Admixture of divergent genomes facilitates hybridization across species in the family Brassicaceae

Summary Hybridization and polyploidization are pivotal to plant evolution. Genetic crosses between distantly related species are rare in nature due to reproductive barriers but how such hurdles can be overcome is largely unknown. Here we report the hybrid genome structure of xBrassicoraphanus, a synthetic allotetraploid of Brassica rapa and Raphanus sativus. We performed cytogenetic analysis and de novo genome assembly to examine chromosome behaviors and genome integrity in the hybrid. Transcriptome analysis was conducted to investigate expression of duplicated genes in conjunction with epigenome analysis to address whether genome admixture entails epigenetic reconfiguration. Allotetraploid xBrassicoraphanus retains both parental chromosomes without genome rearrangement. Meiotic synapsis formation and chromosome exchange are avoided between nonhomologous progenitor chromosomes. Reconfiguration of transcription network occurs, and less divergent cis‐elements of duplicated genes are associated with convergent expression. Genome‐wide DNA methylation asymmetry between progenitors is largely maintained but, notably, B. rapa‐originated transposable elements are transcriptionally silenced in xBrassicoraphanus through gain of DNA methylation. Our results demonstrate that hybrid genome stabilization and transcription compatibility necessitate epigenome landscape adjustment and rewiring of cis–trans interactions. Overall, this study suggests that a certain extent of genome divergence facilitates hybridization across species, which may explain the great diversification and expansion of angiosperms during evolution.


Introduction
Genome hybridization and polyploidization have served as major driving forces in plant evolution (Wendel, 2000;Soltis & Soltis, 2009Van de Peer et al., 2017;Cheng et al., 2018). However, strong hybridization barriers exist in nature to prevent gene flow between different species in plants and animals (Abbott et al., 2013). Several mechanisms have been proposed to explain the postzygotic barriers resulting from genome incompatibility between distantly related species (Lafon-Placette & Kohler, 2015; Dion-Cote & Barbash, 2017). Among them, a 'genome shock' is proposed as one of the critical causes of genome destabilization upon hybridization, restructuring the hybrid genome through changes of chromosomal organization or mobilization of transposable elements (TEs) (McClintock, 1984). Another is a 'transcriptome shock' that incurs extensive changes of parental gene expression patterns in the hybrid (Hegarty et al., 2006;Buggs et al., 2011).
Despite such negative consequences of hybridization between distantly related species, novel species can be naturally or artificially produced on rare occasions while overcoming the hybridization barrier, the mechanism of which is largely unknown. The family Brassicaceae contains a variety of agronomically important crop species such as broccoli, cabbage, cauliflower, oilseed rape, radish and turnip, in addition to the model plant Arabidopsis. Within the genus, Brassica is well known for hybridization between different species (interspecific hybridization). For instance, three diploid species, Brassica rapa (Br; AA), B. nigra (BB) and B. oleracea (Bo; CC), can hybridize with each other, generating allotetraploid species B. napus (AACC), B. juncea (AABB) and B. carinata (BBCC), as epitomized by the model of the 'Triangle of U' (Nagaharu, 1935).
We hypothesized that allopolyploidization events have somewhat ameliorated deleterious shock phenomena such as genome and transcriptome shocks, and thereby overcome an intrinsic hybridization barrier between distantly related species. Here we report the genome structure, chromosome behaviors and transcriptome/epigenome profiles of xB. We observed inhibition of meiotic nonhomologous interactions, adjustment of homoeologous gene expressions and gain of DNA methylation. All these probably contribute to genome stability and transcription network compatibility in xB. This study further proposes the possible mechanisms by which two divergent genomes can successfully merge into a novel species during the evolution of angiosperms.

Materials and Methods
Plant materials xBrassicoraphanus cv BB1 (xB), Brassica rapa L. cv  and Raphanus sativus L. cv WK10039 (Rs) were chosen as primary plant materials for genome and cytogenetic analyses. xB was originally derived from the microspore culture of a synthetic hybrid between commercial cultivars B. rapa cv Jeonseung and R. sativus cv Taebaek, and maintained for > 10 generations by selfpollination (Lee et al., 2011;Park et al., 2020). Br and Rs are inbred lines whose genome sequences are available (Wang et al., 2011;Jeong et al., 2016), propagated by self-pollination. Sterilized seeds were germinated and grown on 19 Murashige & Skoog (MS) medium (Duchefa, Haarlem, the Netherlands) in a growth chamber under 16 h of fluorescent light at 20 AE 10 lmol m À2 s À1 and 22°C for 14 d. The seedlings including shoots and roots were harvested together for whole genomesequencing (seq), RNA-seq, bisulfite (BS)-seq, chromatin immunoprecipitation (ChIP)-seq and small RNA-seq. For tissuespecific transcriptome analysis, RNA was extracted from leaf, hypocotyl and root tissue of seedlings and from opened flowers of Br, Rs and xB. For cold treatment, 14 d after sowing, seedlings of Br, Rs and xB were grown at 4°C for 5 wk. RNA was extracted and stored at À20°C until use.

Genome sequencing, assembly and genome size estimation
Paired-end and mate-pair sequencing libraries with insert sizes of 200 bp, 400 bp, 3 kb, 8 kb, 5 kb, 10 kb and 15 kb were constructed using KAPA Library Prep Kit (Roche, Bazel, Switzerland) and Illumina Mate Pair Library Kit (Illumina, San Diego, CA, USA) following the manufacturers' instructions (Table S1). The libraries were sequenced on an Illumina HiSeq 2000 platform. Prokaryotic sequences, duplicated reads, low-quality reads and lowfrequency reads were filtered out (Table S1). The preprocessed sequences were assembled using SOAPDENOVO2 (Luo et al., 2012) with the best k-mer values for each library. To increase the length of scaffolds, serial scaffolding processes were carried out using SOAPDENOVO2 (Luo et al., 2012) and SSPACE (Boetzer et al., 2011). Gaps in the scaffolds were reduced further using SOAPDENOVO GAPCLOSER (Luo et al., 2012) and PLATANUS (Kajitani et al., 2014) (Table S2). Completeness of the final assembly was validated using BUSCO v.5.2.2 (Simao et al., 2015) with the embryophyta ortholog database. In the k-mer analysis, counting k-mer occurrence of 19mers was performed using JELLYFISH (Marcais & Kingsford, 2011). The genome size of xB was estimated by flow cytometry analysis (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) as previously described (Huang et al., 2013). Genome data were visualized with CIRCOS (Krzywinski et al., 2009).

Chloroplast genome assembly
The chloroplast genome was de novo assembled from the 19 coverage of whole-genome sequencing reads. The chloroplast genome was annotated with GESEQ (Tillich et al., 2017) and manually curated. The chloroplast genome was visualized using ORGANELLARGENOMEDRAW (Lohse et al., 2013).

Assignment of scaffolds to A xB and R xB subgenomes
Whole-genome sequencing reads of Rs and Br from the Brassica Database (BRAD) were mapped to the xB scaffolds using BOWTIE (Langmead et al., 2009). The number of mapped reads was counted and the scaffolds were assigned to A xB and R xB subgenomes, based on a comparison of the number of parental reads (A xB subgenome: > 99% ratio of mapped reads from Br; R xB subgenome: > 99% ratio of mapped reads from Rs). Next, assigned xB scaffolds were anchored to the reference chromosomes of Br and Rs to build xB pseudochromosomes.

Gene and TE annotation
Gene annotations of xB and Rs were performed following the previous annotation pipeline with minor modifications (Kim et al., 2014). Briefly, the annotation pipeline consisted of repeat masking, mapping of different protein sequence sets and mapping of RNA-seq reads. Independent ab initio predictions were performed with AUGUSTUS (Stanke et al., 2008). The EVIDENCEMODELER (Haas et al., 2008) software combines ab initio gene predictions with protein and transcript alignments into weighted consensus gene structures. Gene annotation of Br was downloaded from Ensembl plant (ftp://ftp.ensemblgenomes.org/ pub/plants/release-31/gff3/brassica_rapa/) and an additional 1700 genes were annotated using EXONERATE (Slater & Birney, 2005). Functional annotation was performed through BLASTP against the SwissProt and Plant RefSeq databases. TE-related repeat sequences were predicted by REPEATMODELER (Smit & Hubley, 2008) and REPEATMASKER (Smit et al., 2015).

Fluorescence in situ hybridization (FISH) analysis
The sequences of 5S rDNA, 45S rDNA, RsCent1, RsCent2, BrCent1, BrCent2, RsSTRa, RsSTRb, BrSTRa, BrSTRb and telomere were used as probes (Table S3). The probes were labeled by nick translation with different fluorochromes. Root mitotic chromosome spreads and FISH procedures were performed according to a previous method (Waminal & Kim, 2012). For directly labeled probes, slides were immediately used for FISH after fixation with 4% paraformaldehyde, without subsequent pepsin and RNase pretreatment. Images were captured with an Olympus BX53 fluorescence microscope equipped with a Leica DFC365 FS CCD camera and processed using CYTOVISION v.7.2 (Leica Microsystems, Wetzlar, Germany).

Resynthesized allodiploid and allotetraploid xBrassicoraphanus plants
Resynthesized allodiploid xBrassicoraphanus plants were produced from a cross between Br as a seed parent and Rs as a pollen donor. Thirty-day-old immature hybrid ovules were cultured on 19 MS medium supplemented with 2% (w/v) sucrose and 0.8% (w/v) plant agar. The plates were placed at 24°C in a growth chamber for 2 wk and then seedlings were vernalized at 4°C in a cold chamber for 4 wk with 16 h : 8 h, light : dark. The plants were transferred to pots in the glasshouse with the same light conditions. A 0.3% colchicine solution was applied to the shoot apical meristem for 2 d to induce chromosome doubling.

Resynthesized allodiploid and allotetraploid B. napus plants
Resynthesized allodiploid B. napus plants were produced from a cross between Br as a seed parent and B. oleracea var. Capitata as a pollen donor. Ovary culture was performed as described by Inomata (1977) with modifications. Ovaries at 4 d after pollination were explanted on 19 MS medium supplemented with 5% (w/v) sucrose, 300 mg l À1 casein hydrolysate and 0.8% (w/v) plant agar at 24°C in a growth chamber. Four weeks after explantation, hybrid ovules were transferred to B5 medium with vitamin supplemented with 2% (w/v) sucrose, 300 mg l À1 casein hydrolysate and 0.8% (w/v) plant agar. Ovules were incubated in the dark at 24°C for 3 d under 16 h : 8 h, light : dark conditions. Seedlings were vernalized at 4°C in a cold chamber for 4 wk under 16 h : 8 h, light : dark. The plants were transferred to pots in the glasshouse with the same light conditions. A 0.3% colchicine solution was applied to the shoot apical meristems for 2 d to obtain allotetraploids.

Production of antibody and immunolocalization of meiotic proteins
The coding regions of BrASY1 and BrZYP1 genes were PCRamplified from cDNA of young flowering buds from Br (Table S4). The fragments of BrASY1 (708 bp) and BrZYP1 (1332 bp) were inserted into the pET-28a expression vector (Novagen, Darmstadt, Germany) and transformed into Escherichia coli Rosetta2 (DE3) strains (Novagen). The transformed E. coli cells were grown at 30°C in 1 l of LB medium in the presence of 50 µg ml À1 of kanamycin and 50 µg ml À1 of chloramphenicol until an OD 600 of 0.4 was reached. Recombinant protein expression was induced with 1 mM isopropyl b-Dthiogalactopyranoside at 16°C for 16 h. Cells were centrifuged (4°C) at 6700 g for 15 min and the pellet was resuspended in 100 ml of ice-cold column buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10% glycerol, 0.1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride (PMSF)). Cells were lysed by sonication for 5 min on ice (output power, 4; duty cycle, 50%; Branson Sonifer 250; Branson). Inclusion bodies were collected by centrifugation (4°C) at 9300 g for 25 min and dissolved in 4 M urea. The soluble lysate was purified with a 5 ml HisTrap FF column (GE Healthcare, Piscataway, NJ, USA) with a linear gradient of ice-cold column buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10% glycerol, 0.1 mM dithiothreitol, 250 mM imidazole). The purified BrASY1 and BrZYP1 proteins were used to produce polyclonal antibodies from rabbit and rat, respectively, by Youngin Frontier (Korea), and the quality of the antibody was validated by western blotting. Immunolocalization was performed as described by Chelysheva et al. (2013). In brief, primary antibodies anti-BrASY1 and anti-BrZYP1 were used at a dilution of 1 : 250 in PBST (0.1% Triton-X 100 in 19 PBS) containing 1% BSA and the secondary antibodies (goat antirabbit IgG H&L, Alexa Fluor 488 and donkey antirat IgG H&L, Alexa Fluor 594) were used at a dilution of 1 : 500. Images were captured with an Axioskop2 microscope equipped with an Axiocam 506 color CCD camera (Carl Zeiss, Jena, Germany) and processed using Adobe PHOTOSHOP CS6 (Adobe Systems Inc., Mountain View, CA, USA).

Identification of homoeologous gene pairs
The reciprocal best BLAST hit method was used to determine orthologous gene pairs with over 80% identity and coverage between A and R genomes. The regions that share a common order of five or more orthologs in A and R scaffolds with a distance cutoff of 50 genes were defined as syntenic regions. Then, the reciprocal best BLAST hit method was performed again on the genes within the syntenic regions of A and R subgenomes, and the homoeologous gene pairs were established. Pairwise sequence alignment using CLUSTALW (Larkin et al., 2007) was performed to remove gene pairs with low sequence identity and coverage. Finally, only gene pairs with > 80% identity and > 80% coverage were defined as syntenic homoeologs between A and R subgenomes.

RNA-seq analysis
Total RNA was extracted with RNeasy Plant Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. The DNase-treated RNA samples, including two replicates for each of seedling, leaf, hypocotyl and flower, and one replicate for root of xB and its progenitors, were used for constructing RNA-seq libraries (Zhong et al., 2011). RNA-seq was performed on an Illumina HiSeq 2000 platform. The obtained raw reads were filtered using FASTX-TOOLKIT and low-quality reads (Q < 20) were removed. The filtered reads were mapped to the Br, Rs and xB genomes using TOPHAT ) with default parameters (Table S5). The mapped read counts were calculated using HTSEQ (Anders et al., 2015). Genes not expressed in any tissues were excluded from the orthologous or homoeologous gene pair sets for differentially expressed gene (DEG) analysis. Statistical tests of DEGs were performed using EDGER (Robinson et al., 2010) with false discovery rate (FDR) < 0.05 and fragments per kilobase of transcript per million mapped reads (FPKM) log 2 fold change > 1. Orthologous/homoeologous gene expression patterns were categorized as relative expression levels in the context of A Br -A xBr -R xB -R Rs . Differential expressions for every gene pair of A Br vs R Rs , A Br vs A xBr , A xB vs R xB and R xB vs R Rs were assessed and classified into 27 groups of relative expression changes except ambiguous patterns. The Gene Ontology (GO) terms of the xB genome were annotated by BLAST2GO using the nonredundant sequence database from NCBI with e-value parameter < 1e À15 . Statistical comparison of GO term accumulation was conducted using the R package TOPGO (Alexa & Rahnenfuhrer, 2010) with P-values of Fisher's exact test (P < 0.001).

BS-seq analysis
Genomic DNA (5 lg) extracted from 14-d-old seedlings was used to construct the BS-seq library with the KAPA Library Kit (Roche) and EpiTect Bisulfite Kit (Qiagen) according to the manufacturers' instructions. The libraries were sequenced using the Illumina HiSeq 2000. Raw reads were filtered using FASTX-TOOLKIT and low-quality reads (Q < 20) were removed. Reads were mapped onto the xB genome using BISON (Ryan & Ehninger, 2014), with the parameters '--very-sensitive --scoremin "L,-0.6,-0.6" '. Only cytosine sites with 49 coverage read depth were accepted for subsequent analysis. Differentially methylated cytosines (DMCs) and regions (DMRs) were identified as described previously (Kim et al., 2019). In brief, DMCs were identified using Fisher's exact test (P < 0.05) between the levels of methylation in xB and the progenitors Br and Rs. DMRs were identified based on the regions with a length ≥ 200 bp, five or more DMCs, and mean methylation difference ≥ 0.3 for CG, ≥ 0.15 for CHG and ≥ 0.1 for CHH. Regions 1 kb up-and downstream of the gene body and repeat were divided into 50 bp windows, and weighted DNA methylation levels were represented with a metaplot (Schultz et al., 2012). Methylation data were visualized with the Integrated Genome Browser (Freese et al., 2016).

ChIP-seq analysis
ChIP was performed following Lee et al. (2007). Chromatin was immunoprecipitated with antibody against histone H3K9me2 (Abcam, Cambridge, MA, USA). ChIP-seq libraries were constructed as described in the Illumina ChIP Sequencing Kit (Illumina). DNA fragments of c. 600 bp were excised from an agarose gel and amplified for cluster generation and sequencing. All DNA libraries were sequenced on a HiSeq2500 platform (Illumina) with single-end reads. The sequencing reads were qualitycontrolled with FASTX-TOOLKIT and aligned to the xB genome using BOWTIE (Langmead et al., 2009) with parameters '-best -m1'. H3K9me2-enriched regions were defined using SICER (Zang et al., 2009) (window size = 500, gap size = 600, FDR = 0.01) and overlapping regions between two biological replicates were identified using the MergePeaks module of the HOMER software (Heinz et al., 2010).

Northern blot analysis
Total RNA (10 µg) was electrophoresed on a 1% formaldehyde denaturing gel and transferred onto a Hybond N+ membrane (GE Healthcare). The probes BrGypsy, BrCopia and Actin were amplified by PCR and randomly labelled with [a-32 P]dCTP (Perkin Elmer, Waltham, MA, USA) using a Klenow fragment (3 0 ? 5 0 exoÀ) (New England Biolabs, Ipswich, MA, USA). Hybridization was performed at 65°C overnight in the prehybridization solution containing 69 saline sodium citrate buffer, 59 Denhardt's reagent and 1% sodium dodecyl sulfate. After hybridization, the membrane was washed and exposed to an Xray film (Fujifilm, Tokyo, Japan). Primer sequences are detailed in Table S6.

Genomic features of xBrassicoraphanus
xB is a fertile and genetically stable intergeneric allotetraploid synthesized from a cross between Br and Rs. The xB genome was de novo assembled using 195.0 Gb of Illumina shotgun reads ( Fig. 1a; Tables 1, S1). Flow cytometry analysis estimated the size of the xB genome as 998.3 Mb, close to the sum of the Br (485 Mb) and Rs (510 Mb) genomes (Wang et al., 2011;Jeong et al., 2016) (Fig. S2). We assembled 692.8 Mb of sequence covering c. 70% of the xB genome, on which 97.56% of sequence reads (67 343 270/69 029 900) were successfully mapped back. The final assembly contained 87 861 annotated genes with a benchmarking universal single-copy orthologs (BUSCO) completeness score of 99.7% (Tables 1, S7). Repeat sequences accounted for 39.19% (255.8 Mb) of the assembled xB genome with long terminal repeats (LTRs) being predominant (Table S8). The assembled chloroplast genome of xB (153 482 bp) was 99.9% identical to that of Br, indicating its maternal origin ( Fig. S3; Table S9). In the xB genome (692.8 Mb), 335.5 and 343.5 Mb of scaffolds were assigned to the Br and Rs reference genomes (referred to as A Br and R Rs hereafter), respectively (Wang et al., 2011;Jeong et al., 2016), comprising two subgenomes of xB (referred to as A xB and R xB hereafter) (Table 1; Fig. S4). Of 44 432 and 41 733 annotated genes initially assigned to the A xB and R xB subgenomes, a total of 17 393 syntenic homoeologous pairs were identified in 291 synteny blocks with > 80% identity to each other and > 80% coverage. DEGs whose expressions are up-or downregulated relative to the progenitors emerged evenly throughout the xB genome (Fig. 1a). DNA methylation is predominant in repeat-enriched regions at all CG, CHG and CHH (H = A, T or C) contexts (Figs 1a, S5). DMRs are regions where DNA methylation levels in xB are significantly different (absolute difference > 0.3 for CG, > 0.15 for CHG and > 0.1 for CHH) from those of Br and Rs, and c. 60.2% of hyper-DMRs are confined to repeat regions ( Fig. S5; Table S10). Approximately 75.8% of H3K9me2 repressive histone marks are also enriched in repeat regions (Figs 1a, S6; Table S11). Small RNAs (18-30 nt) are distributed throughout the entire xB genome and significantly associated with DNA methylation (Fig. 1a). A06 chromosome of xB is exemplified in Fig. 1(b), showing distributions of genes, repeats, DNA methylation, H3K9me2 and small RNAs, along with DEGs and DMRs compared to the progenitor (Fig. 1b). Cytological observation revealed a total of 19 chromosome pairs present in xB without aneuploidy and/or chromosome rearrangements (Fig. 1c). Previous studies showed that many synthetic allopolyploid plants such as rapeseed, tobacco and wheat went through massive chromosome reconstruction leading to transgressive gain or loss of chromosomes and/or aneuploidy over generations (Xiong et al., 2011;Zhang et al., 2013;Chen et al., 2018;Sosnowska et al., 2020). However, our findings indicate that xB retains both A Br and R Rs genomes in the single nucleus without structural aberrations, but at the same time experiences substantial changes in transcriptome and epigenome profiles after hybridization.

Avoidance of homoeologous interactions between A and R chromosomes
Interspecific hybridization often involves extensive homoeologous exchanges during meiosis, eventually causing nonhomologous recombination in immediate offspring (Szadkowski et al., 2010(Szadkowski et al., , 2011Xiong et al., 2011;Zhang et al., 2013;Grandont et al., 2014;Chen et al., 2018;Sosnowska et al., 2020). We previously showed that allodiploid xB meiocytes rarely undergo chromosome exchanges during meiosis, as demonstrated by immunolocalization of HUMAN ENHANCER OF INVASION 10 (HEI10), a component of the ZMM complex that mediates meiotic crossover (Park et al., 2020). To verify whether absence of meiotic crossover accompanied by reduction of HEI10 foci is due to the absence of nonhomologous interactions between Br-and Rs-originated chromosomes, we examined synapsis formation of meiotic chromosomes by immunolocalization of ASYNAPTIC1 (ASY1) and ZIPPER1 (ZYP1). ASY1 is the axial/lateral element of meiotic chromosomes loaded onto chromatids before synapsis (Armstrong et al., 2002), and ZYP1 is the central element of synaptonemal complex present in synapsed chromosomes (Higgins et al., 2005). We found that ASY1 was correctly loaded onto the entire axis of all euploid and allodiploid pachytene chromosomes at meiotic prophase I (Fig. 2). ZYP1 also co-localized with ASY1 in all euploid pachytene chromosomes (Fig. 2). Allodiploid B. napus (AC) produced discontinuous stretches of ZYP1 signals, indicating partial synapsis between A and C chromosomes (Fig. S7). Notably, however, ZYP1 was barely associated with allodiploid xB (AR) pachytene chromosomes (Fig. 2) crossover between nonhomologous chromosomes was strongly avoided in xB probably due to the absence of synapsis formation (Park et al., 2020). These findings demonstrate that Br and Rs chromosomes share little structrual similarity, and thus orthologydependent homoeologous interactions are prevented during meiosis while minimizing nonhomologous exchanges, which would otherwise lead to aneuploidy and/or chromosome reshuffling. This also supports our observation that both Br and Rs genomes exist in entirety without losses in allotetraploid xB after hybridization (Fig. 1b).

Homoeologous expression adjustments in xB
It is assumed that speciation between Br and Rs has occurred earlier than between Br and Bo, although exact speciation timing is controversial (Mitsui et al., 2015;Jeong et al., 2016;Kim et al., 2018). Pairwise comparison of coding sequences (CDS) of all orthologs revealed 95.7% sequence identity between Br and Bo within the same genus but 91.9% (Br vs Rs) and 92.0% (Bo vs Rs) across the genera (Fig. 3a,b). The same analysis in the tribe Camelineae also showed similar sequence divergence for interspecific (93.5% for Arabidopsis thaliana vs A. lyrata) and intergeneric (89.7% for A. lyrata vs Capsella rubella, and 90.3% for A. thaliana vs C. rubella) relationships (Fig. 3a,b). Since c. 8.1% sequence divergence exists between Br and Rs orthologous gene pairs, such divergence allowed us to clearly distinguish Br-and Rs-originated transcripts in xB (Fig. 3a,b). In the xB seedling transcriptome, about half of the reads (51.4%) were assigned to A xB and the other half to R xB (48.6%), indicating that both subgenomes contribute equally to the xB transcriptome (Fig. S8a). Similar proportions of A xB and R xB transcripts were also present in four different tissues (leaf, hypocotyl, root and flower; Fig. S8a).
Both Br and Rs genomes are retained, and thus orthologous pairs become homoeologous to each other in xB (Fig. 3c). Among 28 751 genes commonly expressed in Br and xB, the majority were expressed at similar levels but 2703 (9.40%) genes were differentially expressed (> 2-fold) between Br and xB seedlings (1251 upregulated DEGs (up-DEGs) and 1452 downregulated DEGs (down-DEGs) in xB; Fig. 3d,e). Differential expression between Rs and xB was more prominent, with 4767 (20.96%) from 22 741 Rs-derived genes being dissimilarly expressed between Rs and xB seedlings (2395 up-DEGs and 2372 down-DEGs in xB; Fig. 3d,e). In addition, expression levels of Br-originated genes expressed in Br and xB seedlings were more positively correlated (R = 0.9367) than those of Rs-originated genes expressed in Rs and xB seedlings (R = 0.8403). These findings indicate that the majority of genes retain parental gene expression levels in xB, although Br-originated genes have a greater tendency to maintain their parental expression levels than Rs-originated genes. In other words, Br genome retains 'maintenance expression' over Rs, where Br-originated expression levels are preferentially inherited to the xB hybrid genome.
Of 15 376 syntenic gene pairs expressed in xB and its progenitors, 5701 orthologous pairs (37.07%) were differentially expressed (> 2-fold) between Br and Rs seedlings (2440 up-and 3261 down-DEGs in Br relative to Rs; Fig. 3f). This indicates that Br and Rs have distinct expression profiles for phenotypic divergence. In xB seedlings, however, only 3655 (23.77%) homoeologous pairs were differentially expressed (1553 up-and 2102 down-DEGs in A xB relative to R xB ; Fig. 3f). Moreover, expression levels of A xB and R xB homoeologous pairs in xB seedlings were more highly correlated (R = 0.8667) than those of A Br and R Rs orthologous pairs between Br and Rs seedlings (R = 0.7628) (Fig. 3g). This suggests that distinct expressions of many orthologous genes are adjusted to similar levels in the context of homoeologous relationship in xB. Such expression adjustment was also observed in tissue-specific expression profiles (Fig. S8b).

Reconfiguration of transcription network
Previous studies analyzed the changes of expression levels based on the sum of homoeologous pairs in allopolyploids relative to the parents, and determined additive or nonadditive expressions of duplicated genes (Rapp et al., 2009;Grover et al., 2012;Yoo et al., 2014;Li et al., 2020;Shan et al., 2020;Wei et al., 2021). In this study, we further investigated how orthologous pairs were adapted to a new nuclear environment by monitoring changes of expression patterns of homoeologous genes in xB relative to the progenitors (Fig. 4a). After filtering ambiguous patterns, 12 150 out of 15 376 syntenic homoeologous pairs were selected in Br, Rs and xB seedlings and classified into 27 categories of relative expressions (Fig. 4a). Among them, 7631 (62.80%) gene pairs were expressed at similar levels in every genome context, and their expressions are regarded as being 'constant' (gray in Fig. 4a). By contrast, dynamic changes were observed in 1113 (9.16%) homoeologous pairs, which were unequally expressed in xB and also expressed differently from at least one of the progenitor counterparts (yellow in Fig. 4a). It is notable that 1435 (11.81%) pairs showed 'biased' expressions with significant differences between Br and Rs, while maintaining distinct progenitor expression levels in subgenomes A xB and R xB (blue in Fig. 4a). Interestingly, expressions of 1971 (16.22%) homoeologous pairs were adjusted to similar levels in xB, although their expressions were different between A Br and R Rs progenitors (red in Fig. 4a). Such 'convergent' expressions were more prominent for R Rs -originated genes (1483/1971). We assumed that 'convergent' expressions might result from similar cis-regulatory sequences between homoeologous pairs under the same transcriptional control in xB. We analyzed sequence similarities between homoeologous gene pairs of the categories of 'convergent' vs 'biased' expression ( Fig. 4b). CDSs of both 'convergent' and 'biased' homoeologous pairs showed a high level of sequence identity (92.54% vs 92.00%; Fig. 4b). By contrast, the upstream cis-elements were notably divergent between homoeologous pairs. Interestingly, 'convergent' homoeologous pairs shared less diverged cis-element sequences than 'biased' ones (68.42% vs 63.29%; Fig. 4b). These findings support our hypothesis that the upstream regulatory sequences of the orthologs have diverged after speciation but retain essential cis-elements that are probably under control of the same trans-acting regulators in xB. This also suggests that

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New Phytologist both A and R genomes still maintain compatibility in the transcription system to prevent a 'transcriptome shock' (Hegarty et al., 2006;Buggs et al., 2011), but divergence in regulatory elements should entail reconfiguration of the overall expression network in the hybrid genome of xB.

Coordinated expression of homoeologous genes in response to external stimuli
Gene Ontology enrichment analysis was performed for three categories of homoeologous expression -'constant', 'biased' and 'convergent'. 'Constant' homoeologous pairs have enrichment for GO terms such as 'cell differentiation', 'developmental cell growth' and 'cell cycle' (Fig. 5a; Table S12), suggesting that cell function-related genes maintain consistent expression patterns after hybridization. However, the 'biased' homoeologous pairs did not display GO enrichment for specific functions (P > 0.001). Notably, the 'convergent' homoeologous pairs had GO enrichment for diverse responses such as 'response to hormone', 'response to stress', 'response to biotic stimulus' and 'response to abiotic stimulus' (Fig. 5a; Table S12). This suggests that the homoeologous pairs coordinately expressed in response to various stimuli tend to have similar cis-elements, although they are distinctly expressed in the progenitors. Moreover, the motifs of stress-responsive cis-elements such as abscisic acid-responsive element (ABRE; BACGTGK, B = C, G or T; K = G or T) (Lieberman-Lazarovich et al., 2019) and dehydration-responsive element/C-repeat element (DRE/CRT; RCCGAC, R = A or G) (Suzuki et al., 2005) were found abundantly in the upstream sequence of 'convergent' homoeologous pairs (Fig. 5b). This indicates that the genes involved in cellular signaling may require essential cis-elements to properly respond to external stimuli.
We treated 14-d-old seedlings of Br, Rs and xB to cold for 4 wk and monitored expression changes of orthologous/homoeologous genes. Of 15 376 orthologs, 1579 genes were differentially regulated by cold in Br seedlings, with 956 up-DEGs and 623 down-DEGs (Fig. 5c). In cold-treated Rs seedlings, 2378 genes were differentially expressed, with 1093 up-DEGs and 1285 down-DEGs (Fig. 5c). Among them, only small fractions of orthologous genes (182 up-and 91 down-DEGs; 9.75% and 5.01%) were similarly regulated in both Br and Rs (Fig. 5c). In xB seedlings, 2657 genes were differentially regulated by cold   1226 Rs-derived orthologs were differently regulated (562 up-DEGs and 664 down-DEGs in R xB ) after cold treatment (Fig. 5c). Notably, a larger fraction (261 up-and 378 down-DEGs; 27.13% and 35.80%) of A xB and R xB homoeologous pairs were identified as common DEGs in xB (Fig. 5c). These observations indicate that many orthologous/homoeologous pairs are distinctly regulated in Br and Rs progenitors but their expressions are systematically coordinated in the xB hybrid genome in response to cold exposure. We also found that expressions of A Br and R Rs orthologous genes had a weak correlation regardless of expression categories (Fig. 5d). Interestingly, A xB and R xB 'convergent' homoeologous pairs showed a strong correlation (R = 0.620), whereas 'biased' ones did not (R = 0.195) (Fig. 5e). These data suggest that evolutionarily divergent homoeologous pairs still share essential motifs in cis-elements that can be subjected to the same trans-acting regulation, conceivably responsible for coordinated expression in response to environmental cues in hybrids.

Silencing of TEs stabilizes the xB hybrid genome
Resynthesized hybrids often experience epigenetic alterations (Greaves et al., 2015). We investigated methylation profiles in coding genes and repeat regions. In coding regions, DNA

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New Phytologist methylation levels are high in the gene body, decrease towards the 5 0 and 3 0 ends, and increase again beyond translation start and termination sites in all Br, Rs and xB seedlings (Fig. 6a). Notably, A Br and R Rs progenitor genomes have distinct CG methylation patterns in coding genes, with A Br being more densely methylated than R Rs . This methylation asymmetry is inherited to A xB and R xB subgenomes (Fig. 6a). TEs are heavily methylated in general, especially near-complete CG methylation in all species (Fig. 6b). TEs also have higher CHG and CHH methylation levels than coding genes. Interestingly, Br and Rs TEs have distinct CHG methylation profiles, with more CHG methylation at Rs TEs (Fig. 6b). However, such asymmetry is abolished in xB, where Br-derived TEs have an increased CHG methylation level comparable to Rs-derived TEs (Fig. 6b). This suggests that TEs from Br acquired more CHG methylation after hybridization possibly via trans-acting mechanisms. We analyzed small RNAs in Br, Rs and xB seedlings, and found that c. 30-50% of small RNAs were 24 nt RNAs as potential shortinterfering RNAs (siRNAs) (Fig. S9a). siRNAs were highly associated with hyper-DMRs in xB but loosely with hypo-DMRs, indicating a strong correlation between 24 nt RNA and DNA methylation (Fig. 6c). About 12% of 24-nt RNAs from Br and Rs have a pairwise sequence identity and may share the same targets across the genomes (Fig. S9b). Indeed, 10.4% of 24 nt RNAs from xB also have indistinguishable origins (Fig. S9c). This suggests that, in the xB hybrid genome, R xB -originated siRNAs induce gain of CHG methylation at TEs on A xB possibly via RNA-directed DNA methylation (RdDM) (Law & Jacobsen, 2010). DNA transposons are widespread throughout the xB genome with little association with DMRs (Fig. 6d). LTRs that account for c. 30% of repeats (Table S8) were also heavily methylated. Notably, it was clear that LTRs on A xB had higher methylation levels at the CHG context than A Br (Figs 6d, S10, S11). This suggests that DNA methylation profiles have changed in a subgenome-specific manner, for which R xB -originated siRNAs might induce gain of CHG methylation in trans at LTRs of the same kind on A xB . As exemplified in Fig. 6(e), the Gypsy element on A xB was found to have higher CHG methylation levels than A Br at the scaffold level, although CG and CHH methylation levels are nearly identical. Northern blot analysis verified that Copia and Gypsy elements were moderately expressed in Br but silenced in xB seedlings (Fig. 6f). These findings suggest that RdDM-mediated DNA methylation induces TE silencing across subgenomes, which in turn stabilizes the xB hybrid genome.

Discussion
Hybridization barriers serve as a mechanism to prevent gene flow between species (Abbott et al., 2013). In particular, the postzygotic hybridization barrier after fertilization is often manifested as hybrid inviability or sterility (Dion-Cote & Barbash, 2017). Hybrid sterility is generally associated with a failure in meiosis. Normal meiosis requires the formation of synapsis between homologous chromosome pairs, but when they are abolished or formed between multiple and/or nonhomologous chromosomes, the chromosomes segregate abnormally, resulting in sterile gametes and aneuploidy (Martinez-Perez & Colaiacovo, 2009). Aneuploidy and/or chromosome rearrangements are frequently observed in resynthesized allopolyploids between closely related species (Xiong et al., 2011;Zhang et al., 2013;Chen et al., 2018). This is mainly caused by the collinearity/homology between less divergent parental chromosomes. For instance, A1/C1, A2/C2 and parts of A5/C4 (A from Br and C from Bo) chromosomes are homologous to each other (Parkin et al., 2005), and most phenotypic variations and aneuploidy in resynthesized B. napus lines are caused by homoeologous interactions, mostly between nonhomologous chromosomes (Gaeta et al., 2007;Xiong et al., 2011;Grandont et al., 2014). However, the presence of full compliments of both Br and Rs chromosomes in xB demonstrates that a merger of divergent genomes may avoid such harmful interactions, while producing fertile gametes after polyploidization.
The Dobzhansky-Muller model proposes that hybrid incompatibility arises from negative epistatic interactions between the incompatible alleles in nascent hybrids (Moyle & Nakazato, 2010). This may operate as a primary postzygotic barrier, which also appears true for early generations of xB. Most synthetic F 1 individuals of xB, including resynthesized xB plants in this study, display reproductive defects such as pollen inviability and seed abortion (Lee et al., 2011;Shin et al., 2021). Underdeveloped embryos must be in vitro rescued, and thus only a handful of progenies can be finally fixed as stable lines. During this stabilization process over multiple generations, the xB hybrid genome is assumed to have undergone constant transgenerational changes in genome structure and epigenetic landscape until the genome environment settles down to a stable condition. Consequently, deleterious or incompatible alleles have possibly been removed by homoeologous exchanges or silenced by epigenetic mechanisms.
Although Br and Rs genomes are estimated to have diverged 7-22.4 Ma (Mitsui et al., 2015;Jeong et al., 2016;Kim et al., 2018), we cannot rule out the possibility that the two genomes still retain syntenic regions similar enough to trigger homoeologous exchanges. We previously reported that some genomic regions of Br and Rs share structural similarity, particularly between A8 and R8, although most other regions are fragmented and diversified (Park et al., 2020). In addition, we recently showed that several xB lines derived from the same intergeneric cross endured chromosome instability and pollen inviability, while occasionally producing tetrads with an extra micronucleus during microsporogenesis (Shin et al., 2021). This is probably a consequence of homoeologous chromosome exchanges at meiotic prophase I leading to the formation of univalent or multivalent chromosomes (Shin et al., 2021). This suggests that chromosome instability persists in successive generations while generating nonfunctional haploid gametes with aberrant karyotypes. Such chromosome rearrangement may occur in a stochastic manner involving diverse combinations of nonspecific homoeologous interactions, and we presume that once the reorganized genome acquires a stable state by losing a sticky region that is prone to constantly interact with a wrong counterpart, the descendants are able to secure genome stability and finally survive.
Hybridization between species inevitably entails changes in cis-trans interactions bringing about alterations in the transcription network (Hu & Wendel, 2019). Therefore, extensive changes in parental expression profiles are expected, and when such changes are intolerable, the hybrid will undergo a 'transcriptome shock', manifested as hybrid dysgenesis (Martienssen, 2010) or outcrossing depression (Frankham et al., 2011). xB experienced moderate expression changes of progenitor genes after hybridization but still maintains a transcription network between subgenomes compatible enough to generate novel or intermediate phenotypes. Our four-point expression analysis revealed that 'convergent' homoeologs share similar cis-elements, and expression levels of a larger fraction of Rs-derived homoeologs were adjusted to Br-derived ones. This suggests that Br-originated trans-acting factors probably play dominant roles for coregulation of homoeologous pairs in xB (Hu & Wendel, 2019). Notably, stress response-related motifs are enriched in the cis-elements of 'convergent' homoeologs, suggesting that transcriptional regulation is primarily mediated by transacting factors sharing common homoeologous targets that are involved in diverse responses. Such reconfiguration of the transcription network is conceivably crucial to the adaptation of newly synthesized hybrids.
Br has higher gene-body CG methylation levels than Rs, which is inherited to each subgenome in xB. This indicates that differential gene-body methylation is maintained after hybridization and this methylation asymmetry may contribute to 'maintenance expression' of A xB through unknown mechanisms. TEs are heavily methylated in general, but also showed asymmetric CHG methylation between Br and Rs. Intriguingly, Br-originated LTRs gained CHG methylation comparable to Rs LTRs in xB, suggesting that repeat-originated siRNAs trigger hypermethylation via RdDM in trans and TE silencing (Wendel et al., 2016). This may prevent hyperactivation of TEs and subsequent genome destabilization, which would otherwise culminate in a 'genomic shock' as initially proposed by McClintock (1984).
Many previous studies report that global DNA methylation changes occur in allopolyploid plants such as Arabidopsis suecica (Madlung et al., 2002), B. napus (Xu et al., 2009), wheat (Shaked et al., 2001) and rice (Li et al., 2019). Importantly, even autotetraploid rice plants show an increase in CHG and CHH methylation levels at TEs associated with 24 nt siRNA clusters (Zhang et al., 2015), suggesting the role of RdDM for gain of DNA methylation and silencing of TEs and nearby genes. The current model of RdDM delineates the establishment of CHH methylation triggered by repeat-originated small RNAs (Law & Jacobsen, 2010). However, the regulation of CHG methylation via RdDM is largely elusive. One possible mechanism of CHG

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New Phytologist hypermethylation at TEs is that DNA methyltransferase such as chromomethyl transferase 3 is also under the control of RdDM, which may utilize the RdDM components to access either directly or indirectly to targets for CHG methylation. Another is that CHH-methylated regions may provide a unique chromatin signature for subsequent CHG methylation and maintenance of TE silencing. A recent study proposed that silencing of distal LTRs in Arabidopsis and tomato requires an additional phase of CMTmediated CHG methylation following RdDM-mediated CHH methylation (Wang & Baulcombe, 2020). A similar mechanism might have worked in multiple steps for silencing of Br-originated LTRs in xB, for which siRNAs derived from Rs LTRs conceivably play crucial roles for gain of CHG methylation in trans.
It is believed that the more distantly related the species, the stronger the hybridization barrier. Contrary to this assumption, our findings strongly suggest that, as long as the physiology and transcriptional regulatory networks are compatible, a certain extent of genome divergence promotes hybridization between distant species. Therefore, a trade-off between genome divergence and transcriptome compatibility is meaningful to facilitate hybridization between species without causing genome destabilization and/or a conflict in the transcription network. This concept also proposes that interspecific/intergeneric hybridization may occur more frequently in nature than previously thought, and the 'triangle of U' model (Nagaharu, 1935) can be further expanded to the intergeneric level.
After whole genome duplication or hybridization between the different species followed by chromosome doubling (allopolyploidization), polyploid plants generally undergo gradual but substantial genome reconstruction, including massive chromosome rearrangement, differential deletion or retention of duplicated genes and biased genome fragmentation (Cheng et al., 2018). This eventually leads to a decrease in both chromosome number and genome size, with most of the polyploid properties being lost. Extensive changes in genome structure and gene repertoire accompanied by subfunctionalization/neofunctionalization of duplicated genes also contribute to the formation of new species with novel phenotype and function, which sometimes outperform the diploid progenitors with greater ecological fitness. Thus, the evolution of land plants, especially the angiosperms, is not a one-way process. Rather, it is likely to comprise the recurrent cycles of hybridization, diversification, diploidization and reunification among species in the same lineage (Wendel, 2015). Furthermore, understanding the highly dynamic and flexible process of hybridization and polyploidization should provide a clue to Darwin's 'abominable mystery' (Darwin, 1903) questioning the great diversification and expansion of angiosperms within a short geological timeframe. Zhang J, Liu Y, Xia EH, Yao QY, Liu XD, Gao LZ. 2015

Supporting Information
Additional Supporting Information may be found online in the Supporting Information section at the end of the article.