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Keywords:

  • Brassica napus (oilseed rape);
  • homoeologous recombination;
  • meiosis;
  • polyploid formation pathways;
  • synthetic hybrids

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Polyploids can be produced by the union of unreduced gametes or through somatic doubling of F1 interspecific hybrids. The first route is suspected to produce allopolyploid species under natural conditions, whereas experimental data have only been thoroughly gathered for the latter.
  • We analyzed the meiotic behavior of an F1 interspecific hybrid (by crossing Brassica oleracea and B. rapa, progenitors of B. napus) and the extent to which recombined homoeologous chromosomes were transmitted to its progeny. These results were then compared with results obtained for a plant generated by somatic doubling of this F1 hybrid (CD.S0) and an amphidiploid (UG.S0) formed via a pathway involving unreduced gametes; we studied the impact of this method of polyploid formation on subsequent generations.
  • This study revealed that meiosis of the F1 interspecific hybrid generated more gametes with recombined chromosomes than did meiosis of the plant produced by somatic doubling, although the size of these translocations was smaller. In the progeny of the UG.S0 plant, there was an unexpected increase in the frequency at which the C1 chromosome was replaced by the A1 chromosome.
  • We conclude that polyploid formation pathways differ in their genetic outcome. Our study opens up perspectives for the understanding of polyploid origins.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Polyploidy is a widespread phenomenon in angiosperms. Allopolyploids, which carry merged genomes from different species, play an emphasized role in genome evolution (Ramsey & Schemske, 1998; Wendel, 2000). Although some de novo-synthesized or newly formed natural allopolyploid species show structural stability (Liu et al., 2001; Baumel et al., 2002; Mestiri et al., 2010), genetic rearrangements in most polyploids occur rapidly (Pires et al., 2004; Pontes et al., 2004; Soltis et al., 2004; Hegarty et al., 2006; Tate et al., 2009; Petit et al., 2010) via prevalent mechanisms, such as crossovers (COs), between homoeologous chromosomes at meiosis (Chen & Ni, 2006; Leitch & Leitch, 2008; Gaeta & Pires, 2010; Szadkowski et al., 2010).

Allopolyploids differ from homoploids in that they possess two copies of each parental genome. This situation cannot be obtained directly by hybridization of two haploid gametes from different species. One mechanism that is known to induce polyploidy in plants is somatic doubling in mitosis (Ramsey & Schemske, 1998; Comai, 2005). Somatic doubling can occur either spontaneously, as in Primula kewensis, or be induced at a high frequency by artificial means (Ramsey & Schemske, 1998 and references therein). A second mechanism by which polyploids may evolve from diploid plants involves the production of unreduced gametes during meiosis (‘unreduced gamete pathway’), that is, gametes with the same number of chromosomes as the parental plant (Bretagnolle & Thompson, 1995; Ramsey & Schemske, 1998). This pathway is now considered to be the main source of polyploids in the wild. One major type of unreduced gamete is the ‘first division restitution (FDR) gamete’, in which the somatic number of chromosomes is conserved (Bretagnolle & Thompson, 1995). These unreduced FDR gametes can transmit the products of meiotic recombination between homologous chromosomes in diploids (d’Erfurth et al., 2008) and, interestingly, the products of nonhomologous chromosome exchange in haploids of allopolyploid plants (Nicolas et al., 2007). It is thus surprising that, despite the prevalent role of unreduced gametes in allopolyploidy and their potential effect on genome structure, all recent experiments studying early polyploidy generations have focused only on resynthesized plants generated by mitotic genome doubling (Song et al., 1995; Comai et al., 2000; Pires et al., 2004; Wen et al., 2008; Chaudhary et al., 2009; Petit et al., 2010). As a consequence, we still do not know whether the somatic vs unreduced gamete pathways have a different and/or specific impact on the genetic modifications that occur at the onset of polyploid formation.

Rapeseed (Brassica napus, AACC genomes, 2n = 38) is a model allopolyploid plant for the study of meiotic-driven genetic changes (see Pires et al., 2004; Gaeta et al., 2007; reviewed in Gaeta & Pires, 2010; Nicolas et al., 2007, 2009; Szadkowski et al., 2010). Its genomes are highly related to those of Brassica rapa (AA genome, 2n = 20) and Brassica oleracea (CC genome, 2n = 18) (U, 1935). F1 hybrids can be obtained by embryo rescue (Wen et al., 2008) and produce resynthesized allopolyploids from somatic doubling. In these resynthesized B. napus, frequent aberrant meiotic configurations, such as multivalents (Attia & Robbelen, 1986b), are found and numerous genetic changes are detected after a few generations even if each chromosome has a homolog (Parkin & Lydiate, 1997; Udall et al., 2005; Gaeta et al., 2007). These changes are commonly described as translocations or rearrangements even though most are the products of meiotic COs (see Gaeta & Pires, 2010). In a recent study, we showed that the very first meiosis of somatically doubled resynthesized B. napus already acts as a genome blender, with many of the genetic exchanges between A and C chromosomes transmitted to progenies (Szadkowski et al., 2010). However, Brassica species are also able to generate viable unreduced gametes (Olsson & Hagberg, 1955; Heyn, 1977), which may therefore suggest that this pathway is involved in natural B. napus formation. In haploids of natural B. napus that share the same AC structure as F1 hybrids, almost all viable gametes are unreduced, and it has been shown that this haploid structure promotes nonhomologous recombination between chromosomes A and C (Nicolas et al., 2007). The consequences of meiosis in newly formed interspecific F1 hybrids have not yet been studied, but, compared with meiosis in somatically doubled F1 hybrids, may have a major effect on the genetic rearrangements that occur. We therefore aimed to unravel the impact of polyploid formation pathways on genetic rearrangements during the formation of new B. napus.

We first used a genome-wide cytogenetic approach to analyze meiosis in an F1 hybrid. Genetic analysis of the homoeologous chromosome pair A1/C1 in the progeny of the F1 hybrid then allowed comparison with previously published data on somatically doubled resynthesized S0 plants which share the same genetic background (Szadkowski et al., 2010). Lastly, to evaluate the potential of unreduced gametes to create an allopolyploid species, a resynthesized S0 plant, generated by crossing the F1 hybrid with the somatically doubled S0 plant, was also studied using the same approaches.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Production of synthetic hybrids, amphidiploids and crosses

Interspecific hybridization between the doubled haploid lines HDEM (B. oleracea var. botrytis italica, maternal parent) and Z1 (B. rapa var. trilocularis provided by Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, Canada) generated a CxA plant called F1-EMZ (A. M. Chèvre et al., unpublished; see Fig. 1). A progeny of 126 plants was obtained by crossing F1-EMZ as the maternal parent to a B. napus cv Darmor (an oilseed rape winter type line) as the male parent, and was called F1 × Bn.

image

Figure 1. Synthetic Brassica napus plant material produced from an F1 hybrid through somatic doubling or the union of unreduced gametes and the progeny of crosses to a natural B. napus variety. Arrows indicate gamete formation from reduced gametes (continuous lined arrow) to unreduced gametes (double lined arrow). The dotted line represents the somatic doubling process (colchicine).

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As a control, we used a second population that shares the same genetic background. Briefly, the F1-EMZ plant was first colchicine-doubled (as described in Chèvre et al., 1989) and the corresponding S0 synthetic line (CD.S0) was crossed, as female, to B. napus cv Darmor to produce a progeny called CD.S0 × Bn in this article. Eighty-two plants from this population were analyzed previously in Szadkowski et al. (2010); for the current work, we extended the number of individuals analyzed in the CD.S0 × Bn population to 136 and completed the data by adding three new markers that revealed a total of five loci (nia-m032, nia-m086, nia-m096). According to Szadkowski et al. (2010), we excluded plants with marker losses from a complete chromosome (where rearrangements cannot be scored properly), and reduced the population to 130 plants. An additional population (125 plants) was generated by crossing the CD.S0 plant as the paternal parent and the B. napus cv Darmor as maternal parent; the progeny was named Bn × CD.S0.

The CD.S0 plant was used as pollen donor in a cross with F1-EMZ to produce a plant from female unreduced gametes called UG.S0. A population of 108 plants was obtained by crossing UG.S0 as the maternal parent to the same B. napus cv Darmor as the male parent (called UG.S0 × Bn).

Fluorescence in situ hybridization (FISH)

Floral buds were fixed in Carnoy’s solution (ethanol : chloroform : acetic acid, 6 : 3 : 1) for 24 h and stored in 50% ethanol. Genomic In Situ Hybridization (GISH)-like analyses were carried out on meiotic chromosomes for the B. napus synthetic F1 and the UG.S0 amphidiploid at metaphase stage I in pollen mother cells (PMCs). We used a repeated sequence within a Bacterial Artificial Chromosome (BAC) (BoB014O06 from B. oleracea) as a probe that identifies all C chromosomes (Alix et al., 2008) and allows the visualization of the C genome in B. napus. Floral bud fixation, slide preparation and hybridization were performed as described in previous publications (Leflon et al., 2006; Nicolas et al., 2007, 2009; Szadkowski et al., 2010).

Chromosomal counts in progeny of the F1 hybrid crossed with natural B. napus

Flow cytometry was performed on 126 plants from the F1 × Bn population using leaf tissue to assess chromosome number with an accuracy of plus or minus two chromosomes according to Leflon et al. (2006).

Fertility assessment

Male fertility was assessed as the percentage of pollen stained with a 1% aceto-carmine solution. Two flowers and at least 600 pollen grains were analyzed per plant. Seed set was assessed by counting the number of seeds per pollinated flowers fertilized by selfing or crossing. These tests were performed by hand pollination in the glasshouse or open pollination in the field under cages to avoid cross-contamination.

DNA extraction and amplification

DNA was extracted as described by Lombard & Delourme (2001). A1 and C1 linkage groups were built as described in Szadkowski et al. (2010). For structural analysis in populations F1 × Bn, CD.S0 × Bn, Bn × CD.S0 and UG.S0 × Bn, the two linkage groups A1 and C1 are shown in Fig. 2. For structural analysis of F1-EMZ hybrids, CD.S0 and UG.S0 plants, the markers used are shown in Supporting Information Fig. S1. All microsatellite markers are publicly available and originated from the Biotechnology and Biological Sciences Research Council, Swindon, Wiltshire, UK (prefixed with Na, Ol or Ra; Lowe et al., 2004), the CELERA Company, Alameda, CA, USA (prefixed with CB, BRAS or MR; Piquemal et al., 2005), Kim et al. (2009) (prefixed with nia-m) or Agriculture and Agri-Food Canada (prefixed with sN). Markers prefixed with the letters CZ were developed from Arabidopsis and Brassica coding sequences (H. Belcram & B. Chalhoub, unpublished data; primer sequences are available upon request to Genoplante). PCR assays were conducted essentially as described in Delourme et al. (2006). Forward primers were tailed with M13 extensions for revelation with fluorescent technology (Schuelke, 2000). PCR products were analyzed on a 16-capillary ABIPrism 3130xl, as described by Esselink et al. (2004). For a subset of 13 loci, the molecular assay was repeated twice.

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Figure 2. The frequency of marker losses (%) along chromosomes following a first round of meiosis in the resynthesized F1 hybrid of Brassica napus (F1-EMZ, black line) compared with the first meiosis of colchicine-doubled resynthesized B. napus (CD.S0, dotted line). (a) The A1 linkage group and marker loss frequencies. (b) The C1 linkage group and marker loss frequencies. A star next to the marker name indicates a significant difference in marker loss frequency between populations (< 0.05, χ2 test). Centromeres (indicated by the black area on the linkage groups) are positioned according to Pouilly et al. (2008).

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Mapping translocation events on the A1 and C1 chromosomes in the progeny of synthetic plants crossed with natural B. napus

Most primer pairs amplified more than one locus, with individual loci located in homoeologous regions on A1 and C1. We scored the frequency of rearrangements per marker in gametes from the synthetic B. napus by surveying the frequency at which parental bands disappeared (i.e. allele at A1 loci from B. rapa; allele at C1 loci from B. oleracea), given that they are polymorphic with allelic bands from the natural B. napus cv Darmor. For each plant, we scored the number of rearrangements on both the A1 and C1 chromosomes, considering a rearrangement as each contiguous allele loss (one or more allele loss). To compare rearrangement size within the F1 × Bn population (i.e. between chromosomes A1 and C1), we scored intervals of allele loss using three adjacent primer pairs that amplify both homoeologous loci (on the upper arm of A1 and C1; CB10081, nia-m096 and nia-m086), which therefore vary between zero and three adjacent allele losses. To compare rearrangement size between the F1 × Bn and CD.S0 × Bn populations on A1 and C1, we used all markers on both linkage groups and compared the number of contiguous allele losses on A1 or C1 between populations. Comparisons were performed on one allele deletion class vs two or more allele deletion classes using chi-squared tests or two-tailed Fisher exact tests (R software version 2.31, R Foundation for Statistical Computing, Vienna, Austria).

Determination of allele copy number

Fluorescent PCR analysis was used to determine the allele copy number of chromosomes A1 and C1 from the synthetic parents, F1-EMZ, CD.S0 and UG.S0 in the crosses F1 × Bn, CD.S0 × Bn, Bn × CD.S0 and UG.S0 × Bn, respectively. Data collection on markers CB10081, nia- m086 and nia-m096, as well as statistical analyses, were performed as described in Nicolas et al. (2007). Briefly, for each progeny of these crosses, the peak area of the A1- or C1-amplified PCR fragments from the synthetic plant, and the peak area for alleles from B. napus (which was used as an internal PCR control with one copy of A1 and C1 loci), were acquired after electrophoresis using Genemapper software 3.7 (Applied Biosystems, Foster City, CA, USA). For these markers, the ratio of the peak area detected for A1 loci (or C1 loci) of the synthetic plant to the peak area of B. napus cv Darmor should be constant (regardless of the initial amount of DNA from the progeny) and close to unity. The duplication of one allele from the synthetic parent in a progeny will result in a duplication of the ratio between peak areas. Thus, allele loss, single-copy alleles (one A1 or one C1) or double-copy alleles (two copies of A1 or two copies of C1) can be detected in each individual from the three populations. As both the A1 and C1 alleles inherited from the synthetic parent could be distinguished using these three markers, the concurrent loss of an allele and duplication of the homoeologous allele in the gamete from the synthetic plant could be assessed. The robustness of the results was tested using the same statistical analysis and criteria as described in Nicolas et al. (2007).

We modified the protocol slightly from Nicolas et al. (2007) for allele dosage to determine the relative homoeologous allele copy number in the F1-EMZ hybrid, CD.S0 and UG.S0 plants. As an internal PCR quantity control was not available, we tested the ratio between A and C alleles using nine primer pairs which specifically amplify both homoeologous loci in this genetic background (Fig. S1), making it possible to distinguish unfixed rearrangements with one homoeologous allele remaining and three copies of the other allele (‘1A-3C’ or ‘3A-1C’) from the parental situation ‘2A-2C’.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Frequent homoeologous chromosomal associations at meiosis of a newly formed B. napus interspecific F1 hybrid

We first used a cytogenetic approach to evaluate the extent of bivalent formation in the F1 hybrid plant (called F1-EMZ) at metaphase I. We thus assessed the extent to which meiosis in this hybrid plant generates exchanges between the 10 A chromosomes and nine C chromosomes, using a BAC probe which gives a ‘GISH-like’ signal labeling all the C chromosomes (Fig. 3a). This allowed autosyndetic pairs of chromosomes (A–A or C–C pairs) to be distinguished from allosyndetic pairs (A–C pairs). Frequent chromosome associations were observed (in 34 PMCs) even if univalents were found in all cells (11.1% of chromosomes per PMC). On average, 88.9% of chromosomes per PMC formed chiasmatic associations (bivalents or multivalents) and, among these, bivalents were prevalent. In all PMCs, we observed 83.9% of chromosomes in bivalents per PMC, whereas 5.0% of chromosomes were involved in multivalent associations (detected in 23.5% of PMCs). All cells observed showed A–C chromosomal associations (allosyndesis), involving 77.1% of the 19 chromosomes per cell. However, autosyndesis was present in 76.5% of cells and the frequency of autosyndesis was similar within the A and C genomes (involving 14.4% of all A chromosomes and 13.1% of all C chromosomes; > 0.05, χ2 test).

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Figure 3. Meiosis in resynthesized Brassica napus AC and AACC hybrids. Fluorescence in situ hybridization (FISH) analysis of metaphase I pollen mother cells using the bacterial artificial chromosome (BAC) Bob 014O06 that identifies all C chromosomes: the A genome is blue (left and right, stained in 4′,6-diamidino-2-phenylindole), and the C genome is red (right, stained with avidin–Texas red antibody). (a) F1-EMZ plant with 19 chromosomes: nine AC bivalents and one A univalent indicated by (x); (b) UG.S0 × Bn with 19 bivalents, two formed between A and C chromosomes (indicated by an arrow); suspected translocations of C regions on A chromosomes are indicated by a star.

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The large number of A–C and other nonhomologous chiasmatic associations showed that a high level of meiotic recombination occurred at meiosis in the F1 hybrid. Meiotic COs result in reciprocal exchanges of DNA between nonhomologous chromosomes (commonly named rearrangements) that could be transmitted to the progeny, assuming that the F1 hybrid is fertile and produces viable plants.

Reduced fertility in a newly formed B. napus interspecific F1 hybrid

The F1-EMZ hybrid produced anthers with a small amount of viable pollen (0.8% of viable pollen). Crosses were then performed to evaluate the fertility of this hybrid. Very few seeds were obtained from F1-EMZ by selfing (0.4 seeds/100 pollinated flowers) and these were nonviable. A larger seed set was obtained in crosses with natural B. napus (cv Darmor) as the pollen donor, with 24 seeds per 100 hand-pollinated flowers and 21 seeds per 100 flowers in open pollination. We used this progeny (called F1 × Bn) to determine whether meiotic intergenomic exchanges are transmitted to the progeny, focusing on A1 and C1 homoeologous linkage groups. These two linkage groups were chosen because they showed the highest amount of transmitted homoeologous exchanges in previous studies (Gaeta et al., 2007).

Female gametes from the F1 synthetic hybrid transmit homoeologous exchanges generated during meiosis in the newly formed B. napus interspecific F1 hybrid

We first used flow cytometry to ensure that plants of the F1 × Bn progeny, where rearrangements were to be evaluated, had 38 chromosomes (Nicolas et al., 2007). The chromosome number of 126 plantlets was assessed and 74 of these were estimated to have 38 chromosomes (Fig. S2). Of these 74 plants, 66 grew to the second leaf stage and were used for further genetic analysis.

We then analyzed this population with 10 and nine molecular markers spanning A1 and C1, respectively. As detailed in Nicolas et al. (2007) and Szadkowski et al. (2010), to detect the presence of ‘recombined chromosomes’, we looked for plants lacking alleles from the F1 hybrid parent (as a result of disjunction of reciprocal meiotic exchanges). Simultaneous analysis of three pairs of homeologous loci was used to assess the occurrence of exchanges between homoeologous chromosomes. In this approach, the B. napus male parent acts as the ‘genetic tester’ because it shows different alleles from the F1 hybrid.

Marker losses on A1 and/or C1 were detected in 81.8% of the F1 × Bn population. No case of marker loss from a complete chromosome was found in the 66 plants of this population. In addition, when we focused on the three pairs of homoeologous alleles, no concurrent marker loss was found for both A1 and C1 homoeologous alleles. Concurrent loss and duplication of homoeologous loci represented 92.6% of marker loss events (25 of 27 independent cases of deletions analyzed), and no cases of duplication without deletion were found. As this pattern is diagnostic of homoeologous exchanges (see Nicolas et al., 2007), this result indicates that, during meiosis of the F1 hybrid, meiotic COs mainly formed between chromosomes A1 and C1.

COs generate reciprocal exchanges, and so an equal number of marker losses were expected to affect A1 and C1 in the F1 × Bn progeny. Overall, this expectation held true and no statistically significant (> 0.05, χ2 test) distortion between individuals showing genetic changes on A1 and C1 was found: 52% of plants carried marker loss/duplication on A1, whereas 42% carried marker loss/duplication on C1 rearrangements. In addition, 90.9% of A1 marker losses were associated with duplication of C1 alleles, whereas, in the reciprocal case, C1 marker loss and concurrent A1 duplication accounted for 93.8% of cases (> 0.05, χ2 test). This indicated that no or only weak selective constraints biased the transmission of CO products affecting A1 and C1 in the F1 × Bn progeny.

When we examined the distribution of CO products along linkage groups, the frequency of marker losses was homogeneous along A1 but not C1 (from 12.1% to 20.0% on A1; from 0.0% to 21.2% on C1; Fig. 2). The variation along C1 was notably a result of the fact that Na12C08, which maps close to the centromere according to Pouilly et al. (2008), was never lost in the F1 × Bn population. Because a centromeric marker was not available for A1, we do not know whether a similar decrease in CO number at the centromere occurred for this linkage group (Fig. 2). No significant differences (> 0.05, χ2 test) were observed in either the size of the exchanged regions (see Materials and Methods) or the breakpoint position along A1 and C1. Similarly, multiple breakpoints per chromosome were not statistically different between A1 and C1. These accounted for 38.2% and 50.0% of chromosomes with breakpoints, respectively (observed on 34 A1 chromosomes and 28 C1 chromosomes found with breakpoints; > 0.05, χ2 test).

Altogether, these results confirmed that the F1 hybrid can produce viable gametes carrying a large number of genetic changes that mostly result from homoeologous exchanges. We thus used the same methodology to examine whether these rearrangements were more numerous or different from those previously detected in the progeny of resynthesized B. napus produced by somatic colchicine doubling (Szadkowski et al., 2010).

Comparison of transmitted gametes between F1 hybrid and somatically doubled CD.S0 plants sharing a common genetic background

We analyzed a second population of 130 plants produced from the same B. rapa, B. oleracea and B. napus parental genotypes, but via the ‘somatic doubling pathway’ (a colchicine-doubled ‘EMZ’ synthetic line crossed to B. napus cv Darmor called CD.S0 × Bn).

Marker loss in the F1 × Bn population was significantly higher than in the CD.S0 × Bn population, in which only 54.6% of plants lacked alleles from the CD.S0 parent (compared with 81.8% in F1 × Bn; < 0.001, χ2 test). By contrast, a smaller proportion of plants with losses on both A1 and C1 was found in the F1 × Bn relative to the CD.S0 × Bn population (eight of 54 ‘rearranged’ F1 × Bn plants vs 25 of 71 ‘rearranged’ CD.S0 × Bn plants; < 0.05, χ2 test). Overall, this indicated that more plants in the progeny of the F1 hybrid showed marker loss/duplication relative to progeny of the CD.S0 synthetic line. By contrast, proportionally more recombined chromosomes (22.2% vs 43.6% of plants with rearrangements carried multiple independent rearrangements on A1 and C1 in F1 × Bn and CD.S0 × Bn, respectively; < 0.05, χ2 test) were observed in the progeny of the CD.S0 synthetic line.

As shown in Fig. 4, the genetic changes found in the F1 × Bn progeny spanned smaller regions on average than those detected in the CD.S0 × Bn population. This was especially true for the upper arm region of C1. Overall, 12.5% and 48.3% of rearrangements on C1 encompassed four or more markers in the F1 × Bn and CD.S0 × Bn populations, respectively (< 0.001, two-tailed Fisher exact test). In both populations, these marker losses were mainly a result of homoeologous exchanges, with 93.8% and 79% of concurrent deletions and duplications of homoeologous loci for F1 × Bn and CD.S0 × Bn, respectively (> 0.05, two-tailed Fisher exact test). Overall, similar occurrences of multiple breakpoints were observed for A1 and C1 in both populations (> 0.05, χ2 test).

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Figure 4. The most frequent rearrangement types observed following a first round of meiosis in different resynthesized Brassica napus. For rearranged A1 chromosomes (left) or C1 chromosomes (right) identified in the F1 × Bn or CD.S0 × Bn population, the respective frequency (%) is represented for each recombined chromatid type that was found two or more times in the population.

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Genetic structure of a successfully formed amphidiploid from unreduced female gametes and its progeny

The results described above confirmed that F1 hybrid gametes carry products of meiotic COs that are different from those generated during meiosis of somatically doubled plants. To test how this variation may speed up the ‘polyploid ratchet’ proposed by Gaeta & Pires (2010), which predicts that ‘translocation’ heterozygotes progressively accumulate and are prone to increased genomic instability, we characterized one allotetraploid plant (2n = 38), called ‘UG.S0’, produced by crossing F1–EMZ as female and CD.S0 as male (Fig. 1). We then assessed the transmission frequency of genetic changes in this plant (Fig. S3).

The meiotic stability of UG.S0 was first assessed using bivalent formation as the criterion of stability and GISH-like analysis at metaphase I with the C-genome labeling BAC Bob014O06 (18 cells observed; Fig. 3b). In addition to the 18 C chromosomes, the BAC hybridized to some A bivalents, indicating translocations from the C to the A genome (Fig. 3b). In spite of these translocations, most PMCs (83.3%) had an expected number of 19 bivalents, with on average 97.4% of bivalents per PMC, even though univalents and multivalents were commonplace (16.7% of PMCs). However, A–C associations were observed in more than half of the PMCs (55.6%). Overall, slightly more A–C chromosomal associations were found in UG.S0 than in CD.S0, in which 55.0% of PMCs had 19 bivalents (20 observed PMCs) and 40.0% of PMCs had A–C associations (Szadkowski et al., 2010), but this difference was not statistically significant (> 0.05, χ2 tests).

We then assessed whether one of the translocations detected by cytology affected A1 and/or C1. We used a new set of nine primer pairs amplifying alleles along both A1 and C1 (see Fig. S1 for names and respective positions). We first observed that all 18 markers (for the nine primer pairs distinguishing both A1 and C1 alleles) were present in two copies for A1 and C1 loci (see the Materials and Methods section). Thus, any genetic changes affecting A1/C1, if present, should necessarily encompass a complete reciprocal exchange of genetic material between these chromosomes. We then observed that two markers located at the top of A1, which were genetically linked in B. rapa cv Z1, segregated independently from one another in the UG.S0 progeny (crossed with cv Darmor, UG.S0 × Bn, 108 plants). The same observation was true for a pair of markers located in the exact homoeologous region at the top of C1. These results suggest that UG.S0 carried a reciprocal translocation between the distal regions of A1 and C1.

We then assayed the UG.S0 × Bn progeny using the same genetic approaches as for F1 × Bn and CD.S0 × Bn. We observed that 85.2% of plants showed A1 and/or C1 marker losses in this progeny. These losses were mainly a result of COs between homoeologous chromosomes, with 89.8% of marker losses on A1 associated with a duplication of C1 and 89.7% of marker losses on C1 associated with a duplication of A1. However, significantly more plants showed marker loss on C1 (65%) than on A1 (46%; < 0.01, χ2 test). This difference between A1 and C1 persisted even after exclusion of the markers located in the reciprocal translocation detected on A1 and C1 (48% vs 34%; < 0.05, χ2 test).

Overall, the UG.S0 × Bn population showed more genetic changes than the CD.S0 × Bn progeny, even after removing the translocated regions between A1 and C1 (in both populations for a balanced comparison). A significant excess of plants carrying newly formed rearrangements was found in the UG.S0 × Bn population (68.5% vs 53.1% in the CD.S0 × Bn population; < 0.05, χ2 test). Contrary to expectation, the overall increase in the number of rearrangements found in the UG.S0 × Bn population (compared with CD.S0 × Bn) was not a result of increased marker losses in the vicinity of the pre-existing translocation (10.2% vs 11.6% on A1 and 21.3% vs 22.3% on C1 in UG.S0 × Bn vs CD.S0 × Bn populations, respectively; > 0.05, χ2 test), but of a larger number of rearrangements affecting the other chromosome arm (23.1% of marker losses on CB10572 in the UG.S0 × Bn population vs 10.8% for the CD.S0 × Bn population; < 0.05, χ2 test).

When we compared the size of the exchanged regions between populations on either A1 or C1 (including translocations), we found smaller rearrangements in the UG.S0 × Bn population than in the CD.S0 × Bn population for both chromosomes (Fig. S4). Indeed, only 22.0% of genetic changes on A1 encompassed three or more markers (vs 47.9% for the CD.S0 × Bn population; < 0.001, χ2 test), whereas 21.4% of genetic changes on C1 encompassed three or more markers (vs 62.1% for the CD.S0 × Bn population; < 0.001, χ2 test). This reduced size was caused by segregation of the pre-existing translocations, as no difference in rearrangement size was found between populations when markers within these regions were removed from the analysis (> 0.05, χ2 test).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Resynthesized B. napus accumulates homoeologous rearrangements and losses throughout generations (Gaeta & Pires, 2010), from as early as the first meiosis (Szadkowski et al., 2010). In this study, we compared the genetic instability of resynthesized B. napus generated via two distinct pathways (unreduced gametes vs somatically doubled S0 plants), using plant material produced from the same progenitor genotypes. Our results indicate that the resynthesized plants produced from these two pathways show different patterns of genetic rearrangements. In turn, these rearrangements are likely to change the pace at which new rearrangements are expected to form and accumulate in the following generations.

We clearly showed that homoeologous exchanges occurred more frequently during meiosis of the F1 hybrid than in the corresponding CD.S0 colchicine-doubled resynthesized plants, and the resulting recombined chromosomes were transmitted to the progeny of the F1 hybrid by unreduced FDR-like gametes. Indeed, in > 80% of progenies from the F1 hybrid, markers were lost on the A1 and/or C1 chromosomes and allele dosage showed that nine of 10 of these resulted from duplication of the corresponding homoeologous region. Even though A1 and C1 accumulated the largest number of meiotic-driven intergenomic exchanges in resynthesized plants (Gaeta et al., 2007; Szadkowski et al., 2010), we still observed a 1.5-fold increase in the number of rearrangements in the progeny of the F1 hybrid. This could be directly correlated with the frequent chiasmatic associations observed at meiosis of the F1 hybrid (90% of chromosomes per cell). Such a high level of CO formation in B. rapa × B. oleracea hybrids has been reported previously (Prakash & Hinata, 1980; Tsunoda et al., 1980; Attia & Robbelen, 1986a), but our GISH-like analysis provided more precise insight into allosyndetic (between A and C genomes) and autosyndetic (within A and C genomes) pairs. We confirmed that allosyndesis is prevalent in this F1 hybrid (> 75% of chromosomes per cell were involved in bivalent or multivalent formation), even though autosyndesis was detected. The frequency of A–C associations was much higher than in allotetraploid resynthesized S0 plants (20.8 times higher), which is expected because competition for CO formation between homologous and nonhomologous chromosomes does not occur in haploid plants. All our results demonstrate that, compared with somatically doubled plants, the first meiosis in an interspecific hybrid can drastically increase the number of gametes carrying homoeologous translocations. This is in agreement with results from selfed populations, where destabilization of the rDNA locus in self-pollinated lines from unreduced gametes was higher than in somatically doubled lines (T. Książczyk et al., unpublished). In a study on Lilium polyploids, unreduced gametes from F1 hybrid plants were also seen to generate translocations, whereas colchicine-doubled allopolyploids did not (Xie et al., 2010).

Gaeta & Pires (2010) recently introduced the ‘polyploid ratchet’, which predicts that translocations in heterozygote plants generated by homoeologous COs at meiosis will increase genomic instability. In a ratchet-like manner, successive generations will accumulate translocations leading to infertile plants [this is not to be confused with the polyploidy ratchet of Meyers & Levin (2006) that examines the evolution of ploidy within a genus]. According to the theory of Gaeta & Pires (2010), the initial translocation frequency is crucial for the polyploid ratchet to occur. Accordingly, the unreduced gamete pathway from the F1 hybrid would increase the pace of the polyploid ratchet compared with the somatic doubling pathway.

Nevertheless, the size of homoeologous rearrangements in a plant is also an important factor that modulates the pace at which new translocations will appear. Indeed, pre-existing small heterozygous translocations are less prone to destabilize meiosis than are large translocations (Szadkowski et al., 2010). Large translocations in various positions along chromosomes may generate more instability because of the increased probability of COs between large homogenized regions. In connection with this, we observed smaller rearrangements in the F1 hybrid progeny than those transmitted to the progeny of the somatically doubled CD.S0 plant. In addition, rearrangement distributions along chromosomes varied between the F1 × Bn and CD.S0 × Bn populations. For example, no translocation encompassed the centromeric region of C1 in the F1 progeny, whereas it was lost in 16.4% plants of the CD.S0 progeny. In our study, most viable gametes from the F1 hybrid carried 19 chromosomes and, in this respect, transmitted the complete parental chromosome set for centromeric regions. In the CD.S0 progeny, mis-segregation of chromosomes at meiosis leads to gametes carrying pairs of homologs, whilst lacking one homoeolog. This therefore favors larger rearrangements that encompass centromeres, as described in Szadkowski et al. (2010). Taken together, these results indicate that the unreduced gamete and somatic pathways generate different genetic configurations in their respective progeny for processing further meioses. This contrasting situation generated at both meiosis in the F1 hybrid and in its colchicine-doubled counterpart provides an interesting context for testing the influence of pre-existing rearrangements on the emergence of new rearrangements after one generation.

One fertile plant (UG.S0) from the cross between plants from the two pathways carried some translocations and probably one short complete distal reciprocal translocation between chromosomes A1–C1, confirmed by segregation analysis in the progeny. Meiosis in this plant was somewhat irregular, with around half of meiotic cells carrying A–C bivalents. In its progeny, a large number of plants carried rearrangements that mainly resulted from COs between A1 and C1 (90% of observed cases). These translocations were significantly smaller than those described in CD.S0 progeny. Pre-existing translocations influence the frequency of newly formed rearrangements. Indeed, if only markers outside translocated regions are considered, the rearrangement frequency still increased (1.29 times more rearrangements than in the progeny of the S0 somatically doubled plant). Contrary to the observations of Udall et al. (2005), many of the de novo rearrangements formed during meiosis of UG.S0 did not occur in the regions flanking this pre-existing translocation. In contrast, we saw an increase in translocation frequencies on opposite chromosomal arms where large pre-existing translocations were not detected. Thus, the size of pre-existing rearrangements appears to have a critical impact on the instability of genetic regions in their vicinity during meiosis. The relationship between translocated chromosomes and homoeologous CO formation may therefore be extremely complex.

In synthetic B. napus, the ‘polyploid ratchet’ was shown to favor elimination of one genome, because homoeologous marker losses were more frequent on the C genome than on the A genome, in a process of rearrangement fixation from generation to generation (Gaeta et al., 2007). In our study, rearrangements generated from either the F1 hybrid or colchicine-doubled S0 plants (published in Szadkowski et al., 2010) were found as frequently on either A1 or C1. In the UG.S0 plant, however, the difference in structure at the top of A1 and C1 was associated with a strong bias in its gamete transmission, with an excess of C1 marker losses and concurrent duplication of the A1 region. The origin of the bias may be either a larger pre-existing C1 translocation or accumulation of small C1 losses that were not seen at our marker density; selection against unfit genetic configurations may also be acting and cannot be excluded. Although, clearly, analysis of a single plant cannot be used to rule out the average behavior of resynthesized allotetraploids from F1 unreduced gametes, this shows that one single generation hedges chromosomal composition in gametes and illustrates how fast biases can emerge in favor of elimination of one genome copy.

Altogether, these results suggest that the first step in the allopolyploid formation pathway is crucial. It is characterized by different sizes and frequencies of rearrangements and may certainly have different consequences on the pace and extent of the polyploid ratchet. Based on our findings, further work on the unreduced gamete pathway will be necessary to fully address the question of its impact on genetic stability after several generations, and perhaps its potential to escape the B. napus polyploid ratchet.

The genetic pathways which formed natural cultivated B. napus are unknown, but its multiple origins have been confirmed (Song & Osborn, 1992; Allender & King, 2010; Cifuentes et al., 2010). As seen in this study, the genetic outcomes of polyploid formation pathways are different and the largely underexplored unreduced gamete pathway mimics that seen in natural B. napus. For example, translocations seen in B. napus varieties are generally smaller than those seen in somatically doubled plants, and are rarely found on several chromosomes at a time within natural B. napus varieties (Udall et al., 2005; Howell et al., 2008). Likewise, our cytogenetic characterization of the UG.S0 plant, in which only a small number of large-scale translocations are detected, is reminiscent of the few small changes observed by Howell et al. (2008) in domesticated B. napus lines. Natural B. napus varieties seem to share more features in common with the newly formed polyploids obtained via unreduced gametes than with the synthetics produced by somatic doubling. It would thus be tempting to hypothesize that domesticated B. napus may have evolved from unreduced gametes. However, domesticated B. napus certainly had a small population size when it was first selected and certainly underwent breeding for fertility from the very first generations. It is thus conceivable that extensive genome rearrangements that may have first been produced would then have been selected against or swept out of the populations over time by genetic drift. Future sequencing projects examining structural diversity in B. napus and its parental species, in combination with further studies on resynthesized B. napus through unreduced gametes, are thus required to better understand the formation of natural B. napus.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Jean-Claude Letanneur for technical assistance. We thank Biogenouest® (Le Rheu, France) for use of its facilities. Emmanuel Szadkowski was supported by a fellowship from the French Research Ministry (MENRT). This work was carried out with the financial support of the ‘ANR – Agence Nationale de la Recherche – The French National Research Agency’ under the ‘Programme Biodiversité’, project ‘ANR-05-BDIV-015, Effet de la polyploïdie sur la biodiversité et l’évolution du génome des plantes’.

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  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1 Homoeologous markers were used to assess the number of copies of chromosomes A1 and C1 in resynthesized Brassica napus.

Fig. S2 Distribution of estimated chromosome number in individuals from the cross between F1-EMZ and cv Darmor using flow cytometer.

Fig. S3 Comparison between male (Bn × CD.S0) and female (CD.S0 × Bn) meioses from an identical resynthesized Brassica napus plant.

Fig. S4 The most frequent rearrangement types following a first round of meiosis in different resynthesized Brassica napus.

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