Frédérique Eber

The first meiosis of resynthesized Brassica napus, a genome blender

Polyploidy promotes the restructuring of merged genomes within initial generations of resynthesized Brassica napus, possibly caused by homoeologous recombination at meiosis. However, little is known about the impact of the first confrontation of two genomes at the first meiosis which could lead to genome exchanges in progeny. Here, we assessed the role of the first meiosis in the genome instability of synthetic B. napus. We used three different newly resynthesized B. napus plants and established meiotic pairing frequencies for the A and C genomes. We genotyped the three corresponding progenies in a cross to a natural B. napus on the two homoeologous A1 and C1 chromosomes. Pairing at meiosis in a set of progenies with various rearrangements was scored. Here, we confirmed that the very first meiosis of resynthesized plants of B. napus acts as a genome blender, with many of the meiotic-driven genetic changes transmitted to the progenies, in proportions that depend significantly on the cytoplasm background inherited from the progenitors. We conclude that the first meiosis generates rearrangements on both genomes and promotes subsequent restructuring in further generations. Our study advances the knowledge on the timing of genetic changes and the mechanisms that may bias their transmission.

Gene flow from transgenic crops

Gene flow from crops to related wild species must be considered when assessing the potential environmental impact of cultivating genetically modified plants. Evidence of pollen dispersal within species has been found for several crops but little information is available on spontaneous gene flow from crops to related species with simultaneous flowering periods. To study the genetic mechanisms involved, we have developed an intergeneric model of gene flow from transgenic oilseed rape (Brassica napus L.; genotype, AACC; diploid chromosome number, 2n=38) containing one copy of the bar gene, which confers resistance to the herbicide Basta (glufosinate ammonium), to wild radish (Raphanus raphanistrum L.; genotype, RrRr; 2n=18), a widely distributed weed.

Numerous and Rapid Nonstochastic Modifications of Gene Products in Newly Synthesized Brassica napus Allotetraploids

Polyploidization is a widespread process that results in the merger of two or more genomes in a common nucleus. To investigate modifications of gene expression occurring during allopolyploid formation, the Brassica napus allotetraploid model was chosen. Large-scale analyses of the proteome were conducted on two organs, the stem and root, so that >1600 polypeptides were screened. Comparative proteomics of synthetic B. napus and its homozygous diploid progenitors B. rapa and B. oleracea showed that very few proteins disappeared or appeared in the amphiploids (<1%), but a strikingly high number (25–38%) of polypeptides displayed quantitative nonadditive pattern. Nonstochastic gene expression repatterning was found since 99% of the detected variations were reproducible in four independently created amphiploids. More than 60% of proteins displayed a nonadditive pattern closer to the paternal parent B. rapa. Interspecific hybridization triggered the majority of the deviations (89%), whereas very few variations (∼3%) were associated with genome doubling and more significant alterations arose from selfing (∼9%). Some nonadditive proteins behaved similarly in both organs, while others exhibited contrasted behavior, showing rapid organ-specific regulation. B. napus formation was therefore correlated with immediate and directed nonadditive changes in gene expression, suggesting that the early steps of allopolyploidization repatterning are controlled by nonstochastic mechanisms.

Pairing and recombination at meiosis of Brassica rapa (AA) × Brassica napus (AACC) hybrids

Interspecific crosses contribute significantly to plant evolution enabling gene exchanges between species. The efficiency of interspecific crosses depends on the similarity between the implicated genomes as high levels of genome similarity are required to ensure appropriate chromosome pairing and genetic recombination. Brassica napus (AACC) is an allopolyploid, resulting from natural hybridization between Brassica rapa (AA) and Brassica oleracea (CC), both being diploid species derived from a common ancestor. To study the relationships between genomes of these Brassica species, we have determined simultaneously the pairing and recombination pattern of A and C chromosomes during meiosis of AAC triploid hybrids, which result from the interspecific cross between natural B. napus and B. rapa. Different AAC triploid hybrids and their progenies have been analysed using cytogenetic, BAC-FISH, and molecular techniques. In 71% of the pollen mother cells, homologous A chromosomes paired regularly, and usually one chromosome of each pair was transmitted to the progeny. C chromosomes remained mainly univalent, but were involved in homoeologous pairing in 21.5% of the cells, and 13% of the transmitted C chromosomes were either recombined or broken. The rate of transmission of C chromosomes depended on the identity of the particular chromosome and on the way the hybrid was crossed, as the male or as the female parent, to B. napus or to B. rapa. Gene transfers in triploid hybrids are favoured between A genomes of B. rapa and B. napus, but also occur between A and C genomes though at lower rates.

Selection of stable Brassica napus-B. juncea recombinant lines resistant to blackleg (Leptosphaeria maculans). 1. Identification of molecular markers, chromosomal and genomic origin of the introgression

Abstract A scheme of selection combining selfing and backcross was applied to a B. napus line with the blackleg resistance from B. juncea in order to transfer this resistance to a winter oilseed rape variety. Cytogenetic analyses combined with cotyledon blackleg resistance tests at each generation allowed us to obtain a recombinant line showing regular meiotic behavior. The resistance is monogenic and is highly efficient under field conditions. Four-hundred RAPD primers were tested on two segregating populations by bulk segregant analysis. Three markers totally linked to the introgression were identified. The analysis of these markers on both sets of B. napus-B. nigra and B. oleracea-B. nigra addition lines revealed that they are not located on the B4 chromosome of B. nigra, which has already been shown to carry a blackleg resistance gene, but rather on the B8 chromosome. We confirmed that the resistance gene is carried by the B genome of B. juncea. Based on these data, two hypotheses, one involving chromosome rearrangements between the two B genomes of B. nigra and B. juncea, and the other based on a more probable digenic control of the resistance within B. juncea, are discussed.

Homeologous Recombination Plays a Major Role in Chromosome Rearrangements That Occur During Meiosis of Brassica napus Haploids

Chromosomal rearrangements can be triggered by recombination between distinct but related regions. Brassica napus (AACC; 2n = 38) is a recent allopolyploid species whose progenitor genomes are widely replicated. In this article, we analyze the extent to which chromosomal rearrangements originate from homeologous recombination during meiosis of haploid B. napus (n = 19) by genotyping progenies of haploid × euploid B. napus with molecular markers. Our study focuses on three pairs of homeologous regions selected for their differing levels of divergence (N1/N11, N3/N13, and N9/N18). We show that a high number of chromosomal rearrangements occur during meiosis of B. napus haploid and are transmitted by first division restitution (FDR)-like unreduced gametes to their progeny; half of the progeny of Darmor-bzh haploids display duplications and/or losses in the chromosomal regions being studied. We demonstrate that half of these rearrangements are due to recombination between regions of primary homeology, which represents a 10- to 100-fold increase compared to the frequency of homeologous recombination measured in euploid lines. Some of the other rearrangements certainly result from recombination between paralogous regions because we observed an average of one to two autosyndetic A–A and/or C–C bivalents at metaphase I of the B. napus haploid. These results are discussed in the context of genome evolution of B. napus.

Genetic Regulation of Meiotic Cross-Overs between Related Genomes in Brassica napus Haploids and Hybrids

Although the genetic regulation of recombination in allopolyploid species plays a pivotal role in evolution and plant breeding, it has received little recent attention, except in wheat (Triticum aestivum). PrBn is the main locus that determines the number of nonhomologous associations during meiosis of microspore cultured Brassica napus haploids (AC; 19 chromosomes). In this study, we examined the role played by PrBn in recombination. We generated two haploid × euploid populations using two B. napus haploids with differing PrBn (and interacting genes) activity. We analyzed molecular marker transmission in these two populations to compare genetic changes, which have arisen during meiosis. We found that cross-over number in these two genotypes was significantly different but that cross-overs between nonhomologous chromosomes showed roughly the same distribution pattern. We then examined genetic recombination along a pair of A chromosomes during meiosis of B. rapa × B. napus AAC and AACC hybrids that were produced with the same two B. napus genotypes. We observed significant genotypic variation in cross-over rates between the two AAC hybrids but no difference between the two AACC hybrids. Overall, our results show that PrBn changes the rate of recombination between nonhomologous chromosomes during meiosis of B. napus haploids and also affects homologous recombination with an effect that depends on plant karyotype.

Spontaneous hybridization between a male-sterile oilseed rape and two weeds.

Spontaneous interspecific hybrids were produced under natural conditions (pollination by wind and bees) between a male-sterile cybrid Brassica napus (AACC, 2n = 38) and two weeds Brassica adpressa (AdAd, 2n = 14) and Raphanus raphanistrum (RrRr, 2n = 18). After characterization by chromosome counts and isozyme analyses, we observed 512 and 3 734 inter-specific seeds per m2 for the B. napus-B. adpressa and B. napus-R. raphanistrum trials respectively. Most of the hybrids studied had the expected triploid structure (ACX). In order to quantify the frequency of allosyndesis between the genomes involved in the hybrids, their meiotic behavior was compared to a haploid of B. napus (AC). For the B. napus-B. adpressa hybrids, we concluded that probably no allosyndesis occurred between the two parental genomes, and that genetic factors regulating homoeologous chromosome pairing were carried by the B. adpressa genome. For the B. napus-R. raphanistrum hybrids, high chromosome pairing and the presence of multivalents (in 9.16% of the pollen mother cells) indicate that recombination is possible between chromosomes of different genomes. Pollen fertility of the hybrids ranged from 0 to 30%. Blackleg inoculation tests were performed on the three parental species and on the interspecific hybrids. BC1 production with the weeds and with rapeseed was attempted. Results are discussed in regard to the risk assessment of transgenic rapeseed cultivation, F1 hybrid rapeseed variety production, and rapeseed improvement.

Assessment of interspecific hybridization between transgenic oilseed rape and wild radish under normal agronomic conditions.

Abstract In order to assess the hybridization rate between oilseed rape and wild radish under normal agronomic conditions, three 1-ha field experiments were performed. In each case, wild radish plants were transplanted at different densities in the middle, the border, or the margin of the herbicide-tolerant oilseed rape field. Among the 189084 seedlings obtained from seeds harvested on wild radish plants, only one herbicide-tolerant interspecific hybrid (RrRrAC, 2n = 37) was characterized from seeds harvested on an isolated plant growing in the margin of the field. Thus, for the wild radish total harvest, with a 95% confidence limit, the frequency of interspecific hybrids was assessed to range from 10–7 to 3.10–5. Interspecific hybrids were detected in all cases among the smallest seeds with a diameter less than 1.6 mm harvested on oilseed rape, but the highest frequency was obtained from oilseed rape close to wild radish plants growing as clusters in the border or the margin of the field. Most hybrids had the expected triploid genomic structure (ACRr, 2n = 28) except for four amphidiploids (AACCRrRr, 2n = 56) and one hybrid from a wild radish unreduced gamete (ACRrRr, 2n = 37). Among the 73847 seedlings observed on the oilseed rape total harvest, the frequency of interspecific hybrids was assessed to range from 2.10–5to 5.10–4, with a 95% confidence limit. The results are discussed with regard to the type of oilseed rape variety used and the characteristics of the interspecific hybrids.

Polyploid formation pathways have an impact on genetic rearrangements in resynthesized Brassica napus

• Polyploids can be produced by the union of unreduced gametes or through somatic doubling of F(1) 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 F(1) 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.