Centromere sequence-independent but biased loading of subgenome-specific CENH3s in allopolyploid Arabidopsis suecica

Centromere sequence-independent but biased loading of subgenome-specific CENH3s in allopolyploid Arabidopsis suecica | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Centromere sequence-independent but biased loading of subgenome-specific CENH3s in allopolyploid Arabidopsis suecica Andreas Houben, Raheleh Karimi-Ashtiyani, Ali Mohammad Banaei-Moghaddam, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3997508/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Centromeric nucleosomes are determined by the replacement of the canonical histone H3 with the centromere-specific histone H3 (CENH3) variant. Little is known about the centromere organization in allopolyploid species where different subgenome-specific CENH3s and subgenome-specific centromeric sequences coexist. Here, we analyzed the transcription and centromeric localization of subgenome-specific CENH3 variants in the allopolyploid species Arabidopsis suecica. Synthetic A. thaliana x A. arenosa hybrids were generated and analyzed to mimic the early evolution of A. suecica . Our expression analyses indicated that CENH3 has generally higher expression levels in A. arenosa compared to A. thaliana , and this pattern persists in the hybrids. We also demonstrated that despite a different centromere DNA composition, the centromeres of both subgenomes incorporate CENH3 encoded by both subgenomes, but with a positive bias towards A. arenosa -type CENH3. The intermingled arrangement of both CENH3 variants demonstrates centromere plasticity and may be an evolutionary adaption to handle more than one CENH3 variant in the process of allopolyploidization. CENH3 centromere plasticity species evolution haploid uniparental chromosome elimination allopolyploidization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Centromeres are required for the correct segregation of chromosomes. This chromosome domain is the assembly site for the proteinaceous kinetochore complex, which dictates the correct distribution of sister chromatids and their transmission to daughter cells at mitosis and meiosis. Therefore, it was expected that centromeric sequences should have conserved sequence characteristics to be identified by kinetochore proteins. However, it was shown that the function of most centromeres is epigenetically determined and largely independent of the underlying sequences (Talbert and Henikoff 2020 ). Centromeric nucleosomes are distinguished by the replacement of the canonical histone H3 with the centromere-specific histone H3 variant (CENH3, also called CENP-A). This is a mark for the active centromere, which epigenetically determines centromere function in most eukaryotes (Henikoff and Dalal 2005 ). Defects in the centromeric chromatin may lead to missegregate chromosomes, resulting in aneuploidy, a frequently observed phenomenon in cancer (Tomonaga et al. 2003). Besides some diploid species encoding multiple functional CENH3 paralogs, e.g. Hordeum vulgare (Sanei, Pickering et al. 2011 ), Pisum sativum (Neumann, Navrátilová et al. 2012 ), and cowpea (Ishii, Juranić et al. 2020 ), most diploid eukaryotes encode one variant of CENH3. In contrast, in allopolyploid species, each parental subgenome might possess its own type of CENH3 operating in the context of multiple species-specific centromeric sequences. To analyze whether multiple CENH3 variants coexist in a hybrid genetic background, the expression of CENH3s was studied in some allopolyploids or artificial hybrid species. In allotetraploid Oryza species, both CENH3 variants are transcribed, and no CENH3 -type preferential expression pattern was found (Hirsch, Wu et al. 2009 , Li, Lu et al. 2010 ). In three different Brassica allotetraploid species, there was either co-transcription of both parental CENH3 or suppression of one parental CENH3 variant detected (Wang, He et al. 2011 ). On the other hand, uniparental silencing was revealed in an oat-maize chromosome addition line in which the maize-derived CENH3 was silenced (Jin, Melo et al. 2004 ). In the oat x pearl millet combination, despite the biparental expression of CENH3 genes, only the oat-type CENH3 was incorporated into the centromeres of both species in the hybrid embryo (Ishii, Sunamura et al. 2015 ). In a study on the reconstructed wheat chromosome 1B with a hybrid wheat-rye centromere in the background of wheat, it was shown that only the rye-derived centromere part incorporates CENH3 of wheat (Karimi-Ashtiyani, Schubert et al. 2021 ). Even uniparental centromere inactivation due to impaired CENH3 incorporation during early Hordeum vulgare x H. bulbosum hybrid embryogenesis has been demonstrated (Sanei, Pickering et al. 2011 ), underlying the importance of compatible parental centromeres in the process of speciation. Thus, different scenarios exist for how multiple CENH3s are handled in hybrid background. To study the dynamics of multiple CENH3s in Arabidopsis species hybrids, we analyzed the subgenome-specific CENH3 variants in natural and synthetic allopolyploid Arabidopsis suecica as well as A. thaliana x A. arenosa F1 hybrids. A. suecica , a natural hybrid of A. thaliana and A. arenosa , which is estimated to have originated around 16,000 years ago (Novikova, Tsuchimatsu et al. 2017 ), is a valuable model for investigating the genomic and epigenomic changes associated with polyploidization (Comai, Tyagi et al. 2000 ). It offers the ability to replay evolution by producing synthetic hybrids, making it an excellent candidate for such studies. Our analysis revealed that despite a different centromere DNA composition, the centromeres of the investigated allopolyploids incorporate CENH3s of both parental genomes. However, already after the formation of the F1 hybrid, the contribution of A. arenosa -derived CENH3 is higher than that of A. thaliana CENH3. Materials and Methods Plant material and crossing procedure A. thaliana (N3151, 2n = 4x = 20, ecotype Columbia-0 (Col-0), A. arenosa (N3901, 2n = 4x = 32, ecotype Care-1), A. suecica synthetic allopolyploid hybrid N22665 and natural hybrid Sue2 (2n = 4X = 26) were obtained from the European Arabidopsis Stock Centre (NASC). The synthetic allotetraploid A. suecica (N22665) had been produced by the crossing of autotetraploid A. thaliana (N3151) and autotetraploid A. arenosa (N3901) plants. For interspecific crossing to produce F1 hybrids, the closed buds of A. thaliana (N3151) were emasculated, and their stigmas were pollinated with A. arenosa (N3901) pollen. Plants were first grown under an 8 h photoperiod per day, 22˚C/18˚C day/night temperature. After 4 weeks, plants were transferred to long-day conditions (16 h photoperiod per day). Genomic DNA and RNA extraction, PCR and quantitative PCR Genomic DNA was extracted from leaf tissue using a DNAeasy plant mini kit (Qiagen). Total RNA was isolated using the Trizol method (Chomczynski and Sacchi, 1987). The absence of DNA contamination in RNA was confirmed by PCR using ELF1 -specific primers (Suppl. Table 1). The cDNA was synthesized with 1 µg of DNase-treated total RNA using a RevertAid H Minus first-strand cDNA synthesis kit using oligo dT primers (Fermentas). Primers specific for the constitutively expressed Actin 2 gene (At3g18780; Suppl. Table 1) were used as a control for an equal amount of gDNA and cDNA as well as for calibration in quantitative comparisons. The relative transcript level of A. thaliana - and A. arenosa -originated- CENH3 was measured by qPCR using species-specific primers (Suppl. Table 1). 10 µl of PCR mixture contained 1 µl of cDNA template, 5 µl of 2× Power SYBR Green PCR Master Mix (Applied Biosystems), 0.33 mM of the forward and reverse primers for each gene. Reactions were run in an Applied Biosystems 7900HT Fast Real-Time PCR System, and data were analyzed with SDS software v2.2.2. The quantitative PCR was performed using the following conditions: 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and an annealing temperature of 60°C for 60 s. The specificity and efficiency of both primers were determined by qPCR using a dilution series of plasmids of cloned full-length cDNA of A. thaliana and A. arenosa CENH3 genes. A similar Ct value (the PCR cycle at which the fluorescent signal of reporter dye exceeds background level) for an equal amount of plasmid and absence of amplification with the plasmid of the opposite CENH3 variant indicated that both primer pairs can amplify specific transcripts with the same efficiency. Genome size determination by flow cytometry and flow sorting of nuclei The genome size of putative hybrid plants was estimated using Raphanus sativus 'Voran' (1.11 pg/2C, Gatersleben gene bank accession number: RA 34) as an internal reference standard in comparison to the values obtained for their tetraploid parents. For this, roughly 0.5 cm 2 of fresh leaf tissue was chopped together with the internal reference standard in a Petri dish with a sharp razorblade in nuclei isolation buffer (Galbraith, Harkins et al. 1983 ) supplemented with propidium iodide (50 µg/ml) and DNase-free RNase (50 µg/ml). The resulting nuclei suspension was filtered through a 50 µm CellTrics filter (Sysmex-Partec) and measured using a FACStar PLUS cell sorter (BD Biosciences). The means of the nuclear peaks were determined using the software CellQuest (BD Biosciences) and the DNA contents calculated as described (Dolezel, Greilhuber et al. 2007 ).For the parental tetraploid species A. thaliana (N3151) and A. arenosa , we performed 12 and six measurements, respectively. For the putative hybrid plants between one and six independent measurements were done. For sorting of nuclei, leaf tissue was fixed in 4% formaldehyde, washed in Tris buffer, chopped in nuclei isolation buffer LB01 (Dolezel et al. 2007 ) and stained with 4’,6-diamidino-2-phenylindole (DAPI, 1 µg/ml final concentration) as described in (Ahmadli, Kalidass et al. 2023 ). The sorting was performed on BD Influx cell sorter (BD Biosciences) equipped with a 355 nm UV laser using the BD FACS Sortware software. Afterwards, the sorted nuclei suspension was mixed with equal amounts of sucrose buffer on a microscopic slide and dried overnight (for details, see (Ahmadli, Kalidass et al. 2023 )). Slides were either directly used for immunostaining or transferred for longer storage to -20°C. Generation of an A. arenosa CENH3-specific antibody The peptide RTKHFATKSRTGNRTDN was used to generate an A. arenosa CENH3-specific (anti-AaCENH3) polyclonal antibody (Suppl. Figure 1a). Peptide synthesis, immunization of guinea pigs, and peptide affinity purification of antisera were performed by Pineda, Antibody-Service (Berlin, Germany). Fluorescence in situ hybridization and indirect immunostaining A. thaliana (AtCEN) and A. arenosa (AaCEN (Matsushita et al. 2012)) centromere-specific FISH probes were generated by PCR using the primers pairs AtCEN-F/R and AaCEN-F/R, respectively (Suppl. Table 1). PCR was performed with Taq DNA polymerase with a block preheated to 94˚C and an initial denaturation of 94˚C for 2 min followed by 32 cycles of 94˚C for 25 sec, 57˚C for 30 sec, and 72˚C for 40 sec. The probes were labeled with FITC or Cy3 by nick translation. The rabbit HTR12-specific antibody (Abcam, ab72001, (Talbert, Masuelli et al. 2002 )) was used for the detection of A. thaliana CENH3 (anti-AtCENH3). Leaves were fixed in 1x phosphate buffered saline (PBS) containing 4% paraformaldehyde (PFA) for 20 min at room temperature and then immediately washed in 1x PBS twice for 5 min each. Indirect immunostaining and fluorescence in situ hybridization were carried out as described by (Gernand, Demidov et al. 2003 ) and (Ma, Vu et al. 2010 ), respectively. Imaging was performed by using an Olympus BX61 microscope and an ORCA-ER CCD camera (Hamamatsu). All images were collected in greyscale and pseudocoloured with Adobe Photoshop 6 (Adobe). Maximum intensity projections were done with the AnalySIS (Soft Imaging System) program. To achieve an optical resolution of ca. 120 nm, we applied spatial structured illumination microscopy (3D-SIM) using a 63x/1.40 objective of an Elyra PS.1 super-resolution microscope system and the software ZENBlack (Carl Zeiss GmbH). Image stacks were captured separately for each fluorochrome using 561, 488, and 405 nm laser lines for excitation and appropriate emission filters (Weisshart et al. 2016 ; Kubalová et al. 2021). (Weisshart, Fuchs et al. 2016 , Kubalova, Camara et al. 2023 ). The 3D-image stacks were used to generate Suppl. Movie 1 using the Imaris 9.7 (Bitplane) software. Classification of immunosignal patterns To compare the immunostaining patterns of the subgenome-specific CENH3 variants, double immunostaining was performed using rabbit anti-AtCENH3 (red signals) and guinea pig anti-AaCENH3 (green signals) specific antibodies. For this we prepared slides from flow-sorted 2C and 4C leaf nuclei (the two dominating (endopoly)ploidy levels in young leaf tissue) to minimize background signals. In subsequent quantitative immunolabeling experiments, as there were no significant differences observed between 2C and 4C sorted immunolabeled nuclei, we proceeded to analyze them collectively. The immuno signals were classified based on their strength (strong or weak) and absence. The absence of signals was called “0”, strong AtCENH3 or AaCENH3 signals were called “AT” or “AA”, subsequently. Weak AtCENH3 or AaCENH3 signals were called “at” or “aa”, respectively. Therefore, nuclei with unbiased subgenome-specific CENH3 signals were called either ‘AT/AA’ or ‘at/aa’. Nuclei with biased CENH3 signal intensities were called “AT/aa”, “at/AA”, “AT/0”, “at/0”, “AA/0” or “aa/0”. For each genotype, at least 155 immunostained nuclei were characterized (Suppl. Table 2). Results Centromeres of allopolyploid A. suecica incorporate both CENH3 variants , but more from A. arenosa To address whether subgenome-specific CENH3 variants undergo subgenome-specific centromere loading in an allopolyploid species, we used the allotetraploid A. suecica as a model. First, the hybrid nature of A. suecica was confirmed by multicolour FISH using centromere-specific DNA probes for A. thaliana (AtCEN) and A. arenosa (AaCEN) centromeres (Fig. 1 a). After FISH, chromocenters revealed either A. thaliana - or A. arenosa -specific signals. Next, an A. arenosa CENH3-specific antibody (anti-AaCENH3) was produced and tested for specificity with mitotic and interphase chromosomes of A. arenosa (Fig. 1 b, Suppl. Figure 1b). The absence of immunosignals in A. thaliana nuclei confirmed the species specificity of anti-AaCENH3. The species specificity of anti- A. thaliana CENH3 (anti-AtCENH3) was demonstrated as only the centromeres of A. thaliana but not of A. arenosa displayed immunosignals (Fig. 1 b, c). Double immunostaining using both antibodies and subsequent FISH with the A. arenosa centromere-specific probe confirmed that both CENH3 variants are incorporated in all centromeres of A. suecica independent of their origin and sequence (Fig. 1 c). Next, the centromeric localization of both subgenome-specific CENH3 variants was tested in synthetic allotetraploid A. suecica (N22665) plants. Double immunostaining revealed that about 60% of analyzed sorted nuclei incorporated equally both subgenome-specific CENH3s, resulting in either strong (AT/AA) or weak (at/aa) signals indicating an unbiased loading of subgenome-specific CENH3s (Fig. 2 ). In cases with a biased subgenome-specific CENH3 loading (i.e. AT/aa, at/AA, AT/0, at/0, 0/AA or 0/aa), we never found nuclei with strong AtCENH3 signals (i.e. AT/aa and AT/0) (Fig. 2 and Suppl. Table 2). In contrast, about 40% of nuclei, showed strong AaCENH3 signals (i.e. at/AA and 0/AA), indicating a biased loading of A. arenosa subgenome-specific CENH3. To check whether the same bias exists in natural allopolyploid A. suecica , derived from ancient A. thaliana x A. arenosa hybridization events, we performed double immunostaining on sorted nuclei of A. suecica accession “Sue2”. About 70% of analyzed nuclei, had equally incorporated subgenome-specific CENH3s (Fig. 2 ). In cases with a biased subgenome-specific CENH3 loading (i.e. AT/aa, at/AA, AT/0, at/0, 0/AA or 0/aa), no nuclei with strong AtCENH3 signals (i.e. AT/aa and AT/0) were observed (Fig. 2 and Suppl. Table 2). However, about 30% of all nuclei, showed strong AaCENH3 signals (i.e. at/AA and 0/AA), demonstrating a similar biased A. arenosa CENH3 incorporation. To exclude that the observed signal differences of both species-specific CENH3 antibodies were not caused by a lower quality of the AtCENH3 antibody, an additional control experiment was performed with tetraploid A. thaliana , the parent genotype we used for wide hybridization with A. arenosa . The same combination of antibodies resulted in nuclei with about 71% strong (AT/0) and 27% weak (at/0) AtCENH3-specific signals, respectively (Fig. 2 ). Thus, our findings indicate that both subgenome-specific CENH3 variants are incorporated into the centromeres of A. suecica , but with a positive bias towards A. arenosa CENH3. Preferential expression and loading of AaCENH3 become established immediately after hybrid formation Crossing of both species was performed to determine whether the preferential A. arenosa -type CENH3 incorporation is already established in the first generation after the wide hybridization. Only pollination of tetraploid A. thaliana with tetraploid A. arenosa resulted in fertile seeds. As aneuploidy and chromosome elimination were described for crosses between A. thaliana and A. arenosa (Comai, Tyagi et al. 2000 , Wright, Pires et al. 2009 ), flow cytometry was employed to identify successful hybridization events. In an initial pre-screening without an internal reference standard, one (plant 6) out of 30 plants was identified as diploid (Suppl. Figure 2a) while all other plants were confirmed to be tetraploid. Immunostaining with both types of CENH3 antibodies on nuclei of plant 6, confirmed the absence of the pollinator genome, as only up to 10 AtCENH3-specific signal clusters were found (Suppl. Figure 2b). Hence, here all A. arenosa -derived chromosomes were eliminated, likely during hybrid embryogenesis. Uniparental elimination of chromosomes is a common phenomenon in hybrids derived from distantly related species (reviewed in (Ishii, Karimi-Ashtiyani et al. 2015 )). Furthermore, we noticed in the flow cytometric pre-screen an obvious variation in the peak positions between the measurements of the individual plants (Suppl. Figure 2c). Therefore, we determined the genome size of 24 putative F1 hybrids using Raphanus sativus as an internal reference standard and compared the data to values obtained for the tetraploid parents (Suppl. Figure 3a). For tetraploid A. thaliana and A. arenosa , we estimated genome sizes of 0.683 pg/2C and 0.825 pg/2C, respectively, indicating an expected genome size for the interspecific hybrid of 0.754 pg/2C. Among the analyzed plants, 13 plants revealed a genome size deviating by less than 3% from the expected value of a hybrid plant and were considered as euploid hybrid plants. 11 plants showed a deviation from the expected genome size of more than 3%, presumably as a result of aneuploidy. One plant is most likely the product of an A. thaliana selfing event or spontaneous doubling of haploid progeny (plant No. 25, Suppl. Figure 3a, b). Based on the determined DNA contents, leaf nuclei of selected 3-month-old F1 hybrid plants were sorted on slides and analyzed by double immunostaining. About 50% of nuclei showed an unbiased loading of subgenome-specific CENH3 variants (AT/AA and at/aa nuclei). Out of the analyzed nuclei, only 3% showed stronger AtCENH3 signals (i.e. AT/aa, AT/0 and at/0 nuclei). In contrast, about 45% of CENH3-loaded nuclei, showed stronger A. arenosa signals (i.e. at/AA, 0/AA and 0/aa nuclei) (Fig. 2 , Suppl. Table 2), indicating a biased loading of A. arenosa -specific CENH3. The observed increase in the frequency of nuclei with an equal proportion of parental CENH3s (i.e., AT/AA and at/aa nuclei) from F1 to natural hybrids of A. suecica through generations suggests a gradual step-wise adaptation of CENH3 variant loading in allopolyploid Arabidopsis (Fig. 2 , Suppl. Figure 4). Super-resolution microscopy confirmed the mixed composition of both parental CENH3s in F1 hybrid nuclei (Fig. 3 , Suppl. Movie 1). However, the ultrastructure of AtCENH3 and AaCENH3 signals differed but intermingled, suggesting that the subgenome-specific CENH3 variants are preferentially loaded into different centromeric nucleosome arrays. The observed biased CENH3 incorporation prompted us to analyze the transcription of both parental CENH3s . The relative transcript levels of A. thaliana- and A. arenosa -derived CENH3 were quantified and then normalized to ACTIN2 (At3g18780) after qPCR with AaCENH3- and AtCENH3 -specific primer pairs (Fig. 4 , Suppl. Table 1). In all analyzed tissues (young rosette leaves, flower buds, and siliques) of allopolyploid A. suecica (natural hybrid) and synthetic hybrids (F1, older synthetic hybrid), the relative expression of A. arenosa CENH3 was higher in comparison to A. thaliana . In summary, we conclude that the majority (above 90%) of centromeres of A. thaliana x A. arenosa F1 hybrid, synthetic and natural A. suecica incorporate CENH3s of both parental genomes, despite a different centromere DNA composition (Fig. 5 ). However, after the formation of the F1 hybrid, the contribution of A. arenosa -derived CENH3 is higher than that of AtCENH3. Discussion An invaluable plant model for examining the genomic and epigenomic alterations linked to polyploidization is A. suecica , a naturally occurring hybrid of A. thaliana and A. arenosa (Comai, Tyagi et al. 2000 ). It is a great option for these kinds of studies since it provides a means of simulating evolution through the creation of artificial hybrids. Here, we investigated whether distinct CENH3s, which originated from both parental species, incorporate centromere-sequence independently in a hybrid situation. Therefore, we used varying evolutionary ages of A. suecica , ranging from F1 hybrids produced by wide crossing of tetraploid A. thaliana and A. arenosa to resynthesize the species, a synthetic hybrid and natural A. suecica . We found that both CENH3s encoded by either subgenome incorporate into the centromeres of hybrid genotypes, even though the centromeric satellite repeats of both subgenomes share only 58–80% sequence identity (Kamm, Galasso et al. 1995 ). This observation aligns with the findings of Talbert et al. ( 2002 ), who showed that anti-CENH3 of A. thaliana recognizes all centromeres in both synthetic and natural allopolyploid A. suecica . Also, a GFP:: A. arenosa CENH3 construct can functionally replace CENH3 of an A. thaliana null mutant ( cenh3-1) (Ravi, Kwong et al. 2010 ). Analysis of alien CENH3s localization in A. thaliana - and tobacco-tissue cultured cells showed that in contrast to the CENH3 of rice, CENH3 of A. thaliana and tobacco incorporated into the centromeres of both tobacco and A. thaliana while CENH3 of the holocentric Luzula nivea became integrated only partially into the centromeres of A. thaliana cultured cells, suggesting that only evolutionally close CENH3s can target centromeres in alien species (Nagaki, Terada et al. 2010 ). In addition, the replacement of endogenous CENH3 in A. thaliana with CENH3 from related species demonstrated that CENH3 of heterologous species must be related to the A. thaliana CENH3 for functional complementation (Ravi, Kwong et al. 2010 , Maheshwari, Ishii et al. 2017 ). Our observations that A. arenosa –derived CENH3 could localize to the centromeric satellite repeats of A. thaliana and vica versa are in agreement with these findings and confirm the close evolutionary relationship of both parent CENH3s. The coexistence of nuclei with different CENH3 patterns is likely caused by the multi-cell-type composition of leaves which were used for our analysis. Likely, the centromere composition is dynamically organized according to the needs of individual cell and tissue-types during development. In analogy, a tissue-specific CENH3-type composition was also found in barley and cowpea ((Ishii, Karimi-Ashtiyani et al. 2015 , Ishii, Juranic et al. 2020 ). Super-resolution microscopy revealed that AtCENH3 and AaCENH3 signals have an almost similar but not identical distribution in the centromeres of the hybrid plants, suggesting that both CENH3s do not exist as monomers in the same nucleosome. Instead, subgenome-specific CENH3s are loaded into separate nucleosome arrays to form distinct centromeric substructures. This phenomenon of CENH3 variant-specific distribution has also been observed in other plants, such as barley, a wheat 1BL/1RS translocation line, and cowpea, where different CENH3 variants occupy distinct centromeric nucleosomes (Ishii, Karimi-Ashtiyani et al. 2015 , Yuan, Guo et al. 2015 , Ishii, Juranić et al. 2020 , Karimi-Ashtiyani, Schubert et al. 2021 ). The intermingled arrangement of different CENH3 variants in a centromere may be an evolutionary adaption to handle more than one CENH3 variant. In a study focusing on the process of polyploidization, Burns et al. ( 2021 ) found that the speciation process in A. suecica was a gradual and adaptive evolutionary process rather than a sudden or drastic event. This gradual evolution was evidenced by the absence of massive changes in the genome arrangement and mobilization of transposable elements, as well as the lack of subgenome dominance in gene expression (Burns, Mandáková et al. 2021 ). This finding is consistent with the observed increased frequency of unbiasedly loaded nuclei of parental CENH3s from F1 to natural hybrids of A. suecica through generations in our study (Fig. 2 , Supple Fig. 3 ). However, we cannot rule out the possibility that other A. suecica genotypes exhibit different CENH3 patterns, given that natural A. suecica is a product of several founding individuals rather than a single origin (Novikova et al. 2017 ). Our expression analysis revealed that the dynamic of CENH3 expression is similar in parents and their allopolyploid hybrids, with higher expression levels in tissues with rapidly dividing cells. Furthermore, our results indicate that CENH3 has generally higher expression levels in A. arenosa compared to A. thaliana , and this pattern persists in the hybrids. This result is in line with the findings that the A. arenosa subgenome in resynthesized and natural A. suecica allotetraploids is hypomethylated, which may contribute to the observed upregulation of many genes involved in reproduction and adaptation (Jiang, Song et al. 2021 ). In conclusion, our analysis of transcription and centromeric localization of subgenome-specific CENH3 variants in the allopolyploid species A. suecica demonstrates that both parental CENH3 variants are retained, with a gradual increase of equal loading of subgenome-specific CENH3s during the progression from F1 to natural hybrids. This suggests the potential impact of centromere plasticity on establishing stable centromeres, genome integrity and evolution across generations in allopolyploid speciation. Declarations Acknowledgement AH was supported by the German Federal Ministry of Education and Research (FKZ 0315965), RK-A was supported by Iran National Science Foundation (INSF) under project No. 4004919. TI was supported by the JSPS KAKENHI Grant-in-Aid for Scientific Research (C), (Grant No. 22K05572) and JST-FOREST (Grant No. JPMJFR 2001). The excellent technical assistance of Karla Meier and Katrin Kumke (IPK, Gatersleben, Germany) is gratefully acknowledged. Author contributions RK-A contributed to material preparation, performed experiments, collected and analyzed data, and interpreted the results. AMB-M performed expression analysis and interpreted the results. OW contributed to immunostaining and material preparation. JF performed flow cytometry and interpreted the results. VS performed high-resolution microscopy. RK-A wrote the first draft of the manuscript, and all authors provided comments. AH contributed to the study conception and writing. All authors read and approved the final manuscript. Competing Interests The authors have no competing interests to declare that are relevant to the content of this article. References Ahmadli U, Kalidass M, Khaitova LC, Fuchs J, Cuacos M, Demidov D, Zuo S, Pecinkova J, Mascher M, Ingouff M, Heckmann S, Houben A, Riha K, Lermontova I (2023) High temperature increases centromere-mediated genome elimination frequency and enhances haploid induction in Arabidopsis. Plant Commun 4(3):100507 Burns R, Mandáková T, Gunis J, Soto-Jiménez LM, Liu C, Lysak MA, Novikova PY, Nordborg M (2021) Gradual evolution of allopolyploidy in Arabidopsis suecica. 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Commun Biol 3(1):775 Ishii T, Karimi-Ashtiyani R, Banaei-Moghaddam AM, Schubert V, Fuchs J, Houben A (2015) The differential loading of two barley CENH3 variants into distinct centromeric substructures is cell type- and development-specific. Chromosome Res 23(2):277–284 Ishii T, Sunamura N, Matsumoto A, Eltayeb AE, Tsujimoto H (2015) Preferential recruitment of the maternal centromere-specific histone H3 (CENH3) in oat (Avena sativa L.) x pearl millet (Pennisetum glaucum L.) hybrid embryos. Chromosome Res 23(4):709–718 Jiang X, Song Q, Ye W, Chen ZJ (2021) Concerted genomic and epigenomic changes accompany stabilization of Arabidopsis allopolyploids. Nat Ecol Evol 5(10):1382–1393 Jin W, Melo JR, Nagaki K, Talbert PB, Henikoff S, Dawe RK, Jiang J (2004) Maize centromeres: organization and functional adaptation in the genetic background of oat. Plant Cell 16(3):571–581 Kamm A, Galasso I, Schmidt T, Heslop-Harrison JS (1995) Analysis of a repetitive DNA family from Arabidopsis arenosa and relationships between Arabidopsis species. Plant Mol Biol 27(5):853–862 Karimi-Ashtiyani R, Schubert V, Houben A (2021) Only the rye derived part of the 1BL/1RS hybrid centromere incorporates CENH3 of wheat. Front Plant Sci 12:802222 Karimi-Ashtiyani R, Schubert V, Houben A (2021) Only the Rye Derived Part of the 1BL/1RS Hybrid Centromere Incorporates CENH3 of Wheat. 12(2960) Kubalova I, Camara AS, Capal P, Beseda T, Rouillard JM, Krause GM, Holusova K, Toegelova H, Himmelbach A, Stein N, Houben A, Dolezel J, Mascher M, Simkova H, Schubert V (2023) Helical coiling of metaphase chromatids. Nucleic Acids Res 51(6):2641–2654 Li H, Lu L, Heng Y, Qin R, Xing Y, Jin W (2010) Expression of CENH3 alleles in synthesized allopolyploid Oryza species. 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PLoS Genet 8(6):e1002777 Novikova PY, Tsuchimatsu T, Simon S, Nizhynska V, Voronin V, Burns R, Fedorenko OM, Holm S, Säll T, Prat E, Marande W, Castric V, Nordborg M (2017) Genome Sequencing Reveals the Origin of the Allotetraploid Arabidopsis suecica. Mol Biol Evol 34(4):957–968 Ravi M, Kwong PN, Menorca RM, Valencia JT, Ramahi JS, Stewart JL, Tran RK, Sundaresan V, Comai L, Chan SW (2010) The rapidly evolving centromere-specific histone has stringent functional requirements in Arabidopsis thaliana. Genetics 186(2):461–471 Sanei M, Pickering R, Kumke K, Nasuda S, Houben A (2011) Loss of centromeric histone H3 (CENH3) from centromeres precedes uniparental chromosome elimination in interspecific barley hybrids. Proc Natl Acad Sci U S A 108(33):E498–505 Sanei M, Pickering R, Kumke K, Nasuda S, Houben A (2011) Loss of centromeric histone H3 (CENH3) from centromeres precedes uniparental chromosome elimination in interspecific barley hybrids. Proc Natl Acad Sci U S A Talbert PB, Henikoff S (2020) What makes a centromere? Exp Cell Res 389(2) Talbert PB, Masuelli R, Tyagi AP, Comai L, Henikoff S (2002) Centromeric localization and adaptive evolution of an Arabidopsis histone H3 variant. Plant Cell 14(5):1053–1066 Wang G, He Q, Liu F, Cheng Z, Talbert PB, Jin W (2011) Characterization of CENH3 proteins and centromere-associated DNA sequences in diploid and allotetraploid Brassica species. Chromosoma 120(4):353–365 Weisshart K, Fuchs J, Schubert V (2016) Structured illumination microscopy (SIM) and photoactivated localization microscopy (PALM) to analyze the abundance and distribution of RNA polymerase II molecules on flow-sorted Arabidopsis nuclei. Bio-protocol 6:e1725 Wright KM, Pires JC, Madlung A (2009) Mitotic instability in resynthesized and natural polyploids of the genus Arabidopsis (Brassicaceae). Am J Bot 96(9):1656–1664 Yuan J, Guo X, Hu J, Lv Z, Han F (2015) Characterization of two CENH3 genes and their roles in wheat evolution. New Phytol 206(2):839–851 Supplementary Files KarimietalSupplFiguresonly27.2.2024.pptx Suppltable1.docx Suppltable2.docx Suppl.Movie1.mp4 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revisions 08 Apr, 2024 Reviewers agreed at journal 09 Mar, 2024 Reviewers invited by journal 09 Mar, 2024 Editor invited by journal 29 Feb, 2024 Editor assigned by journal 28 Feb, 2024 First submitted to journal 27 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3997508","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":277751743,"identity":"23e39cae-e06a-49a0-a4fd-dbcccc8d8db3","order_by":0,"name":"Andreas Houben","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIiWNgGAWjYBACAwYGNiBlw8PAAxNiB4swMDbg15IG1ZIAxMzEaTnMQLwWc/Yeswcfd5yXMec5wPzi5w+bfPlm5mNAEQbZfhxaLHvOmBvOPHObx7K3gc2yJyHNcsNhtnSgCIPxTBzWGNzIMZPmbbvNY3Ce/5sBT8JhAwNmHpAIQ+KGA3i0/G07B9TCwGb4J+G/gXwz/zegCEPifnxaGNsO8BicbWB+zJNwwIDhMA8bUARoCw6/GJw5Vm7Y25bMY9lzgI1ZJi3ZwOAwmzlQRMJ4Bi5bjjdve/Czzc7enCeB+eMbGzsD+fbmZ0ARG9l+HN5H6AVGkAQSXwKnSmQtzB8IKxsFo2AUjIKRCABUeVjG0c4iigAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-3419-239X","institution":"IPK Gatersleben","correspondingAuthor":true,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Houben","suffix":""},{"id":277751744,"identity":"5753f6d6-e27e-4291-aef5-6dc87d7e5bb3","order_by":1,"name":"Raheleh Karimi-Ashtiyani","email":"","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":false,"prefix":"","firstName":"Raheleh","middleName":"","lastName":"Karimi-Ashtiyani","suffix":""},{"id":277751745,"identity":"7534b2cf-ca84-4614-92f4-5a6d09fb681d","order_by":2,"name":"Ali Mohammad Banaei-Moghaddam","email":"","orcid":"","institution":"Tehran University: University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"Mohammad","lastName":"Banaei-Moghaddam","suffix":""},{"id":277751746,"identity":"9701d589-fd03-4000-a686-a0711c6d7346","order_by":3,"name":"Takayoshi Ishii","email":"","orcid":"","institution":"Tottori University Graduate School of Agriculture Faculty of Agriculture: Tottori Daigaku Daigakuin Nogaku Kenkyuka Nogakubu","correspondingAuthor":false,"prefix":"","firstName":"Takayoshi","middleName":"","lastName":"Ishii","suffix":""},{"id":277751747,"identity":"f12bc75b-1567-41e1-9d45-08b2e2faf6f8","order_by":4,"name":"Oda Weiss","email":"","orcid":"","institution":"Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK): Leibniz-Institut fur Pflanzengenetik und Kulturpflanzenforschung (IPK)","correspondingAuthor":false,"prefix":"","firstName":"Oda","middleName":"","lastName":"Weiss","suffix":""},{"id":277751748,"identity":"a4286b4b-cb5c-41b1-919f-1231bf3fa0c4","order_by":5,"name":"Jörg Fuchs","email":"","orcid":"","institution":"Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK): Leibniz-Institut fur Pflanzengenetik und Kulturpflanzenforschung (IPK)","correspondingAuthor":false,"prefix":"","firstName":"Jörg","middleName":"","lastName":"Fuchs","suffix":""},{"id":277751749,"identity":"5672be69-b31a-4761-988c-3e28a90efe57","order_by":6,"name":"Veit Schubert","email":"","orcid":"","institution":"Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK): Leibniz-Institut fur Pflanzengenetik und Kulturpflanzenforschung (IPK)","correspondingAuthor":false,"prefix":"","firstName":"Veit","middleName":"","lastName":"Schubert","suffix":""}],"badges":[],"createdAt":"2024-02-28 18:42:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3997508/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3997508/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52628314,"identity":"51bfe2bf-ceb5-40d6-8d37-7df07c2ab8fe","added_by":"auto","created_at":"2024-03-13 18:27:53","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":118390,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVerification of the hybrid nature of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. suecica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and specificity of species-specific CENH3 antibodies. \u003c/strong\u003e(a) The allopolyploid nature of \u003cem\u003eA. suecica\u003c/em\u003e, confirmed by FISH on sorted 2C nuclei of \u003cem\u003eA. suecica\u003c/em\u003e using a combination of \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eA. arenosa\u003c/em\u003ecentromere-specific DNA probes. (b) Specificity of an \u003cem\u003eA. arenosa\u003c/em\u003eCENH3-specific antibody and anti-\u003cem\u003eA. thaliana\u003c/em\u003e CENH3 (anti-AtCENH3) confirmed by indirect immunostaining on mitotic chromosomes of \u003cem\u003eA. arenosa\u003c/em\u003eand \u003cem\u003eA. thaliana\u003c/em\u003e. (c) FISH with an \u003cem\u003eA. arenosa\u003c/em\u003e centromere-specific probe after immunostaining confirmed that both variants of CENH3 are centromere incorporated irrespectively of the underlying centromere sequence. Bar represents 2 µm\u003c/p\u003e","description":"","filename":"Slide1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3997508/v1/806b1cbcc568b4f2d287d790.jpg"},{"id":52627846,"identity":"b0baf20d-9cb4-4ae4-8505-732b8a430054","added_by":"auto","created_at":"2024-03-13 18:19:53","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":89160,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSequence-independent but biased loading of parental CENH3 variants in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. thaliana x A. arenosa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e F1, synthetic and natural \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. suecica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e plants.\u003c/strong\u003e(a) Frequencies of distribution patterns of subgenome-specific CENH3 immunosignals on sorted nuclei. (b) Typical examples of labelled nuclei with different CENH3 distribution patterns. Bar represents 2 µm\u003c/p\u003e","description":"","filename":"Slide2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3997508/v1/46a1910e5787bdec346084f6.jpg"},{"id":52627845,"identity":"3736cd57-a790-4e0f-82d4-889eb298d798","added_by":"auto","created_at":"2024-03-13 18:19:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":61367,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eA. thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. arenosa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e CENH3s are loaded into neighbouring nucleosome arrays of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. arenosa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e x \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e F1 centromeres.\u003c/strong\u003e (a) Interphase distribution of anti-AtCENH3 (in red) and anti-AaCENH3 (in green) signals analyzed by\u003cstrong\u003e \u003c/strong\u003esuper-resolution microscopy (3D-SIM) (see also Suppl. Movie 1). Bar represents 2 µm. (b’,’’) Further enlarged centromeres\u003cem\u003e.\u003c/em\u003e Bar represents 0.2 µm\u003c/p\u003e","description":"","filename":"Slide3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3997508/v1/9a76d74751d12495a9c33d59.jpg"},{"id":52627843,"identity":"c4ba97e3-f772-4ed3-9bff-72834b68f2bd","added_by":"auto","created_at":"2024-03-13 18:19:53","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":53998,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression analysis of CENH3s in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. thaliana x A. arenosa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e F1, synthetic and natural \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. suecica \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eplants\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e. \u003c/strong\u003e\u003c/em\u003eThe expression levels of CENH3 genes originating from \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eA. arenosa\u003c/em\u003e were investigated in parents, and their hybrids using parent-specific-CENH3 primers in different tissues of young rosette leaves, flower buds and siliques. The relative transcript levels of CENH3 were quantified and normalized to \u003cem\u003eACTIN2\u003c/em\u003e (At3g18780). The relative magnitude of transcription was calculated by the following formula: R= 2^\u003csup\u003e(CtCENH3–CtActin2)\u0026nbsp; \u003c/sup\u003e(Livak and Schmittgen, 2001), where R= relative expression level. Bars represent the means of the relative transcript level of CENH3 compared to \u003cem\u003eActin2\u003c/em\u003e. Error bars represent the standard deviation between three biological replicates.\u003c/p\u003e","description":"","filename":"Slide4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3997508/v1/e44963cf5d07880d79acde19.jpg"},{"id":52627851,"identity":"951149f7-f8c1-48a6-ab67-2d4c02ca27c3","added_by":"auto","created_at":"2024-03-13 18:19:54","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":74825,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModel of the centromere sequence-independent but biased centromere loading of subgenome-specific CENH3s in allopolyploid \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. suecica. \u003c/strong\u003e\u003c/em\u003eThe majority of centromeres of \u003cem\u003eA. thaliana\u003c/em\u003e x \u003cem\u003eA. arenosa\u003c/em\u003e F1 hybrid, synthetic and natural \u003cem\u003eA. suecica\u003c/em\u003e incorporate CENH3s of both parental genomes, despite a different DNA centromere composition. In nuclei with biased loading of subgenome-specific CENH3, the \u003cem\u003eA. arenosa\u003c/em\u003e-derived CENH3 is dominant. After several generations, the frequency of biasedly loaded subgenome-specific CENH3 nuclei decreases, and both subgenome-specific CENH3s load more balanced to the centromeres of natural \u003cem\u003eA. suecica\u003c/em\u003e plants.\u003c/p\u003e","description":"","filename":"Slide5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3997508/v1/ab88aa0160e64106eeb5322a.jpg"},{"id":52628642,"identity":"25fca1cc-05e6-41a6-a44d-ab6dce91fa67","added_by":"auto","created_at":"2024-03-13 18:35:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":711719,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3997508/v1/33e15402-e08a-46d7-b1ee-1efe4d181587.pdf"},{"id":52627850,"identity":"b502514d-41c2-4251-a97b-bf8122c01706","added_by":"auto","created_at":"2024-03-13 18:19:54","extension":"pptx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":3720948,"visible":true,"origin":"","legend":"","description":"","filename":"KarimietalSupplFiguresonly27.2.2024.pptx","url":"https://assets-eu.researchsquare.com/files/rs-3997508/v1/78abfce5374863fa2eff8a59.pptx"},{"id":52627849,"identity":"f66eaf9f-718c-41bf-9f1a-de6c4f833a78","added_by":"auto","created_at":"2024-03-13 18:19:54","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":13019,"visible":true,"origin":"","legend":"","description":"","filename":"Suppltable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3997508/v1/22eac8eeea239f696e280293.docx"},{"id":52627848,"identity":"30e0d2fb-b998-4016-bd80-48b7962ffe08","added_by":"auto","created_at":"2024-03-13 18:19:54","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":17992,"visible":true,"origin":"","legend":"","description":"","filename":"Suppltable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-3997508/v1/56cc7154022c0c6b1dd2a984.docx"},{"id":52627852,"identity":"295f82bc-22e4-4d6b-bddc-33f6b239ff1f","added_by":"auto","created_at":"2024-03-13 18:19:55","extension":"mp4","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":30003730,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Movie1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3997508/v1/ea1d2edd3ca0ddf85639f03a.mp4"}],"financialInterests":"","formattedTitle":"Centromere sequence-independent but biased loading of subgenome-specific CENH3s in allopolyploid Arabidopsis suecica","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCentromeres are required for the correct segregation of chromosomes. This chromosome domain is the assembly site for the proteinaceous kinetochore complex, which dictates the correct distribution of sister chromatids and their transmission to daughter cells at mitosis and meiosis. Therefore, it was expected that centromeric sequences should have conserved sequence characteristics to be identified by kinetochore proteins. However, it was shown that the function of most centromeres is epigenetically determined and largely independent of the underlying sequences (Talbert and Henikoff \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Centromeric nucleosomes are distinguished by the replacement of the canonical histone H3 with the centromere-specific histone H3 variant (CENH3, also called CENP-A). This is a mark for the active centromere, which epigenetically determines centromere function in most eukaryotes (Henikoff and Dalal \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Defects in the centromeric chromatin may lead to missegregate chromosomes, resulting in aneuploidy, a frequently observed phenomenon in cancer (Tomonaga et al. 2003).\u003c/p\u003e \u003cp\u003eBesides some diploid species encoding multiple functional CENH3 paralogs, e.g. \u003cem\u003eHordeum vulgare\u003c/em\u003e (Sanei, Pickering et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), \u003cem\u003ePisum sativum\u003c/em\u003e (Neumann, Navr\u0026aacute;tilov\u0026aacute; et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and cowpea (Ishii, Juranić et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), most diploid eukaryotes encode one variant of CENH3. In contrast, in allopolyploid species, each parental subgenome might possess its own type of CENH3 operating in the context of multiple species-specific centromeric sequences. To analyze whether multiple CENH3 variants coexist in a hybrid genetic background, the expression of CENH3s was studied in some allopolyploids or artificial hybrid species. In allotetraploid \u003cem\u003eOryza\u003c/em\u003e species, both CENH3 variants are transcribed, and no \u003cem\u003eCENH3\u003c/em\u003e-type preferential expression pattern was found (Hirsch, Wu et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Li, Lu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In three different \u003cem\u003eBrassica\u003c/em\u003e allotetraploid species, there was either co-transcription of both parental CENH3 or suppression of one parental CENH3 variant detected (Wang, He et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). On the other hand, uniparental silencing was revealed in an oat-maize chromosome addition line in which the maize-derived CENH3 was silenced (Jin, Melo et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In the oat x pearl millet combination, despite the biparental expression of CENH3 genes, only the oat-type CENH3 was incorporated into the centromeres of both species in the hybrid embryo (Ishii, Sunamura et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In a study on the reconstructed wheat chromosome 1B with a hybrid wheat-rye centromere in the background of wheat, it was shown that only the rye-derived centromere part incorporates CENH3 of wheat (Karimi-Ashtiyani, Schubert et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Even uniparental centromere inactivation due to impaired CENH3 incorporation during early \u003cem\u003eHordeum vulgare\u003c/em\u003e x \u003cem\u003eH. bulbosum\u003c/em\u003e hybrid embryogenesis has been demonstrated (Sanei, Pickering et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), underlying the importance of compatible parental centromeres in the process of speciation. Thus, different scenarios exist for how multiple CENH3s are handled in hybrid background.\u003c/p\u003e \u003cp\u003eTo study the dynamics of multiple CENH3s in Arabidopsis species hybrids, we analyzed the subgenome-specific CENH3 variants in natural and synthetic allopolyploid \u003cem\u003eArabidopsis suecica\u003c/em\u003e as well as \u003cem\u003eA. thaliana\u003c/em\u003e x \u003cem\u003eA. arenosa\u003c/em\u003e F1 hybrids. \u003cem\u003eA. suecica\u003c/em\u003e, a natural hybrid of \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eA. arenosa\u003c/em\u003e, which is estimated to have originated around 16,000 years ago (Novikova, Tsuchimatsu et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), is a valuable model for investigating the genomic and epigenomic changes associated with polyploidization (Comai, Tyagi et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). It offers the ability to replay evolution by producing synthetic hybrids, making it an excellent candidate for such studies. Our analysis revealed that despite a different centromere DNA composition, the centromeres of the investigated allopolyploids incorporate CENH3s of both parental genomes. However, already after the formation of the F1 hybrid, the contribution of \u003cem\u003eA. arenosa\u003c/em\u003e-derived CENH3 is higher than that of \u003cem\u003eA. thaliana\u003c/em\u003e CENH3.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePlant material and crossing procedure\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eA. thaliana\u003c/em\u003e (N3151, 2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;20, ecotype Columbia-0 (Col-0), \u003cem\u003eA. arenosa\u003c/em\u003e (N3901, 2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;32, ecotype Care-1), \u003cem\u003eA. suecica\u003c/em\u003e synthetic allopolyploid hybrid N22665 and natural hybrid Sue2 (2n\u0026thinsp;=\u0026thinsp;4X\u0026thinsp;=\u0026thinsp;26) were obtained from the European Arabidopsis Stock Centre (NASC). The synthetic allotetraploid \u003cem\u003eA. suecica\u003c/em\u003e (N22665) had been produced by the crossing of autotetraploid \u003cem\u003eA. thaliana\u003c/em\u003e (N3151) and autotetraploid \u003cem\u003eA. arenosa\u003c/em\u003e (N3901) plants. For interspecific crossing to produce F1 hybrids, the closed buds of \u003cem\u003eA. thaliana\u003c/em\u003e (N3151) were emasculated, and their stigmas were pollinated with \u003cem\u003eA. arenosa\u003c/em\u003e (N3901) pollen. Plants were first grown under an 8 h photoperiod per day, 22˚C/18˚C day/night temperature. After 4 weeks, plants were transferred to long-day conditions (16 h photoperiod per day).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGenomic DNA and RNA extraction, PCR and quantitative PCR\u003c/h2\u003e \u003cp\u003eGenomic DNA was extracted from leaf tissue using a DNAeasy plant mini kit (Qiagen). Total RNA was isolated using the Trizol method (Chomczynski and Sacchi, 1987). The absence of DNA contamination in RNA was confirmed by PCR using \u003cem\u003eELF1\u003c/em\u003e-specific primers (Suppl. Table\u0026nbsp;1). The cDNA was synthesized with 1 \u0026micro;g of DNase-treated total RNA using a RevertAid H Minus first-strand cDNA synthesis kit using oligo dT primers (Fermentas). Primers specific for the constitutively expressed \u003cem\u003eActin 2\u003c/em\u003e gene (At3g18780; Suppl. Table\u0026nbsp;1) were used as a control for an equal amount of gDNA and cDNA as well as for calibration in quantitative comparisons. The relative transcript level of \u003cem\u003eA. thaliana\u003c/em\u003e- and \u003cem\u003eA. arenosa\u003c/em\u003e-originated-\u003cem\u003eCENH3\u003c/em\u003e was measured by qPCR using species-specific primers (Suppl. Table\u0026nbsp;1). 10 \u0026micro;l of PCR mixture contained 1 \u0026micro;l of cDNA template, 5 \u0026micro;l of 2\u0026times; Power SYBR Green PCR Master Mix (Applied Biosystems), 0.33 mM of the forward and reverse primers for each gene. Reactions were run in an Applied Biosystems 7900HT Fast Real-Time PCR System, and data were analyzed with SDS software v2.2.2. The quantitative PCR was performed using the following conditions: 95\u0026deg;C for 10 min, followed by 40 cycles at 95\u0026deg;C for 15 s and an annealing temperature of 60\u0026deg;C for 60 s. The specificity and efficiency of both primers were determined by qPCR using a dilution series of plasmids of cloned full-length cDNA of \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eA. arenosa CENH3\u003c/em\u003e genes. A similar \u003cem\u003eCt\u003c/em\u003e value (the PCR cycle at which the fluorescent signal of reporter dye exceeds background level) for an equal amount of plasmid and absence of amplification with the plasmid of the opposite CENH3 variant indicated that both primer pairs can amplify specific transcripts with the same efficiency.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eGenome size determination by flow cytometry and flow sorting of nuclei\u003c/h2\u003e \u003cp\u003eThe genome size of putative hybrid plants was estimated using \u003cem\u003eRaphanus sativus\u003c/em\u003e 'Voran' (1.11 pg/2C, Gatersleben gene bank accession number: RA 34) as an internal reference standard in comparison to the values obtained for their tetraploid parents. For this, roughly 0.5 cm\u003csup\u003e2\u003c/sup\u003e of fresh leaf tissue was chopped together with the internal reference standard in a Petri dish with a sharp razorblade in nuclei isolation buffer (Galbraith, Harkins et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1983\u003c/span\u003e) supplemented with propidium iodide (50 \u0026micro;g/ml) and DNase-free RNase (50 \u0026micro;g/ml). The resulting nuclei suspension was filtered through a 50 \u0026micro;m CellTrics filter (Sysmex-Partec) and measured using a FACStar\u003csup\u003ePLUS\u003c/sup\u003e cell sorter (BD Biosciences). The means of the nuclear peaks were determined using the software CellQuest (BD Biosciences) and the DNA contents calculated as described (Dolezel, Greilhuber et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).For the parental tetraploid species \u003cem\u003eA. thaliana\u003c/em\u003e (N3151) and \u003cem\u003eA. arenosa\u003c/em\u003e, we performed 12 and six measurements, respectively. For the putative hybrid plants between one and six independent measurements were done.\u003c/p\u003e \u003cp\u003eFor sorting of nuclei, leaf tissue was fixed in 4% formaldehyde, washed in Tris buffer, chopped in nuclei isolation buffer LB01 (Dolezel et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and stained with 4\u0026rsquo;,6-diamidino-2-phenylindole (DAPI, 1 \u0026micro;g/ml final concentration) as described in (Ahmadli, Kalidass et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The sorting was performed on BD Influx cell sorter (BD Biosciences) equipped with a 355 nm UV laser using the BD FACS Sortware software. Afterwards, the sorted nuclei suspension was mixed with equal amounts of sucrose buffer on a microscopic slide and dried overnight (for details, see (Ahmadli, Kalidass et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)). Slides were either directly used for immunostaining or transferred for longer storage to -20\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGeneration of an\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eA. arenosa\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCENH3-specific antibody\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe peptide RTKHFATKSRTGNRTDN was used to generate an \u003cem\u003eA. arenosa\u003c/em\u003e CENH3-specific (anti-AaCENH3) polyclonal antibody (Suppl. Figure\u0026nbsp;1a). Peptide synthesis, immunization of guinea pigs, and peptide affinity purification of antisera were performed by Pineda, Antibody-Service (Berlin, Germany).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eFluorescence\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003ein situ\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehybridization and indirect immunostaining\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eA. thaliana\u003c/em\u003e (AtCEN) and \u003cem\u003eA. arenosa\u003c/em\u003e (AaCEN (Matsushita et al. 2012)) centromere-specific FISH probes were generated by PCR using the primers pairs AtCEN-F/R and AaCEN-F/R, respectively (Suppl. Table\u0026nbsp;1). PCR was performed with \u003cem\u003eTaq\u003c/em\u003e DNA polymerase with a block preheated to 94˚C and an initial denaturation of 94˚C for 2 min followed by 32 cycles of 94˚C for 25 sec, 57˚C for 30 sec, and 72˚C for 40 sec. The probes were labeled with FITC or Cy3 by nick translation.\u003c/p\u003e \u003cp\u003eThe rabbit HTR12-specific antibody (Abcam, ab72001, (Talbert, Masuelli et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2002\u003c/span\u003e)) was used for the detection of \u003cem\u003eA. thaliana\u003c/em\u003e CENH3 (anti-AtCENH3). Leaves were fixed in 1x phosphate buffered saline (PBS) containing 4% paraformaldehyde (PFA) for 20 min at room temperature and then immediately washed in 1x PBS twice for 5 min each. Indirect immunostaining and fluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization were carried out as described by (Gernand, Demidov et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and (Ma, Vu et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), respectively. Imaging was performed by using an Olympus BX61 microscope and an ORCA-ER CCD camera (Hamamatsu). All images were collected in greyscale and pseudocoloured with Adobe Photoshop 6 (Adobe). Maximum intensity projections were done with the AnalySIS (Soft Imaging System) program. To achieve an optical resolution of ca. 120 nm, we applied spatial structured illumination microscopy (3D-SIM) using a 63x/1.40 objective of an Elyra PS.1 super-resolution microscope system and the software ZENBlack (Carl Zeiss GmbH). Image stacks were captured separately for each fluorochrome using 561, 488, and 405 nm laser lines for excitation and appropriate emission filters (Weisshart et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kubalov\u0026aacute; et al. 2021). (Weisshart, Fuchs et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Kubalova, Camara et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The 3D-image stacks were used to generate Suppl. Movie 1 using the Imaris 9.7 (Bitplane) software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eClassification of immunosignal patterns\u003c/h2\u003e \u003cp\u003eTo compare the immunostaining patterns of the subgenome-specific CENH3 variants, double immunostaining was performed using rabbit anti-AtCENH3 (red signals) and guinea pig anti-AaCENH3 (green signals) specific antibodies. For this we prepared slides from flow-sorted 2C and 4C leaf nuclei (the two dominating (endopoly)ploidy levels in young leaf tissue) to minimize background signals. In subsequent quantitative immunolabeling experiments, as there were no significant differences observed between 2C and 4C sorted immunolabeled nuclei, we proceeded to analyze them collectively. The immuno signals were classified based on their strength (strong or weak) and absence. The absence of signals was called \u0026ldquo;0\u0026rdquo;, strong AtCENH3 or AaCENH3 signals were called \u0026ldquo;AT\u0026rdquo; or \u0026ldquo;AA\u0026rdquo;, subsequently. Weak AtCENH3 or AaCENH3 signals were called \u0026ldquo;at\u0026rdquo; or \u0026ldquo;aa\u0026rdquo;, respectively. Therefore, nuclei with unbiased subgenome-specific CENH3 signals were called either \u0026lsquo;AT/AA\u0026rsquo; or \u0026lsquo;at/aa\u0026rsquo;. Nuclei with biased CENH3 signal intensities were called \u0026ldquo;AT/aa\u0026rdquo;, \u0026ldquo;at/AA\u0026rdquo;, \u0026ldquo;AT/0\u0026rdquo;, \u0026ldquo;at/0\u0026rdquo;, \u0026ldquo;AA/0\u0026rdquo; or \u0026ldquo;aa/0\u0026rdquo;. For each genotype, at least 155 immunostained nuclei were characterized (Suppl. Table\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCentromeres of allopolyploid\u003c/b\u003e \u003cb\u003eA. suecica\u003c/b\u003e \u003cb\u003eincorporate both CENH3 variants\u003c/b\u003e, \u003cb\u003ebut more from\u003c/b\u003e \u003cb\u003eA. arenosa\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo address whether subgenome-specific CENH3 variants undergo subgenome-specific centromere loading in an allopolyploid species, we used the allotetraploid \u003cem\u003eA. suecica\u003c/em\u003e as a model. First, the hybrid nature of \u003cem\u003eA. suecica\u003c/em\u003e was confirmed by multicolour FISH using centromere-specific DNA probes for \u003cem\u003eA. thaliana\u003c/em\u003e (AtCEN) and \u003cem\u003eA. arenosa\u003c/em\u003e (AaCEN) centromeres (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). After FISH, chromocenters revealed either \u003cem\u003eA. thaliana\u003c/em\u003e- or \u003cem\u003eA. arenosa\u003c/em\u003e-specific signals. Next, an \u003cem\u003eA. arenosa\u003c/em\u003e CENH3-specific antibody (anti-AaCENH3) was produced and tested for specificity with mitotic and interphase chromosomes of \u003cem\u003eA. arenosa\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, Suppl. Figure\u0026nbsp;1b). The absence of immunosignals in \u003cem\u003eA. thaliana\u003c/em\u003e nuclei confirmed the species specificity of anti-AaCENH3. The species specificity of anti-\u003cem\u003eA. thaliana\u003c/em\u003e CENH3 (anti-AtCENH3) was demonstrated as only the centromeres of \u003cem\u003eA. thaliana\u003c/em\u003e but not of \u003cem\u003eA. arenosa\u003c/em\u003e displayed immunosignals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, c). Double immunostaining using both antibodies and subsequent FISH with the \u003cem\u003eA. arenosa\u003c/em\u003e centromere-specific probe confirmed that both CENH3 variants are incorporated in all centromeres of \u003cem\u003eA. suecica\u003c/em\u003e independent of their origin and sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, the centromeric localization of both subgenome-specific CENH3 variants was tested in synthetic allotetraploid \u003cem\u003eA. suecica\u003c/em\u003e (N22665) plants. Double immunostaining revealed that about 60% of analyzed sorted nuclei incorporated equally both subgenome-specific CENH3s, resulting in either strong (AT/AA) or weak (at/aa) signals indicating an unbiased loading of subgenome-specific CENH3s (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In cases with a biased subgenome-specific CENH3 loading (i.e. AT/aa, at/AA, AT/0, at/0, 0/AA or 0/aa), we never found nuclei with strong AtCENH3 signals (i.e. AT/aa and AT/0) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Suppl. Table\u0026nbsp;2). In contrast, about 40% of nuclei, showed strong AaCENH3 signals (i.e. at/AA and 0/AA), indicating a biased loading of \u003cem\u003eA. arenosa\u003c/em\u003e subgenome-specific CENH3.\u003c/p\u003e \u003cp\u003eTo check whether the same bias exists in natural allopolyploid \u003cem\u003eA. suecica\u003c/em\u003e, derived from ancient \u003cem\u003eA. thaliana\u003c/em\u003e x \u003cem\u003eA. arenosa\u003c/em\u003e hybridization events, we performed double immunostaining on sorted nuclei of \u003cem\u003eA. suecica\u003c/em\u003e accession \u0026ldquo;Sue2\u0026rdquo;. About 70% of analyzed nuclei, had equally incorporated subgenome-specific CENH3s (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In cases with a biased subgenome-specific CENH3 loading (i.e. AT/aa, at/AA, AT/0, at/0, 0/AA or 0/aa), no nuclei with strong AtCENH3 signals (i.e. AT/aa and AT/0) were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Suppl. Table\u0026nbsp;2). However, about 30% of all nuclei, showed strong AaCENH3 signals (i.e. at/AA and 0/AA), demonstrating a similar biased \u003cem\u003eA. arenosa\u003c/em\u003e CENH3 incorporation. To exclude that the observed signal differences of both species-specific CENH3 antibodies were not caused by a lower quality of the AtCENH3 antibody, an additional control experiment was performed with tetraploid \u003cem\u003eA. thaliana\u003c/em\u003e, the parent genotype we used for wide hybridization with \u003cem\u003eA. arenosa\u003c/em\u003e. The same combination of antibodies resulted in nuclei with about 71% strong (AT/0) and 27% weak (at/0) AtCENH3-specific signals, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Thus, our findings indicate that both subgenome-specific CENH3 variants are incorporated into the centromeres of \u003cem\u003eA. suecica\u003c/em\u003e, but with a positive bias towards \u003cem\u003eA. arenosa\u003c/em\u003e CENH3.\u003c/p\u003e\n\u003ch3\u003ePreferential expression and loading of AaCENH3 become established immediately after hybrid formation\u003c/h3\u003e\n\u003cp\u003eCrossing of both species was performed to determine whether the preferential \u003cem\u003eA. arenosa\u003c/em\u003e-type CENH3 incorporation is already established in the first generation after the wide hybridization. Only pollination of tetraploid \u003cem\u003eA. thaliana\u003c/em\u003e with tetraploid \u003cem\u003eA. arenosa\u003c/em\u003e resulted in fertile seeds. As aneuploidy and chromosome elimination were described for crosses between \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eA. arenosa\u003c/em\u003e (Comai, Tyagi et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Wright, Pires et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), flow cytometry was employed to identify successful hybridization events. In an initial pre-screening without an internal reference standard, one (plant 6) out of 30 plants was identified as diploid (Suppl. Figure\u0026nbsp;2a) while all other plants were confirmed to be tetraploid. Immunostaining with both types of CENH3 antibodies on nuclei of plant 6, confirmed the absence of the pollinator genome, as only up to 10 AtCENH3-specific signal clusters were found (Suppl. Figure\u0026nbsp;2b). Hence, here all \u003cem\u003eA. arenosa\u003c/em\u003e-derived chromosomes were eliminated, likely during hybrid embryogenesis. Uniparental elimination of chromosomes is a common phenomenon in hybrids derived from distantly related species (reviewed in (Ishii, Karimi-Ashtiyani et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)). Furthermore, we noticed in the flow cytometric pre-screen an obvious variation in the peak positions between the measurements of the individual plants (Suppl. Figure\u0026nbsp;2c). Therefore, we determined the genome size of 24 putative F1 hybrids using \u003cem\u003eRaphanus sativus\u003c/em\u003e as an internal reference standard and compared the data to values obtained for the tetraploid parents (Suppl. Figure\u0026nbsp;3a). For tetraploid \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eA. arenosa\u003c/em\u003e, we estimated genome sizes of 0.683 pg/2C and 0.825 pg/2C, respectively, indicating an expected genome size for the interspecific hybrid of 0.754 pg/2C. Among the analyzed plants, 13 plants revealed a genome size deviating by less than 3% from the expected value of a hybrid plant and were considered as euploid hybrid plants. 11 plants showed a deviation from the expected genome size of more than 3%, presumably as a result of aneuploidy. One plant is most likely the product of an \u003cem\u003eA. thaliana\u003c/em\u003e selfing event or spontaneous doubling of haploid progeny (plant No. 25, Suppl. Figure\u0026nbsp;3a, b).\u003c/p\u003e \u003cp\u003eBased on the determined DNA contents, leaf nuclei of selected 3-month-old F1 hybrid plants were sorted on slides and analyzed by double immunostaining. About 50% of nuclei showed an unbiased loading of subgenome-specific CENH3 variants (AT/AA and at/aa nuclei). Out of the analyzed nuclei, only 3% showed stronger AtCENH3 signals (i.e. AT/aa, AT/0 and at/0 nuclei). In contrast, about 45% of CENH3-loaded nuclei, showed stronger \u003cem\u003eA. arenosa\u003c/em\u003e signals (i.e. at/AA, 0/AA and 0/aa nuclei) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Suppl. Table\u0026nbsp;2), indicating a biased loading of \u003cem\u003eA. arenosa\u003c/em\u003e-specific CENH3. The observed increase in the frequency of nuclei with an equal proportion of parental CENH3s (i.e., AT/AA and at/aa nuclei) from F1 to natural hybrids of \u003cem\u003eA. suecica\u003c/em\u003e through generations suggests a gradual step-wise adaptation of CENH3 variant loading in allopolyploid Arabidopsis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Suppl. Figure\u0026nbsp;4). Super-resolution microscopy confirmed the mixed composition of both parental CENH3s in F1 hybrid nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Suppl. Movie 1). However, the ultrastructure of AtCENH3 and AaCENH3 signals differed but intermingled, suggesting that the subgenome-specific CENH3 variants are preferentially loaded into different centromeric nucleosome arrays.\u003c/p\u003e \u003cp\u003eThe observed biased CENH3 incorporation prompted us to analyze the transcription of both parental \u003cem\u003eCENH3s\u003c/em\u003e. The relative transcript levels of \u003cem\u003eA. thaliana-\u003c/em\u003e and \u003cem\u003eA. arenosa\u003c/em\u003e-derived \u003cem\u003eCENH3\u003c/em\u003e were quantified and then normalized to \u003cem\u003eACTIN2\u003c/em\u003e (At3g18780) after qPCR with \u003cem\u003eAaCENH3-\u003c/em\u003e and \u003cem\u003eAtCENH3\u003c/em\u003e-specific primer pairs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Suppl. Table\u0026nbsp;1). In all analyzed tissues (young rosette leaves, flower buds, and siliques) of allopolyploid \u003cem\u003eA. suecica\u003c/em\u003e (natural hybrid) and synthetic hybrids (F1, older synthetic hybrid), the relative expression of \u003cem\u003eA. arenosa CENH3\u003c/em\u003e was higher in comparison to \u003cem\u003eA. thaliana\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn summary, we conclude that the majority (above 90%) of centromeres of \u003cem\u003eA. thaliana\u003c/em\u003e x \u003cem\u003eA. arenosa\u003c/em\u003e F1 hybrid, synthetic and natural \u003cem\u003eA. suecica\u003c/em\u003e incorporate CENH3s of both parental genomes, despite a different centromere DNA composition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, after the formation of the F1 hybrid, the contribution of \u003cem\u003eA. arenosa\u003c/em\u003e-derived CENH3 is higher than that of AtCENH3.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAn invaluable plant model for examining the genomic and epigenomic alterations linked to polyploidization is \u003cem\u003eA. suecica\u003c/em\u003e, a naturally occurring hybrid of \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eA. arenosa\u003c/em\u003e (Comai, Tyagi et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). It is a great option for these kinds of studies since it provides a means of simulating evolution through the creation of artificial hybrids. Here, we investigated whether distinct CENH3s, which originated from both parental species, incorporate centromere-sequence independently in a hybrid situation. Therefore, we used varying evolutionary ages of \u003cem\u003eA. suecica\u003c/em\u003e, ranging from F1 hybrids produced by wide crossing of tetraploid \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eA. arenosa\u003c/em\u003e to resynthesize the species, a synthetic hybrid and natural \u003cem\u003eA. suecica\u003c/em\u003e. We found that both CENH3s encoded by either subgenome incorporate into the centromeres of hybrid genotypes, even though the centromeric satellite repeats of both subgenomes share only 58\u0026ndash;80% sequence identity (Kamm, Galasso et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). This observation aligns with the findings of Talbert et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), who showed that anti-CENH3 of \u003cem\u003eA. thaliana\u003c/em\u003e recognizes all centromeres in both synthetic and natural allopolyploid \u003cem\u003eA. suecica\u003c/em\u003e. Also, a GFP::\u003cem\u003eA. arenosa\u003c/em\u003e CENH3 construct can functionally replace CENH3 of an \u003cem\u003eA. thaliana\u003c/em\u003e null mutant (\u003cem\u003ecenh3-1)\u003c/em\u003e (Ravi, Kwong et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Analysis of alien CENH3s localization in \u003cem\u003eA. thaliana\u003c/em\u003e- and tobacco-tissue cultured cells showed that in contrast to the CENH3 of rice, CENH3 of \u003cem\u003eA. thaliana\u003c/em\u003e and tobacco incorporated into the centromeres of both tobacco and \u003cem\u003eA. thaliana\u003c/em\u003e while CENH3 of the holocentric \u003cem\u003eLuzula nivea\u003c/em\u003e became integrated only partially into the centromeres of \u003cem\u003eA. thaliana\u003c/em\u003e cultured cells, suggesting that only evolutionally close CENH3s can target centromeres in alien species (Nagaki, Terada et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In addition, the replacement of endogenous CENH3 in \u003cem\u003eA. thaliana\u003c/em\u003e with CENH3 from related species demonstrated that CENH3 of heterologous species must be related to the \u003cem\u003eA. thaliana\u003c/em\u003e CENH3 for functional complementation (Ravi, Kwong et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Maheshwari, Ishii et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our observations that \u003cem\u003eA. arenosa\u003c/em\u003e\u0026ndash;derived CENH3 could localize to the centromeric satellite repeats of \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003evica versa\u003c/em\u003e are in agreement with these findings and confirm the close evolutionary relationship of both parent CENH3s. The coexistence of nuclei with different CENH3 patterns is likely caused by the multi-cell-type composition of leaves which were used for our analysis. Likely, the centromere composition is dynamically organized according to the needs of individual cell and tissue-types during development. In analogy, a tissue-specific CENH3-type composition was also found in barley and cowpea ((Ishii, Karimi-Ashtiyani et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Ishii, Juranic et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSuper-resolution microscopy revealed that AtCENH3 and AaCENH3 signals have an almost similar but not identical distribution in the centromeres of the hybrid plants, suggesting that both CENH3s do not exist as monomers in the same nucleosome. Instead, subgenome-specific CENH3s are loaded into separate nucleosome arrays to form distinct centromeric substructures. This phenomenon of CENH3 variant-specific distribution has also been observed in other plants, such as barley, a wheat 1BL/1RS translocation line, and cowpea, where different CENH3 variants occupy distinct centromeric nucleosomes (Ishii, Karimi-Ashtiyani et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Yuan, Guo et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Ishii, Juranić et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Karimi-Ashtiyani, Schubert et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The intermingled arrangement of different CENH3 variants in a centromere may be an evolutionary adaption to handle more than one CENH3 variant.\u003c/p\u003e \u003cp\u003eIn a study focusing on the process of polyploidization, Burns et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) found that the speciation process in \u003cem\u003eA. suecica\u003c/em\u003e was a gradual and adaptive evolutionary process rather than a sudden or drastic event. This gradual evolution was evidenced by the absence of massive changes in the genome arrangement and mobilization of transposable elements, as well as the lack of subgenome dominance in gene expression (Burns, Mand\u0026aacute;kov\u0026aacute; et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This finding is consistent with the observed increased frequency of unbiasedly loaded nuclei of parental CENH3s from F1 to natural hybrids of \u003cem\u003eA. suecica\u003c/em\u003e through generations in our study (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Supple Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, we cannot rule out the possibility that other \u003cem\u003eA. suecica\u003c/em\u003e genotypes exhibit different CENH3 patterns, given that natural \u003cem\u003eA. suecica\u003c/em\u003e is a product of several founding individuals rather than a single origin (Novikova et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur expression analysis revealed that the dynamic of CENH3 expression is similar in parents and their allopolyploid hybrids, with higher expression levels in tissues with rapidly dividing cells. Furthermore, our results indicate that CENH3 has generally higher expression levels in \u003cem\u003eA. arenosa\u003c/em\u003e compared to \u003cem\u003eA. thaliana\u003c/em\u003e, and this pattern persists in the hybrids. This result is in line with the findings that the \u003cem\u003eA. arenosa\u003c/em\u003e subgenome in resynthesized and natural \u003cem\u003eA. suecica\u003c/em\u003e allotetraploids is hypomethylated, which may contribute to the observed upregulation of many genes involved in reproduction and adaptation (Jiang, Song et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn conclusion, our analysis of transcription and centromeric localization of subgenome-specific CENH3 variants in the allopolyploid species \u003cem\u003eA. suecica\u003c/em\u003e demonstrates that both parental CENH3 variants are retained, with a gradual increase of equal loading of subgenome-specific CENH3s during the progression from F1 to natural hybrids. This suggests the potential impact of centromere plasticity on establishing stable centromeres, genome integrity and evolution across generations in allopolyploid speciation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAH was supported\u0026nbsp;by\u0026nbsp;the German Federal Ministry of Education and Research (FKZ 0315965), RK-A was supported by Iran National Science Foundation (INSF) under project No. 4004919. TI was supported by the JSPS KAKENHI Grant-in-Aid for Scientific Research (C), (Grant No. 22K05572) and \u0026nbsp;JST-FOREST (Grant No. JPMJFR 2001).\u0026nbsp;The excellent technical assistance of Karla Meier and Katrin Kumke (IPK, Gatersleben, Germany) is gratefully acknowledged.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRK-A contributed to material preparation, performed experiments, collected and analyzed data, and interpreted the results. AMB-M performed expression analysis and interpreted the results. OW contributed to immunostaining and material preparation. JF performed flow cytometry and interpreted the results. VS performed high-resolution microscopy. RK-A wrote the first draft of the manuscript, and all authors provided comments. AH contributed to the study conception and writing. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhmadli U, Kalidass M, Khaitova LC, Fuchs J, Cuacos M, Demidov D, Zuo S, Pecinkova J, Mascher M, Ingouff M, Heckmann S, Houben A, Riha K, Lermontova I (2023) High temperature increases centromere-mediated genome elimination frequency and enhances haploid induction in Arabidopsis. Plant Commun 4(3):100507\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurns R, Mand\u0026aacute;kov\u0026aacute; T, Gunis J, Soto-Jim\u0026eacute;nez LM, Liu C, Lysak MA, Novikova PY, Nordborg M (2021) Gradual evolution of allopolyploidy in Arabidopsis suecica. 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New Phytol 206(2):839\u0026ndash;851\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-molecular-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plan","sideBox":"Learn more about [Plant Molecular Biology](https://www.springer.com/journal/11103)","snPcode":"11103","submissionUrl":"https://submission.nature.com/new-submission/11103/3","title":"Plant Molecular Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"CENH3, centromere plasticity, species evolution, haploid, uniparental chromosome elimination, allopolyploidization","lastPublishedDoi":"10.21203/rs.3.rs-3997508/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3997508/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCentromeric nucleosomes are determined by the replacement of the canonical histone H3 with the centromere-specific histone H3 (CENH3) variant. Little is known about the centromere organization in allopolyploid species where different subgenome-specific CENH3s and subgenome-specific centromeric sequences coexist. Here, we analyzed the transcription and centromeric localization of subgenome-specific CENH3 variants in the allopolyploid species \u003cem\u003eArabidopsis suecica.\u003c/em\u003e Synthetic \u003cem\u003eA. thaliana\u003c/em\u003e x \u003cem\u003eA. arenosa\u003c/em\u003e hybrids were generated and analyzed to mimic the early evolution of \u003cem\u003eA. suecica\u003c/em\u003e. Our expression analyses indicated that CENH3 has generally higher expression levels in \u003cem\u003eA. arenosa\u003c/em\u003e compared to \u003cem\u003eA. thaliana\u003c/em\u003e, and this pattern persists in the hybrids. We also demonstrated that despite a different centromere DNA composition, the centromeres of both subgenomes incorporate CENH3 encoded by both subgenomes, but with a positive bias towards \u003cem\u003eA. arenosa\u003c/em\u003e-type CENH3. The intermingled arrangement of both CENH3 variants demonstrates centromere plasticity and may be an evolutionary adaption to handle more than one CENH3 variant in the process of allopolyploidization.\u003c/p\u003e","manuscriptTitle":"Centromere sequence-independent but biased loading of subgenome-specific CENH3s in allopolyploid Arabidopsis suecica","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-13 18:19:48","doi":"10.21203/rs.3.rs-3997508/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2024-04-08T17:25:34+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-03-09T22:24:49+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-09T19:09:49+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant Molecular Biology","date":"2024-03-01T03:07:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-28T06:00:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Molecular Biology","date":"2024-02-27T09:42:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-molecular-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plan","sideBox":"Learn more about [Plant Molecular Biology](https://www.springer.com/journal/11103)","snPcode":"11103","submissionUrl":"https://submission.nature.com/new-submission/11103/3","title":"Plant Molecular Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f27dc23e-91a3-4c0d-8e0e-5440722ab102","owner":[],"postedDate":"March 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-21T01:30:55+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-13 18:19:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3997508","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3997508","identity":"rs-3997508","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}