In the framework of a gene flow assessment, we investigated the natural hybridization rate between Gossypium hirsutum (AADD genome) and G. herbaceum (AA genome). The latter species, a diploid progenitor of G. hirsutum, is spontaneously present in South Africa. Reciprocal crosses were performed without emasculation between G. herbaceum and G. hirsutum. Neither examination of the morphological characteristics nor flow cytometry analysis of the 335 plants resulting from the G. hirsutum × G. herbaceum cross showed any hybrid features. Of the 148 plants produced from the G. herbaceum × G. hirsutum cross, three showed a hybrid phenotype, and their hybrid status was confirmed by SSR markers. Analysis of DNA content by flow cytometry and morphological traits clearly showed that two of these plants were triploid (AAD). The third plant had a flow cytometry DNA content slightly higher than G. hirsutum. In addition, its morphological characteristics (plant architecture, presence and size of petal spots, leaf shape) led us to conclude that this plant was AAAD thus resulting from fertilization with an unreduced AA gamete of the female G. herbaceum parent. Fluorescent In Situ Hybridization (FISH) and meiotic behavior confirmed this hypothesis. To the best of our knowledge, this is the first description of such gametes in G. herbaceum, and it opens new avenues in breeding programs. Furthermore, this plant material could provide a useful tool for studying the expression of genes duplicated in the A and D cotton genome.

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 F-1 hybrid (CD.S-0) and an amphidiploid (UG.S-0) 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 F-1 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.S-0 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.

Transposable elements (TEs) represent a major component of plant genomes and may provide the host genome with the necessary plasticity for its adaptative response. Polyploidy has played a major role in the evolution of plants (Wendel, 2000) and its success can be explained by the fact that a polyploid genome is not only the strict addition of the progenitor genomes, but that polyploidisation induces structural and functional modifications which represent important sources of novelty (Adams and Wendel, 2005). Our study aims at evaluating the implication of TEs in the genomic modifications that occur during the formation of a polyploid genome. Our plant model is oilseed rape (Brassica napus, AACC) originating from interspecific hybridization between B. rapa (AA) and B. oleracea (CC), and we dispose of newly synthesized B. napus allotetraploids resulting from independent AA x CC crosses. We developed sequence-specific amplification polymorphism (S-SAP) markers anchored in two different families of Brassica-specific TEs, an Athila-like retrotransposon and a MITE, which are contrasting TEs regarding their genomic organisation, dynamics of evolution, and mode of replication. Comparison of the SSAP profiles and characterization of the polymorphism identified in the newly synthesized allotetraploids will provide new insights into the putative role of TEs during the creation of an allopolyploid species.

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.

Given their tremendous importance for correct chromosome segregation, the number and distribution of crossovers are tightly controlled during meiosis. In this review, we give an overview of crossover formation in polyploid Brassica hybrids and haploids that illustrates or underscores several aspects of crossover control. We first demonstrate that multiple targets for crossover formation (i.e. different but related chromosomes or duplicated regions) are sorted out during meiosis based on their level of relatedness. In euploid Brassica napus (AACC; 2n = 38), crossovers essentially occur between homologous chromosomes and only a few of them form between homeologues. The situation is different in B. napus haploids in which crossovers preferentially occur between homeologous chromosomes and a few can then form between more divergent duplicated regions. We then provide evidence that the frequency of crossovers between a given pair of chromosomes is influenced by the karyotypic and genetic composition of the plants that undergo meiosis. For instance, genetic evidence indicates that the number of crossovers between exactly the same pairs of homologous A chromosomes gets a boost in Brassica digenomic tetraploid (AACC) and triploid (AAC) hybrids. Increased autosyndesis within B. napus haploids as compared to monoploid B. rapa and B. oleracea is another illustration of this process. All these observations may suggest that polyploidization overall boosts up crossover machinery and/or that the number of crossovers is modulated through inter-bivalents or univalent-bivalent cross-talk effects. The last part of this review gives an up-to-date account of what we know about the genetic control of homologous and homeologous crossover formation among Brassica species.

The value of Katanning Early Maturing (KEM) breeding lines from Western Australia, derived from Brassica napus × B. juncea crosses, was assessed as a source of germplasm for resistance to blackleg disease (caused by Leptosphaeria maculans) in spring-type oilseed rape cultivars. The stability of blackleg resistance in these KEM lines was related to key cytological characteristics to determine why there are poor levels of introgression of this resistance into progeny. Promising recombinant KEM lines were crossed with the spring-type B. napus cv. Dunkeld, which has useful polygenic resistance to blackleg, and screened for resistance. The lines were analyzed cytologically for pairing of bivalents in each generation to aid in the selection of stable recombinant lines. KEM recombinant lines showing regular meiotic behavior and a high level of blackleg resistance were obtained for the first time. We also showed that the stable introgression of the B. juncea resistance from the KEM lines into a 'Dunkeld' background was possible. Inoculation of selfing and backcross populations with isolates of L. maculans having different AvrLm genes indicated that the B. juncea resistance gene, Rlm6, had been introgressed into a B. napus spring-type cultivar carrying polygenic resistance. The combination of both resistances would enhance the overall effectiveness of resistance against L. maculans. This is clearly needed in Australia and France where cultivars relying upon single dominant gene-based resistance for their effectiveness have proved not durable.

The assessment of gene flow from crop species to weeds has found a new emphasis over the last years because of the marketing of transgenic crops and the possible selective advantage that crop (trans)gene may confer to the weeds. Several studies focused on the F1 interspecific hybrid production but few data are available on the factors affecting the genetic structure of advanced generations. It depends on the genomic structure of the species concerned as well as on the degree of their genome homology that affect the occurrence of intergenomic recombination. Oilseed rape ( Brassica napus , AACC, 2 n = 38)-wild radish ( Raphanus raphanistrum , RrRr, 2 n = 18), a distantly related weed, is a good model to address such questions. From seven male sterile oilseed rape lines carrying an herbicide tolerance transgene, F1 interspecific hybrids and four advanced generations were produced under field conditions with wild radish as pollinator. Observation of hybrid chromosome numbers across four generations revealed a high variability, especially in the “BC1” generation. A regression model was fitted in order to describe the relationship between parent and offspring chromosome numbers. The effects of generation, transgenic line and selection pressure on the mean relationship were investigated. The first two factors had an influence on the rate of decrease of chromosome numbers, whereas selection pressure resulted in the presence of an additional chromosome in the herbicide treated plants. The model provided a convenient framework for analysing how chromosome numbers evolve over successive hybridization events and it may prove useful as a basis for simulation-based approaches.

Blackleg (stem canker) caused by the fungus Leptosphaeria maculans is one of the most damaging diseases of oilseed rape (Brassica napus). Crop relatives represent a valuable source of "new" resistance genes that could be used to diversify cultivar resistance. B. rapa, one of the progenitors of B. napus, is a potential source of new resistance genes. However, most of the accessions are heterozygous so it is impossible to directly detect the plant genes conferring specific resistance due to the complex patterns of avirulence genes in L. maculans isolates. We developed a strategy to simultaneously characterize and introgress resistance genes from B. rapa, by homologous recombination, into B. napus. One B. rapa plant resistant to one L. maculans isolate was used to produce B. rapa backcross progeny and a resynthesized B. napus plant from which a population of doubled haploid lines was derived after crossing with natural B. napus. We then used molecular analyses and resistance tests on these populations to identify and map the resistance genes and to characterize their introgression from B. rapa into B. napus. Three specific genes conferring resistance to L. maculans (Rlm1, Rlm2 and Rlm7) were identified in B. rapa. Comparisons of genetic maps showed that two of these genes were located on the R7 linkage group, in a region homologous to the region on linkage group N7 in B. napus, where these genes have been reported previously. The results of our study offer new perspectives for gene introgression and cloning in Brassicas.

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.

Polyploidization is a major evolutionary process in eukaryotes. In plants, genetic and epigenetic changes occur rapidly after formation of allopolyploids. Hybridization, rather than genome doubling itself, is considered as the main cause for the resulting differential gene expression. We studied the consequences of genome doubling alone in an autopolyploid model, by comparing two-dimensional gel electrophoresis (2-DE) gels of haploid, diploid, and tetraploid Brassica oleracea cabbages. Two fully homozygous lines, HDEM and RC, as well as two organs, leaf and stem, were studied. For the 558 common spots found present in all the 29 2-DE gels of the experiment, inter-organ and -genotype differences were the major sources of the variation in protein amounts: 41 and 10-13 %, respectively. HDEM leaf and stem proteomes were not significantly affected by the ploidy level, since no qualitative variation was detected and since the number of quantitative variations could be due to chance. For RC, no qualitative variations were observed, but a few spots were significantly variable in protein amount. However, the number of inter-ploidy variations was of the same range as the number of intra-ploidy variations. In conclusion, whatever the ploidy level, leaf and stem proteomes remained globally unchanged in both cabbage lines.

A major objective of breeders using the Ogu-INRA cytoplasmic male sterility (cms) system in rapeseed (Brassica napus L.) is to obtain double low restorer lines with a shorter introgression and a good agronomic value. The development of low glucosinolate content (low GC) restorer lines often occurs through the deletion of a part of the introgression. One of these lines has lost the radish Pgi-2 allele expression, without recovering that of the rapeseed Pgi-2 allele. This line shows a defect in the meiotic transmission of the restorer gene Rfo and a very poor agronomic value. We initiated a programme to force non-spontaneous recombination between this Rfo-carrying introgression and the rapeseed homologous chromosome from a low GC B. napus line. Gamma ray irradiation was used to induce chromosome breakage just prior meiosis aiming at just such a recombination. Low GC cms plants were crossed with the pollen of irradiated plants that were heterozygous for this introgression. The F(2) families were scored for their vigour, transmission rate of Rfo and female fertility. One family of plants, R2000, showed an improved behaviour for these three traits. This family presented a unique combination of molecular markers when compared to other rapeseed restorers analysed, which suggests that the recombination event allowed the recovery of B. oleracea genetic information that was originally replaced by the radish introgression in the original restorers. This resulted in a duplicated region (originating from radish and B. oleracea) on the chromosome carrying the introgression in the R2000 family.

For diversification of specific resistance genes to Leptosphaeria maculans, Brassica rapa (AA, 2n=20) populations were screened. One resistant plant was selected within a forage population. A BC1 progeny was produced after two crosses of this plant to a susceptible B. rapa doubled haploid line and screened with six isolates. Resistance to different isolates proves to be under mono or oligogenic control. Resistance genes are organized in a cluster. The same B. rapa plant was crossed twice to ‘Darmor’ oilseed rape variety, susceptible at the cotyledon stage but partially resistant at the adult stage. The BC1 oilseed rape progeny was analyzed with two isolates and showed the same results as in B. rapa BC1 progeny. From 2n=38 resistant BC1 plants, two additional backcrosses were performed in which the different resistant genes were separated through recombination. The results will be analyzed according to breeding for a durable resistance.

One of the main concerns about the commercial release of transgenic crops is the likelihood of transgene spread from cultivated species into wild relatives. This question is relevant for oilseed rape/canola (Brassica napus, AACC, 2n=38), as this species is partially allogamous with several wild relatives that are often sympatric with oilseed rape production. A workshop sponsored by the European Science Foundation (11-13 June 2001, Rennes, France) was held: (i) to identify the main weeds present in European and North American countries; (ii) to review results on the ability of oilseed rape to hybridize and backcross with wild relatives; (iii) to review the usefulness and limitations of the tools available for monitoring interspecific hybridization and gene introgression; and (iv) to provide recent results on modelling of gene flow.

The assessment of the likelihood of transgene spread from cultivated species to their wild relatives is relevant for oilseed rape (Brassica napus, AACC, 2n=38) as it is partially allogamous and presents numerous wild relatives growing nearby in cultivated areas and having an overlapping of the flowering period. We have assessed the probability of F1 interspecific hybrid formation between oilseed rape and three main weeds encountered in France, wild mustard (Sinapis arvensis, SarSar, 2n=18), hoary mustard (Hirschfeldia incana, AdAd, 2n=14) and wild radish (Raphanus raphanistrum, RrRr, 2n=18), under optimal conditions. Because of the higher frequency of interspecific hybrids observed with this latter weed, complementary field experiments were carried out under normal agronomic conditions. The F1 hybrids obtained showed different genomic structures, either 2n=28, or 2n=37, or 2n=56. These different hybrids were cultivated in the presence of wild radish and the succeeding generations were studied.

Precise control of chromosome pairing is vital for conferring meiotic, and hence reproductive, stability in sexually reproducing polyploids. Apart from the Ph1 locus of wheat that suppresses homeologous pairing, little is known about the activity of genes that contribute to the cytological diploidization of allopolyploids. In oilseed rape (Brassica napus) haploids, the amount of chromosome pairing at metaphase I (MI) of meiosis varies depending on the varieties the haploids originate from. In this study, we combined a segregation analysis with a maximum-likelihood approach to demonstrate that this variation is genetically based and controlled mainly by a gene with a major effect. A total of 244 haploids were produced from F(1) hybrids between a high- and a low-pairing variety (at the haploid stage) and their meiotic behavior at MI was characterized. Likelihood-ratio statistics were used to demonstrate that the distribution of the number of univalents among these haploids was consistent with the segregation of a diallelic major gene, presumably in a background of polygenic variation. Our observations suggest that this gene, named PrBn, is different from Ph1 and could thus provide complementary information on the meiotic stabilization of chromosome pairing in allopolyploid species.

Fertility and reproductive stability in polyploids depend on the precise control of chromosome pairing at meiosis, In this study, meiotic pairing configurations are related to isozyme segregation ratios in F, hybrids between colchicine-induced autotetraploids of kale. An immediate diploid-like pairing regime is associated with tetrasomic segregation. Although the mechanisms driving such a behaviour have yet to be identified, pairing preferences are not responsible for the cytological diploidization of autotetraploid kales.

The process of introgression between a transgenic crop modified for better agronomic characters and a wild relative could lead potentially to increased weediness and adaptation to the environment of the wild species. However, the formation of hybrid and hybrid progeny could be associated with functional imbalance and low fitness, which reduces the risk of gene escape and establishment of the wild species in the field. Our work compares the fitness components of parents and different types of backcross in the sixth generation of hybrids between transgenic oilseed rape (Brassica napus, AACC, 2n = 38) resistant to the herbicide glufosinate and wild radish (Raphanus raphanistrum, RrRr, 2n = 18). The backcross with oilseed rape cytoplasm (OBC) has a fitness value 100 times lower than that of the backcross with wild radish cytoplasm (RBC). The herbicide-resistant RBC has similar growth to the susceptible RBC, but final male and female fitness values are two times lower. In turn, susceptible RBC exhibit similar fitness to the control wild radishes. The relative fitnesses of the different types are the same whether or not they grow under competitive conditions. The consequence on fitness of the chromosome location of the transgene conferring resistance and the relevance of these results to the impact of gene flow on the environment are discussed.

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.