buffer contains 800 mM KCl, 200 mM NaCl, 150 mM PIPES, 20 mM EGTA, 5 mM 229 EDTA, 1 M NaOH, pH 6.8), 1x polyamines (1,000 x polyamines contains 0.15 M spermine 230 and 0.5 M spermidine), 1x dithiothreitol (1,000 x dithiothreitol contains 1.0 M DTT, 0.01 M

231 NaOAc), 0.32 M sorbitol and 99 µl ddH2O was rapidly combined with 25 µl of freshly

232 prepared 20% ammonium sulfate in ddH2O and 25 µl 20% sodium sulfate (anhydrous) in

233 ddH2O. One volume of activated PAA gel mix was mixed with two volumes of flow-sorted 234 nuclei on a microscopic slide coated with aminoalkylsilane (Sigma-Aldrich, Darmstadt,

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235 Germany) and stirred gently with the pipette tip. The PAA drop was covered with a clean 236 glass coverslip and let to polymerize at 37°C for ~40 min. The coverslip was removed and

237 silane-coated slides with acrylamide pad were washed three times with 1x MBA in Coplin jar 238 to remove unpolymerized acrylamide. 239 240 Immuno-staining and fluorescence in situ hybridization (FISH) 241 In order to visualize centromeric regions, slides with PAA pad were washed in blocking 242 buffer (phosphate buffer, 1% Triton X-100, 1 mM EDTA) at RT for 1 h, after which 100 µl of 243 blocking buffer was added per slide and covered with parafilm for 10 min. Next, 50 µl of 244 diluted anti-OsCenH3 primary antibody (1:100) (Nagaki et al., 2004) was added, covered 245 with parafilm and incubated at 4°C in a humid chamber for 12 hrs. The slides were then 246 washed in a wash buffer (phosphate buffer, 0.1% Tween, 1 mM EDTA) three times for 15 247 min and once for 10 min in 2x Saline-Sodium Citrate buffer (2x SSC) and fixed in 1% (v/v) 248 formaldehyde in 2 x SSC for 30 min at RT. After the fixation, slides were washed 3 x 15 min 249 in 2x SSC at RT and used for FISH. 250 Hybridization mix (35 µl) containing 50% formamide, 2x SSC and 400 ng of directly

251 labelled telomere oligo-probe ([CCCTAAA]4) was added onto a slide and covered by a glass 252 coverslip. The slides were denatured at 94 °C for 6 min and incubated in a humid chamber at 253 37 °C overnight. Post-hybridization washing steps comprised of 5 min wash in 2x SSC, 254 stringent washes 2 x 15 min in 0.1 x SSC at 37°C, 2 x 15 min in 2x SSC at 37 °C, 2 x 15 min 255 2x SSC at RT, and 1 x 15 min in 4x SSC at RT. After washing, 100 µl of secondary antibody 256 diluted 1:250 in blocking buffer (5 % bovine serum albumin with 0.1 % Tween dissolved in 257 phosphate buffer, secondary antibody anti-Rabbit Alexa Fluor 546 (ThermoFisher 258 Scientific/Invitrogen) was added, covered with parafilm and incubated at 37 °C for 3h. 259 Finally, the preparation was washed in 4x SSC (3 x 15 min, RT) and in phosphate buffer (3 x 260 15 min, RT). PAA pad was mounted in 30 µl of Vectashield with DAPI (Vector 261 Laboratories), covered with a glass coverslip, and sealed with nail polish. 262 263 Confocal microscopy and image analysis 264 Images were acquired using Leica TCS SP8 STED 3X confocal microscope (Leica 265 Microsystems) equipped with 63x/1.4 NA Oil Plan-Apochromat objective (z-stacks, pinhole 266 Airy) equipped by Leica LAS-X software with Leica Lightning module. Image stacks were 267 captured separately for each fluorochrome using 647 nm, 561 nm, 488 nm, and 405 nm laser 268 lines for excitation and appropriate emission filters. Typically, image stacks of 100 slides on

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269 average with 0.2 µm spacing were acquired. 3D models of microscopic images, colocalization 270 analysis and volume calculations were performed using Imaris 9.2 software (Bitplane, Oxford 271 Instruments, Zurich, Switzerland). To estimate colocalization signals, Imaris software’s 272 ‘Colocalization’ function based on Pearson’s correlation coefficient were used (Manders et 273 al., 1992). The region of interest (ROI) was individually determined for each nucleus and 274 each channel. Importantly, setting ROI ensures, the ‘layer by layer’ correlation, so negative 275 colocalization of green channel of EdU signals representing another layer is prevented. The 276 volume of each nucleus and EdU signals were detected based on primary intensity of 277 fluorophores obtained after microscopic analysis. Imaris function ‘Surface’ and ‘Spot 278 detection’ was used for modeling the centromere - telomere arrangements. Chanel contrast 279 was adjusted using ‘Chanel Adjustment’ and videos were created using ‘Animation function’. 280 Between 100 and 150 nuclei were analyzed per each plant species. 281 282 283 Results 284 As we have studied plant species differing in genome sizes and root morphology, 285 experimental protocols had to be individually optimized. While EdU concentration and 286 incubation times were the same for all species, microscopic analysis of root tips after EdU 287 labelling revealed differences in the area of meristematic zones (Fig. 1). This information was 288 useful for the excision of meristematic regions to prepare suspensions of meristem cell nuclei. 289 Other critical step of the preparation of nuclei suspensions was the extent of mechanical 290 homogenization, which affected the nuclei integrity and yield (Table 1). The thick maize roots 291 were chopped in nuclei isolation buffer using a razor blade to obtain sufficient amounts of 292 nuclei suitable for flow cytometry. 293 DNA replication kinetics in interphase nuclei of root tip meristems during cell cycle 294 was evaluated after EdU incorporation into replicating DNA. Bivariate flow cytometric 295 analysis EdU vs. DAPI fluorescence resulted in typical “horseshoe” dotplot patterns, which 296 made it possible to unambiguously distinguish the nuclei at G1 and G2 phases of the cell 297 cycle and in early, middle and late S phase (Fig. 2). Fluorescent detection of incorporated 298 EdU was useful not only for flow cytometric analysis and nuclei sorting, but also for 299 microscopy. 300 301 DNA replication kinetics

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302 Microscopic analysis of EdU fluorescence in cell nuclei revealed different replication 303 patterns specific for early, middle and late S phase. Weak discrete signals were typical for 304 early DNA replication stage, while speckled signals concentrated in particular areas were 305 characteristic for late DNA replication stages. Strong signals dispersed throughout the whole 306 nuclei were observed in nuclei at middle S phase (Fig. 3). Overlaps between heterochromatin 307 regions and EdU signals at late S phase indicated that these regions were replicated later than 308 euchromatin. EdU signals were also detected inside nucleoli as discrete and well visible spots 309 during early and middle S phase. Clear signals were also seen at the periphery of nucleoli at 310 middle and late S phase (Supplementary Fig. 1), suggesting the replication of 45S rDNA loci. 311 312 Replication timing of centromeric and telomeric regions 313 Replication timing of centromeric and telomeric regions was determined after 314 microcopy of nuclei at different stages of S phase and based on the overlap of EdU and 315 immunofluorescent signals at centromeric regions and FISH signals at telomeric regions (Fig. 316 4). Although the replication of centromeric regions initiated at early S phase, the highest 317 number of centromeric regions underwent replication during middle and late S phase (Fig. 4, 318 5, Supplementary Fig. 2, 3, 4). In contrast, prevalent replication of telomeric sequences was 319 observed during early and middle S phase. This replication pattern of both chromosome 320 domains was observed in all species (Fig. 4, 5, Supplementary Fig. 5, 4, 7) except of rye, 321 where a minor difference was observed in replication dynamics of telomeric sequences. 322 Telomeric regions of rye comprise large heterochromatic blocks (Appels et al., 1978; 323 Evtushenko et al., 2016) and our results showed, that DNA loci closely connected to 324 heterochromatic block (probably flanking regions) were replicated at late S phase, while 325 telomeres located out of heterochromatin were replicated earlier, during middle S phase (Fig. 326 4B, D, E). 327 The difference in phasing centromere and telomere replication had an impact on the 328 overall replication pattern of S phase nuclei as highlighted by EdU (Supplementary videos 1, 329 2, 3). The first replication signals in the nuclei were concentrated at telomeric regions, while 330 the opposite pole of nucleus where centromeres were localized lacked EdU signals. In 331 analogy, heterochromatin blocks and regions around centromeres replicated during late S 332 phase, whereas the telomeres at the opposite pole of nuclei lacked the replication signals. A 333 majority of nuclear DNA replicated during middle S phase, resulting in strong EdU signals 334 spread across the whole nuclear volume (Supplementary Video 4, 5, 6). The images captured 335 in rye are shown in Figure 4; supplementary Figures 2 – 7 show the images captured in the

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336 remaining species. The analysis of FISH signals by the Imaris software (Fig. 5) showed that 337 co-localization between EdU and centromere fluorescence channels increased linearly from 338 early S phase to middle and late S phase. 339 340 Chromosome positioning in interphase nuclei 341 The localization of centromeres and telomeres during the course of S phase was used 342 to infer chromosome positioning in interphase nuclei. In total, we analyzed 700 nuclei (100 343 nuclei for each species) with spherical shapes which are typical for the meristem cells of the 344 root meristems. We confirmed regular Rabl configuration, when centromeres and telomeres 345 localize at opposite nuclear poles, in large genomes of wheat, oat, rye, barley, as well as in 346 Brachypodium distachyon with a small genome. On the other hand, chromosomes of rice with 347 a small genome and maize with relatively large genome did not assume proper Rabl 348 configuration (Fig. 6). The given interphase chromosome arrangement was stable throughout 349 the interphase in all species (Fig. 7, Supplementary Fig. 8, Supplementary Video 1 – 12). 350 In a majority of species, the number of fluorescence signals from centromeres and 351 telomeres corresponded to the number of mitotic chromosomes. The only exception was rice, 352 where the Rabl configuration was not observed. Here, the telomeric and centromeric signals 353 constituted large clustered signals randomly distributed in the nucleoplasm (Fig. 6, 354 Supplementary Video 7, 8, 9). In maize, the centromeres clustered in one region of nuclei, but 355 telomeres were randomly dispersed over the whole nucleoplasm (Fig. 6, Supplementary 356 Video 10, 11, 12). 357 358 Discussion 359 There is a growing interest to understand the principles of genome organization and its 360 dynamics in three-dimensional space of interphase nuclei at various levels: from DNA fibers 361 up to individual chromosomes and their domains. A range of studies focused on interphase 362 chromosome positioning in plants (e.g. Pecinka et al., 2004; Idziak et al., 2015). However, 363 except a study on maize (Bass et al., 2015), chromosome positioning was not followed 364 throughout the interphase, from G1 to G2 phase of the cell cycle. In order to fill these gaps, 365 we analyzed spatiotemporal patterns of DNA replication and positioning of interphase 366 chromosomes during cell cycle in root tip meristems of seven Poaceae species. They included 367 important and evolutionary related crops differing in genome size and Brachypodium 368 distachyon, a model wild species with a small genome.

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369 A popular approach to study patterns of DNA replication in different parts of cell 370 nuclei was microscopic detection of thymidine analogues incorporated into the newly 371 synthesized DNA (Gilbert et al., 2010; Bryant and Aves 2011; Bass et al., 2014; Bass et al., 372 2015). In mammals, early replication was observed in the interior regions of nuclei, while late 373 replication occurred mostly at nuclear periphery (Li et al., 2001; Pope and Gilbert 2013). In 374 plants, differences in replication patterns at early, middle and late S phase were revealed by 375 fluorescently labelled thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU) (Hayashi et al., 376 2013; Bass et al., 2014; Bass et al., 2015; Dvořáčková et al., 2018). However, with a few 377 exceptions, the patterns of nuclear DNA replication were analyzed only in plants with small 378 and moderate genome sizes, such as Arabidopsis, rice and maize (Hayashi et al., 2013; Bass 379 et al., 2014; Bass et al., 2015; Dvořáčková et al., 2018). 380 Earlier, Cortes et al. (1980) used thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) 381 in Allium cepa to observe a high coincidence between constitutive heterochromatin, including 382 pericentromeric regions and late replicating DNA, which possesses a large genome. In barley, 383 another species with a large genome, Jasencakova et al. (2001) found that DNA replication 384 started at rDNA loci, continued at euchromatin and centromeric regions and was completed at 385 pericentromeric heterochromatin. Our observations obtained after EdU labelling of newly 386 replicated DNA agree with the results obtained in maize by Bass et al. (2015). Early S phase 387 nuclei were characterized by localized weak signals, strong signals dispersed throughout the 388 whole nuclei were observed in the nuclei at middle S phase and speckled signals concentrated 389 in particular areas were observed at late DNA replication stages. As we studied seven species 390 differing considerably in genome size, our results indicate that this pattern of DNA replication 391 is general and does not depend on the amount of nuclear DNA. 392 We combined EdU labelling with the localization of telomere and centromere regions 393 by FISH to provide a more detailed view on DNA replication kinetics. We observed opposite 394 replication timing of telomeres and centromeres in all seven plant species, where the highest 395 intensity of early replication was observed in gene-dense chromosome termini, while the 396 highest intensity of late replicating DNA was typical for pericentromeric regions. In middle S 397 phase, replication was almost evenly dispersed along the entire chromosomes and only 398 slightly increased in the interstitial regions of chromosome arms. These findings confirmed 399 the recent results made in Arabidopsis and maize obtained by Repli-seq analysis and 400 corresponded to the gene density of their chromosome profiles (Wear et al., 2017, Zynda et 401 al., 2017; Concia et al., 2018). Kwasniewska et al. (2018) showed that terminal parts of 402 barley chromosomes replicated in early S phase, whole chromosomes were covered with EdU

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403 signal at middle S phase and centromeric parts of chromosomes were replicated in late S 404 phase. The authors reasoned that this chromosome replication profile implied presence of 405 transcriptionally active genes in the terminal parts of chromosomes and inactive 406 heterochromatin in the centromeric regions. 407 Two main types of arrangement were described for chromosomes in interphase nuclei 408 of plants: Rabl configuration and Rosette-like structure (Rabl 1885; Francz et al., 2002; Tiang 409 et al., 2012). The Rabl configuration, where centromeres and telomeres localize at opposite 410 nuclear poles is considered typical for plants with large genomes. However, this does not 411 seem to be a general rule and in some plants with relatively large genomes such maize and 412 sorghum, Rabl configuration was not confirmed (Anamthawat-Jónsson and Heslop-Harrison 413 1990; Schubert and Shaw 2011; Tiang et al., 2012). The Rosette structure, where the 414 centromeres are located at the nuclear periphery whereas the telomeres congregate around 415 nucleolus was described only in Arabidopsis (Armstrong et al., 2001; Fransz et al., 2002). 416 This work revealed a stable arrangement of centromeres and telomeres throughout the 417 interphase. Based on the positions of telomeres and centromeres, we confirmed Rabl 418 configuration in Brachypodium distachyon with a small genome, and in barley, rye, oat and 419 wheat with large genomes. We also confirmed, that chromosomes in rice with a small genome 420 and maize with a moderate genome did not assume Rabl configuration, confirming the results 421 of Dong and Jiang (1998) and Santos and Shaw (2004) obtained by FISH on interphase 422 nuclei. Some of the centromeric signals clustered at specific region of nucleoplasm, indicating 423 a tendency to Rabl-like polarized organization, but telomeric signals were dispersed. One 424 reason for the irregular distribution of rice and maize interphase chromosomes could be the 425 presence of acrocentric chromosomes as hypothesized by Idziak et al., (2015) who observed 426 disrupted Rabl configuration in Brachypodium stacei and B. hybridum whose karyotypes 427 comprise acrocentric chromosomes. 428 To conclude, our study indicates that spatiotemporal pattern of DNA replication 429 timing during S phase in plants is conserved and does not depend on the amount of nuclear 430 DNA. While the positioning of interphase chromosomes is stable throughout cell cycle, there 431 seems to be more complex relation between interphase chromosome positioning and genome 432 size. The observations by other authors that chromosome positioning may differ between 433 tissues and even within tissue of the same plant indicates that interphase chromosome 434 configuration is not a simple consequence of chromosome orientation in the preceding mitosis 435 and that it is controlled by so far unknown factors. 436

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437 438 Acknowledgements 439 We are grateful to Dr. Kateřina Malínská for advice on confocal microscopy and we thank 440 Ms. Zdeňka Dubská and Bc. Jitka Weiserová for excellent technical assistance. We 441 acknowledge the core facility CELLIM of CEITEC, which has been supported by the Czech- 442 BioImaging large RI project funded by MEYS CR, grant award LM2015062 for providing 443 imaging facility. This work was supported by the Czech Science Foundation (grant award 17 - 444 14048S) and by the ERDF project "Plants as a tool for sustainable global development" (grant 445 award CZ.02.1.01/0.0/0.0/16_019/0000827). 446 447 Literature 448 Anamthawat-Jónsson K, Heslop-Harrison JS. 1990. Centromeres, telomeres and chromatin in 449 the interphase nucleus of cereals. Caryologia 43, 205-213.

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597 598 Legends to figures 599 Table 1 600 Parameters for nuclei suspension preparation after EdU pulse for individual species. 601 602 Figure 1

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603 Root meristematic zones of seven Poaceae species. Roots were incubated with 20 mM EdU 604 for 30 min and EdU incorporated into replicating DNA was detected by Alexa Fluor 488 605 (green color). Nuclei were stained with DAPI (blue color). 606 607 Figure 2 608 Bivariate flow cytometric analysis of cell cycle in rye (A) and rice (B). Roots of young 609 seedlings were incubated with 20 mM EdU for 30 min. EdU in isolated nuclei was detected 610 by Alexa Fluor 488 (green color) and their DNA was stained with DAPI (blue). x axis 611 represents relative DNA content estimated as the intensity of DAPI fluorescence (linear 612 scale). y axis shows the extent of EdU incorporation into newly synthesized DNA quantified 613 by Alexa Fluor 488 fluorescence intensity (log scale). Red boxes in the dot plot show G1- and 614 G2-phase nuclei, green boxes highlight the early, middle and late S phase. 615 616 Figure 3 617 Maximum intensity projection of rye nuclei in 3D in different phases of cell cycle. EdU 618 (green color) was incorporated during a 30 min pulse into the newly synthesized DNA. G1 619 and G2 phases lack green signals of EdU. Changes in DNA replication pattern during early, 620 middle and late S phase are clearly visible. Note the replication of heterochromatin regions. 621 DNA was stained by DAPI (blue color). 622 623 Figure 4 624 DNA replication in rye nuclei and replication timing of centromeric and telomeric sequences. 625 Colocalization of signals specific to telomeres (red) and centromeres (pink) with EdU signals 626 corresponding to replicated DNA (green) was used to describe the pattern of replication 627 timing. In early S phase, centromeric signals (A) as well as telomeric signals which are 628 connected with heterochromatin regions (B, red rectangle) did not colocalize with EdU. 629 Telomeric sequences which were not localized in heterochromatin (B, white rectangle) co- 630 localized with EdU signals, pointing to ongoing replication process. Colocalization of EdU 631 and cetromeric signals is visible in middle and late S phase (C, D). Similarly, telomeric 632 sequences connected with heterochromatin regions (red rectangle) colocalized with EdU in 633 mid and late S-phase (E, G). 634 635 Figure 5

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636 Replication timing of centromeric and telomeric sequences. Replication time was obtained 637 after colocalization analysis and volume calculations of 3D models of microscopic images by 638 Imaris 9.2 software. The columns represent percent of colocalized volume of signals from 639 telomeric and centromeric regions and EdU signals. 640 641 Figure 6 642 Chromosome positioning in G1 nuclei estimated based on the positions of telomeres and 643 centromeres. Centromeres were labelled using CenH3 antibody (yellow), telomeres were 644 visualized by FISH with an oligonucleotide probe (red color). Nuclear DNA was stained with 645 DAPI (blue color). 646 647 Figure 7 648 Orientation of chromosomes in interphase nuclei of rye. Centromeres were labelled using 649 CenH3 immunostaining (yellow), telomeres were visualized by FISH with oligonucleotide 650 probe (red color). Nuclear DNA was stained with DAPI (blue). 651 652 653 Supplementary data 654 655 Supplementary Figure S1 656 Replication of 45S rDNA in oat. EdU signals (green) and 45S rDNA FISH signals (red) 657 detected inside the nucleoli (white dashed lines) at early and middle S phase and in the 658 periphery of nucleoli at middle and late S phase. 659 660 Supplementary Figure S2 661 Non-colocalization of CenH3 in early S phase in seven species. Immunofluorescent detection 662 of CenH3 (pink) and EdU signals corresponding to replicating DNA (green) were visualized 663 in Brachypodium distachyon (A), Oryza sativa (B), Zea mays (C), Hordeum vulgare (D), 664 Secale cereale (E), Avena sativa (F) and Triticum aestivum (G). Non-colocalized signals are 665 shown in white rectangles. 666 667 Supplementary Figure S3 668 Colocalization of CenH3 in middle S phase in seven species. Immunofluorescent detection of 669 CenH3 (pink) and EdU signals corresponding to replicating DNA (green) were visualized in

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670 Brachypodium distachyon (A), Oryza sativa (B), Zea mays (C), Hordeum vulgare (D), Secale 671 cereale (E), Avena sativa (F) and Triticum aestivum (G). colocalization is shown by white 672 rectangles. 673 674 Supplemetary Figure S4 675 Colocalization of CenH3 in late S phase in seven selected species. Immunofluorescent 676 detection of CenH3 (pink) and EdU signals corresponding to replicating DNA (green) were 677 visualized in Brachypodium distachyon (A), Oryza sativa (B), Zea mays (C), Hordeum 678 vulgare (D), Secale cereale (E), Avena sativa (F) and Triticum aestivum (G). colocalization is 679 shown by white rectangle, 680 681 Supplementary Figure S5 682 Colocalization of telomeres in early S phase in seven selected species. FISH signals specific 683 to telomeres (red) and with EdU signals corresponding to replicating DNA (green) were 684 visualized in Brachypodium distachyon (A), Oryza sativa (B), Zea mays (C), Hordeum 685 vulgare (D), Secale cereale (E), Avena sativa (F) and Triticum aestivum (G). colocalization is 686 shown by white rectangle and is visible as yellow color in merged pictures. 687 688 Supplementary Figure S6 689 Colocalization of telomeres in middle S phase in seven selected species. FISH signals specific 690 to telomeres (red) and with EdU signals corresponding to replicating DNA (green) were 691 visualized in Brachypodium distachyon (A), Oryza sativa (B), Zea mays (C), Hordeum 692 vulgare (D), Secale cereale (E), Avena sativa (F) and Triticum aestivum (G). colocalization is 693 shown by white rectangle and is visible as yellow color in merged pictures. 694 695 Supplementary Figure S7 696 Non-colocalization of telomeres in late S phase in seven selected species. FISH signals 697 specific to telomeres (red) and with EdU signals corresponding to replicating DNA (green) 698 were visualized in Brachypodium distachyon (A), Oryza sativa (B), Zea mays (C), Hordeum 699 vulgare (D), Secale cereale (E), Avena sativa (F) and Triticum aestivum (G). colocalization is 700 shown by white rectangle. 701 702 Supplementary Figure S8

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703 Models of 3D chromosome positioning in interphase nuclei of Brachypodium distachyon (A), 704 Oryza sativa (B), Zea mays (C), Hordeum vulgare (D), Secale cereale (E), Avena sativa (F) 705 and Triticum aestivum (G). The volume of nuclei (grey) was modeled based on the primary 706 intensity of DAPI staining. Telomeres (red) were detected based on the intensity of 707 fluorescently labelled telomeres and centromeres (yellow) were detected based on the primary 708 intensity of CenH3. 709 710 Supplementary Figure S9 711 Maximum intensity projection of 3D nuclei from different stages of interphase in 712 Brachypodium distachyon (A), Oryza sativa (B), Zea mays (C), Hordeum vulgare (D), Secale 713 cereale (E), Avena sativa (F) and Triticum aestivum (G). All nuclei were counterstand with 714 DAPI (blue) and early, middle and late phase shows replication pattern visualized by EdU 715 (green). 716 717 Supplementary Video 1 718 Barley nucleus in early S phase. Centromeres were visualized using immunolabelling CenH3 719 (yellow), telomeres were visualized after FISH with an oligonucleotide probe (red) and 720 replicating DNA was labelled with EdU (green). Nuclear DNA was stained with DAPI (blue). 721 722 Supplementary Video 2 723 Barley nucleus in middle S phase. Centromeres were visualized using immunolabelling 724 CenH3 (yellow), telomeres were visualized after FISH with an oligonucleotide probe (red) 725 and replicating DNA was labelled with EdU (green). Nuclear DNA was stained with DAPI 726 (blue). 727 728 Supplementary Video 3 729 Barley nucleus in late S phase. Centromeres were visualized using immunolabelling CenH3 730 (yellow), telomeres were visualized after FISH with an oligonucleotide probe (red) and 731 replicating DNA was labelled with EdU (green). Nuclear DNA was stained with DAPI (blue). 732 733 Supplementary Video 4 734 Rye nucleus in early S phase. Centromeres were visualized using immunolabelling CenH3 735 (yellow), telomeres were visualized after FISH with an oligonucleotide probe (red) and 736 replicating DNA was labelled with EdU (green). Nuclear DNA was stained with DAPI (blue).

23 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021857; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

737 738 Supplementary Video 5 739 Rye nucleus in middle S phase. Centromeres were visualized using immunolabelling CenH3 740 (yellow), telomeres were visualized after FISH with an oligonucleotide probe (red) and 741 replicating DNA was labelled with EdU (green). Nuclear DNA was stained with DAPI (blue). 742 743 Supplementary Video 6 744 Rye nucleus in late S phase. Centromeres were visualized using immunolabelling CenH3 745 (yellow), telomeres were visualized after FISH with an oligonucleotide probe (red) and 746 replicating was labelled with EdU (green). Nuclear DNA was stained with DAPI (blue). 747 748 Supplementary Video 7 749 Rice nucleus in G1 phase. Centromeres were visualized using immunolabelling CenH3 750 (yellow) and telomeres were visualized after FISH with an oligonucleotide probe (red). 751 Nuclear DNA was stained with DAPI (blue). 752 753 Supplementary Video 8 754 Rice nucleus in middle S phase. Centromeres were visualized using immunolabelling CenH3 755 (yellow), telomeres were visualized after FISH with an oligonucleotide probe (red) and 756 replicating DNA was labelled with EdU (green). Nuclear DNA was stained with DAPI (blue). 757 758 Supplementary Video 9 759 Rice nucleus in G2 phase. Centromeres were visualized using immunolabelling CenH3 760 (yellow) and telomeres were visualized after FISH with an oligonucleotide probe (red). 761 Nuclear DNA was stained with DAPI (blue). 762 763 Supplementary Video 10 764 Maize nucleus in G1 phase. Centromeres were visualized using immunolabelling CenH3 765 (yellow) and telomeres were visualized after FISH with an oligonucleotide probe (red). 766 Nuclear DNA was stained with DAPI (blue). 767 768 Supplementary Video 11 769 Maize nucleus in middle S phase. Centromeres were visualized using immunolabelling 770 CenH3 (yellow), telomeres were visualized after FISH with an oligonucleotide probe (red)

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771 and replicating DNA was labelled with EdU (green). Nuclear DNA was stained with DAPI 772 (blue). 773 774 Supplementary Video 12 775 Maize nucleus in G2 phase. Centromeres were visualized using immunolabelling CenH3 776 (yellow) and telomeres were visualized after FISH with an oligonucleotide probe (red). 777 Nuclear DNA was stained with DAPI (blue). 778

25 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021857; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021857; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021857; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021857; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021857; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021857; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021857; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021857; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Table 1: Parameters for the nuclei suspension preparation

Plant species Number of roots Homogenisation [rpm] Time [s] Brachypodium distachyon 100 10000 13 Oryza sativa 100 14500 13 Zea mays 50 Chopped by razor blade - Hordeum vulgare 70 14500 13 Secale cereale 70 14500 13 Avena sativa 70 24000 13 Triticum aestivum 70 20000 13