Meiotic recombination is a key biological process in plant evolution and breeding, as it generates genetic diversity in each generation through the formation of crossovers (COs). However, due to their importance in genome stability, COs are highly regulated in frequency and distribution. We previously demonstrated that this strict regulation of COs can be modified, both in terms of CO frequency and distribution, in allotriploid Brassica hybrids (2n = 3x = 29; AAC) resulting from a cross between Brassica napus (2n = 4x = 38; AACC) and Brassica rapa (2n = 2x = 20; AA). Using the recently updated B. napus genome now including pericentromeres, we demonstrated that COs occur in these cold regions in allotriploids, as close as 375 kb from the centromere. Reverse transcription quantitative PCR (RT-qPCR) of various meiotic genes indicated that Class I COs are likely involved in the increased recombination frequency observed in allotriploids. We also demonstrated that this modified recombination landscape can be maintained via successive generations of allotriploidy (odd ploidy level). This deregulated meiotic behavior reverts to strict regulation in allotetraploid (even ploidy level) progeny in the second generation. Overall, we provide an easy way to manipulate tight recombination control in a polyploid crop.

Meiotic recombination is a key biological process in plant evolution and breeding, as it generates novel genetic diversity at each generation. However, due to its importance in chromosome segregation and genomic stability, crossovers are highly regulated in both frequency and distribution. We previously demonstrated that this strict regulation is not a fatality and that it can be naturally modified (3.6-fold increased frequency and altered distribution) in an allotriploid Brassica hybrid (2 n =3 x =29; AAC), resulting from a cross between B. napus (2 n =4 x =38; AACC) and B. rapa (2 n =2 x =20; AA). Taking advantage of the recently updated Brassica napus genome assembly, which now includes the pericentromeric regions, we unambiguously demonstrated that crossovers occur in these normally cold regions in allotriploids, with the presence of crossovers as close as 375 kb from the centromere. We deciphered that this modified recombination landscape (both frequency and distribution) can be maintained in successive generations of allotriploidy, with even a slight increase of crossover frequency. We also showed that this deregulated meiotic behavior may revert back to a strictly regulated one when recovering an allotetraploid progeny in the second generation. Overall, we provide here for the first time a practical and natural way to switch on and off the tight recombination control in a polyploid crop. We also discuss the potential role of this modified regulation of recombination in polyploid speciation success.### Competing Interest StatementThe authors have declared no competing interest.

Summary Meiotic recombination is a major evolutionary process generating genetic diversity at each generation in sexual organisms. However, this process is highly regulated, with the majority of crossovers lying in the distal chromosomal regions that harbor low DNA methylation levels. Even in these regions, some islands without recombination remain, for which we investigated the underlying causes. Genetic maps were established in two Brassica napus hybrids to detect the presence of such large nonrecombinant islands. The role played by DNA methylation and structural variations in this local absence of recombination was determined by performing bisulfite sequencing and whole genome comparisons. Inferred structural variations were validated using either optical mapping or oligo fluorescence in situ hybridization. Hypermethylated or inverted regions between Brassica genomes were associated with the absence of recombination. Pairwise comparisons of nine B. napus genome assemblies revealed that such inversions occur frequently and may contain key agronomic genes such as resistance to biotic stresses. We conclude that such islands without recombination can have different origins, such as DNA methylation or structural variations in B. napus. It is thus essential to take into account these features in breeding programs as they may hamper the efficient combination of favorable alleles in elite varieties.

Summary Allopolyploids have globally higher fitness than their diploid progenitors; however, by comparison, most resynthesized allopolyploids have poor fertility and highly unstable genome. Elucidating the evolutionary processes promoting genome stabilization and fertility is thus essential to comprehend allopolyploid success. Using the Brassica model, we mimicked the speciation process of a nascent allopolyploid species by resynthesizing allotetraploid Brassica napus and systematically selecting for euploid individuals over eight generations in four independent allopolyploidization events with contrasted genetic backgrounds, cytoplasmic donors, and polyploid formation type. We evaluated the evolution of meiotic behavior and fertility and identified rearrangements in S1 to S9 lineages to explore the positive consequences of euploid selection on B. napus genome stability. Recurrent selection of euploid plants for eight generations drastically reduced the percentage of aneuploid progenies as early as the fourth generation, concomitantly with a decrease in number of newly fixed homoeologous rearrangements. The consequences of homoeologous rearrangements on meiotic behavior and seed number depended strongly on the genetic background and cytoplasm donor. The combined use of both self‐fertilization and recurrent euploid selection allowed identification of genomic regions associated with fertility and meiotic behavior, providing complementary evidence to explain B. napus speciation success.

Meiotic recombination is the main tool used by breeders to generate biodiversity, allowing genetic reshuffling at each generation. It enables the accumulation of favorable alleles while purging deleterious mutations. However, this mechanism is highly regulated with the formation of one to rarely more than three crossovers, which are not randomly distributed. In this study, we showed that it is possible to modify these controls in oilseed rape (Brassica napus, AACC, 2n = 4x = 38) and that it is linked to AAC allotriploidy and not to polyploidy per se. To that purpose, we compared the frequency and the distribution of crossovers along A chromosomes from hybrids carrying exactly the same A nucleotide sequence, but presenting three different ploidy levels: AA, AAC and AACC. Genetic maps established with 202 SNPs anchored on reference genomes revealed that the crossover rate is 3.6-fold higher in the AAC allotriploid hybrids compared to AA and AACC hybrids. Using a higher SNP density, we demonstrated that smaller and numerous introgressions of B. rapa were present in AAC hybrids compared to AACC allotetraploid hybrids, with 7.6 Mb vs. 16.9 Mb on average and 21 B. rapa regions per plant vs. nine regions, respectively. Therefore, this boost of recombination is highly efficient to reduce the size of QTL carried in cold regions of the oilseed rape genome, as exemplified here for a QTL conferring blackleg resistance.

[This corrects the article DOI: 10.3389/fpls.2018.00368.].

SUMMARYAllopolyploids have globally higher fitness than their diploid progenitors however, by comparison, most resynthesized allopolyploids have poor fertility and highly unstable genome. Elucidating the evolutionary processes promoting genome stabilization and fertility is thus essential to comprehend allopolyploid success.Using theBrassicamodel, we mimicked the speciation process of a nascent allopolyploid species by resynthesizing allotetraploidB. napusand systematically selecting for euploid individuals over eight generations in four independent allopolyploidization events with contrasted genetic backgrounds, cytoplasmic donors and polyploid formation type. We evaluated the evolution of meiotic behavior, fertility and identified rearrangements in S1 to S9 lineages, to explore the positive consequences of euploid selection onB. napusgenome stability.Recurrent selection of euploid plants for eight generations drastically reduced the percentage of aneuploid progenies as early as the fourth generation, concomitantly with a quasi disappearance of newly fixed homoeologous rearrangements. The consequences of homoeologous rearrangements on meiotic behavior and seed number strongly depended on the genetic background and cytoplasm donor.The combined use of both self-fertilisation and outcrossing as well as recurrent euploid selection, allowed identification of genomic regions associated with fertility and meiotic behavior, providing complementary evidence to explainB. napusspeciation success.

Meiotic crossovers (COs) are essential for proper chromosome segregation and the reshuffling of alleles during meiosis. In WT plants, the number of COs is usually small, which limits the genetic variation that can be captured by plant breeding programs. Part of this limitation is imposed by proteins like FANCM, the inactivation of which results in a 3-fold increase in COs in Arabidopsis thaliana. Whether the same holds true in crops needed to be established. In this study, we identified EMS induced mutations in FANCM in two species of economic relevance within the genus Brassica. We showed that CO frequencies were increased in fancm mutants in both diploid and tetraploid Brassicas, Brassica rapa and Brassica napus respectively. In B. rapa, we observed a 3-fold increase in the number of COs, equal to the increase observed previously in Arabidopsis. In B. napus we observed a lesser but consistent increase (1.3-fold) in both euploid (AACC) and allohaploid (AC) plants. Complementation tests in A. thaliana suggest that the smaller increase in crossover frequency observed in B. napus reflects residual activity of the mutant C copy of FANCM. Altogether our results indicate that the anti-CO activity of FANCM is conserved across the Brassica, opening new avenues to make a wider range of genetic diversity accessible to crop improvement.

Allopolyploidy, which results from the merger and duplication of two divergent genomes, has played a major role in the evolution and diversification of flowering plants. The genomic changes that occur in resynthesized or natural neopolyploids have been extensively studied, but little is known about the effects of the reproductive mode in the initial generations that may precede its successful establishment. To truly reflect the early generations of a nascent polyploid, two resynthesized allotetraploid Brassica napus populations were obtained for the first time by open pollination. In these populations, we detected a much lower level of aneuploidy (third generation) compared with those previously published populations obtained by controlled successive selfing. We specifically studied 33 resynthesized B. napus individuals from our two open pollinated populations, and showed that meiosis was affected in both populations. Their genomes were deeply shuffled after allopolyploidization: up to 8.5 and 3.5% of the C and A subgenomes were deleted in only two generations. The identified deletions occurred mainly at the distal part of the chromosome, and to a significantly greater extent on the C rather than the A subgenome. Using Fluorescent In Situ Hybridization (BAC-FISH), we demonstrated that four of these deletions corresponded to fixed translocations (via homeologous exchanges). We were able to evaluate the size of the structural variations and their impact on the whole genome size, gene content, and allelic diversity. In addition, the evolution of fertility was assessed, to better understand the difficulty encountered by novel polyploid individuals before the putative formation of a novel stable species.

The effect of gene location within a crop genome on its transfer to a weed genome remains an open question for gene flow assessment. To elucidate this question, we analyzed advanced generations of intergeneric hybrids, derived from an initial pollination of known oilseed rape varieties (Brassica napus, AACC, 2n = 38) by a local population of wild radish (Raphanus raphanistrum, RrRr, 2n = 18). After five generations of recurrent pollination, 307 G5 plants with a chromosome number similar to wild radish were genotyped using 105 B. napus specific markers well distributed along the chromosomes. They revealed that 49.8% of G5 plants carried at least one B. napus genomic region. According to the frequency of B. napus markers (0–28%), four classes were defined: Class 1 (near zero frequency), with 75 markers covering ∼70% of oilseed rape genome; Class 2 (low frequency), with 20 markers located on 11 genomic regions; Class 3 (high frequency), with eight markers on three genomic regions; and Class 4 (higher frequency), with two adjacent markers detected on A10. Therefore, some regions of the oilseed rape genome are more prone than others to be introgressed into wild radish. Inheritance and growth of plant progeny revealed that genomic regions of oilseed rape could be stably introduced into wild radish and variably impact the plant fitness (plant height and seed number). Our results pinpoint that novel technologies enabling the targeted insertion of transgenes should select genomic regions that are less likely to be introgressed into the weed genome, thereby reducing gene flow.

Meiotic recombination by crossovers (COs) is tightly regulated, limiting its key role in producing genetic diversity. However, while COs are usually restricted in number and not homogenously distributed along chromosomes, we show here how to disrupt these rules in Brassica species by using allotriploid hybrids (AAC, 2n = 3x = 29), resulting from the cross between the allotetraploid rapeseed (B. napus, AACC, 2n = 4x = 38) and one of its diploid progenitors (B. rapa, AA, 2n = 2x = 20). We produced mapping populations from different genotypes of both diploid AA and triploid AAC hybrids, used as female and/or as male. Each population revealed nearly 3,000 COs that we studied with SNP markers well distributed along the A genome (on average 1 SNP per 1.25 Mbp). Compared to the case of diploids, allotriploid hybrids showed 1.7 to 3.4 times more overall COs depending on the sex of meiosis and the genetic background. Most surprisingly, we found that such a rise was always associated with (i) dramatic changes in the shape of recombination landscapes and (ii) a strong decrease of CO interference. Hybrids carrying an additional C genome exhibited COs all along the A chromosomes, even in the vicinity of centromeres that are deprived of COs in diploids as well as in most studied species. Moreover, in male allotriploid hybrids we found that Class I COs are mostly responsible for the changes of CO rates, landscapes and interference. These results offer the opportunity for geneticists and plant breeders to dramatically enhance the generation of diversity in Brassica species by disrupting the linkage drag coming from limits on number and distribution of COs.

Meiotic recombination by crossovers (COs) is tightly regulated, limiting its key role in producing genetic diversity. While one obligate CO occurs per pair of homologs, ensuring their proper segregation during meiosis, rarely more than three are formed and their distribution is not homogenous along chromosomes. In plants, Whole Genome Duplication (WGD) was highlighted to result in a boost of the number of COs between homologous chromosomes, which may contribute to the success of allopolyploid species by generating enhanced allelic combinations. However, the consequences on COs distribution as well as the regulation of this phenomenon are poorly understood. We investigated here the striking case of Brassica allotriploids (AAC, 2n=3x=29), resulting from crosses between B. napus (AACC, 2n=4x=38) and its B. rapa progenitor (AA, 2n=2x=20), showing far higher CO rates between A homologs than diploids and allotetraploids due to specific additional C chromosomes. From several populations developed, we assessed the homologous recombination in AA diploids and hybrids carrying either an additional complete C genome (9 C chromosomes) or specific C chromosomes, through the genotyping of 204 SNP markers well distributed along A chromosomes (one SNP each 1.2 Mb). Compared to what was previously known, we showed that the presence of the C genome in AAC allotriploids leads to a very substantial increase of COs all along the A chromosomes, especially in the vicinity of centromeres that are normally deprived of COs. We also demonstrated that the addition of a C09 chromosome originating either from B. oleracea (CC, 2n=2x=18) or B. napus have contrasted effects on recombination. This latter result points out that regulation of homologous recombination in AAC allotriploids may have changed in the allopolyploid B. napus, as a result of the 7,500 years of genome coevolution in a polyploid context. Together, our findings provide new insights on homologous recombination in allopolyploids as well as the opportunity to break the linkage disequilibrium in the rapeseed breading programs by using allotriploids.

Meiotic homologous recombination by crossovers (COs) is the main mechanism responsible for mixing genetic diversity in plant breeding. However, loci separations are limited due to the strict regulation of the rates and distribution of recombination events along the chromosomes. Indeed, rarely more than two COs occur between homologous chromosomes per meiosis and their distribution is not homogenous in all chromosomic regions, such as the pericentromeric regions that are free of recombination. Exceptions to this rule were observed and linked to different factors such as polyploidy, genetic background or sex meiosis. Accordingly, it was shown that in allotriploid hybrids (AAC, 2n=29), resulting from crosses between Brassica napus (AACC, 2n=38) and B. rapa(AA, 2n=20), COs get a boost along the A genome compared to diploid hybrids (AA, 2n=20). However, the impact on COs distribution as well as the effects of different genetic backgrounds and sex meiosis were still unclear. To that purpose, progenies deriving from 3 diploid and 3 triploid hybrids were used, enabling to study the distribution of 3000 COs per hybrid by the genotyping of 200 SNPs well distributed along A chromosomes (one SNP each 1.2 Mb). Compared to what was previously known, we showed that the presence of the haploid C genome modifies the COs distribution, by generating new recombining regions within A chromosomes. In addition, significant variations were observed between triploid hybrids depending on the genetic background and sex meiosis. These findings may enable to combine new loci by modifying interference and linkage disequilibrium.

Constitutive genomes of allopolyploid species evolve throughout their life span. However, the consequences of long-term alterations on the interdependency between each original genome have not been established. Here, we attempted an approach corresponding to subgenome extraction from a previously sequenced natural allotetraploid, offering a unique opportunity to evaluate plant viability and structural evolution of one of its diploid components. We employed two different strategies to extract the diploid AA component of the Brassica napus variety 'Darmor' (AACC, 2n = 4x = 38) and we assessed the genomic structure of the latest AA plants obtained (after four to five rounds of selection), using a 60K single nucleotide polymorphism Illumina array. Only one strategy was successful and the diploid AA plants that were structurally characterized presented a lower proportion of the B. napus A subgenome extracted than expected. In addition, our analyses revealed that some genes lost in a polyploid context appeared to be compensated for plant survival, either by conservation of genomic regions from B. rapa, used in the initial cross, or by some introgressions from the B. napus C subgenome. We conclude that as little as c. 7500 yr of coevolution could lead to subgenome interdependency in the allotetraploid B. napus as a result of structural modifications.

Background: There are two strategies available for understanding structural and/or functional modifications that took place during the stabilization of polyploid species. The first one allows assessment of events that arose immediately after the formation of a polyploid species by crossing and doubling its parental genomes in order to produce synthetic forms. The second one tries to elucidate the changes that occurred since the polyploid species was created by extracting in the polyploidy one of its parental genome. Objectives: In the present study, we tried to identify the structural rearrangements that occurred since the origin (about 8000 years ago) of oilseed rape (Brassica napus, AACC, 2n=38), which is a natural hybrid between B. rapa (AA, 2n=20) and B. oleracea (CC, 2n=18). To that purpose, we produced an original plant material in which the B. napus A subgenome was extracted. Methods: We used two methods to extract the diploid AA genome from B. napus. Firstly, AAC F1 interspecific hybrids (produced by crosses between B. napus var Darmor and B. rapa) were backcrossed three times to B. napus, and AAC plants were selected at each generation. Secondly, the initial AAC F1 hybrids were crossed to B. rapa and plants with AA genomes were selected for, selfed and also backcrossed to B. napus. After four cycles of such crossing, we selected AA plants with mainly the A genome of B. napus. Using the 60k SNP Illumina microarray and the sequence of B. napus genomes var Darmor, we assessed the genomic structure of the so far extracted B. napus A subgenome. Result: We found that the backcrosses of AAC F1 interspecific hybrids to B. napus (first strategy) could not permit to eliminate the C chromosomes by selfing since the progenies were male sterile. The second strategy allowed production of AA plants with a regular meiosis. We expected more than 68% of Darmor A genome in this plant. To validate this assessment, genomic structure was established by SNP analysis using markers specific of A genome of Darmor and of the B. rapa variety used in the initial crosses. The homozygous or heterozygous stage of each marker physically anchored was determined. Additionally, CDarmor genome regions introduced by homeologous recombination were characterized. Conclusions: From this original material, it will be possible to determine the comparative evolution of the A genome in a diploid and polyploid genetic background. The first data seem to indicate that rearrangements are too large and/or too frequent to obtain 100% A genome of B. napus at the diploid stage. However, functional analyses will allow identification of the rearrangement impacts.

Production of allohexaploid Brassica (2n = AABBCC) is a promising goal for plant breeders due to the potential for hybrid heterosis and useful allelic contributions from all three of the Brassica genomes present in the cultivated diploid (2n = AA, 2n = BB, 2n = CC) and allotetraploid (2n = AABB, 2n = AACC, and 2n = BBCC) crop species (canola, cabbages, mustards). We used high-throughput SNP molecular marker assays, flow cytometry, and fluorescent in situ hybridization (FISH) to characterize a population of putative allohexaploids derived from self-pollination of a hybrid from the novel cross (B. napus × B. carinata) × B. juncea to investigate whether fertile, stable allohexaploid Brassica can be produced. Allelic segregation in the A and C genomes generally followed Mendelian expectations for an F2 population, with minimal nonhomologous chromosome pairing. However, we detected no strong selection for complete 2n = AABBCC chromosome complements, with weak correlations between DNA content and fertility (r(2) = 0.11) and no correlation between missing chromosomes or chromosome segments and fertility. Investigation of next-generation progeny resulting from one highly fertile F2 plant using FISH revealed general maintenance of high chromosome numbers but severe distortions in karyotype, as evidenced by recombinant chromosomes and putative loss/duplication of A- and C-genome chromosome pairs. Our results show promise for the development of meiotically stable allohexaploid lines, but highlight the necessity of selection for 2n = AABBCC karyotypes.