Stable resynthesized Brassica napus lines show similar meiotic behaviour to established B. napus

Stable resynthesized Brassica napus lines show similar meiotic behaviour to established B. napus | 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 Stable resynthesized Brassica napus lines show similar meiotic behaviour to established B. napus Vinita Ramtekey, Elizabeth Ihien Katche, Mariana Baez, Zhenling Lv, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7700382/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Apr, 2026 Read the published version in Chromosome Research → Version 1 posted 7 You are reading this latest preprint version Abstract Brassica napus (rapeseed/canola) is an allotetraploid (AACC, 2 n = 4 x = 38) resulting from spontaneous hybridization between B. rapa (AA, 2 n = 2 x = 20) and B. oleracea (CC, 2 n = 2 x = 18). Although established B. napus is meiotically stable, resynthesized lines (2 n = AACC) produced by hybridizing between progenitor species B. rapa and B. oleracea are usually meiotically unstable, and show frequent chromosomal rearrangements caused by homoeologous recombination between the A and C genomes. Previously, we identified resynthesized rapeseed lines showing contrasting levels of homoeologous recombination, as assessed by genotyping for copy number variants. Here, we aimed to characterise meiotic chromosome pairing behaviour in fifteen resynthesized lines representing putatively stable, unstable and intermediate types. Putatively stable lines showed predominantly normal meiosis (average 91% bivalent formation), while putatively unstable lines showed frequent abnormalities such as multivalent formation (average 60% bivalent formation). Univalents were unexpectedly rare in Metaphase I. Surprisingly, all intermediate resynthesized lines showed either stable or unstable-type meiotic behaviour. A1-C1 specific probes revealed that stable lines showed approximately 18% A-C pairing (7/40 pollen mother cells), not significantly different to the 13% A-C pairing (5/40 pollen mother cells) in established B. napus , but in contrast to the unstable line with 46% A-C pairing (25/54 pollen mother cells). Our results suggest that differences in multivalent formation frequencies and homoeologous A-C pairing differentiate stable and unstable lines, confirm the production of meiotically stable synthetic B. napus , and provide a basis for further investigation of genetic factors contributing to this effect. meiotic stability cytogenetics chromosome pairing behaviour rapeseed oligo-FISH Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Polyploidization is a major driving force in evolution and speciation (Leitch and Leitch 2008 ), and is particularly common in angiosperms (Jiao et al., 2011 ). Polyploids often exhibit genome buffering, enhanced heterozygosity, and novel phenotypic and genotypic variation compared to their diploid counterparts, which can be attributed to various interconnected mechanisms such as genome rearrangements, altered gene dosage, gene expression and regulation and epigenetic modification (Pelé et al., 2018 ; Doyle & Coate, 2019 ; Heslop-Harrison et al., 2023 ). Polyploids are usually categorised as either autopolyploid (sets of chromosomes originating from the same species), or allopolyploid (two or more genomes derived from different species) (Kihara and Ono, 1926 ). Allopolyploids which arise from hybridization between two different sets of chromosomes (genomes) face the major challenge of differentiating between ancestrally related (homoeologous) chromosome copies (Pelé et al., 2018 ). During the course of evolution, the major adaptation that has been observed in established allopolyploids is their ability to distinguish between homoeologous and homologous chromosomes when choosing meiotic recombination partners. Prevention of extensive pairing between non-homologous (homoeologous or otherwise) chromosomes is critical for ensuring regular segregation of chromosomes into subsequent gametes without putative issues such as loss of chromosomes or chromosome segments, which is potentially detrimental to plant fertility and viability (Mercier et al., 2015 ). However, how established allopolyploids stabilise meiosis is still unknown in most taxa (Bomblies, 2023 ). There are several ways in which allopolyploids might stabilise meiosis. Crossovers between homoeologous chromosomes could be prevented completely, resolved in such a way that no recombination can occur between homoeologues, or simply made extremely rare due to strong preference for homologous over homoeologous crossovers, even in the absence of specific genetic factors that suppress pairing between homoeologous chromosomes (Bomblies, 2023 ). In allohexaploid bread wheat, which is one of the most extensively studied species with respect to understanding of the establishment of meiotic stability in allopolyploids, the Ph1 gene acts not only to suppress homoeologous chromosome recombination but also to promote homologous recombination (Riley & Chapman, 1958 ; Griffiths et al., 2006 ; Bhullar et al., 2014 ). Other genetic factors acting to prevent homoeologous recombination have been identified in Arabidopsis suecica (Henry et al., 2014 ) and Brassica napus (Jenczewski et al., 2003 ; Liu et al., 2006 ; Higgins et al., 2021 ), although the mechanism of action of these genetic factors is so far unknown. Jenczewski et al., 2003 identified a genetic factor PrBn which influences homoeologous crossover frequency in B. napus haploids, and Nicolas et al., 2009 observed differences in homologous recombination frequency in allotriploid Brassica hybrids (AAC) produced with different PrBn types, suggesting PrBn could have dosage sensitive effects on recombination. However, the gene corresponding to this locus has not yet been identified or fully functionally characterized. Recently, Gonzalo et al., ( 2019 ) demonstrated that reduced expression of MSH4 , belonging to the ZMM-group of class I crossover pathway proteins, significantly decreases the occurrence of homoeologous recombination, while having minimal impact on homologous recombination in B. napus . However, the specific impact on partner choice in the context of pairing or recombination regulation in Brassica is still uncertain. As suggested by earlier QTL mapping results (Jenczewski et al., 2003 ; Liu et al., 2006 ), an integrated system of multiple genes is most likely associated with meiotic stabilization in B. napus , such that the molecular basis of meiotic stability involves polygenic adaptation to allopolyploidy. Neo-allopolyploids and resynthesized hybrids are a useful model with which to investigate mechanisms underlying meiotic stability. In most newly synthesised allopolyploids (produced by crosses between lower ploidy parents), meiosis is associated with numerous abnormalities, including but not limited to incorrect synapsis, homoeologous recombination, chromosome bridges, and chromosome mis-segregation (anaphase I). These meiotic abnormalities can lead to aneuploidy, chromosome rearrangements and deletions and duplications of chromosome segments, which may result in loss of fertility and viability in subsequent generations (Bomblies, 2023 ; Pelé et al., 2018 ; Xiong et al., 2021 ). Studies of synthetic hybrids have previously investigated cytological causes of meiotic stability. Madlung et al., ( 2005 ) observed about 30% meiotic abnormalities in the form of chromosome breakage, bridges and rearrangements in synthetic Arabidopsis allopolyploids, indicating increased meiotic instability compared to their parents (10%). Similarly, Chéron et al., 2024 suggested that associations between incorrect recombination partners and homoeologous recombination contribute to meiotic instability in neo-synthetic allopolyploid A. suecica . Recently, synthetic Brassica napus has emerged as an important model system to study meiosis in allopolyploids ( Katche & S. Mason, 2023; Bomblies, 2023 ). Allotetraploid B. napus (AACC, 2 n = 4 x = 38) is the product of natural interspecific hybridization coupled with polyploidization between diploid ancestors of Brassica rapa (AA, 2 n = 2 x = 20) and Brassica oleracea (CC, 2 n = 2 x = 18) around 7500 years ago (U, 1935; Chalhoub et al., 2014 ). Established B. napus is a relatively meiotically and genomically stable allopolyploid which shows diploid-like meiosis, including predominantly homologous recombination even in the presence of homoeologous chromosomes (Jenczewski et al., 2003 ). By contrast, synthetic B. napus (formed by either B. rapa × B. oleracea or B. oleracea × B. rapa ) is usually meiotically unstable, but the cause is still unknown (reviewed by Katche & Mason, 2023 ). Cytogenetic, molecular and genome sequencing studies have revealed that resynthesized B. napus often display genetic changes as well as homoeologous rearrangements (Gaeta et al., 2007 ; Xiong et al., 2011 ; Chalhoub et al., 2014 ; Xiong et al., 2021 ; Katche, et al., 2023a ; Katche et al., 2023b ; Davis et al., 2023 ), leading to genomic copy number variants (deletions, duplications, and translocation) as well as presence/absence variation (Katche et al., 2023a ; Schiessl et al., 2019 ). Such variants are most common in chromosomes which are structurally conserved (syntenic along the whole length of the chromosome) between subgenomes, such as A1 and C1, and A2 and C2 (Xiong et al., 2021 ; Higgins et al., 2021 ). Unlike resynthesized B. napus , established cultivars show rarer or lower rates of homoeologous rearrangements (Parkin et al., 1995 ; Sharpe et al., 1995 ; Howell et al., 2008 ; Udall et al., 2005 ). In the past decades many cytogenetic studies have extensively confirmed with the help of fluorescently labelled probes as well BAC-FISH that homoeologous recombination between the closely related A and C subgenomes is a major feature of resynthesized B. napus (Howell et al., 2008 ; Xiong et al., 2021 ; Xiong & Chris Pires, 2011 ). In our previous work, we identified several synthetic B. napus lines from different genetic backgrounds which accumulate no or very few new copy number variants after many generations of self-pollination and are therefore putatively stable (Katche et al., 2023a ), unlike all previously produced resynthesized B. napus lines (Katche et al., 2023b ). In the present study, we aimed to investigate and characterise meiosis in these lines and compare between these putatively “stable” and “unstable” resynthesized B. napus types as well as established B. napus . We hypothesised that meiosis in the putatively stable lines would be normal and similar to meiosis as established B. napus , in contrast to unstable resynthesized B. napus , and aimed to characterise meiotic progression in order to better elucidate the cytological mechanisms responsible for these differences. Material and Methods Plant material Production of the resynthesized winter B. napus allotetraploid (AACC) lines used in the present research is described in Girke et al., ( 2012 ) and Jesske et al., ( 2013 ). Lines were produced by crossing different genotypes of B. rapa with (AA) with B. oleracea (CC). The Girke et al., ( 2012 ) material consists of domesticated resynthesized B. napus lines derived from hybridization between vegetable-type B. rapa (AA) and B. oleracea (CC), while the Jesske et al., ( 2013 ) material comprised wild C genome species crossed with cultivated B. rapa lines. Katche et al., ( 2023a ) later characterized these lines as putatively stable or unstable based on frequencies of novel and inherited CNVs in each line. For the first year of flower bud collection, plants were grown at Justus Liebig University Giessen, Germany, as described by Katche et al., ( 2023a ). In the subsequent generation, only putatively stable and unstable resynthesized lines with a minimum of three seeds per line were germinated in quick-pots during the year of 2024. Germinated plants at the 4–6 leaf stage were vernalized at 4–6°C for minimum 14–16 weeks (December 2023 to March 2024) in a controlled environment room at Campus Klein Altendorf, the University of Bonn field station. After vernalization, plants were transferred to the glasshouse and grown in 10 L pots under heated glasshouse conditions (minimum 20°C day and 16°C night, but up to 35°C on hot days in late summer) at the University of Bonn, Poppelsdorf from April to September 2024. In the present study we selected fifteen resynthesized B. napus lines from Katche et al., ( 2023a ) and categorised these into three groups based on CNV data: 1) no novel CNVs = “stable”, 2) 1–3 novel CNVs = “intermediate”, and 3) > 8 CNVs = “unstable” (Table S1 ). For the putatively stable lines (with the exception of R76), the maternal parent was B. oleracea and the paternal parent was B. rapa , with different subspecies for both parents (Table S1 ). For the putatively unstable lines (with the exception of OLY21), the maternal parent was B. rapa , while paternal parent was B. oleracea (Table S1 ). The putatively highly stable lines were > S3 generation, while the putatively highly unstable were either S1 or S2 generation (Katche et al., 2023a ). As a reference for stable meiosis, we used B. napus cv. Drakkar (received from INRAE, France). Additionally, we selected one putatively stable (L16) and unstable (OLY21) resynthesized line to investigate meiotic progression and A1/C1 pairing, as these lines did not have any apparent fixed translocations involving chromosomes A1 and C1 (Katche et al., 2023a ). Seed fertility in putatively stable and unstable resynthesized lines Three individual plants per genotype were bagged using microperforated plastic bags to encourage self-pollinated seed set. The average number of total self-pollinated seed set was measured for each of the three plants per genotype to assess seed fertility differences between putatively stable and unstable resynthesized lines. Inflorescence fixation, slide preparation and meiotic observation Immature unopened flower buds were collected in Carnoy’s I fixative solution (absolute ethanol: glacial acetic acid 3:1, v/v) for 24–48 hours at room temperature to fix the cells at the respective meiotic stages (Windham et al., 2020 ). Later, the fixed flower buds were transferred to 50% ethanol for further downstream analysis as well as long term storage. Out of six anthers, one anther was squashed and stained with 1% acetocarmine to identify meiotic stages while the remaining five anthers were used for slide preparation via enzymatic digestion (composition of 1% pectyolase Y-23 and 2% cellulase “Onozuka” R-10 in 0.1M citrate buffer)(Kirov et al., 2014 ). Slide preparation involved submerging the remaining five anthers in an enzymatic mixture of 20 µl in a 0.5 ml tube and incubating for 1–1.5 hours at 37°C followed by washes with 70% ethanol. Subsequently, anthers were scrambled with a needle in 60–80 µl of 70% ethanol to form a white musky coloration, which gives a pellet upon quick centrifugation (25–30 s) using a benchtop centrifuge (FastGene® Mini Centrifuge (NG002P)). The pellet contains meiocytes as well as some somatic tissue (anther wall and tapetum). The ethanol supernatant is removed and pellet dried. Once the pellet is dried, we add 100% GAA (approximately 25–40 µl depending on the pellet size) and vortex thoroughly. Finally, we use the dropping method to prepare slides containing meiocytes and incubate for another 5 minutes in a humid chamber at room temperature (RT) to evenly spread the meiocytes. A drop of Vectashield® antifade mounting medium containing 4’, 6- diamidino-2-phenylindole (DAPI) (H-1200-10, Vector Laboratories) was used on slides to stain and visualize the chromosomes inside meiocytes for cells undergoing meiosis). These slides were finally used to investigate chromosome pairing behaviour at diakinesis of prophase I of meiosis I from male meiocytes. In order to understand the overall meiosis progression of the putatively stable and unstable resynthesized lines, we focused on metaphase I, anaphase I, telophase I, metaphase II, and anaphase II/telophase II. Visualization of meiocytes was performed using an inverted fluorescence microscope (Zeiss Axio Imager M2) with a 40 × and 100 × oil immersion lens and up to 10 000 × total magnification. Images were captured using the Zeiss software ZEN Blue (version 3.2), which was also used for cropping, size adjustment and contrast optimization of microscopic images. A minimum of 30 cells per line was analysed for a robust interpretation of meiotic chromosome behaviour at diakinesis. Chromosome configurations were scored as bivalent, univalent or multivalent at diakinesis. Other chromosomal aberrations such as anaphase bridges, laggards or chromosome fragments at anaphase/telophase stages of meiosis I and meiosis II were also observed. Probe labelling, oligo-FISH and imaging In order to clearly identify univalent, bivalent and multivalent chromosomes, DNA from centromere repeat-specific sequences CentBrI and CentBrII (Xiong and Chris Pires, 2011 ) were labelled with Cy5-dUTP, using a nick translation kit (11745808910, Roche) as described by (Kato et al., 2006 ). Specific labelling of A1 and C1 was done using A1-specific oligo probes labelled with ATT0550 (red) and C1-specific oligo probes labelled with ATT0488 (green) (by Arbor Biosciences, via BioCat GmbH, Germany). FISH was performed according to Han et al., ( 2015 ) and Montenegro et al., ( 2022 ) with slight modification. We used all three DNA probes at the same time on a single slide. Hybridization started with fixing the slides in Carnoy’s I solution for 30 minutes followed by dehydration with 100% ethanol for 15 minutes. Once the slides dried out, we treated with pepsin (50 µg/ml) for 30 minutes at 37°C in a humid chamber followed by two successive washes with 2 × SSC buffer (saline sodium citrate buffer) at RT. Later we treated slides with 4% formaldehyde and incubated for 10 minutes at RT, followed by two successive washes with 2 × SSC buffer then dehydration with an ethanol series (70% and 100% ethanol). After these treatments, we air dried the slides for at least an hour and applied the hybridization mixture to the meiotic cell zone on the slides. The hybridization mixture comprised of 50% formaldehyde, 10% dextran sulphate, 2 × SSC buffer, 200 ng A1 and C1 oligo probes and 50 ng of centromere probes. Slides were incubated at 75°C for 5–7 minutes after adding hybridization mixture to the slides and later hybridized for 2 days at 37°C in a moisture chamber, in the dark. After a long incubation, we washed slides once with 2 × SSC buffer at RT and at 55°C for about 20 minutes followed by another wash at RT with 2 × SSC buffer. Once the slides partially dried, the Vectashield® antifade mounting medium containing DAPI (H-1200-10, Vector Laboratories) was used on slides to capture the images using an inverted fluorescence microscope (Zeiss Axio Imager M2) with a 40 × and 100 × oil immersion lens with different filters and software described above. A minimum of 30 microscopic images/line were analysed. Univalent, bivalent or multivalent behaviour of chromosomes A1/C1 was scored at diakinesis. Statistical Data Analysis Statistical analysis was performed in R studio v 4.4.1 (R Core Team (2024) https://stat.ethz.ch/pipermail/r-announce/2024/000704.html ). Visualization plots for seed fertility and meiotic chromosome pairing behaviour (bivalent, univalent and multivalent) of putatively stable and unstable lines were produced using the “ggplot2” package available in R (Wickham H, 2016 ). A Shapiro-Wilk normality test was carried out to assess normality of seed fertility and meiosis confirmation data. Seed set was normally distributed, so we performed ANOVA followed by Tukey’s HSD test to check for significant differences between putatively stable and unstable resynthesized lines. Meiotic configuration data was not normally distributed, so a non-parametric Kruskal-Wallis test was used to check for significant differences between groups and between genotypes followed by a pairwise Dunn’s post hoc test for significant comparisons with multiple testing correction using the Bonferroni method. The adjusted p-values were used to cluster groups and genotypes into sets by significant differences between them, which were indicated by different letters in the plot. Fisher’s exact test for count data was used to detect the significance for A1/C1 homoeologous pairing between putatively stable and unstable resynthesized lines. The Pearson correlation coefficients were computed by using ‘corrplot’ package available in R (Wei & Simko, 2024 ). Results Putatively stable resynthesize lines exhibited higher seed set, in contrast to unstable lines Putatively stable lines showed higher seed set. Total self-pollinated seeds ranged from 499 to 2720 (on an average 1181) per plant across putatively stable resynthesized lines which was significantly higher (Fig. 1 , Table S2 , ANOVA, P = 0.01, followed by Tukey’s HSD P < 0.01) than seed set in unstable lines, which ranged from 0 to 444 seeds per plant (average 52) (Table S2 ). Putatively stable resynthesized B. napus lines exhibited similar chromosome pairing behaviour to established B. napus , over two generations, in contrast to unstable lines Putatively stable lines displayed meiotic chromosome pairing behaviour which was statistically similar to that of established B. napus cultivar “Drakkar” in terms of univalent, bivalent and multivalent frequencies (p > 0.05, Fig. 2 , Table S3). The putatively stable group showed higher frequencies of bivalents (91% on an average), than the putatively unstable lines (60% on average; Fig. 2 , Table S3; p < 0.001). There was a significant difference between univalent frequencies in stable lines (2%, p < 0.001) compared to unstable lines (1%), as well as for multivalent formation (3% in established and stable, p < 0.0001) compared to unstable lines (13%) (Table S3; Fig. 2 ). In the first experimental year and generation, putatively stable lines exhibited 1% univalent frequency and 3% multivalent frequency on average, while unstable lines averaged 1% univalent frequency and 12% multivalent frequency (Table S3). However, only the average multivalent frequencies showed statistically significant differences between putatively stable and established B. napus as well as between putatively stable and unstable resynthesized lines (p < 0.001 for stable and for established, Fig. S1 ). Similar results were observed in the subsequent generation in the resynthesized lines, where the subsequent generation was produced from self-pollinated seeds collected from the previous generation plants, except where univalent frequencies were also found to be significantly different between established and stable and the unstable lines (Fig. S2 , Table S3, p < 0.0001). In the first experimental year, the average meiotic pairing behaviour of the putatively stable resynthesized lines was 0.41 I + 17.10 II + 0.66 multivalents, in contrast to putatively unstable lines with 0.28 I + 11.52 II + 2.33 multivalents. Similar results were observed in the second year in the following generation of progeny, with 0.25 I + 17.81 II + 0.36 multivalents across the putatively stable lines and 1.07 I + 10.78 II + 2.48 multivalents in the putatively unstable resynthesized lines. As we hypothesized, the putatively stable resynthesized lines behaved similarly to established B. napus “Drakkar”, where we observed 0.26 I + 17.30 II + 0.52 multivalents (Table S3). “Intermediate" lines with 1–3 CNVs fell into two distinct meiotic behaviour classes (stable and unstable) Five lines, which were classed as intermediate based on the presence of 1–3 novel CNVs, exhibited either “stable” or “unstable” phenotypic behaviour in terms of chromosome pairing. Out of the five intermediate lines, two (R8 and H327) showed normal bivalent formation (86% and 93% respectively), similar to the putatively stable lines. By contrast, the other three lines (R140, J122 and H4) exhibited lower bivalent frequencies (54%, 63% and 66% respectively), similar to the unstable lines. Based on present phenotypic results, we re-categorized these intermediate lines accordingly as putatively stable and unstable lines (Fig. 2 ). Genotype-specific differences between lines within the “stable” group and within the “unstable” group for chromosome pairing behaviour at diakinesis We observed statistically significant differences of chromosome pairing behaviour between some of the putatively stable resynthesized lines (Fig. S3, S5) as well as between some of the putatively unstable resynthesized lines (Fig. S4, S6). Specifically, H327 (originally identified as intermediate) showed significantly lower bivalent frequencies than stable lines J154 and R8 in the first generation, although the other lines were not significantly different from each other (Fig. S3). Significant differences between multiple stable lines were also observed for multivalent and univalent frequencies (Fig. S3). However, in the next generation, there were no significant differences observed between putatively stable resynthesized lines for bivalent or multivalent frequencies, and only L16 had significantly higher univalent frequencies (Fig. S5). For the putatively unstable resynthesized lines, only OLY21 was statistically significantly different to the other four lines for bivalent frequencies in the first generation (Fig. S4) and K241 in the second generation (Fig. S6), although there were more differences in both generations for multivalent and univalent frequencies (Fig. S4, S6). Meiotic progression in stable resynthesized lines is similar to that in established B. napus , but contrasts with that of putatively unstable resynthesized lines We looked into the different stages of meiosis I and meiosis II to understand the meiotic progression in different putatively stable and unstable lines of resynthesized rapeseed and in established rapeseed. Here, we observed relatively normal and regular meiosis in putatively stable line “L16”, which was quite similar with that of established B. napus “Drakkar”, but differed with that of putatively unstable resynthesized line “OLY21” (Fig. 3 ). Specifically, the unstable line showed higher frequencies of multivalents at diakinesis, as previously mentioned (Fig. 2 , Table S3), and these multivalents were not resolved but were carried forward to metaphase I. Subsequently at anaphase I, we observed another type of structural chromosome aberration in the unstable line, namely “anaphase bridges”, which can result from crossovers between non-homologous chromosomes where two centromeres end up on the same chromatid. As well, unequal segregation ratios of chromosomes were observed at telophase I in the unstable line, indicating failure to segregate chromosomes correctly to different poles. Chromosome laggards were also observed at metaphase II and anaphase II/telophase II of meiosis II (Fig. 3 ), which can represent acentric fragments following non-homologous crossovers or univalent chromosomes which do not correctly segregate to the poles. Non-homologous multivalents involving chromosomes A1 and C1 were common but much more frequent in unstable resynthesized lines compared to established and stable lines Homoeologous pairing is one of the most probable causes for meiotic instability, therefore we analysed more specifically associations between chromosome pairs A1 and C1, which have the highest known homoeology in the B. napus genome (Parkin et al. 2003). We applied oligo painting probes specific to the entire chromosome lengths of A1 and C1 as well as centromere-specific probes in order to analyse the frequency of A1-C1 associations in diakinesis. The average frequency of A1 - C1 pairing was approximately 13% in established B. napus “Drakkar”, not significantly different from the approximately 18% inferred A1-C1 pairing in putatively stable resynthesized line “L16”, but contrasting significantly with the 46% frequency observed in putatively unstable line “OLY21” (p < 0.05, Fisher’s exact test for count data; Table S4 and Fig. 4 ). Correlations between meiotic behaviour and seed fertility Across all lines assessed, seed fertility was significantly positively correlated with bivalent formation (r = 0.88, p = 0.00066), and significantly negatively correlated with both univalent formation (r = -0.80, p = 0.0058) and multivalent formation (r =-0.81, p = 0.0048). Correlations within each of the “stable” and “unstable” groups (five lines each) were not significant. Unsurprisingly, meiotic configuration frequencies were also strongly significantly correlated across all lines: multivalents and bivalents were correlated at r = -0.96 (p < 0.0001), bivalents and univalents at r = -0.80 (p = 0.0053), and univalents and multivalents at r = 0.67 (p = 0.034) (Fig. 5 ). Discussion Here, we provide the first confirmation of stable meiosis in a subset of resynthesized rapeseed lines ( B. rapa × B. oleracea ), comparable to that of established B. napus . By contrast, unstable resynthesized rapeseed lines were characterized by high frequencies of multivalents: pairing between highly similar homoeologous chromosomes A1 and C1 occurred in half of all PMCs, suggesting most multivalent formation could be attributed to pairing between homoeologous chromosomes. Additional aberrations such as acentric chromosome fragments, anaphase I chromosome bridges and unequal chromosome segregation in the unstable resynthesized lines then occurred putatively as a consequence of these non-homologous multivalents, and lower fertility was also observed in our unstable resynthesized lines compared to our stable lines. All individual resynthesized lines could be qualitatively categorised into “stable” and “unstable” on the basis of diakinesis chromosome pairing behaviour. This qualitative (stable/unstable) rather than quantitative (more or less stable on a spectrum) classification suggests a single major gene effect might differentiate these two categories, potentially supporting previous studies suggesting that one locus might be predominantly responsible for the meiotic stability trait in B. napus (Jenczewski et al., 2003 ; Higgins et al., 2021 ). However, we also detected some minor but significant meiotic differences between genotypes within each of the stable and unstable groups (so between stable genotypes or between unstable genotypes), which may suggest a more complex genetic background to this trait, as also observed by Katche et al., ( 2023a , 2023b ) based on frequencies of non-homologous recombination outcomes (CNVs), and as supported by previous QTL mapping results (Liu et al., 2006 ; Higgins et al., 2021 ). Similar results were reported by Heneen et al., ( 1995 ), who observed differences in bivalent and multivalent frequencies in four different resynthesized B. napus lines depending upon the genetic background of the parental species, and by Katche et al. ( 2023b ) who found differences in putative meiotic stability between early-generation synthetic lines with different combinations of shared B. rapa and B. oleracea parents. Our results provide an excellent basis to further investigate the genetic mechanisms underlying meiotic stability in allopolyploid Brassica napus . We observed low frequencies of meiotic abnormalities in established B. napus as well as the stable resynthesized lines, but high frequencies of meiotic abnormalities in the unstable resynthesized lines (> 2 multivalents per PMC on average), and some minor genotype-specific differences within the “stable” and “unstable” groups. Stable resynthesized lines and established B. napus Drakkar showed similar frequencies of bivalents (both 91%), univalents (2% and 1%) and multivalents (3% in both) at diakinesis. Similar meiotic chromosome pairing behaviour in established cultivars of B. napus has been reported in other studies (Jenczewski et al., 2003 ; Udall et al., 2005 ; Sheidai et al., 2006 ). Genomics studies have also frequently observed evidence for non-homologous recombination between the A and C subgenomes in B. napus (Chalhoub et al., 2014 ; Mason et al., 2017 ). We observed no significant difference between stable and unstable lines for univalent frequency in the first generation, but highly significant differences in multivalent frequency in both generations, suggesting that multivalent formation plays a major role in meiotic and genomic instability. Similar to our results, Xiong et al., ( 2021 ) observed low frequencies of univalents in resynthesized rapeseed lines. These multivalents are most likely predominantly due to A-C chromosome associations (Hasterok et al., 2005 ; Leflon et al., 2006 ; Yang et al., 2017 ). Autosyndetic pairing (e.g., A01 – A02) is possible, and has been observed at low frequencies in haploid Brassica rapa (2 n = x = 10, A) and B. oleracea (2 n = x = 9, C) (Armstrong & Keller, 1981 , 1982 ) as well as in AABC, BBAC and CCAB hybrid types where at least one of the A and C genomes is haploid (Mason et al., 2010 ), but allosyndesis and intergenomic exchanges between A and C chromosome occur much more frequently in synthetic Brassica hybrids (Yang et al., 2017 ; Gaebelein et al. 2019 ; Quezada-Martinez et al. 2022 ). Meiotic behaviour is substantially more irregular in most of the resynthesized B. napus produced to date than in established B. napus , with some supporting cytogenetic observations of meiosis (Szadkowski et al., 2010 ) and with genetic (Song et al., 1995 ; Szadkowski et al., 2010 ; Katche et al., 2023a ; Katche et al., 2023b ), genomic (Chalhoub et al., 2014 ; Samans et al., 2018 ) and cytogenetic (Xiong et al., 2011 ) evidence for resulting frequent non-homologous recombination between the A and C genomes. We assessed progression through meiotic stages such as diakinesis, metaphase, anaphase and/or telophase in order to determine if stage-specific failures to complete normal and regular meiosis and develop functional gametocytes were present. Our detailed comparative meiotic progression study between putatively stable (L16) and unstable (OLY21) resynthesized lines identified regular meiotic progression in the stable resynthesized line, similar to established cultivar “Drakkar”. By contrast, higher frequencies of multivalents at diakinesis and misalignment of bivalents at metaphase I were observed in the unstable resynthesized line, suggesting that homologous and homoeologous chromosomes were unable to sort, synapse and properly recombine in the early stages of prophase. Similarly, (Grandont et al., 2014 ) reported early and effective sorting of homologous and homoeologous chromosomes in early prophase in two different B. napus accessions (although these accessions showed different frequencies of homoeologous A-C recombination as allohaploids). Subsequently, meiotic irregularities resulting from early meiosis carry forward through meiosis I and meiosis II. In the unstable line, multivalents which are unable to resolve putatively continue to anaphase I and/or telophase I in the form of anaphase bridges, where the chromosomes are unable to segregate equally to opposite poles, along with chromosome fragments and laggards which putatively result from univalents. These univalents/chromosome laggards carry forward to metaphase II and anaphase II and/or telophase II. By contrast, the stable line showed relatively normal progression through anaphase I/telophase I to anaphase II/telophase II without chromosomal abnormalities, indicating that the initial prophase I step is essential to maintain stable and regular meiosis, as has also been previously proposed (Grandont et al., 2014 ). Studies in ACC hybrids, which also have frequent multivalent formation as well as high numbers of univalents from the haploid genome, also show similar meiotic issues:Yang et al., ( 2017 ) reported partially tangled chromosomes at diakinesis, jumbled alignment at metaphase I with frequent chromosome bridges, unequal segregation and laggards in anaphase I and anaphase II, in contrast to parental lines diploid B. oleracea and allotetraploid B. napus , where the chromosomes were paired as bivalents, orderly aligned at the equatorial plate in metaphase and equally segregated. Meiotic configurations comprising univalents and especially multivalents could be due to mispairing and misalignment of homologous chromosomes as well as pairing between homoeologous chromosomes, leading to unbalanced gametes. We also observed lower total seed fertility in the putatively unstable resynthesized lines, which may result from these meiotic aberrations. Our results are similar toKatche et al., ( 2023a ) and Katche et al., ( 2023b ) who observed that resynthesized lines with lower CNVs showed higher seed set while resynthesized lines with higher CNVs exhibited poor seed fertility. While statistically similar frequencies of A1/C1 homoeologous associations were observed in stable resynthesized lines (18%) and established B. napus (13%), nearly half (46%) of PMCs in the unstable resynthesized line showed A1/C1 associations. Homoeologous chromosomes A1 and C1 share collinearity and homology along the length of the chromosome, which leads to higher chances of homoeologous recombination than between other, more rearranged A/C genome homoeologous (Higgins et al., 2021 ; Udall et al., 2005 ). Analysis of these A1/C1 associations therefore allowed us to estimate homoeologous pairing and make general assumptions. High frequencies of homoeologous recombination between the A and C genomes have also been observed in other studies of resynthesized rapeseed (Perkin et al., 1995; Sharpe et al., 1995 ; Udall et al., 2005 ), supporting our results.Xiong et al., ( 2021 ) reported 49–57% A1/C1 homoeologous pairing in resynthesized B. napus in both the S1 and S11 generations, very similar to our current observations. We observed lower but still relatively high frequencies of A1/C1 homoeologous associations in the putatively stable resynthesized line and established cultivar. Frequent homoeologous translocations in established B. napus were reported as early as 1995 (Parkin et al., 1995 ; Sharpe et al., 1995 ), although at lower rates than in synthetic B. napus , suggesting that B. napus , as a relatively young allopolyploid, is not fully able to prevent homoeologous pairing between the A and C genomes (Jenczewski & Alix, 2004 ; Sourdille & Jenczewski, 2021 ). Possibly, the presence of pre-existing homoeologous recombination events in either Drakkar or in our putatively stable resynthesized line may also have contributed to this observation. Similar to this,(Udall et al., 2005 ) observed pre-existing homoeologous translocations between A1 and C1 based on marker studies in a DH mapping population, and pre-existing A-C translocations are also known to cause multivalent formation in Brassica when present in translocation heterozygotes (Osborn et al., 2003 ; Mwathi et al., 2019 ). However, the use of chromosome oligo painting for the entire length of chromosomes A1 and C1 in the present study probably excludes the pre-existence of large-scale (> 1 Mb) translocations, as these would have been visible cytogenetically; such translocations were also not visible from SNP array data (Katche et al., 2023a ). Overall, our results indicate that the putatively stable resynthesized line preferences homologous recombination over homoeologous recombination similarly to established B. napus . Although we only assessed one line of each of the stable, unstable and established types for A1/C1 homoeologous recombination, such that we cannot completely rule out genotype-specific factors, it is highly likely that this finding of homoeologous pairing reduction could be a general event at least to the set of stable synthetic lines we investigated in the present study. Previous studies have also suggested that A/C pairing occurs between all homoeologous chromosome regions genome-wide in direct relation to degree of homoeology in a mostly genotype-independent fashion (Nicolas et al., 2007 , 2009 , 2012 ; Mason et al., 2014 ), supporting a general mechanism acting to prevent homoeologous recombination in established but not novel resynthesized lines. We observed major differences between the stable and unstable lines as well as minor but statistically significant differences within each of the groups of putatively stable and unstable resynthesized lines for chromosome pairing behaviour. This indicates individual genotype-specific differences between resynthesized lines, which could be the result of differences in specific genotypic factors inherited from the parental species used for development of these resynthesized lines (Heneen et al., 1995 ; Szadkowski et al., 2010 ). Some differences may however also be due to aneuploidy in specific plants, particularly in the unstable lines: lack of a homologous chromosome pairing partner or a third homologous copy of a chromosome will lead to increased univalent and multivalent frequencies, but this was difficult to confirm in this material due to the sometimes highly complex meiotic configurations observed in the unstable lines. Our results confirm that the genetic background of the parental species plays a crucial and essential role in meiotic stability. Our observation is a further validation of (Katche et al., 2023b ) where genome stability was genotype dependent and inherited genetic factors from both the diploid parental species affected accumulation of copy number variants. All our stable lines were later generation lines and our unstable lines were all early generation, such that further investigation of generational effects on stability may also be warranted. It is unclear from previous studies of resynthesized and synthetic Brassica if meiotic stability can improve generationally when starting from homozygous lines, where no segregation for allelic variation is possible. In studies where later-generation, putatively more homozygous lines show better meiotic stability than earlier generation lines (Tian et al., 2010 in B. rapa by B. carinata allohexaploids, as well as; Katche et al., 2023a ), selection putatively occurs in the early generations, such that unstable lines fail to produce seeds and are not able to be propagated to later generations, while lines which are initially more stable produce more seeds and survive to later generations. Xiong et al., ( 2021 ) also observed no significant difference in non-homologous chromosome pairing frequency over 11 generations in homozygous resynthesized B. napus (20% in the S1 and 16% in the S11 generation). Nevertheless, substantial changes are also possible in synthetic hybrids as a result of “genome shock” resulting from the hybridization event, such as transcriptional regulation changes (Gaeta et al., 2007 ), chromosome rearrangements (Szadkowski et al., 2010 ), activation of transposable elements (Zou et al., 2011 ) and proteomic changes (Albertin et al., 2006 ), all of which may create novel genetic variation which may be acted on by selection from early to late generation lines and provide a mechanism for stabilisation of meiosis. Further investigation would be necessary to confirm this hypothesis. We observed no major differences between the two generations we grew in our study, but we did find that in the unstable lines, univalent frequency in the second experimental year was significantly higher than in the first experimental year generation. Szadkowski et al., ( 2010 ) also observed high frequencies of univalents in S1 resynthesized B. napus , where this was attributed to aneuploidy (absence of a homologous pairing partner is expected to result in a univalent or trivalent) although Xiong et al., ( 2021 ) did not observe many univalents in their synthetic B. napus lines. Mwathi et al., ( 2019 ) also found that meiotically unstable homozygous Brassica allohexaploids showed increased frequencies of chromosome rearrangements and reduced fertility from one generation to the next, and hypothesised that initial chromosome rearrangements also resulted in more rearrangements. Similar effects may be responsible for the increased meiotic instability observed from one generation to the next in our unstable resynthesized lines. Conclusions In our study putatively stable resynthesized lines appeared to show higher seed fertility, normal and regular meiosis with low frequencies of aberrations in the form of univalents, multivalents, acentric chromosome fragments, anaphase bridges and laggards. Although these events were still observed at low frequencies, these frequencies were similar to those observed in established B. napus cultivar “Drakkar”, suggesting that a low level of meiotic abnormalities might be normal for B. napus , in line with previous studies (Sheidai et al., 2006 ). Lines could be grouped into “putatively stable” and “putatively unstable” based on large differences in bivalent and multivalent frequency, possibly supporting a major gene effect, with minor differences also between lines. Our results suggest that whether or not a resynthesized line is meiotically stable is highly genotype dependent. We also confirmed that non-homologous pairing is common in both “stable” resynthesized lines and established B. napus , but is observed at much higher frequencies in unstable resynthesized lines, where it also putatively causes additional meiotic abnormalities as meiosis progresses. Our study conclusively demonstrates meiotic stability in resynthesized B. napus for the first time, and provides a useful basis to further investigate genetic factors related to this stability mechanism. Declarations Conflict of interest The authors declare no conflict of interest. Author Contribution VR carried out the experiments, generated material for the second-year experimental generation with assistance and input from ZL and MB, analysed the data, prepared figures and tables and drafted the manuscript. EIK generated the material for the first-year experimental generation and data necessary for this project, provided input into project planning and critically revised the manuscript. 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Proceedings of the National Academy of Sciences, 92 (17), 7719–7723. https://pubmed.ncbi.nlm.nih.gov/7644483/ Sourdille, P., & Jenczewski, E. (2021). Homoeologous exchanges in allopolyploids: how Brassica napus established self-control! The New Phytologist , 229 (6), 3041–3043. https://doi.org/10.2307/27001280 Szadkowski, E., Eber, F., Huteau, V., Lodé, M., Huneau, C., Belcram, H., Coriton, O., Manzanares-Dauleux, M. J., Delourme, R., King, G. J., Chalhoub, B., Jenczewski, E., & Chèvre, A. M. (2010). The first meiosis of resynthesized Brassica napus , a genome blender. New Phytologist , 186 (1), 102–112. https://doi.org/10.1111/j.1469-8137.2010.03182.x Tian, E., Jiang, Y., Chen, L., Zou, J., Liu, F., & Meng, J. (2010). Synthesis of a Brassica trigenomic allohexaploid ( B. carinata × B. rapa ) de novo and its stability in subsequent generations. Theoretical and Applied Genetics , 121 (8), 1431–1440. https://doi.org/10.1007/s00122-010-1399-1 Udall, J. A., Quijada, P. A., & Osborn, T. C. (2005). Detection of chromosomal rearrangements derived from homeologous recombination in four mapping populations of Brassica napus L. Genetics , 169 (2), 967–979. https://doi.org/10.1534/genetics.104.033209 Wei, T. & Simko, V. (2024). R package 'corrplot': Visualization of a Correlation Matrix. (Version 0.95), https://github.com/taiyun/corrplot . Wickham H (2016). ggplot2: Elegant Graphics for Data Analysis. Springer- Verlag New York. ISBN 978-3-319-24277-4, https://ggplot2.tidyverse.org . Windham, M. D., Pryer, K. M., Poindexter, D. B., Li, F. W., Rothfels, C. J., & Beck, J. B. (2020). A step-by-step protocol for meiotic chromosome counts in flowering plants: A powerful and economical technique revisited. Applications in Plant Sciences , 8 (4), e11342. https://doi.org/10.1002/aps3.11342 Xiong, Z., & Chris Pires, J. (2011). Karyotype and identification of all homoeologous chromosomes of allopolyploid Brassica napus and its diploid progenitors. Genetics , 187 (1), 37–49. https://doi.org/10.1534/genetics.110.122473 Xiong, Z., Gaeta, R. T., Edger, P. P., Cao, Y., Zhao, K., Zhang, S., & Chris Pires, J. (2021). Chromosome inheritance and meiotic stability in allopolyploid Brassica napus . G3: Genes, Genomes, Genetics , 11 (2), jkaa011. https://doi.org/10.1093/G3JOURNAL/JKAA011 Xiong, Z., Gaeta, R. T., & Pires, J. C. (2011). Homoeologous shuffling and chromosome compensation maintain genome balance in resynthesized allopolyploid Brassica napus . Proceedings of the National Academy of Sciences , 108 (19), 7908–7913. https://doi.org/10.1073/pnas.1014138108 Yang, Y., Wei, X., Shi, G., Wei, F., Braynen, J., Zhang, J., Tian, B., Cao, G., & Zhang, X. (2017). Molecular and cytological analyses of A and C genomes at meiosis in synthetic allotriploid Brassica hybrids (ACC) between B. napus (AACC) and B. oleracea (CC). Journal of Plant Biology , 60 (2), 181–188. https://doi.org/10.1007/s12374-016-0221-2 Zou, J., Fu, D., Gong, H., Qian, W., Xia, W., Pires, J. C., Li, R., Long, Y., Mason, A. S., Yang, T. J., Lim, Y. P., Park, B. S., & Meng, J. (2011). De novo genetic variation associated with retrotransposon activation, genomic rearrangements and trait variation in a recombinant inbred line population of Brassica napus derived from interspecific hybridization with Brassica rapa . The Plant Journal , 68 (2), 212–224. https://doi.org/10.1111/j.1365-313X.2011.04679.x Additional Declarations No competing interests reported. Supplementary Files 20250923suppfigures.pdf 20250908supplementarytables.zip SupplementaryMaterialLegends.docx Cite Share Download PDF Status: Published Journal Publication published 07 Apr, 2026 Read the published version in Chromosome Research → Version 1 posted Editorial decision: Revision requested 09 Mar, 2026 Reviews received at journal 24 Oct, 2025 Reviewers agreed at journal 07 Oct, 2025 Reviewers invited by journal 05 Oct, 2025 Editor assigned by journal 30 Sep, 2025 Submission checks completed at journal 25 Sep, 2025 First submitted to journal 24 Sep, 2025 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-7700382","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":530208668,"identity":"229e6b09-5c7a-4dd6-bc25-dc8a4f814807","order_by":0,"name":"Vinita Ramtekey","email":"","orcid":"","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Vinita","middleName":"","lastName":"Ramtekey","suffix":""},{"id":530208669,"identity":"e09d2222-e914-4b50-bd05-a5671af62654","order_by":1,"name":"Elizabeth Ihien Katche","email":"","orcid":"","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Elizabeth","middleName":"Ihien","lastName":"Katche","suffix":""},{"id":530208670,"identity":"6be9a322-e759-414d-b3bd-5f5c7a68e93a","order_by":2,"name":"Mariana Baez","email":"","orcid":"","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Mariana","middleName":"","lastName":"Baez","suffix":""},{"id":530208671,"identity":"bf99f8ac-93c6-4a47-bac9-e8608591fe14","order_by":3,"name":"Zhenling Lv","email":"","orcid":"","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Zhenling","middleName":"","lastName":"Lv","suffix":""},{"id":530208672,"identity":"3d69ac70-b390-4b31-8775-3b2cbb457fa1","order_by":4,"name":"Annaliese S. 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17:26:07","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":217311,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7700382/v1/0fa2116037f500d332f2d264.html"},{"id":93709529,"identity":"70cab046-6d40-4f03-843d-0001a2407d4d","added_by":"auto","created_at":"2025-10-16 17:26:07","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":91904,"visible":true,"origin":"","legend":"\u003cp\u003eSeed fertility (total seeds per plant) in putatively stable and unstable resynthesized lines.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7700382/v1/daa56a71aa58fd6150a32657.jpeg"},{"id":93709532,"identity":"fbe55e3c-e152-45c9-888e-b9960df0aa0b","added_by":"auto","created_at":"2025-10-16 17:26:07","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":433650,"visible":true,"origin":"","legend":"\u003cp\u003eMeiotic chromosome pairing behaviour in established \u003cem\u003eBrassica napus\u003c/em\u003e cultivar “Drakkar” and in putatively stable and unstable resynthesized \u003cem\u003eB. napus\u003c/em\u003e lines. Letters indicate significant differences between lines for each of bivalent, univalent and multivalent frequencies (Kruskal-Wallis test followed by Dunn’s post hoc test, p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7700382/v1/04294d54fbdef6285830abd0.jpeg"},{"id":93710088,"identity":"2bdddbe3-1edf-4afb-a051-7f9fbe0863d6","added_by":"auto","created_at":"2025-10-16 17:42:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":408123,"visible":true,"origin":"","legend":"\u003cp\u003eMeiotic progression in established, putatively stable and putatively unstable resynthesized \u003cem\u003eBrassica napus.\u003c/em\u003e Green arrows: bivalent, blue arrows: univalent, orange arrow: multivalent, yellow arrows: anaphase bridge, white arrows: chromosome laggards\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7700382/v1/30916f0f98abc2ea2a19a1ec.png"},{"id":93709542,"identity":"a27eabbd-4742-4676-9750-0699938625d9","added_by":"auto","created_at":"2025-10-16 17:26:07","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":98917,"visible":true,"origin":"","legend":"\u003cp\u003eA1-C1 chromosome pairing associations at diakinesis in established, putatively stable and unstable resynthesized \u003cem\u003eBrassica napus\u003c/em\u003e lines.\u003cstrong\u003e \u003c/strong\u003eA1: red signal, C1: green signal, centromere: purple signal,\u003cstrong\u003e \u003c/strong\u003eblue arrow: univalent, dotted circle: A1-C1 trivalent (A1: univalent, C1: bivalent)\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7700382/v1/91c48c8c3e5354aaa9359c87.jpeg"},{"id":93709638,"identity":"7212287b-248b-46c0-b5de-9533a94c6c07","added_by":"auto","created_at":"2025-10-16 17:34:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":33678,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelations between different meiotic configuration and seed fertility in resynthesized \u003cem\u003eB. napus\u003c/em\u003e (Pearson correlation, * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7700382/v1/380aa2fc1c61783f9d1eacb6.png"},{"id":106809176,"identity":"c8d17cd2-88e8-4129-a45c-189d52b643db","added_by":"auto","created_at":"2026-04-13 16:07:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2137678,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7700382/v1/8f181cbf-0ae2-41a5-a035-e59367f8f7e7.pdf"},{"id":93709639,"identity":"09318f82-5dda-49b4-a4c5-2418922c8857","added_by":"auto","created_at":"2025-10-16 17:34:07","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":471558,"visible":true,"origin":"","legend":"","description":"","filename":"20250923suppfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7700382/v1/cd7383f764933217a63dada5.pdf"},{"id":93709534,"identity":"d3180881-0f85-4e11-bdae-7c21fa4cddce","added_by":"auto","created_at":"2025-10-16 17:26:07","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":100996,"visible":true,"origin":"","legend":"","description":"","filename":"20250908supplementarytables.zip","url":"https://assets-eu.researchsquare.com/files/rs-7700382/v1/44f9446e89e9a9b1bda976d8.zip"},{"id":93709531,"identity":"ed9961d9-2efc-426a-92a4-a6ae61954969","added_by":"auto","created_at":"2025-10-16 17:26:07","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":15238,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-7700382/v1/ed4eb0f027771d6e0d548227.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Stable resynthesized Brassica napus lines show similar meiotic behaviour to established B. napus","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePolyploidization is a major driving force in evolution and speciation (Leitch and Leitch \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and is particularly common in angiosperms (Jiao et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Polyploids often exhibit genome buffering, enhanced heterozygosity, and novel phenotypic and genotypic variation compared to their diploid counterparts, which can be attributed to various interconnected mechanisms such as genome rearrangements, altered gene dosage, gene expression and regulation and epigenetic modification (Pel\u0026eacute; et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Doyle \u0026amp; Coate, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Heslop-Harrison et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Polyploids are usually categorised as either autopolyploid (sets of chromosomes originating from the same species), or allopolyploid (two or more genomes derived from different species) (Kihara and Ono, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1926\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAllopolyploids which arise from hybridization between two different sets of chromosomes (genomes) face the major challenge of differentiating between ancestrally related (homoeologous) chromosome copies (Pel\u0026eacute; et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). During the course of evolution, the major adaptation that has been observed in established allopolyploids is their ability to distinguish between homoeologous and homologous chromosomes when choosing meiotic recombination partners. Prevention of extensive pairing between non-homologous (homoeologous or otherwise) chromosomes is critical for ensuring regular segregation of chromosomes into subsequent gametes without putative issues such as loss of chromosomes or chromosome segments, which is potentially detrimental to plant fertility and viability (Mercier et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, how established allopolyploids stabilise meiosis is still unknown in most taxa (Bomblies, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThere are several ways in which allopolyploids might stabilise meiosis. Crossovers between homoeologous chromosomes could be prevented completely, resolved in such a way that no recombination can occur between homoeologues, or simply made extremely rare due to strong preference for homologous over homoeologous crossovers, even in the absence of specific genetic factors that suppress pairing between homoeologous chromosomes (Bomblies, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In allohexaploid bread wheat, which is one of the most extensively studied species with respect to understanding of the establishment of meiotic stability in allopolyploids, the \u003cem\u003ePh1\u003c/em\u003e gene acts not only to suppress homoeologous chromosome recombination but also to promote homologous recombination (Riley \u0026amp; Chapman, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1958\u003c/span\u003e; Griffiths et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Bhullar et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Other genetic factors acting to prevent homoeologous recombination have been identified in \u003cem\u003eArabidopsis suecica\u003c/em\u003e (Henry et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and \u003cem\u003eBrassica napus\u003c/em\u003e (Jenczewski et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Higgins et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), although the mechanism of action of these genetic factors is so far unknown. Jenczewski et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2003\u003c/span\u003e identified a genetic factor \u003cem\u003ePrBn\u003c/em\u003e which influences homoeologous crossover frequency in \u003cem\u003eB. napus\u003c/em\u003e haploids, and Nicolas et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2009\u003c/span\u003e observed differences in homologous recombination frequency in allotriploid \u003cem\u003eBrassica\u003c/em\u003e hybrids (AAC) produced with different \u003cem\u003ePrBn\u003c/em\u003e types, suggesting \u003cem\u003ePrBn\u003c/em\u003e could have dosage sensitive effects on recombination. However, the gene corresponding to this locus has not yet been identified or fully functionally characterized. Recently, Gonzalo et al., (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) demonstrated that reduced expression of \u003cem\u003eMSH4\u003c/em\u003e, belonging to the ZMM-group of class I crossover pathway proteins, significantly decreases the occurrence of homoeologous recombination, while having minimal impact on homologous recombination in \u003cem\u003eB. napus\u003c/em\u003e. However, the specific impact on partner choice in the context of pairing or recombination regulation in \u003cem\u003eBrassica\u003c/em\u003e is still uncertain. As suggested by earlier QTL mapping results (Jenczewski et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), an integrated system of multiple genes is most likely associated with meiotic stabilization in \u003cem\u003eB. napus\u003c/em\u003e, such that the molecular basis of meiotic stability involves polygenic adaptation to allopolyploidy.\u003c/p\u003e\u003cp\u003eNeo-allopolyploids and resynthesized hybrids are a useful model with which to investigate mechanisms underlying meiotic stability. In most newly synthesised allopolyploids (produced by crosses between lower ploidy parents), meiosis is associated with numerous abnormalities, including but not limited to incorrect synapsis, homoeologous recombination, chromosome bridges, and chromosome mis-segregation (anaphase I). These meiotic abnormalities can lead to aneuploidy, chromosome rearrangements and deletions and duplications of chromosome segments, which may result in loss of fertility and viability in subsequent generations (Bomblies, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Pel\u0026eacute; et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xiong et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Studies of synthetic hybrids have previously investigated cytological causes of meiotic stability. Madlung et al., (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) observed about 30% meiotic abnormalities in the form of chromosome breakage, bridges and rearrangements in synthetic \u003cem\u003eArabidopsis\u003c/em\u003e allopolyploids, indicating increased meiotic instability compared to their parents (10%). Similarly, Ch\u0026eacute;ron et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e suggested that associations between incorrect recombination partners and homoeologous recombination contribute to meiotic instability in neo-synthetic allopolyploid \u003cem\u003eA. suecica\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eRecently, synthetic \u003cem\u003eBrassica napus\u003c/em\u003e has emerged as an important model system to study meiosis in allopolyploids ( Katche \u0026amp; S. Mason, 2023; Bomblies, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Allotetraploid \u003cem\u003eB. napus\u003c/em\u003e (AACC, 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4\u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;38) is the product of natural interspecific hybridization coupled with polyploidization between diploid ancestors of \u003cem\u003eBrassica rapa\u003c/em\u003e (AA, 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2\u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20) and \u003cem\u003eBrassica oleracea\u003c/em\u003e (CC, 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2\u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;18) around 7500 years ago (U, 1935; Chalhoub et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Established \u003cem\u003eB. napus\u003c/em\u003e is a relatively meiotically and genomically stable allopolyploid which shows diploid-like meiosis, including predominantly homologous recombination even in the presence of homoeologous chromosomes (Jenczewski et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). By contrast, synthetic \u003cem\u003eB. napus\u003c/em\u003e (formed by either \u003cem\u003eB. rapa\u003c/em\u003e \u0026times; \u003cem\u003eB. oleracea\u003c/em\u003e or \u003cem\u003eB. oleracea\u003c/em\u003e \u0026times; \u003cem\u003eB. rapa\u003c/em\u003e) is usually meiotically unstable, but the cause is still unknown (reviewed by Katche \u0026amp; Mason, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Cytogenetic, molecular and genome sequencing studies have revealed that resynthesized \u003cem\u003eB. napus\u003c/em\u003e often display genetic changes as well as homoeologous rearrangements (Gaeta et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Xiong et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Chalhoub et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Xiong et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Katche, et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e; Katche et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e; Davis et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), leading to genomic copy number variants (deletions, duplications, and translocation) as well as presence/absence variation (Katche et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e; Schiessl et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Such variants are most common in chromosomes which are structurally conserved (syntenic along the whole length of the chromosome) between subgenomes, such as A1 and C1, and A2 and C2 (Xiong et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Higgins et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Unlike resynthesized \u003cem\u003eB. napus\u003c/em\u003e, established cultivars show rarer or lower rates of homoeologous rearrangements (Parkin et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Sharpe et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Howell et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Udall et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In the past decades many cytogenetic studies have extensively confirmed with the help of fluorescently labelled probes as well BAC-FISH that homoeologous recombination between the closely related A and C subgenomes is a major feature of resynthesized \u003cem\u003eB. napus\u003c/em\u003e (Howell et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Xiong et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Xiong \u0026amp; Chris Pires, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn our previous work, we identified several synthetic \u003cem\u003eB. napus\u003c/em\u003e lines from different genetic backgrounds which accumulate no or very few new copy number variants after many generations of self-pollination and are therefore putatively stable (Katche et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e), unlike all previously produced resynthesized \u003cem\u003eB. napus\u003c/em\u003e lines (Katche et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). In the present study, we aimed to investigate and characterise meiosis in these lines and compare between these putatively \u0026ldquo;stable\u0026rdquo; and \u0026ldquo;unstable\u0026rdquo; resynthesized \u003cem\u003eB. napus\u003c/em\u003e types as well as established \u003cem\u003eB. napus\u003c/em\u003e. We hypothesised that meiosis in the putatively stable lines would be normal and similar to meiosis as established \u003cem\u003eB. napus\u003c/em\u003e, in contrast to unstable resynthesized \u003cem\u003eB. napus\u003c/em\u003e, and aimed to characterise meiotic progression in order to better elucidate the cytological mechanisms responsible for these differences.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant material\u003c/h2\u003e\u003cp\u003eProduction of the resynthesized winter \u003cem\u003eB. napus\u003c/em\u003e allotetraploid (AACC) lines used in the present research is described in Girke et al., (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and Jesske et al., (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Lines were produced by crossing different genotypes of \u003cem\u003eB. rapa\u003c/em\u003e with (AA) with \u003cem\u003eB. oleracea\u003c/em\u003e (CC). The Girke et al., (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) material consists of domesticated resynthesized \u003cem\u003eB. napus\u003c/em\u003e lines derived from hybridization between vegetable-type \u003cem\u003eB. rapa\u003c/em\u003e (AA) and \u003cem\u003eB. oleracea\u003c/em\u003e (CC), while the Jesske et al., (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) material comprised wild C genome species crossed with cultivated \u003cem\u003eB. rapa\u003c/em\u003e lines. Katche et al., (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e) later characterized these lines as putatively stable or unstable based on frequencies of novel and inherited CNVs in each line.\u003c/p\u003e\u003cp\u003eFor the first year of flower bud collection, plants were grown at Justus Liebig University Giessen, Germany, as described by Katche et al., (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). In the subsequent generation, only putatively stable and unstable resynthesized lines with a minimum of three seeds per line were germinated in quick-pots during the year of 2024. Germinated plants at the 4\u0026ndash;6 leaf stage were vernalized at 4\u0026ndash;6\u0026deg;C for minimum 14\u0026ndash;16 weeks (December 2023 to March 2024) in a controlled environment room at Campus Klein Altendorf, the University of Bonn field station. After vernalization, plants were transferred to the glasshouse and grown in 10 L pots under heated glasshouse conditions (minimum 20\u0026deg;C day and 16\u0026deg;C night, but up to 35\u0026deg;C on hot days in late summer) at the University of Bonn, Poppelsdorf from April to September 2024.\u003c/p\u003e\u003cp\u003eIn the present study we selected fifteen resynthesized \u003cem\u003eB. napus\u003c/em\u003e lines from Katche et al., (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e) and categorised these into three groups based on CNV data: 1) no novel CNVs = \u0026ldquo;stable\u0026rdquo;, 2) 1\u0026ndash;3 novel CNVs = \u0026ldquo;intermediate\u0026rdquo;, and 3)\u0026thinsp;\u0026gt;\u0026thinsp;8 CNVs = \u0026ldquo;unstable\u0026rdquo; (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). For the putatively stable lines (with the exception of R76), the maternal parent was \u003cem\u003eB. oleracea\u003c/em\u003e and the paternal parent was \u003cem\u003eB. rapa\u003c/em\u003e, with different subspecies for both parents (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). For the putatively unstable lines (with the exception of OLY21), the maternal parent was \u003cem\u003eB. rapa\u003c/em\u003e, while paternal parent was \u003cem\u003eB. oleracea\u003c/em\u003e (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The putatively highly stable lines were \u0026gt;\u0026thinsp;S3 generation, while the putatively highly unstable were either S1 or S2 generation (Katche et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). As a reference for stable meiosis, we used \u003cem\u003eB. napus\u003c/em\u003e cv. Drakkar (received from INRAE, France). Additionally, we selected one putatively stable (L16) and unstable (OLY21) resynthesized line to investigate meiotic progression and A1/C1 pairing, as these lines did not have any apparent fixed translocations involving chromosomes A1 and C1 (Katche et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSeed fertility in putatively stable and unstable resynthesized lines\u003c/h3\u003e\n\u003cp\u003eThree individual plants per genotype were bagged using microperforated plastic bags to encourage self-pollinated seed set. The average number of total self-pollinated seed set was measured for each of the three plants per genotype to assess seed fertility differences between putatively stable and unstable resynthesized lines.\u003c/p\u003e\n\u003ch3\u003eInflorescence fixation, slide preparation and meiotic observation\u003c/h3\u003e\n\u003cp\u003eImmature unopened flower buds were collected in Carnoy\u0026rsquo;s I fixative solution (absolute ethanol: glacial acetic acid 3:1, v/v) for 24\u0026ndash;48 hours at room temperature to fix the cells at the respective meiotic stages (Windham et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Later, the fixed flower buds were transferred to 50% ethanol for further downstream analysis as well as long term storage. Out of six anthers, one anther was squashed and stained with 1% acetocarmine to identify meiotic stages while the remaining five anthers were used for slide preparation via enzymatic digestion (composition of 1% pectyolase Y-23 and 2% cellulase \u0026ldquo;Onozuka\u0026rdquo; R-10 in 0.1M citrate buffer)(Kirov et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Slide preparation involved submerging the remaining five anthers in an enzymatic mixture of 20 \u0026micro;l in a 0.5 ml tube and incubating for 1\u0026ndash;1.5 hours at 37\u0026deg;C followed by washes with 70% ethanol. Subsequently, anthers were scrambled with a needle in 60\u0026ndash;80 \u0026micro;l of 70% ethanol to form a white musky coloration, which gives a pellet upon quick centrifugation (25\u0026ndash;30 s) using a benchtop centrifuge (FastGene\u0026reg; Mini Centrifuge (NG002P)). The pellet contains meiocytes as well as some somatic tissue (anther wall and tapetum). The ethanol supernatant is removed and pellet dried. Once the pellet is dried, we add 100% GAA (approximately 25\u0026ndash;40 \u0026micro;l depending on the pellet size) and vortex thoroughly. Finally, we use the dropping method to prepare slides containing meiocytes and incubate for another 5 minutes in a humid chamber at room temperature (RT) to evenly spread the meiocytes. A drop of Vectashield\u0026reg; antifade mounting medium containing 4\u0026rsquo;, 6- diamidino-2-phenylindole (DAPI) (H-1200-10, Vector Laboratories) was used on slides to stain and visualize the chromosomes inside meiocytes for cells undergoing meiosis). These slides were finally used to investigate chromosome pairing behaviour at diakinesis of prophase I of meiosis I from male meiocytes. In order to understand the overall meiosis progression of the putatively stable and unstable resynthesized lines, we focused on metaphase I, anaphase I, telophase I, metaphase II, and anaphase II/telophase II. Visualization of meiocytes was performed using an inverted fluorescence microscope (Zeiss Axio Imager M2) with a 40 \u0026times; and 100 \u0026times; oil immersion lens and up to 10 000 \u0026times; total magnification. Images were captured using the Zeiss software ZEN Blue (version 3.2), which was also used for cropping, size adjustment and contrast optimization of microscopic images. A minimum of 30 cells per line was analysed for a robust interpretation of meiotic chromosome behaviour at diakinesis. Chromosome configurations were scored as bivalent, univalent or multivalent at diakinesis. Other chromosomal aberrations such as anaphase bridges, laggards or chromosome fragments at anaphase/telophase stages of meiosis I and meiosis II were also observed.\u003c/p\u003e\n\u003ch3\u003eProbe labelling, oligo-FISH and imaging\u003c/h3\u003e\n\u003cp\u003eIn order to clearly identify univalent, bivalent and multivalent chromosomes, DNA from centromere repeat-specific sequences CentBrI and CentBrII (Xiong and Chris Pires, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) were labelled with Cy5-dUTP, using a nick translation kit (11745808910, Roche) as described by (Kato et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Specific labelling of A1 and C1 was done using A1-specific oligo probes labelled with ATT0550 (red) and C1-specific oligo probes labelled with ATT0488 (green) (by Arbor Biosciences, via BioCat GmbH, Germany). FISH was performed according to Han et al., (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and Montenegro et al., (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) with slight modification. We used all three DNA probes at the same time on a single slide. Hybridization started with fixing the slides in Carnoy\u0026rsquo;s I solution for 30 minutes followed by dehydration with 100% ethanol for 15 minutes. Once the slides dried out, we treated with pepsin (50 \u0026micro;g/ml) for 30 minutes at 37\u0026deg;C in a humid chamber followed by two successive washes with 2 \u0026times; SSC buffer (saline sodium citrate buffer) at RT. Later we treated slides with 4% formaldehyde and incubated for 10 minutes at RT, followed by two successive washes with 2 \u0026times; SSC buffer then dehydration with an ethanol series (70% and 100% ethanol). After these treatments, we air dried the slides for at least an hour and applied the hybridization mixture to the meiotic cell zone on the slides. The hybridization mixture comprised of 50% formaldehyde, 10% dextran sulphate, 2 \u0026times; SSC buffer, 200 ng A1 and C1 oligo probes and 50 ng of centromere probes. Slides were incubated at 75\u0026deg;C for 5\u0026ndash;7 minutes after adding hybridization mixture to the slides and later hybridized for 2 days at 37\u0026deg;C in a moisture chamber, in the dark. After a long incubation, we washed slides once with 2 \u0026times; SSC buffer at RT and at 55\u0026deg;C for about 20 minutes followed by another wash at RT with 2 \u0026times; SSC buffer. Once the slides partially dried, the Vectashield\u0026reg; antifade mounting medium containing DAPI (H-1200-10, Vector Laboratories) was used on slides to capture the images using an inverted fluorescence microscope (Zeiss Axio Imager M2) with a 40 \u0026times; and 100 \u0026times; oil immersion lens with different filters and software described above. A minimum of 30 microscopic images/line were analysed. Univalent, bivalent or multivalent behaviour of chromosomes A1/C1 was scored at diakinesis.\u003c/p\u003e\n\u003ch3\u003eStatistical Data Analysis\u003c/h3\u003e\n\u003cp\u003eStatistical analysis was performed in R studio v 4.4.1 (R Core Team (2024) \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://stat.ethz.ch/pipermail/r-announce/2024/000704.html\u003c/span\u003e\u003cspan address=\"https://stat.ethz.ch/pipermail/r-announce/2024/000704.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Visualization plots for seed fertility and meiotic chromosome pairing behaviour (bivalent, univalent and multivalent) of putatively stable and unstable lines were produced using the \u0026ldquo;ggplot2\u0026rdquo; package available in R (Wickham H, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A Shapiro-Wilk normality test was carried out to assess normality of seed fertility and meiosis confirmation data. Seed set was normally distributed, so we performed ANOVA followed by Tukey\u0026rsquo;s HSD test to check for significant differences between putatively stable and unstable resynthesized lines. Meiotic configuration data was not normally distributed, so a non-parametric Kruskal-Wallis test was used to check for significant differences between groups and between genotypes followed by a pairwise Dunn\u0026rsquo;s post hoc test for significant comparisons with multiple testing correction using the Bonferroni method. The adjusted p-values were used to cluster groups and genotypes into sets by significant differences between them, which were indicated by different letters in the plot. Fisher\u0026rsquo;s exact test for count data was used to detect the significance for A1/C1 homoeologous pairing between putatively stable and unstable resynthesized lines. The Pearson correlation coefficients were computed by using \u0026lsquo;corrplot\u0026rsquo; package available in R (Wei \u0026amp; Simko, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003ePutatively stable resynthesize lines exhibited higher seed set, in contrast to unstable lines\u003c/h2\u003e\u003cp\u003ePutatively stable lines showed higher seed set. Total self-pollinated seeds ranged from 499 to 2720 (on an average 1181) per plant across putatively stable resynthesized lines which was significantly higher (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, ANOVA, P\u0026thinsp;=\u0026thinsp;0.01, followed by Tukey\u0026rsquo;s HSD P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) than seed set in unstable lines, which ranged from 0 to 444 seeds per plant (average 52) (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePutatively stable resynthesized\u003c/b\u003e \u003cb\u003eB. napus\u003c/b\u003e \u003cb\u003elines exhibited similar chromosome pairing behaviour\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eto established\u003c/b\u003e \u003cb\u003eB. napus\u003c/b\u003e, \u003cb\u003eover two generations, in contrast to unstable lines\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePutatively stable lines displayed meiotic chromosome pairing behaviour which was statistically similar to that of established \u003cem\u003eB. napus\u003c/em\u003e cultivar \u0026ldquo;Drakkar\u0026rdquo; in terms of univalent, bivalent and multivalent frequencies (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Table S3). The putatively stable group showed higher frequencies of bivalents (91% on an average), than the putatively unstable lines (60% on average; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Table S3; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). There was a significant difference between univalent frequencies in stable lines (2%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to unstable lines (1%), as well as for multivalent formation (3% in established and stable, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared to unstable lines (13%) (Table S3; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the first experimental year and generation, putatively stable lines exhibited 1% univalent frequency and 3% multivalent frequency on average, while unstable lines averaged 1% univalent frequency and 12% multivalent frequency (Table S3). However, only the average multivalent frequencies showed statistically significant differences between putatively stable and established \u003cem\u003eB. napus\u003c/em\u003e as well as between putatively stable and unstable resynthesized lines (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for stable and for established, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Similar results were observed in the subsequent generation in the resynthesized lines, where the subsequent generation was produced from self-pollinated seeds collected from the previous generation plants, except where univalent frequencies were also found to be significantly different between established and stable and the unstable lines (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, Table S3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the first experimental year, the average meiotic pairing behaviour of the putatively stable resynthesized lines was 0.41 I\u0026thinsp;+\u0026thinsp;17.10 II\u0026thinsp;+\u0026thinsp;0.66 multivalents, in contrast to putatively unstable lines with 0.28 I\u0026thinsp;+\u0026thinsp;11.52 II\u0026thinsp;+\u0026thinsp;2.33 multivalents. Similar results were observed in the second year in the following generation of progeny, with 0.25 I\u0026thinsp;+\u0026thinsp;17.81 II\u0026thinsp;+\u0026thinsp;0.36 multivalents across the putatively stable lines and 1.07 I\u0026thinsp;+\u0026thinsp;10.78 II\u0026thinsp;+\u0026thinsp;2.48 multivalents in the putatively unstable resynthesized lines. As we hypothesized, the putatively stable resynthesized lines behaved similarly to established \u003cem\u003eB. napus\u003c/em\u003e \u0026ldquo;Drakkar\u0026rdquo;, where we observed 0.26 I\u0026thinsp;+\u0026thinsp;17.30 II\u0026thinsp;+\u0026thinsp;0.52 multivalents (Table S3).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e\u0026ldquo;Intermediate\" lines with 1\u0026ndash;3 CNVs fell into two distinct meiotic behaviour classes (stable and unstable)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFive lines, which were classed as intermediate based on the presence of 1\u0026ndash;3 novel CNVs, exhibited either \u0026ldquo;stable\u0026rdquo; or \u0026ldquo;unstable\u0026rdquo; phenotypic behaviour in terms of chromosome pairing. Out of the five intermediate lines, two (R8 and H327) showed normal bivalent formation (86% and 93% respectively), similar to the putatively stable lines. By contrast, the other three lines (R140, J122 and H4) exhibited lower bivalent frequencies (54%, 63% and 66% respectively), similar to the unstable lines. Based on present phenotypic results, we re-categorized these intermediate lines accordingly as putatively stable and unstable lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eGenotype-specific differences between lines within the \u0026ldquo;stable\u0026rdquo; group and within the \u0026ldquo;unstable\u0026rdquo; group for chromosome pairing behaviour at diakinesis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe observed statistically significant differences of chromosome pairing behaviour between some of the putatively stable resynthesized lines (Fig. S3, S5) as well as between some of the putatively unstable resynthesized lines (Fig. S4, S6). Specifically, H327 (originally identified as intermediate) showed significantly lower bivalent frequencies than stable lines J154 and R8 in the first generation, although the other lines were not significantly different from each other (Fig. S3). Significant differences between multiple stable lines were also observed for multivalent and univalent frequencies (Fig. S3). However, in the next generation, there were no significant differences observed between putatively stable resynthesized lines for bivalent or multivalent frequencies, and only L16 had significantly higher univalent frequencies (Fig. S5). For the putatively unstable resynthesized lines, only OLY21 was statistically significantly different to the other four lines for bivalent frequencies in the first generation (Fig. S4) and K241 in the second generation (Fig. S6), although there were more differences in both generations for multivalent and univalent frequencies (Fig. S4, S6).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMeiotic progression in stable resynthesized lines is similar to that in established\u003c/b\u003e \u003cb\u003eB. napus\u003c/b\u003e, \u003cb\u003ebut contrasts with that of putatively unstable resynthesized lines\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe looked into the different stages of meiosis I and meiosis II to understand the meiotic progression in different putatively stable and unstable lines of resynthesized rapeseed and in established rapeseed. Here, we observed relatively normal and regular meiosis in putatively stable line \u0026ldquo;L16\u0026rdquo;, which was quite similar with that of established \u003cem\u003eB. napus\u003c/em\u003e \u0026ldquo;Drakkar\u0026rdquo;, but differed with that of putatively unstable resynthesized line \u0026ldquo;OLY21\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Specifically, the unstable line showed higher frequencies of multivalents at diakinesis, as previously mentioned (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Table S3), and these multivalents were not resolved but were carried forward to metaphase I. Subsequently at anaphase I, we observed another type of structural chromosome aberration in the unstable line, namely \u0026ldquo;anaphase bridges\u0026rdquo;, which can result from crossovers between non-homologous chromosomes where two centromeres end up on the same chromatid. As well, unequal segregation ratios of chromosomes were observed at telophase I in the unstable line, indicating failure to segregate chromosomes correctly to different poles. Chromosome laggards were also observed at metaphase II and anaphase II/telophase II of meiosis II (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003e), which can represent acentric fragments following non-homologous crossovers or univalent chromosomes which do not correctly segregate to the poles.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eNon-homologous multivalents involving chromosomes A1 and C1 were common but much more frequent in unstable resynthesized lines compared to established and stable lines\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHomoeologous pairing is one of the most probable causes for meiotic instability, therefore we analysed more specifically associations between chromosome pairs A1 and C1, which have the highest known homoeology in the \u003cem\u003eB. napus\u003c/em\u003e genome (Parkin et al. 2003). We applied oligo painting probes specific to the entire chromosome lengths of A1 and C1 as well as centromere-specific probes in order to analyse the frequency of A1-C1 associations in diakinesis. The average frequency of A1 - C1 pairing was approximately 13% in established \u003cem\u003eB. napus\u003c/em\u003e \u0026ldquo;Drakkar\u0026rdquo;, not significantly different from the approximately 18% inferred A1-C1 pairing in putatively stable resynthesized line \u0026ldquo;L16\u0026rdquo;, but contrasting significantly with the 46% frequency observed in putatively unstable line \u0026ldquo;OLY21\u0026rdquo; (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fisher\u0026rsquo;s exact test for count data; Table S4 and Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCorrelations between meiotic behaviour and seed fertility\u003c/h3\u003e\n\u003cp\u003eAcross all lines assessed, seed fertility was significantly positively correlated with bivalent formation (r\u0026thinsp;=\u0026thinsp;0.88, p\u0026thinsp;=\u0026thinsp;0.00066), and significantly negatively correlated with both univalent formation (r = -0.80, p\u0026thinsp;=\u0026thinsp;0.0058) and multivalent formation (r =-0.81, p\u0026thinsp;=\u0026thinsp;0.0048). Correlations within each of the \u0026ldquo;stable\u0026rdquo; and \u0026ldquo;unstable\u0026rdquo; groups (five lines each) were not significant. Unsurprisingly, meiotic configuration frequencies were also strongly significantly correlated across all lines: multivalents and bivalents were correlated at r = -0.96 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), bivalents and univalents at r = -0.80 (p\u0026thinsp;=\u0026thinsp;0.0053), and univalents and multivalents at r\u0026thinsp;=\u0026thinsp;0.67 (p\u0026thinsp;=\u0026thinsp;0.034) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we provide the first confirmation of stable meiosis in a subset of resynthesized rapeseed lines (\u003cem\u003eB. rapa\u003c/em\u003e \u0026times; \u003cem\u003eB. oleracea\u003c/em\u003e), comparable to that of established \u003cem\u003eB. napus\u003c/em\u003e. By contrast, unstable resynthesized rapeseed lines were characterized by high frequencies of multivalents: pairing between highly similar homoeologous chromosomes A1 and C1 occurred in half of all PMCs, suggesting most multivalent formation could be attributed to pairing between homoeologous chromosomes. Additional aberrations such as acentric chromosome fragments, anaphase I chromosome bridges and unequal chromosome segregation in the unstable resynthesized lines then occurred putatively as a consequence of these non-homologous multivalents, and lower fertility was also observed in our unstable resynthesized lines compared to our stable lines. All individual resynthesized lines could be qualitatively categorised into \u0026ldquo;stable\u0026rdquo; and \u0026ldquo;unstable\u0026rdquo; on the basis of diakinesis chromosome pairing behaviour. This qualitative (stable/unstable) rather than quantitative (more or less stable on a spectrum) classification suggests a single major gene effect might differentiate these two categories, potentially supporting previous studies suggesting that one locus might be predominantly responsible for the meiotic stability trait in \u003cem\u003eB. napus\u003c/em\u003e (Jenczewski et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Higgins et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, we also detected some minor but significant meiotic differences between genotypes within each of the stable and unstable groups (so between stable genotypes or between unstable genotypes), which may suggest a more complex genetic background to this trait, as also observed by Katche et al., (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e) based on frequencies of non-homologous recombination outcomes (CNVs), and as supported by previous QTL mapping results (Liu et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Higgins et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similar results were reported by Heneen et al., (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), who observed differences in bivalent and multivalent frequencies in four different resynthesized \u003cem\u003eB. napus\u003c/em\u003e lines depending upon the genetic background of the parental species, and by Katche et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e) who found differences in putative meiotic stability between early-generation synthetic lines with different combinations of shared \u003cem\u003eB. rapa\u003c/em\u003e and \u003cem\u003eB. oleracea\u003c/em\u003e parents. Our results provide an excellent basis to further investigate the genetic mechanisms underlying meiotic stability in allopolyploid \u003cem\u003eBrassica napus\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eWe observed low frequencies of meiotic abnormalities in established \u003cem\u003eB. napus\u003c/em\u003e as well as the stable resynthesized lines, but high frequencies of meiotic abnormalities in the unstable resynthesized lines (\u0026gt;\u0026thinsp;2 multivalents per PMC on average), and some minor genotype-specific differences within the \u0026ldquo;stable\u0026rdquo; and \u0026ldquo;unstable\u0026rdquo; groups. Stable resynthesized lines and established \u003cem\u003eB. napus\u003c/em\u003e Drakkar showed similar frequencies of bivalents (both 91%), univalents (2% and 1%) and multivalents (3% in both) at diakinesis. Similar meiotic chromosome pairing behaviour in established cultivars of \u003cem\u003eB. napus\u003c/em\u003e has been reported in other studies (Jenczewski et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Udall et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Sheidai et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Genomics studies have also frequently observed evidence for non-homologous recombination between the A and C subgenomes in \u003cem\u003eB. napus\u003c/em\u003e (Chalhoub et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Mason et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). We observed no significant difference between stable and unstable lines for univalent frequency in the first generation, but highly significant differences in multivalent frequency in both generations, suggesting that multivalent formation plays a major role in meiotic and genomic instability. Similar to our results, Xiong et al., (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) observed low frequencies of univalents in resynthesized rapeseed lines. These multivalents are most likely predominantly due to A-C chromosome associations (Hasterok et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Leflon et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Autosyndetic pairing (e.g., A01 \u0026ndash; A02) is possible, and has been observed at low frequencies in haploid \u003cem\u003eBrassica rapa\u003c/em\u003e (2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;x\u0026thinsp;=\u0026thinsp;10, A) and \u003cem\u003eB. oleracea\u003c/em\u003e (2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;x\u0026thinsp;=\u0026thinsp;9, C) (Armstrong \u0026amp; Keller, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1981\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1982\u003c/span\u003e) as well as in AABC, BBAC and CCAB hybrid types where at least one of the A and C genomes is haploid (Mason et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), but allosyndesis and intergenomic exchanges between A and C chromosome occur much more frequently in synthetic \u003cem\u003eBrassica\u003c/em\u003e hybrids (Yang et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Gaebelein et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Quezada-Martinez et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Meiotic behaviour is substantially more irregular in most of the resynthesized \u003cem\u003eB. napus\u003c/em\u003e produced to date than in established \u003cem\u003eB. napus\u003c/em\u003e, with some supporting cytogenetic observations of meiosis (Szadkowski et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and with genetic (Song et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Szadkowski et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Katche et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e; Katche et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e), genomic (Chalhoub et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Samans et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and cytogenetic (Xiong et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) evidence for resulting frequent non-homologous recombination between the A and C genomes. We assessed progression through meiotic stages such as diakinesis, metaphase, anaphase and/or telophase in order to determine if stage-specific failures to complete normal and regular meiosis and develop functional gametocytes were present. Our detailed comparative meiotic progression study between putatively stable (L16) and unstable (OLY21) resynthesized lines identified regular meiotic progression in the stable resynthesized line, similar to established cultivar \u0026ldquo;Drakkar\u0026rdquo;. By contrast, higher frequencies of multivalents at diakinesis and misalignment of bivalents at metaphase I were observed in the unstable resynthesized line, suggesting that homologous and homoeologous chromosomes were unable to sort, synapse and properly recombine in the early stages of prophase. Similarly, (Grandont et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) reported early and effective sorting of homologous and homoeologous chromosomes in early prophase in two different \u003cem\u003eB. napus\u003c/em\u003e accessions (although these accessions showed different frequencies of homoeologous A-C recombination as allohaploids). Subsequently, meiotic irregularities resulting from early meiosis carry forward through meiosis I and meiosis II.\u003c/p\u003e\u003cp\u003eIn the unstable line, multivalents which are unable to resolve putatively continue to anaphase I and/or telophase I in the form of anaphase bridges, where the chromosomes are unable to segregate equally to opposite poles, along with chromosome fragments and laggards which putatively result from univalents. These univalents/chromosome laggards carry forward to metaphase II and anaphase II and/or telophase II. By contrast, the stable line showed relatively normal progression through anaphase I/telophase I to anaphase II/telophase II without chromosomal abnormalities, indicating that the initial prophase I step is essential to maintain stable and regular meiosis, as has also been previously proposed (Grandont et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Studies in ACC hybrids, which also have frequent multivalent formation as well as high numbers of univalents from the haploid genome, also show similar meiotic issues:Yang et al., (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) reported partially tangled chromosomes at diakinesis, jumbled alignment at metaphase I with frequent chromosome bridges, unequal segregation and laggards in anaphase I and anaphase II, in contrast to parental lines diploid \u003cem\u003eB. oleracea\u003c/em\u003e and allotetraploid \u003cem\u003eB. napus\u003c/em\u003e, where the chromosomes were paired as bivalents, orderly aligned at the equatorial plate in metaphase and equally segregated. Meiotic configurations comprising univalents and especially multivalents could be due to mispairing and misalignment of homologous chromosomes as well as pairing between homoeologous chromosomes, leading to unbalanced gametes. We also observed lower total seed fertility in the putatively unstable resynthesized lines, which may result from these meiotic aberrations. Our results are similar toKatche et al., (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e) and Katche et al., (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e) who observed that resynthesized lines with lower CNVs showed higher seed set while resynthesized lines with higher CNVs exhibited poor seed fertility. While statistically similar frequencies of A1/C1 homoeologous associations were observed in stable resynthesized lines (18%) and established \u003cem\u003eB. napus\u003c/em\u003e (13%), nearly half (46%) of PMCs in the unstable resynthesized line showed A1/C1 associations. Homoeologous chromosomes A1 and C1 share collinearity and homology along the length of the chromosome, which leads to higher chances of homoeologous recombination than between other, more rearranged A/C genome homoeologous (Higgins et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Udall et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Analysis of these A1/C1 associations therefore allowed us to estimate homoeologous pairing and make general assumptions. High frequencies of homoeologous recombination between the A and C genomes have also been observed in other studies of resynthesized rapeseed (Perkin et al., 1995; Sharpe et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Udall et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), supporting our results.Xiong et al., (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported 49\u0026ndash;57% A1/C1 homoeologous pairing in resynthesized \u003cem\u003eB. napus\u003c/em\u003e in both the S1 and S11 generations, very similar to our current observations. We observed lower but still relatively high frequencies of A1/C1 homoeologous associations in the putatively stable resynthesized line and established cultivar. Frequent homoeologous translocations in established \u003cem\u003eB. napus\u003c/em\u003e were reported as early as 1995 (Parkin et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Sharpe et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), although at lower rates than in synthetic \u003cem\u003eB. napus\u003c/em\u003e, suggesting that \u003cem\u003eB. napus\u003c/em\u003e, as a relatively young allopolyploid, is not fully able to prevent homoeologous pairing between the A and C genomes (Jenczewski \u0026amp; Alix, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Sourdille \u0026amp; Jenczewski, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Possibly, the presence of pre-existing homoeologous recombination events in either Drakkar or in our putatively stable resynthesized line may also have contributed to this observation. Similar to this,(Udall et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) observed pre-existing homoeologous translocations between A1 and C1 based on marker studies in a DH mapping population, and pre-existing A-C translocations are also known to cause multivalent formation in \u003cem\u003eBrassica\u003c/em\u003e when present in translocation heterozygotes (Osborn et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Mwathi et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, the use of chromosome oligo painting for the entire length of chromosomes A1 and C1 in the present study probably excludes the pre-existence of large-scale (\u0026gt;\u0026thinsp;1 Mb) translocations, as these would have been visible cytogenetically; such translocations were also not visible from SNP array data (Katche et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). Overall, our results indicate that the putatively stable resynthesized line preferences homologous recombination over homoeologous recombination similarly to established \u003cem\u003eB. napus\u003c/em\u003e. Although we only assessed one line of each of the stable, unstable and established types for A1/C1 homoeologous recombination, such that we cannot completely rule out genotype-specific factors, it is highly likely that this finding of homoeologous pairing reduction could be a general event at least to the set of stable synthetic lines we investigated in the present study. Previous studies have also suggested that A/C pairing occurs between all homoeologous chromosome regions genome-wide in direct relation to degree of homoeology in a mostly genotype-independent fashion (Nicolas et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Mason et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), supporting a general mechanism acting to prevent homoeologous recombination in established but not novel resynthesized lines.\u003c/p\u003e\u003cp\u003eWe observed major differences between the stable and unstable lines as well as minor but statistically significant differences within each of the groups of putatively stable and unstable resynthesized lines for chromosome pairing behaviour. This indicates individual genotype-specific differences between resynthesized lines, which could be the result of differences in specific genotypic factors inherited from the parental species used for development of these resynthesized lines (Heneen et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Szadkowski et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Some differences may however also be due to aneuploidy in specific plants, particularly in the unstable lines: lack of a homologous chromosome pairing partner or a third homologous copy of a chromosome will lead to increased univalent and multivalent frequencies, but this was difficult to confirm in this material due to the sometimes highly complex meiotic configurations observed in the unstable lines. Our results confirm that the genetic background of the parental species plays a crucial and essential role in meiotic stability. Our observation is a further validation of (Katche et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e) where genome stability was genotype dependent and inherited genetic factors from both the diploid parental species affected accumulation of copy number variants.\u003c/p\u003e\u003cp\u003eAll our stable lines were later generation lines and our unstable lines were all early generation, such that further investigation of generational effects on stability may also be warranted. It is unclear from previous studies of resynthesized and synthetic \u003cem\u003eBrassica\u003c/em\u003e if meiotic stability can improve generationally when starting from homozygous lines, where no segregation for allelic variation is possible. In studies where later-generation, putatively more homozygous lines show better meiotic stability than earlier generation lines (Tian et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2010\u003c/span\u003e in \u003cem\u003eB. rapa\u003c/em\u003e by \u003cem\u003eB. carinata\u003c/em\u003e allohexaploids, as well as; Katche et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e), selection putatively occurs in the early generations, such that unstable lines fail to produce seeds and are not able to be propagated to later generations, while lines which are initially more stable produce more seeds and survive to later generations. Xiong et al., (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) also observed no significant difference in non-homologous chromosome pairing frequency over 11 generations in homozygous resynthesized \u003cem\u003eB. napus\u003c/em\u003e (20% in the S1 and 16% in the S11 generation). Nevertheless, substantial changes are also possible in synthetic hybrids as a result of \u0026ldquo;genome shock\u0026rdquo; resulting from the hybridization event, such as transcriptional regulation changes (Gaeta et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), chromosome rearrangements (Szadkowski et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), activation of transposable elements (Zou et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and proteomic changes (Albertin et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), all of which may create novel genetic variation which may be acted on by selection from early to late generation lines and provide a mechanism for stabilisation of meiosis. Further investigation would be necessary to confirm this hypothesis.\u003c/p\u003e\u003cp\u003eWe observed no major differences between the two generations we grew in our study, but we did find that in the unstable lines, univalent frequency in the second experimental year was significantly higher than in the first experimental year generation. Szadkowski et al., (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) also observed high frequencies of univalents in S1 resynthesized \u003cem\u003eB. napus\u003c/em\u003e, where this was attributed to aneuploidy (absence of a homologous pairing partner is expected to result in a univalent or trivalent) although Xiong et al., (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) did not observe many univalents in their synthetic \u003cem\u003eB. napus\u003c/em\u003e lines. Mwathi et al., (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) also found that meiotically unstable homozygous \u003cem\u003eBrassica\u003c/em\u003e allohexaploids showed increased frequencies of chromosome rearrangements and reduced fertility from one generation to the next, and hypothesised that initial chromosome rearrangements also resulted in more rearrangements. Similar effects may be responsible for the increased meiotic instability observed from one generation to the next in our unstable resynthesized lines.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn our study putatively stable resynthesized lines appeared to show higher seed fertility, normal and regular meiosis with low frequencies of aberrations in the form of univalents, multivalents, acentric chromosome fragments, anaphase bridges and laggards. Although these events were still observed at low frequencies, these frequencies were similar to those observed in established \u003cem\u003eB. napus\u003c/em\u003e cultivar \u0026ldquo;Drakkar\u0026rdquo;, suggesting that a low level of meiotic abnormalities might be normal for \u003cem\u003eB. napus\u003c/em\u003e, in line with previous studies (Sheidai et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Lines could be grouped into \u0026ldquo;putatively stable\u0026rdquo; and \u0026ldquo;putatively unstable\u0026rdquo; based on large differences in bivalent and multivalent frequency, possibly supporting a major gene effect, with minor differences also between lines. Our results suggest that whether or not a resynthesized line is meiotically stable is highly genotype dependent. We also confirmed that non-homologous pairing is common in both \u0026ldquo;stable\u0026rdquo; resynthesized lines and established \u003cem\u003eB. napus\u003c/em\u003e, but is observed at much higher frequencies in unstable resynthesized lines, where it also putatively causes additional meiotic abnormalities as meiosis progresses. Our study conclusively demonstrates meiotic stability in resynthesized \u003cem\u003eB. napus\u003c/em\u003e for the first time, and provides a useful basis to further investigate genetic factors related to this stability mechanism.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eVR carried out the experiments, generated material for the second-year experimental generation with assistance and input from ZL and MB, analysed the data, prepared figures and tables and drafted the manuscript. EIK generated the material for the first-year experimental generation and data necessary for this project, provided input into project planning and critically revised the manuscript. MB developed and tested the oligo painting A1/C1 chromosome-specific probes and critically revised the manuscript. ASM conceptualised the project, acquired funding, supervised VR and EIK and assisted with writing and critical revisions of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eVR was supported by a PhD scholarship funded by the Indian Council of Agricultural Research (Netaji Subhas-ICAR International Fellowship), New Delhi, India. Project funding from the German Research Council (DFG, MA6473/2\u0026thinsp;\u0026minus;\u0026thinsp;1) is gratefully acknowledged.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll data generated by this project is available in the manuscript and supplementary material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlbertin, W., Balliau, T., Brabant, P., Ch\u0026egrave;vre, A. M., Eber, F., Malosse, C., \u0026amp; Thiellement, H. (2006). 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Homoeologous shuffling and chromosome compensation maintain genome balance in resynthesized allopolyploid \u003cem\u003eBrassica napus\u003c/em\u003e. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e, \u003cem\u003e108\u003c/em\u003e(19), 7908\u0026ndash;7913. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1014138108\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1014138108\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang, Y., Wei, X., Shi, G., Wei, F., Braynen, J., Zhang, J., Tian, B., Cao, G., \u0026amp; Zhang, X. (2017). Molecular and cytological analyses of A and C genomes at meiosis in synthetic allotriploid Brassica hybrids (ACC) between \u003cem\u003eB. napus\u003c/em\u003e (AACC) and \u003cem\u003eB. oleracea\u003c/em\u003e (CC). \u003cem\u003eJournal of Plant Biology\u003c/em\u003e, \u003cem\u003e60\u003c/em\u003e(2), 181\u0026ndash;188. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12374-016-0221-2\u003c/span\u003e\u003cspan address=\"10.1007/s12374-016-0221-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZou, J., Fu, D., Gong, H., Qian, W., Xia, W., Pires, J. C., Li, R., Long, Y., Mason, A. S., Yang, T. J., Lim, Y. P., Park, B. S., \u0026amp; Meng, J. (2011). De novo genetic variation associated with retrotransposon activation, genomic rearrangements and trait variation in a recombinant inbred line population of \u003cem\u003eBrassica napus\u003c/em\u003e derived from interspecific hybridization with \u003cem\u003eBrassica rapa\u003c/em\u003e. \u003cem\u003eThe Plant Journal\u003c/em\u003e, \u003cem\u003e68\u003c/em\u003e(2), 212\u0026ndash;224. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-313X.2011.04679.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-313X.2011.04679.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chromosome-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"chrs","sideBox":"Learn more about [Chromosome Research](http://link.springer.com/journal/10577)","snPcode":"10577","submissionUrl":"https://submission.nature.com/new-submission/10577/3","title":"Chromosome Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"meiotic stability, cytogenetics, chromosome pairing behaviour, rapeseed, oligo-FISH","lastPublishedDoi":"10.21203/rs.3.rs-7700382/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7700382/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eBrassica napus\u003c/em\u003e (rapeseed/canola) is an allotetraploid (AACC, 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4\u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;38) resulting from spontaneous hybridization between \u003cem\u003eB. rapa\u003c/em\u003e (AA, 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2\u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20) and \u003cem\u003eB. oleracea\u003c/em\u003e (CC, 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2\u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;18). Although established \u003cem\u003eB. napus\u003c/em\u003e is meiotically stable, resynthesized lines (2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;AACC) produced by hybridizing between progenitor species \u003cem\u003eB. rapa\u003c/em\u003e and \u003cem\u003eB. oleracea\u003c/em\u003e are usually meiotically unstable, and show frequent chromosomal rearrangements caused by homoeologous recombination between the A and C genomes. Previously, we identified resynthesized rapeseed lines showing contrasting levels of homoeologous recombination, as assessed by genotyping for copy number variants. Here, we aimed to characterise meiotic chromosome pairing behaviour in fifteen resynthesized lines representing putatively stable, unstable and intermediate types. Putatively stable lines showed predominantly normal meiosis (average 91% bivalent formation), while putatively unstable lines showed frequent abnormalities such as multivalent formation (average 60% bivalent formation). Univalents were unexpectedly rare in Metaphase I. Surprisingly, all intermediate resynthesized lines showed either stable or unstable-type meiotic behaviour. A1-C1 specific probes revealed that stable lines showed approximately 18% A-C pairing (7/40 pollen mother cells), not significantly different to the 13% A-C pairing (5/40 pollen mother cells) in established \u003cem\u003eB. napus\u003c/em\u003e, but in contrast to the unstable line with 46% A-C pairing (25/54 pollen mother cells). Our results suggest that differences in multivalent formation frequencies and homoeologous A-C pairing differentiate stable and unstable lines, confirm the production of meiotically stable synthetic \u003cem\u003eB. napus\u003c/em\u003e, and provide a basis for further investigation of genetic factors contributing to this effect.\u003c/p\u003e","manuscriptTitle":"Stable resynthesized Brassica napus lines show similar meiotic behaviour to established B. napus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-16 17:26:03","doi":"10.21203/rs.3.rs-7700382/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-09T13:44:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-24T16:35:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"327522989863007343844463087458279367627","date":"2025-10-07T17:09:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-05T17:04:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-30T16:02:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-25T06:01:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chromosome Research","date":"2025-09-24T06:58:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chromosome-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"chrs","sideBox":"Learn more about [Chromosome Research](http://link.springer.com/journal/10577)","snPcode":"10577","submissionUrl":"https://submission.nature.com/new-submission/10577/3","title":"Chromosome Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4f2a090d-372c-4ef5-a3a2-c5cb6a0e4776","owner":[],"postedDate":"October 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T16:03:36+00:00","versionOfRecord":{"articleIdentity":"rs-7700382","link":"https://doi.org/10.1007/s10577-026-09799-1","journal":{"identity":"chromosome-research","isVorOnly":false,"title":"Chromosome Research"},"publishedOn":"2026-04-07 15:58:16","publishedOnDateReadable":"April 7th, 2026"},"versionCreatedAt":"2025-10-16 17:26:03","video":"","vorDoi":"10.1007/s10577-026-09799-1","vorDoiUrl":"https://doi.org/10.1007/s10577-026-09799-1","workflowStages":[]},"version":"v1","identity":"rs-7700382","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7700382","identity":"rs-7700382","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}