Nondisjunction and Unequal Spindle Organization Accompany the Drive of 2 Aegilops Speltoides B Chromosomes

bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Nondisjunction and unequal spindle organization accompany the drive of 2 Aegilops speltoides B chromosomes

3 4 DanDan Wu a,d, Alevtina Ruban a, Jörg Fuchs a, Jiri Macas b, Petr Novák b, 5 Magdalena Vaio c, YongHong Zhou d and Andreas Houben a,e 6 7 a Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, 8 06466 Stadt Seeland, Germany 9 b Biology Centre, Czech Academy of Sciences, Ceske Budejovice, Czech Republic 10 cDepartment of Plant Biology, College of Agronomy, 12900 Montevideo, Uruguay 11 d Triticeae Institute, Sichuan Agricultural University, 611130, Chengdu, China 12 e corresponding author, ORCID: 0000-0003-3419-239X; email: houben@ipk- 13 gatersleben.de; IPK Gatersleben, D-06466 Seeland, Germany 14 15 16 17

18 Abstract 19 Supernumerary B chromosomes (Bs), which are often preferentially inherited, 20 deviating from usual Mendelian segregation. This chromosome drive is one of the 21 most important features of Bs. Here we analyzed the drive mechanism of Aegilops 22 speltoides Bs and provide direct insight into its cellular mechanism. Comparative 23 genomics resulted in the identification of the tandem repeat AesTR-183 of Ae. 24 speltoides Bs, which also can be found on the Bs of Ae. mutica and rye, was used to 25 track Bs during microgametogenesis. Nondisjunction of CENH3-positive, tubulin 26 interacting B sister chromatids and an asymmetric spindle during first pollen grain 27 mitosis are likely components of the accumulation process. A quantitative flow 28 cytometric approach revealed, that independent on the number of Bs present in the 29 mother plant Bs accumulate in the generative nuclei with more than 93%. Nine of 30 eleven tested (peri)centromeric repeats were shared by A and B chromosomes. A 31 common origin of the drive process in Poaceae is likely.

32 bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

34 Keywords 35 supernumerary B chromosome, chromosome drive, chromosome nondisjunction, 36 centromere, pollen grain mitosis, chromosome segregation, asymmetric spindle, flow 37 cytometry, Aegilops speltoides 38 39

40 Introduction 41 Supernumerary B chromosomes (Bs) are considered as dispensable chromosomes, 42 which under standard conditions do not confer advantages on the organisms that 43 harbour them. Bs are found in thousands of fungi, plant and animal species and are 44 thought to stand for a specific type of selfish DNA (Jones and Rees, 1982). Recent 45 advances in deciphering the sequence composition of B chromosomes provided a 46 unique opportunity for the analysis of the origin and evolution of this enigmatic 47 genome component. It was shown that the Bs of several plant and animal species 48 harbour gene-derived sequences. This allowed to trace their origin from duplicated 49 fragments of multiple A chromosomes of their host species (reviewed in (Ruban et 50 al., 2017). Hence, Bs could be considered as a by-product of standard chromosome 51 (As) evolution. 52 53 Often the transmission rate of B chromosomes is higher than 0.5, not obeying the 54 Mendelian law of equal segregation. The resulting transmission advantage is 55 collectively referred to as ‘drive’. The maximum number of Bs tolerated by the host 56 varies between species and depends on a balance between the B chromosome 57 drive, and the negative effects, especially on fertility and vigour, caused by B 58 chromosomes if they occur in higher numbers (Bougourd and Jones, 1997). Among 59 the relatively small number of B chromosome-positive species that have been 60 investigated, relative to the total number of known species, there is a variety of 61 mechanisms of B chromosome drive that occurs before, during or after meiosis, while 62 in some cases drive has not been found (reviewed in (Austin et al., 2009; Burt and 63 Trivers, 2006; Carlson, 1988; Houben, 2017; Jones, 1991; Jones, 2018)). Besides 64 drive, the non-Mendelian inheritance of Bs could also be affected by mitotic and 65 meiotic instabilities. 66 bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

67 The drive of the rye (Secale cereale L.) B chromosome is one of the best-analyzed 68 accumulation mechanisms amongst supernumerary chromosomes. Rye Bs do not 69 separate at anaphase of the first pollen grain mitosis and mostly are included in the 70 generative nucleus. In the second pollen mitosis, the Bs segregate normally into both 71 sperm nuclei (Hasegawa, 1934). Nondisjunction of rye Bs is likely caused by 72 extended cohesion of the B sister pericentromeres. Because of unequal spindle 73 formation at the first pollen mitosis, nondisjoined B chromatids preferentially become 74 located toward the generative pole and get consequently included into the generative 75 nucleus. The failure to resolve pericentromeric cohesion is under the control of the B- 76 specific nondisjunction control region (reviewed in (Houben, 2017)). A comparable 77 accumulation mechanism takes place in the female gametophytes of rye 78 (Hakansson, 1948). 79 80 In Aegilops speltoides Tausch (syn. Triticum speltoides (Tausch) Gren.), an annual 81 diploid Poaceae (genome type: S), besides the seven pairs of A chromosomes, 82 plants with a maximum number of eight additional Bs were reported (Raskina et al., 83 2011). The particular number of Bs is constant in all aerial parts within the same 84 individual. Interestingly, B chromosomes are completely absent in the roots 85 (Mendelson and Zohary, 1972; Ruban et al., 2014). Based on FISH experiments 86 using Spelt I and 5S rDNA probes the B of Ae. speltoides is considered to be derived 87 from the A chromosomes 1, 4 and 5 (Belyayev and Raskina, 2013; Friebe et al., 1995; 88 Raskina et al., 2011). 89 90 Reciprocal crosses between 0B and +B plants indicated that the drive of Bs occurs 91 during the first mitosis in the male gametophyte while there is no accumulation of Bs 92 during the development of female gametophytes (Mendelson and Zohary, 1972). The 93 non-Mendelian transmission of Bs cannot be solely explained by irregular 94 segregation during meiosis. The net increase of B number in the progeny of 0B x 1B 95 crosses can only be accounted for the directed nondisjunction of the Bs to the 96 generative nucleus during first pollen mitosis as in rye. 97

98 Here we analyzed the drive of Ae. speltoides B chromosomes and provide direct 99 insight into its cellular mechanism. We first employed comparative genomics to 100 identify a B-specific repeat, which we used to track the B chromosomes during bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

101 microgametogenesis. It was found that sister chromatid nondisjunction and an 102 asymmetric spindle during first pollen grain mitosis are components of the B 103 chromosome accumulation process. To determine the B accumulation rate in sperm 104 nuclei, we developed a novel flow cytometric approach that allowed the differentiation 105 between vegetative and sperm nuclei as well as the quantification of B chromosomes 106 inside the nuclei. Utilizing this method, we found that independent of the number of B 107 chromosomes present in the mother plant, Bs accumulate in the generative nuclei to 108 more than 93%. The prerequisites for the drive process seems to be common in 109 Poaceae, thus enabling independent origins of Bs in different lineages within the 110 family.

111 112

113 Results

114 Identification of a conserved B-specific repeat suitable to trace B 115 chromosomes in Ae. speltoides, Ae. mutica and rye 116 To find a B-specific marker suitable to track segregation of Ae. speltoides Bs during 117 microgametogenesis by FISH, genomic 0B and +B DNA were paired-end Illumina 118 sequenced and the corresponding reads were characterized by comparative 119 similarity-based clustering using the RepeatExplorer pipeline (Novák et al., 2013). 120 Highly and moderately abundant repeats represented by clusters containing at least 121 0.01% of analyzed reads were found to make up about 77% of the genome. They 122 were mostly composed of LTR-retrotransposons, transposons and satellite repeats, 123 with overall proportions of individual repeat types being similar in the 0B/+B 124 genotypes (Suppl. Table 1). Comparative cluster analysis revealed four tandem 125 repeats with monomers ranging from 86 to 1090 bp, which were found to be 126 potentially enriched on B chromosomes (Table 1, Figure 1A-B). The B-specific 127 candidate repeats were PCR amplified from +B genotype, cloned and verified by 128 sequencing. In contrast to AesTR-183 and AesTR-205, AesTR-126 and AesTR-148 129 could also be amplified by using 0B genotype DNA as template (Suppl. Figure 1). 130 FISH with cloned probes resulted in an exclusively B-specific localization of AesTR- 131 183 and AesTR-205, while AesTR-126 and AesTR-148 signals were present on A 132 and B chromosomes (Figure1C-D). In all analyzed populations, a B-specific 133 distribution of AesTR-183 hybridization signals was found along the long B bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

134 chromosome arm, while AesTR-205 clusters occur on both arms (Figure 1D). Ae. 135 speltoides from the Katzir population possess in addition a morphological variant of 136 the B, presumably lacking the terminal region of the long arm (Ruban et al., 2014). In 137 this metacentric B variant, AesTR-183 repeats are present on both arms (Suppl. 138 Figure 2). 139 140 Notably, the tandem repeat AesTR-183 with a monomer length of 1090 bp displayed 141 also B-specific signals in the closely related species Aegilops mutica (Figure 1E). In 142 +B rye, AesTR-183 signals are localized in the nondisjunction-controlling region of 143 the B chromosome (Figure 1F). To test the presence of this repeat on the Bs of a 144 more distantly related grass species, AesTR-183 probe was hybridized to a Lolium 145 perenne accession carrying two Bs. However, no B-specific signal was found (data 146 not shown). Hence, three species from the Triticeae encode a conserved B-specific 147 repeat that can be used for the identification of Bs. BLAST (Altschul et al., 1990) 148 search revealed 82% similarity between a part of AesTR-183 and a 489 bp long 149 region on chromosome 1B of Triticum aestivum (accession LS992081.1). To estimate 150 the relationships of corresponding sequences, a phylogenetic tree was constructed 151 based on sequences of PCR amplified AesTR-183 monomers from Ae. speltoides, 152 Ae. mutica and rye, including the similar sequence from wheat chromosome 1B 153 (Suppl. Figure 3). The obtained phylogenetic tree placed the analyzed sequences in 154 three clades according to the species of origin. The much longer branch lengths in 155 the case of rye-derived sequences indicates higher substitution rate. Surprisingly, the 156 sequence from wheat chromosome 1B appeared as sister group to the rye B-located 157 sequences, while it would rather be expected to cluster together with Ae. speltoides- 158 derived sequences due to closer evolutionary relationships of wheat and Ae. 159 speltoides genomes (Bernhardt et al., 2017). Interestingly, the diversity of AesTR-183 160 repeat monomers in Ae. mutica was much lower than in Ae. speltoides and rye. 161 162

163 Flow cytometry enables to determine the accumulation of B chromosomes in 164 sperm nuclei 165 To quantify the accumulation of Ae. speltodies B chromosomes during pollen 166 development we applied a combination of flow cytometry, immunostaining and FISH. 167 Differences in size and shape of nuclei isolated from mature pollen grains (Figure 2A) bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

168 of a 0B plant allowed the separation of vegetative and sperm nuclei by plotting the 169 fluorescence intensity (DNA content) against the forward scatter signal (FSC) 170 representing particle size. The round-shaped and larger vegetative nuclei appear in 171 the upper fraction while the spindle-shaped smaller sperm nuclei were found in the 172 lower fraction (Figure 2B). To confirm the flow-cytometric separation of both types of 173 nuclei we sorted both fractions and used the nuclei for immunolabelling with an 174 antibody against histone H3K27me3 which was shown to be preferentially enriched 175 in vegetative nuclei in rye (Houben et al., 2011). Indeed, an intense immunolabelling 176 of nuclei was only observed on samples derived from the upper fraction representing 177 the vegetative nuclei (Figure 2C) but not on sperm nuclei (Figure 2D). Thus, both 178 types of nuclei can easily be differentiated flow cytometrically. 179 180 In nuclei suspensions of pollen derived from plants with Bs, additional nuclei 181 populations were observed in the dot plots (Figure 2E, Suppl. Figure 4B-G). 182 Depending on the number of Bs in the mother plant we detected one to three 183 additional nuclei populations at similar forward scatter signal intensities as detected 184 for the sperm nuclei of the 0B plants (Suppl. Figure 4A-H) but with a higher 185 fluorescence intensity indicating an increased DNA content due to the presence of 186 additional B chromosomes. The number of Bs represented by the individual 187 populations could be estimated by the distance between the fractions of vegetative 188 nuclei, which appeared unchanged, and the corresponding fraction of sperm nuclei. 189 In case of a 1B plant, one additional population of sperm nuclei appeared at a 190 position that indicates the presence of two B chromosomes. However, the majority of 191 sperms showed no Bs (Suppl. Figure 4B). This observation is explainable by the fact 192 that the B chromosome appears as a univalent during metaphase I leading either to 193 laggards mostly being excluded from the new daughter nuclei or to the migration of 194 the single B chromosome to one of the two poles. In both cases the second meiotic 195 division occurs normally, only in the latter case two of the four spores contain one B 196 chromosome each (Mendelson and Zohary, 1972) (Suppl. Figure 5). Consequently, 197 sperm nuclei formed after two rounds of mitotic divisions contain no or two B 198 chromosomes. In our experiments we detected 25% of sperm nuclei containing 2Bs 199 (Table 2). In pollen of 2B plants almost exclusively sperm nuclei with two Bs were 200 detected (Suppl. Figure 4C). In this case the two Bs frequently form bivalents during 201 meiosis I and segregate normally. At the end of meiosis spores with one B bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

202 chromosome each are formed (Mendelson and Zohary, 1972) which result in sperm 203 nuclei with two chromosomes (Suppl. Figure 5). 3B plants produced pollen with more 204 or less equal sized fractions of sperm nuclei with two and four B chromosomes 205 (Suppl. Figure 4D) similarly as plants with 5Bs where nuclei with four and six B 206 chromosomes (Suppl. Figure 4E) were detectable. For 3B plants the occurrence of 207 univalents, bivalents and trivalents was described (Mendelson and Zohary, 1972). 208 Resolving of these pairing structures preferentially results in spores with either one or 209 two B chromosomes finally leading to sperm nuclei with two or four Bs (Suppl. Figure 210 5). However, with a much lower frequency also sperm nuclei with no and six Bs could 211 be detected (Suppl. Figure 4D). In plants with 5 Bs the number of potential pairing 212 configurations further increases. However, preferably either two or three Bs migrate 213 to opposite poles during meiosis I finally resulting in sperm nuclei with either four or 214 six Bs. In pollen of plants with 4Bs and 6Bs preferentially sperm nuclei with the same 215 number of B chromosomes as in the corresponding mother plant were produced and 216 with a lower frequency nuclei with two and six or four and eight Bs, respectively 217 (Suppl. Figure 4F-G). Plants with four or six Bs preferentially perform regular 218 segregations during meiosis independent on the pairing configuration (bivalents up to 219 hexavalents) (Mendelson and Zohary, 1972). Deviations from this normal segregation 220 may lead to a decrease or increase of B chromosomes in sperm nuclei compared to 221 the situation in the mother plant (Table 2). 222 223 To measure the efficiency of the B accumulation, vegetative and sperm nuclei from 224 mature pollen of individual +B plants were separately sorted onto microscopic slides 225 first (red and blue frames in Figure 2E). Application of immunolabelling using anti- 226 histone H3K27me3 to identify the vegetative nuclei in combination with FISH using 227 the B-specific repeat AesTR-183 to score for the presence of Bs (Figure 2F-G) 228 confirmed the purity of the individual populations. In order to evaluate if the B 229 chromosome-containing fractions with a FSC signal intensity similar to that of the 230 vegetative nuclei (light blue frame in Figure 2E and Suppl. Figure 4B-H) exclusively 231 contain sperm nuclei or also vegetative nuclei with Bs we sorted the upper (light blue) 232 and lower (dark blue) fractions of sperm nuclei separately. Only the upper sperm 233 nuclei fractions (light blue) contained additionally a low percentage of vegetative 234 nuclei harboring Bs (Figure 2H) in a range of 1.7% to 6.1%. Based on these 235 cytological evaluations in combination with the flow cytometric profiles we were able bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

236 to precisely determine the transmission of Bs to sperm nuclei (Table 2). Independent 237 of the number of Bs present in the mother plant Bs accumulate in the sperm nuclei 238 with more than 93% as a result of an unequal segregation of Bs during the first pollen 239 mitosis. 240 241 Since in rye a structural change of the B chromosome impaired its ability to drive 242 (Endo et al., 2008; Müntzing, 1945 ), we included Ae. speltoides plants from the 243 Katzir population with a metacentric B variant in our analysis (Suppl. Figure 2B). 244 However, based on flow cytometry, this B-variant shows a comparable frequency of 245 accumulation in sperm nuclei (Suppl. Figure 4H). Hence, the changes involved in the 246 formation of this B-variant did not affect the process behind the control of its 247 preferential transmission to sperm nuclei. 248 249

250 B chromosomes preferentially distribute in the generative nucleus of the 251 bicellular pollen grain 252 To obtain visual insights into the cellular mechanism of B chromosome drive in Ae. 253 speltoides, we analyzed the dynamics of chromosomes at microgametogenesis. 254 Anther tissue sections were used to maintain the natural arrangement of 255 chromosomes and to avoid pollen wall background signals. The B-specific probe 256 AesTR-183 was used to trace the segregation of Bs during pollen formation. 257 258 Products of the pollen mitosis I were analyzed first. Due to differences in the 259 chromatin condensation and size of generative and vegetative nuclei (Borg et al., 260 2009; Tanaka, 1997), both types of nuclei are easily distinguishable. Condensed 261 generative nuclei locate closely toward the pollen wall. 120 bicellular pollen grains of 262 plants possessing 2Bs were analyzed. In 85% of pollen, the Bs were observed in the 263 generative nucleus only (Figure 3A). Micronuclei containing Bs were found in close 264 vicinity to the generative nucleus in 3.3% of pollen (Figure 3B). 3.3% of pollen 265 revealed B-specific signals in vegetative nuclei only (Figure 3C). 1.7% of pollen 266 revealed Bs in the generative nucleus and in micronuclei (Figure 3D). 6.7% of pollen 267 showed B-specific signals in both, generative and vegetative nuclei (Figure 3E). In all 268 10 analyzed mature tricellular pollen grains, B-specific signals were never observed 269 in vegetative nuclei, while all of the sperm nuclei revealed signals; indicating a normal bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

270 segregation of Bs at second pollen grain mitosis (Figure 3F). Thus, Bs accumulate in 271 the generative nucleus during the first pollen mitosis. The observed values of the B 272 chromosome accumulation in tissue sections are in close agreement with the flow 273 cytometric data. 274 275 Directed nondisjunction of Ae. speltoides Bs is accompanied by unequal cell 276 division during pollen mitosis 277 To decipher the mechanism behind the preferential accumulation of B chromosomes 278 in the generative nucleus, tubulin and CENH3 antibodies were applied at all stages of 279 the first pollen mitosis of B chromosome-containing plants. The centromere-specific 280 histone variant H3 (CENH3) was selected because it was shown that its loss results 281 in the failure of chromosome segregation in mammals (Howman et al., 2000) and 282 plants (Sanei et al., 2011). The centromeres of all anaphase A and B chromosomes 283 were equally CENH3 labeled and revealed an interaction with microtubules. During 284 early anaphase, the peripheral generative spindle bundle is blunt and the pole is 285 nearly in contact with the pollen cortex. By contrast, the interior spindle bundle is long 286 and pointed (Figure 4A). In late anaphase, a nondisjoined lagging chromosome with 287 attached spindle fibers was observed (Figure 4B). During telophase, the generative 288 nucleus contained a higher number of CENH3 signals indicating the presence of Bs 289 (Figure 4C). Comparing the distance between the spindle midzone and the 290 centromere positions of the generative (6.82 µm) versus the vegetative nucleus (7.97 291 µm) revealed that in most cases the spindle midzone is more proximal to the 292 generative nucleus (n = 10, t-test P<0.05) (Figure 4D).

293 In order to quantify the centromere size as a means for centromere activity (Zhang 294 and Dawe, 2012), the size of immunosignals was determined after anti-CENH3 295 labeling and B-specific in situ hybridizations of somatic mitotic +B metaphase cells. 296 No major differences in CENH3 signal size were found when we compared A and B 297 centromeres, although the amount of CENH3 slightly varied between the individual 298 chromosomes (Figure 5A-B). Similarly, we did not observe differences in CENH3 299 signal size using meiotic prophase cells. Thus, a different centromere activity of B 300 chromosomes could be excluded as the reason for the nondisjunction of Bs, since 301 the centromeres of all chromosomes are CENH3-positive and interact with tubulin. 302 303 bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

304 The (peri)centromere composition differs between A and B chromosomes of 305 Ae. speltoides

306 Next, we asked whether the sequence composition differs between A and B 307 (peri)centromeres. Eleven (peri)centromere-specific repeats, originally described for 308 wheat were identified in Ae. speltoides. The corresponding FISH probes were 309 generated by PCR using genomic DNA of Ae. speltoides carrying B chromosomes 310 (Suppl. Table 2,) and hybridized to mitotic metaphase chromosomes of +B plants 311 (Figure 6, Suppl. Figure 6, Table 3). Four of the eleven probes (RCS2, 6C6-3, 6C6-4 312 and CRW2) resulted in signals of similar intensities on A and B chromosomes (Figure 313 6A, Suppl. Figure 6A-C). Four probes (Quinta-LTR, 192 bp, Weg 1-LTR and CCS1) 314 showed stronger hybridization signals on the A chromosomes (Figure 6B, Suppl. 315 Figure 6D-F), and pBs301 and the centromeric satellite repeat (CenSR) co-localized 316 with the pericentromeres of As but were not detectable on Bs (Figure 6C, Suppl 317 Figure 6G). In contrast, TailI revealed an enriched signal accumulation at 318 centromeres of Bs (Figure 6D). No obvious differences in the distribution and 319 intensity of the used (peri)centromeric probes were found between the different A 320 chromosomes of Ae. speltoides. 321 322 To evaluate the centromeric localization of the identified repeats in more detail we 323 used naturally extended pachytene chromosomes for anti-CENH3 staining and 324 subsequent FISH with the corresponding repeats. AesTR-183 was additionally used 325 to distinguish between A and B chromosomes. Quinta-LTR, 192 bp, Weg 1-LTR 326 (Figure 6B’, Suppl. Figure 6D’-E’) signals partly overlapped with CENH3-marked 327 chromatin and extended into pericentromeric regions. CCS1 completely overlapped 328 with CENH3 signals in A chromosomes showed reduced centromere labeling in B 329 chromosomes (Suppl. Figure 6F’). No overlapping with CENH3-signals was found for 330 the A chromosome-specific pBs301 and CenSR (Figure 6C’, Suppl. Figure 6G’). TailI 331 coincided with CENH3 and showed stronger centromere signals on Bs than on As 332 (Figure 6D’). Although nine of the eleven (peri)centromeric candidate sequences co- 333 localize with CENH3 signals, CENH3 never encompassed fully the area covered by 334 centromere repeats. We conclude that all A centromere sequences except pBs301 335 and the CenSR are also present in the centromeres of the Bs. Hence, an intraspecific 336 origin of the B centromere is likely. However, the contrasting abundance of Quinta- bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

337 LTR, 192 bp, Weg 1- LTR and CCS1 signals indicates that the (peri)centromere of 338 the B diversified. 339

340 Discussion

341 AesTR-183, a conserved B chromosome-specific repeat 342 The high copy-repeat fraction of the Ae. speltoides genome is mostly composed of 343 LTR-retrotransposons, transposons and satellite repeats, with overall proportions of 344 individual repeat types being similar in the 0B/+B genotypes. Among four identified B- 345 located repeats in Ae. speltoides only AesTR-183 and AesTR-126 showed similarity 346 to already known repeats in other species according to BLAST results. AesTR-126 347 appeared to be identical to a part of the T. aestivum clone pTa-713, a repetitive 348 sequence positively tested in FISH on wheat chromosomes (KC290900.1) (Badaeva 349 et al., 2015). AesTR-183 shows 84% similarity to the rye B-specific tandem repeat 350 Sc26c38 which occurs within the nondisjunction control region of the rye B (Klemme 351 et al., 2013). In addition, we discovered an AesTR-183-like repeat in the Bs of Ae. 352 mutica. Unknown is, whether the Bs of Ae. speltoides and Ae. mutica possess like 353 rye a repeat-enriched nondisjunction control region. The existence of a B 354 chromosome conserved repeat is intriguing and provokes the question whether the 355 AesTR-183 repeat has acquired a function in Bs. Possibly, the ancestor of all three 356 species possessed an AesTR-183-positive B chromosome, which evolved further in 357 rye and Aegilops, but kept the AesTR-183 sequence. The phylogenetic analysis of 358 AesTR-183 repeat monomer sequences rather support this theory, as the divergence 359 of B-specific repeat in this case coincides with divergence of the species themselves 360 (Bernhardt et al., 2017). The similar but much shorter sequence detected on wheat 361 chromosome 1B does not follow this pattern and seems to be independent from the 362 B-specific sequences. 363 364

365 A combination of nondisjunction and unequal spindle formation at first pollen 366 mitosis might promote the accumulation of Bs in sperm nuclei 367 We provide direct insight into the cellular basis of the drive mechanism of the B 368 chromosome. The flow-cytometrically estimated frequencies of B chromosome 369 accumulation correspond with the cytologically determined one using tissue sections bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

370 of developing pollen grains (93.9% versus 95%). However, one advantage of the 371 cytological approach is the observation of micronuclei, which escaped from detection 372 in the flow cytometrical approach. On the other hand, flow cytometry allows the rapid 373 analysis of a large number of pollen nuclei of different plants. This approach seems 374 to be applicable also in other plant species with B chromosomes provided the 375 structural and morphological differences between vegetative and sperm nuclei allow 376 a clear differentiation, and the size of the B chromosomes is large enough to 377 separate individual fractions with different B chromosome numbers. 378 379 More than 93% of sperm nuclei accumulated Bs independent on the number of Bs 380 present in the mother plant. In +6B plants, sperm nuclei with up to 8Bs were found. 381 Eight reflects the maximum number of Bs detected in Ae. speltoides. Thus, embryos 382 with a B number above 8 are not formed or do not develop due to the negative effect 383 of Bs in a high number. It would be of interest to investigate pollen of +8B plants to 384 see if at least sperm nuclei with more than eight Bs are produced. However, plants 385 with 8Bs were not found during this study and are probably male sterile. 386 387 An important hint regarding the mechanism of chromosome accumulation comes 388 from our finding that the microtubule spindle of Ae. speltoides is asymmetrical during 389 first pollen mitosis, in accordance with previous studies in other species (Banaei- 390 Moghaddam et al., 2012; Borg et al., 2009; Jin et al., 2005; Tanaka, 1997; Twell, 391 2011). Considering the asymmetric geometry of the spindle at first pollen grain 392 mitosis, it is likely, that the inclusion of Bs into the generative nucleus is caused by 393 the fact that the equatorial plate is nearer to the generative pole as suggested by 394 Jones (1991). Spindle asymmetry as a component of the B chromosome drive 395 process has been suggested also for the lily Lilium callosum (Kimura and Kayano, 396 1961), the Asteraceae Crepis capillaris (Rutishauser and Rothlisberger, 1966) and 397 the grasshopper Myrmeleotettix maculatus (Hewitt, 1976). Alternatively, due to a 398 higher pulling force on the B centromere toward the generative pole, Bs may 399 preferentially accumulate in the generative nucleus (Banaei-Moghaddam et al., 400 2012). 401

402 Mitotic nondisjunction and lagging chromatids could be explained by centromere 403 inactivity (Sanei et al., 2011). However, as the centromeres of lagging Bs show no bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

404 reduction of CENH3 signals and interact with spindle microtubules, centromere 405 inactivity is unlikely the reason behind the nondisjunction process. In rye, the failure 406 to separate CENH3-positive B sister pericentromeres during pollen grain mitosis is 407 under the control of the nondisjunction control region (Banaei-Moghaddam et al., 408 2012; Endo et al., 2008). Also in maize, centromere function and B chromosome 409 nondisjunction are two independent processes, but the region required for 410 nondisjunction resides in the centromeric region (Han et al., 2007). It is likely, that in 411 Ae. speltoides and rye the cohesion between B sister chromatids during first pollen 412 mitosis is stronger than the microtubule traction force required for separation of 413 chromatids. At second pollen grain mitosis, B sister chromatids disjoin and a normal 414 segregation of Bs occurs. The proposed mechanism of B chromosome accumulation 415 is summarized in Figure 7.

416 417 Notably, the B chromosome accumulation mechanism in Ae. speltoides and rye is 418 similar. In both species, nondisjunction of CENH3-positive B sister chromatids occurs 419 and an asymmetric spindle during first pollen grain mitosis are likely components of 420 the accumulation process. Assuming that the Bs of rye and Ae. speltoides originated 421 independently, the comparable drive mechanisms in both species likely evolved in 422 parallel. 423 424 425 Similarities in the (peri)centromere composition of As and Bs suggest an 426 intraspecific origin of B chromosomes in Ae. speltoides

427 The observed generally similar sequence composition of the A and B centromeres of 428 Ae. speltoides could be considered as evidence of an intraspecific A chromosome- 429 derived origin of the B chromosome. Nine of eleven tested (peri)centromeric 430 sequences were shared by both types of chromosomes. Only CenSR and pBs301 431 did not reveal a hybridization signal in the B centromeres. However, copy number 432 differences assumed by different FISH signal sizes were also found for the repeats 433 Quinta-LTR, 192 bp, Weg 1- LTR, CCS1 and Taill. One might imagine that the B 434 centromere evolved from a standard A chromosome centromere and the repeats 435 rearranged in the newly formed B chromosome. A comparable situation was also 436 observed for the B chromosome centromeres of the daisy Brachycome bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

437 dichromosomatica (Houben et al., 2001), maize (Jin et al., 2005), rye (Banaei- 438 Moghaddam et al., 2012) and the grasshopper Xyleus discoideus angulatus 439 (Bernardino et al., 2017). Likely is that the B-specific centromere composition is 440 functionally involved in the regulation of chromosome segregation to ensure the 441 maintenance of Bs in natural populations. 442 443 444 Conclusions 445 Drive is the key for understanding supernumerary B chromosomes. Nondisjunction of 446 CENH3-positive, tubulin interacting B sister chromatids and an asymmetric spindle 447 during first pollen grain mitosis are likely components of the B chromosome drive 448 process. A quantitative flow cytometric approach demonstrated that independent of 449 the number of Bs present in the mother plant, B chromosomes accumulate in the 450 generative nuclei with more than 93%. The prerequisites for the drive process seems 451 to be common in Poaceae, thus enabling independent origins of Bs in different 452 lineages within the family. 453 454

455 Methods

456 Plant materials and cultivation 457 Following accessions were used: Aegilops speltoides from Syria (PI 487238, USDA- 458 ARS, USA) and Israel (Katzir,TS 89; Technion, 2-36; Ramat Hanadiv, 2-46, Institute 459 of Evolution, University of Haifa, Israel) (Belyayev and Raskina, 2013); Aegilops 460 mutica (KU-12008, Plant Germplasm Institute, Kyoto University, Japan) (Ohta and 461 Saruhashi, 1999); Triticum aestivum ‘Chinese Spring’ (TRI 12922, IPK-Gatersleben 462 Genebank, Germany); Secale cereale inbred line 7415 (Jimenez et al., 1994) and 463 Lolium perenne accession G7 with Bs (Taylor and Evans, 1977). Plants were 464 cultivated under greenhouse conditions (16 h light, 22°C day/16°C night). 465

466 Isolation and sequencing of genomic DNA 467 Genomic DNA from leaf tissue was purified with DNeasy Plant Mini Kit (Qiagen, 468 Germany). Using the Illumina HiSeq 2500 system, we sequenced 2x 101 bp genomic 469 DNA from 0B and 2B plants from the Ae. speltoides Katzir accession. In total 171 bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

470 million Illumina 101 bp paired end reads were generated having an output of residues 471 of 17 Gbp. All sequences are publicly available under PRJEB29862 (ENA).

472

473 Identification of B-specific repeats using similarity-based read clustering 474 Identification of Ae. speltoides B chromosome-specific repeats was performed by 475 similarity-based clustering of Illumina reads as implemented in the RepeatExplorer 476 pipeline (Novák et al., 2010; Novák et al., 2013). The analysis was done in a 477 comparative mode, using as its input a randomly sampled fraction of 1.4 million 478 paired-end reads from 0B and +B samples, which corresponds to 0.03x genome 479 coverage. Following the clustering of the pooled reads and annotation of repeats 480 represented by the largest clusters containing at least 0.01% of analyzed reads, 481 proportions of reads in each cluster from 0B and +B samples were determined by 482 parsing read names where genotypes of origin were encoded by specific tags. This 483 allowed for identification of repeats enriched on B chromosomes as the clusters with 484 increased proportions of reads from +B sample compared to 0B. Monomer lengths 485 and consensus sequences of newly identified satellite repeats were determined using 486 TAREAN (Novák et al., 2017). PCR primers were designed for consensus sequences 487 of identified B-repeats (Suppl.Table 2 and 3). Control primers were designed for the 488 T. aestivum 18S ribosomal RNA coding part of rDNA 25S-18S intergenic region 489 EcoRI-BamHI fragment (GenBank accession X07841.1). 0B/+B genomic DNA was 490 used as template and PCR products were cloned using CloneJET PCR Cloning Kit 491 (Thermo Fisher Scientific, Germany) and verified by sequencing (GeneBank, AesTR- 492 126 MK283665,AesTR-148 MK283666, AesTR-183 MK283667, AesTR-205 493 MK283668). 494 495

496 Molecular phylogenetic analysis of AesTR-183-like repeats 497 AesTR-183-like sequences were amplified by PCR from genomic DNA of Ae. mutica, 498 S. cereale and three Ae. speltoides accessions (Katzir, Technion, Ramat Hanadiv) all 499 containing B chromosomes. Sequences were analyzed and assembled by 500 Sequencher 5.4.6 (Gene Codes Corp., Ann Arbor, MI, USA). These sequences 501 including a similar region of wheat chromosome 1B (accession LS992081.1, position 502 432930807- 432930254) were aligned with ClustalW as implemented in Unipro bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

503 UGENE v1.31.0 (Okonechnikov et al., 2012). When necessary, alignments were 504 edited manually. jModelTest2 (Darriba et al., 2012) was used to estimate best-fit 505 nucleotide substitution model resulting in selection of HKY + G out of 88 tested 506 models according to the Bayesian information criterion (BIC). Phylogenetic analysis 507 was performed in MrBayes v3.2.6 (Ronquist and Huelsenbeck, 2003) by running two 508 parallel Metropolis-coupled Markov chain Monte Carlo analyses with four chains each 509 for 2 million generations. Tree sampling was conducted every 500 generations. The 510 consensus tree was generated after removal of 25% of samples from the beginning 511 of the chain as burn-in. The final tree was visualized with FigTree v1.4.3 512 (http://tree.bio.ed.ac.uk). 513

514 Flow sorting of sperm and vegetative nuclei 515 Mature pollen grains from anthers at dehiscence stage were collected and the 516 corresponding nuclei were isolated by applying the filter bursting method (Kron and 517 Husband, 2012) using CellTrics disposable filters of 100 μm and 20 μm (SYSMEX). 518 The resulting nuclei suspension was stained with propidium iodide (50 µg/ml, PI) and 519 analyzed on a BD Influx (Becton Dickinson) by blotting the PI fluorescence against 520 the forward scatter signal. Based on differences in the DNA content and nuclear size, 521 populations of vegetative and sperm nuclei without and with Bs could be 522 differentiated. The individual fractions were flow-sorted into Eppendorf tubes and 523 fixed with 4% formaldehyde in 5% sucrose buffer (10 mM Tris, 50 mM KCl, 2 mM

524 MgCl2, 5% sucrose, 0.05% Tween, pH 7.5) for 10 min. Subsequently, 15-20 µl of the 525 nuclei suspensions were applied on microscopic slides and dried overnight before 526 performing immunostaining and FISH.

527

528 Preparation of tissue sections and chromosome spreads 529 For the preparation of tissue sections, post-meiotic anthers (5 - 5.5 mm in length) 530 were fixed in 4% paraformaldehyde prepared in 1x MTSB buffer as described in 531 (Houben et al., 2011). In brief, for embedding, the anthers were infiltrated first with a 532 graded series of PEG-wax in absolute ethanol solutions (1/2, 1/1, 2/1, (v/v)), 12 h for 533 each at 37°C. Finally, anthers were infiltrated with pure PEG-wax for 12 h at 37°C 534 and transferred into casting moulds. 10 µm tissue sections were cut on histological 535 microtome Leica RM 2265 (Leica Biosystems) and placed on poly-L-lysine coated bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

536 microscopic slides. The preparation of meiotic chromosomes was performed 537 according (Badaeva et al., 2017). Meiotic anthers (~1.5 mm in length) were fixed in 538 3:1 (ethanol: acetic acid, v/v) for one hour. Meiocytes were squeezed out of the 539 anthers on a slide in 7 µl 45% acetic acid with the help of a needle and squashed 540 between slide and coverslip. The coverslips of liquid nitrogen frozen slides were 541 removed and slides were kept in 1×PBS at 4°C for immunostaining and FISH. 542 The preparation of mitotic chromosomes from the apical meristem of three days old 543 seedlings was performed according to Aliyeva-Schnorr et al. (2015) by dropping the 544 cell suspension on the slides. The slides were stored until use in 96% ethanol at 4℃. 545

546 Immunostaining and FISH 547 Slides carrying the anther tissue sections were washed in 96% ethanol twice for 10

548 min, in 90% ethanol twice for 10 min, and finally in dH2O twice for 5 min. After an 800 549 Watt microwave treatment of the slides for 60 seconds (Chelysheva et al., 2010) in 550 10 mM sodium citrate buffer, slides were immediately transferred into a 1×MTSB for 551 5 min. Mouse anti-α tubulin (Sigma, cat. no. T 9026, diluted at 1:200), rabbit anti- 552 grass CENH3 (Sanei et al., 2011) (diluted at 1:3000) and rabbit anti-histone 553 H3K27me3 (Millipore, #07-449, diluted at 1:100) were used as primary antibodies. 554 Anti-mouse Alexa 488 (Molecular Probes, cat. no. #A11001, diluted in 1:200) and 555 anti-rabbit Cy3 (Jackson/ Dianova, cat. no. 111-165-144, diluted in 1:300) were used 556 as secondary antibodies. To combine FISH and immunostaining, slides were rinsed

557 with dH2O and placed in 2×SSC for 5 min before FISH. FISH was performed after 558 post-fixation of specimens in 3:1 fixative for 10 min, washes in 2 × SSC twice and 559 final fixation in 3% paraformaldehyde. 50 μl hybridization mixture per slide were used 560 and the denaturing of tissue sections was done for 5 min on a hot plate at 80°C. For 561 multiple rounds of FISH, FISH signal stripping was performed as described by 562 (Heslop-Harrison et al., 1992; Shibata et al., 2009). In brief, after removing the 563 coverslips the slides were rinsed in 0.1 × SSC at room temperature for 2 × 5 min. 564 Next, slides were incubated in shaking jars at 42 °C for 30 min in each solution: once 565 in 0.1 × SSC, twice in probe stripping solution [0.05% (v/v) Tween-20, 50% (v/v) 566 formamide in 0.1 × SSC] and once in 4T solution [0.05% (v/v) Tween-20 in 4 × SSC]. 567 Finally, specimens were fixed in 4% formaldehyde for 10 min, dehydrated in an 568 ethanol series and air-dried. 569 bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

570 Generation of FISH probes 571 The wheat (peri)centromere-specific probes 192 bp (Ito et al., 2004), 6C6-3&4 572 (Zhang et al., 2004), CCS1 (Aragón-Alcaide et al., 1996), centromere satellite repeat 573 (CenSR) (Li et al., 2013), CRW2 (Liu et al., 2008), pBs301(Cheng and Murata, 2003), 574 RCS2 (Liu et al., 2008), Quinta-LTR (Li et al., 2013), TaiII (Kishii et al., 2001) and 575 Weg 1-LTR (Li et al., 2013) were generated by PCR (Suppl. Table 2). Nine of the 576 eleven probes are part of a (peri)centromeric Ty3/gypsy-type retrotransposon. The 577 corresponding positions along the retrotransposon are shown in Suppl. Figure 7. 578 Plant genomic DNA was extracted by the CTAB method (Murray and Thompson, 579 1980). The 25 µl PCR reactions contained following components: 2.5 μl 10×PCR

580 buffer (20 mM MgCl2), 1.5 μl dNTP mix (10 mM), 1 μl of each primer (10 mM), 0.2 μl

581 Taq (1U) polymerase (Quiagen), 50 ng gDNA and dH2O. Purified PCR products as 582 well as plasmid clones of the newly identified repeats, the rye B-specific repeat 583 D1100 (Sandery et al., 1990) and 5S rDNA clone pTa794 (Gerlach and Dyer, 1980) 584 were directly labelled with dUTP-ATTO-647, dUTP-ATTO-550 or dUTP-ATTO-488 585 using a nick translation labelling kit (Jena Bioscience, http://www. 586 jenabioscience.com). 587

588 Microscopy and image analysis 589 The specimens were analyzed using an Olympus BX61 microscope equipped with an 590 ORCA-ER CCD camera (Hamamatsu, Japan). Deconvolution microscopy was 591 employed to remove out-of-focus information. Maximum intensity projections were 592 processed with the program software CellSens dimension (Olympus). All images 593 were collected in grey scale and pseudo-coloured with Adobe Photoshop CS 594 (Adobe). The ImageJ (https://imagej.nih.gov/ij/download.html) particle analyzis tool 595 was used to measure the size of CENH3 signals. Graphs showing the CENH3 signal 596 size and the length of spindel microtubules were prepared with the Rstudio ggplot2 597 package. 598

599 Supplemental material 600 The following materials are available in the online version of this article. 601 Additional figures bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

602 Figure S1. PCR amplification of AesTR-126, AesTR-148, AesTR-183 and AesTR- 603 205 repeats from Ae. speltoides genomic DNA of 0B and +B plants. Figure S2. 604 Distribution of the B-specific probes AesTR-183 (magenta) and AesTR-205 (green) 605 on the two B chromosome variants of Ae. speltoides. Figure S3. Phylogeny of 606 AesTR-183-like sequence. Figure S4. Flow cytometry efficiently showed B 607 chromosome accumulation in sperm nuclei of +B plants compared with a 0B plant. 608 Figure S5. Scheme of the B chromosome transmission process in the male 609 gametogenesis of Ae. speltoides exemplified for plants with 1B, 2B and 3B 610 chromosomes. Figure S6. Sequence composition of Ae. speltoides A and B 611 (peri)centromeres. Figure S7. Schemata of a Ty3/gypsy retrotransposon showing the 612 positions of the used (peri)centromeric FISH probes. 613

614 Additional Table 615 Table S1. Repeat composition of Ae. speltoides 0B and +B genotypes estimated 616 from clustering analysis of Illumina reads. Table S2. PCR primers used for 617 amplification of AesTR-126, AesTR-148, AesTR-183 and AesTR-205 repeats, control 618 primers for 18S rDNA and primers for (peri)centromeric sequences. Table S3. 619 Consensus monomer sequences of AesTR-126, AesTR-148, AesTR-183 and 620 AesTR-205 determined by TAREAN analysis and used to design the primers. 621

622 Acknowledgements 623 We thank Olga Raskina (Institute of Evolution, University of Haifa, Israel) and John 624 Harper (Aberystwyth University, UK) for providing Ae. speltoides and L. perenne, 625 respectively. We thank Katrin Kumke and Oda Weiss for technical assistance, as well 626 as Nadine Bernhardt, Frank Blattner and Ingo Schubert for insightful discussions. 627 This work was supported by the China Scholarship Council (Grant No. 628 201606910015) (to DW), the Czech Academy of Sciences (Grant No. 629 RVO:60077344) (to PN, JM) and the Deutsche Forschungsgemeinschaft DFG (Grant 630 No. HO1779/26-1) (to AH). 631 The authors declare that they have no competing interests.

632 bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

633 Author contributions

634 DW, AR, JF, YZ and AH designed the study, interpreted the data and wrote the 635 manuscript. MV, PN, JM performed the bioinformatics analysis. JF conducted the 636 flow cytometry. All authors read and approved the final manuscript.

637

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766 major role in centromeric structure. Plant J. 73:952-965. 767 https://doi.org/10.1111/tpj.12086 768 Liu, Z., W. Yue, D. Li, R.R.-C. Wang, X. Kong, K. Lu, G. Wang, Y. Dong, W. Jin, and 769 X. Zhang. 2008. Structure and dynamics of retrotransposons at wheat 770 centromeres and pericentromeres. Chromosoma. 117:445-456. 771 https://doi.org/10.1007/s00412-008-0161-9 772 Müntzing, A. 1945 Cytological studies of extra fragment chromosomes in rye. II. 773 Transmission and multiplication of standard fragments and iso-fragments. 774 Hereditas. 31:457-477. https://doi.org/10.1111/j.1601-5223.1945.tb02763.x 775 Mendelson, D., and D. Zohary. 1972. Behavior and transmission of supernumerary 776 chromosomes in Aegilops speltoides. Heredity. 29:329-339. 777 https://doi.org/10.1038/hdy.1972.97 778 Murray, M.G., and W.F. Thompson. 1980. Rapid isolation of high molecular weight 779 plant DNA. Nucleic Acids Res. 8:4321-4326. 780 https://doi.org/10.1093/nar/8.19.4321 781 Novák, P., P. Neumann, and J. Macas. 2010. Graph-based clustering and 782 characterization of repetitive sequences in next-generation sequencing data. 783 BMC Bioinformatis. 11:378-389. https://doi.org/10.1186/1471-2105-11-378 784 Novák, P., P. Neumann, J. Pech, J. Steinhaisl, and J. Macas. 2013. RepeatExplorer: 785 a Galaxy-based web server for genome-wide characterization of eukaryotic 786 repetitive elements from next generation sequence reads. Bioinformatics 787 (Oxford, England). 29:792-793. https://doi.org/10.1093/bioinformatics/btt054 788 Novák, P., L.Á. Robledillo, A. Koblížková, I. Vrbová, P. Neumann, and J. Macas. 789 2017. TAREAN: a computational tool for identification and characterization of 790 satellite DNA from unassembled short reads. Nucleic Acids Res. 45:111. 791 https://doi.org/10.1093/nar/gkx257 792 Ohta, S., and Y. Saruhashi. 1999. Geographical distribution of B chromosomes in 793 Aegilops mutica Boiss., a wild relative of wheat. Hereditas. 130:177-183. 794 https://doi.org/10.1111/j.1601-5223.1999.00177.x 795 Okonechnikov, K., O. Golosova, and M. Fursov. 2012. Unipro UGENE: a unified 796 bioinformatics toolkit. Bioinformatics (Oxford, England). 28:1166-1167. 797 https://doi.org/10.1093/bioinformatics/bts091 bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

798 Raskina, O., L. Brodsky, and A. Belyayev. 2011. Tandem repeats on an eco- 799 geographical scale: outcomes from the genome of Aegilops speltoides. 800 Chromosome Res. 19:607-623. https://doi.org/10.1007/s10577-011-9220-9 801 Ronquist, F., and J.P. Huelsenbeck. 2003. MrBayes 3: Bayesian phylogenetic 802 inference under mixed models. Bioinformatics (Oxford, England). 19:1572- 803 1574. https://doi.org/10.1093/bioinformatics/btg180 804 Ruban, A., J. Fuchs, A. Marques, V. Schubert, A. Soloviev, O. Raskina, E. Badaeva, 805 and A. Houben. 2014. B Chromosomes of Aegilops speltoides are enriched in 806 organelle genome-derived sequences. PLoS One. 9:e90214. 807 https://doi.org/10.1371/journal.pone.0090214 808 Ruban, A., T. Schmutzer, U. Scholz, and A. Houben. 2017. How next-generation 809 sequencing has aided our understanding of the sequence composition and 810 origin of B chromosomes. Genes. 8: 294-308. 811 https://doi.org/10.3390/genes8110294 812 Rutishauser, A., and E. Rothlisberger. 1966. Boosting mechanism of B-chromosomes 813 in Crepis capilaris. Chromosomes Today. 1:28-30. 814 Sandery, M.J., J.W. Forster, R. Blunden, and R.N. Jones. 1990. Identification of a 815 family of repeated sequences on the rye B chromosome. Genome. 33:908- 816 913. https://doi.org/10.1139/g90-137 817 Sanei, M., R. Pickering, K. Kumke, S. Nasuda, and A. Houben. 2011. Loss of 818 centromeric histone H3 (CENH3) from centromeres precedes uniparental 819 chromosome elimination in interspecific barley hybrids. P Natl Aca Sci. 820 108:E498-E505. https://doi.org/10.1073/pnas.1103190108 821 Shibata, F., K. Sahara, Y. Naito, and Y. Yasukochi. 2009. Reprobing Multicolor Fish 822 Preparations in Lepidopteran Chromosome. Zoo Sci. 26:187-190. 823 https://doi.org/10.2108/zsj.26.187 824 Tanaka, I. 1997. Differentiation of generative and vegetative cells in angiosperm 825 pollen. Sex Plant Reprod. 10:1-7. https://doi.org/10.1007/s004970050060 826 Taylor, I.B., and G.M. Evans. 1977. Genotypic control of homoeologous chromosome 827 association in Lolium temulentum X Lolium perenne interspecific hybrids. 828 Chromosoma. 62:57-67. https://doi.org/10.1007/bf00328440 829 Twell, D. 2011. Male gametogenesis and germline specification in flowering plants. 830 Sex Plant Reprod. 24:149-160. https://doi.org/10.1007/s00497-010-0157-5 bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

831 Zhang, H., and R.K. Dawe. 2012. Total centromere size and genome size are 832 strongly correlated in ten grass species. Chromosome Res. 20:403-412. 833 https://doi.org/10.1007/s10577-012-9284-1 834 Zhang, P., W. Li, J. Fellers, B. Friebe, and B.S. Gill. 2004. BAC-FISH in wheat 835 identifies chromosome landmarks consisting of different types of transposable 836 elements. Chromosoma. 112:288-299. https://doi.org/10.1007/s00412-004- 837 0273-9

838

839 List of abbreviations 840

A A chromosome B B chromosome BAD Background aggregate and debris percentage CENH3 Centromere histone H3 ChIP Chromatin immunoprecipitation CV Coefficient of variation DAPI 4´,6-diamidino-2-phenylindole DNA Deoxyribonucleic acid FISH Fluorescence in situ hybridization FSC Forward scatter signal Gen Generative nucleus H3K27me3 Trimethylated histone H3 at lysine 27 Micro Micronucleus MTSB Microtubule stabilization buffer PCR Polymerase chain reaction PI Propidium iodide RNA Ribonucleic acid Sp Sperm nucleus

SSC Saline sodium citrate buffer Veg Vegetative nucleus 841

842 Figures 843 bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

844 Figure 1 845 Identification and characterization of B chromosome enriched sequences. (A) In silico 846 identification of repeat clusters containing B-enriched sequences. The enrichment is 847 revealed by calculating the ratio of reads derived from +B genotype to the total 848 number of reads (+B and 0B) in the cluster. Clusters showing partial enrichment 849 (AesTR-126 and 148) or specificity to B chromosomes (AesTR-183 and 205) are 850 labelled. (B) Graph representation of read similarities in B-enriched repeat clusters. 851 Individual reads from 0B and +B plants are represented by green and red nodes, 852 respectively. Nodes are placed according to sequence similarity, where similar 853 sequences are close together and connected with edges (gray lines). Circular or 854 globular structure of these graphs is typical for repeats with tandem arrangement in 855 the genome. (C-F) Chromosomal distribution of B-enriched repeats in Ae. speltoides, 856 Ae. mutica and S. cereale. (C) Localization of AesTR-126, AesTR-148, AesTR-183, 857 AesTR-205 and 5S rDNA on mitotic metaphase chromosomes of Ae. speltoides from 858 Tartus (Syria) revealed by FISH. (D) Showing the hybridization patterns of FISH 859 probes individually. (E) Co-localization of AesTR-183 (magenta) and the S. cereale B- 860 specific probe D1100 (in green) in S. cereale with 2Bs. (F) Localization of AesTR-183 861 in Ae. mutica with 2Bs. The Bs are marked with arrowheads.

862 863 Figure 2 864 Flow cytometry showed high efficiency of B chromosome drive. (A-D) Vegetative 865 (Veg, red) and sperm (Sp, blue) nuclei of 0B mature pollen were separated flow 866 cytometrically and the purity of both fractions was verified by H3K27me3 867 immunostaining. (E-F) Vegetative (red) and sperm (blue) nuclei of +5B mature pollen 868 were sorted and subjected to H3K27me3 immunostaining and B-specific FISH using 869 AesTR-183. (A) PI stained mature pollen grain from 0B plant contained round 870 vegetative nucleus and two spindle shaped sperm nuclei. (B) Dot plot displaying the 871 relative fluorescence (DNA content; x-axis) versus forward scatter signal (particle 872 size; y-axis) intensities. (C) Sorted vegetative nuclei fraction (red) was abundantly 873 labelled by anti-H3K27me3 (in green). (D) Sperm nuclei fraction (blue) showed no 874 anti-H3K27me3 signal. (E) Dot plot of +5B plant pollen with fraction of vegetative 875 (red) and two fractions of different fluorescence intensities of sperm nuclei (blue). (F) 876 Sorted vegetative nuclei labelled by H3K27me3 showed no B-specific FISH signals. 877 (G-H) Sorted sperm nuclei after immunostainig with H3K27me3 and FISH with the B- bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

878 specific probe AesTR-183. While in the lower part (blue) of the sperm nuclei fractions 879 exclusively B-carrying sperm nuclei (G) were detected the upper fraction (light blue) 880 contained also a low percentage of vegetative nuclei with B chromosomes (H). 881 882 Figure 3 883 B chromosomes of Ae. speltoides preferentially accumulate in the generative nucleus 884 during first pollen grain mitosis. Tissue sections after FISH with the B-specific repeat 885 AesTR-183 (magenta). (A) 85% of binucleated pollen possess Bs in generative 886 nuclei (Gen) only. (B) 3.33% of binucleated pollen possess Bs in micronuclei (Micro) 887 only. (C) 3.33% of binucleated pollen possess Bs in vegetative nuclei (Veg). (D) 888 1.67% of binucleated pollen possess Bs in generative and micronuclei. (E) 6.67% of 889 binucleated pollen possess Bs in generative and vegetative nuclei. (H) In mature 890 pollen both sperm nuclei possess Bs. DAPI is showen in blue. 891 892 Figure 4 893 The accumulation of Bs during pollen mitosis of Ae. speltoides +2B. (A-C) CENH3 894 (magenta) and α-tubulin (green) immunolabelling patterns at different stages of first 895 pollen mitosis. (A) Formation of asymmetric spindle at early anaphase. The 896 generative spindle (Gen) is short and blunt and the vegetative spindle (Veg) is long 897 and sharp. (B) At late anaphase, nondisjunction of lagging chromosomes with 898 positive CENH3 signals attached to the tubulin (insert shows further enlarged CENH3 899 signals). (C) Pollen at late anaphase forming an asymmetric spindle midzone. Note 900 the unequal number of CENH3 signals. (D) The dots indicate the distance between 901 the spindle midzone and the individual centromeres of the generative nucleus (red) or 902 vegetative nucleus (blue). X-axis: individual cells, Y-axis: distance. 903 904 Figure 5 905 Size of CENH3 immunosignals does not differ between mitotic A and B 906 chromosomes of Ae. speltoides. (A) Mitotic metaphase of a 3B Ae. speltoides plant 907 after CENH3 immunostaining (green) and FISH with the B-specific probe AesTR-183 908 (magenta). Bs are marked with arrowheads. Scale bar equals 10 µm. (B) Each dot 909 represents the CENH3 signal size of an individual sister chromatid (As in black, Bs in 910 magenta). X-axis: individual cells, Y-axis: CENH3 signal size. 911 bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

912 Figure 6 913 Sequence composition of Ae. speltoides A and B (peri)centromeres. (A-D) FISH 914 patterns of RCS2, Quinta-LTR, pBs301 and TailI (green) as well as of the B-specific 915 repeat AesTR-183 (magenta) on mitotic metaphase chromosomes. White arrows 916 indicate Bs. (B’-D’) Meiotic pachytene chromosomes labelled with the corresponding 917 (peri)centromeric probes (except for RCS2) and with antibodies against CENH3 918 (blue). Schematic chromosomes represent typical (peri)centromeric distributions of 919 the corresponding sequences on A and B chromosomes. Hybridization patterns of 920 the remaining (peri)centromeric sequences are shown in Suppl. Figure 6. DAPI 921 staining shown in grey. Scale bars equal 10 µm.

922 923 Figure 7 924 Model to explain the accumulation of Ae. speltoides B chromosomes during male 925 gametophyte development. At first pollen mitosis, metaphase chromosomes are 926 located toward the pollen cortex and an asymmetric spindle is formed. The peripheral 927 generative (Gen) spindle bundle is blunt and the interior spindle bundle toward the 928 vegetative pole (Veg) is longer and pointed. In contrast to the separated A sister 929 chromatids, the cohesion of B chromatids stays unresolved and Bs migrate 930 preferentially to generative nuclei. The placement of Bs toward the generative 931 nucleus is probably realized because the tubulin midzone is closer to the generative 932 pole and lagging Bs are included in the generative nucleus as the nuclear membrane 933 is formed. Alternatively, a higher spindle tension force toward the generative pole 934 might preferentially pull the Bs to the generative nucleus. Unlike As, nondisjoined B 935 chromatids locate preferentially at a position close to the spindle midzone. 936 937

938 Tables 939 940 Table 1 Repeats significantly enriched on the B chromosomes of Ae. speltoides. 941 942 Table 2 Quantification of B chromosome accumulation in sperm nuclei. 943 944 Table 3 Characterization of the employed (peri)centromeric probes. 945 bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

2n=14+0B A mature pollen 2n=14+5B F B Sp E Veg Sp

100 µm 103 Veg C G Veg

FSC

D H

2 Sp 10 Sp 4B 6B

5 10 DAPI H3K27me3 5 10 DAPI H3K27me3 AesTR-183 bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/547612; this version posted February 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table 1

Repeats significantly enriched on B chromosomes.

Repeat Type Monomer [bp] Cluster Reads Genome % 0B +B 0B +B AesTR-126 tandem 86 126 1688 3528 0.123 0.258

AesTR-148 tandem 791 148 934 1943 0.068 0.142

AesTR-183 tandem 1090 183 1 730 0.000 0.053

AesTR-205 tandem 270 205 0 358 0.000 0.026

Table 2

Quantification of B chromosome accumulation in sperm nuclei.

Mother plant Drive (%)1 Frequency of Bs in sperm nuclei (%)2 (number of nuclei 0B 2B 4B 6B 8B counted) 1B 94.4 (407) 75.1 24.9 2B 93.9 (770) 1.9 98.1 3B 98.3 (754) 6.5 48.0 39.5 5.9 4B 97.9 (660) 2.1 14.4 74.2 9.2 5B 96.3 (589) 1.4 57.3 41.4 6B n.d. 2.5 24.6 61.7 11.2

1 Bs drive efficiency calculated based on vegetative nuclei without Bs after flow sorting, immunostaining and FISH

2 frequencies were quantified flow cytometrically

Table 3 (Peri)centromeric FISH probes.

(peri)centromeric repeat Signal intensity and size1 CENH3 interaction2 Sequence type

As Bs ChIP FISH in (original pachytene of paper) Ae.speltoides 192 bp (Ito et al. 2004) ** * + + Ty3/gypsy LTR 6C6-3 (Zhang et al. 2004) *** *** + + Ty3/gypsy cereba gag 6C6-4 (Zhang et al. 2004) *** *** + + Ty3/gypsy cereba gag CCS1 (Aragon-Alcaide et al. 1996) *** * + + Ty3/gypsy LTRs Centromeric satellite repeats ** - - - centromeric satellite (CenSR) (Li et al., 2013) CRW2 (Liu et al. 2008) *** *** + + Ty3/gypsy cereba LTR pBs301 (Cheng & Murata 2003) ** - + - Ty3/gypsy family RCS2 (Liu et al. 2008) *** *** + - Ty3/gypsy family Quinta-LTR (Li et al., 2013) ** * + + Ty3/gypsy Quinta LTR Tail-family (Kishii et al., 2001) * *** n.d. + tandem repeat Weg 1-LTR (Li et al., 2013) ** * + + Ty3/gypsy Weg 1-LTR

1 “*” represents the appearance of signal, “-“ means no signal detected

2 “+” represents (peri)centromere colocolized with CENH3 antibody, “-“ means no interaction, “n.d.” no data available