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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.
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Duplication of genes at different time period, through recurrent and frequent polyploidization events, have played a major role in plant evolution, adaptation and diversification. Interestingly, some of the ancestral duplicated genes (referred as paleologs), have been maintained for millions of years, and there is still a poor knowledge of the reasons of their retention, especially when testing the phenotypic effect of individual copies by using functional genetic approaches. To fill this gap, we performed functional genetic (CRISPR-Cas9), physiological, transcriptomic and evolutionary studies to finely investigate this open question, taking the example of the petC gene (involved in cytochrome b6/f and thus impacting photosynthesis) that is present in four paleologous copies in the oilseed crop Brassica napus. RNA-Seq and selective pressure analyses suggested that all paleologous copies conserved the same function and that they were all highly transcribed. Thereafter, the Knock Out (K.O.) of one, several or all petC copies highlighted that all paleologous copies have to be K.O. to suppress the gene function. In addition, we could determine that phenotypic effects in single and double mutants could only be deciphered in high light conditions. Interestingly, we did not detect any significant differences between single mutants K.O. for either the A03 or A09 copy (despite being differentially transcribed), or even between mutants for a single or two petC copies. Altogether, this work revealed that petC paleologs have retained their ancestral function and that the retention of these copies is explained by their compensatory role, especially in optimal environmental conditions.
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Brassica napus , Brassica napus/genética , Genoma de Planta/genética , Genes de Plantas/genética , Genes Duplicados/genética , PoliploidiaRESUMO
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.
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Brassica napus , Brassica napus/genética , Cromossomos de Plantas , Epigenômica , Genoma de Planta , Hibridização in Situ Fluorescente , Melhoramento VegetalRESUMO
Meganucleases are rare cutting enzymes that can generate DNA modifications and are part of the plant genome editing toolkit although they lack versatility. Here, we evaluated the use of two meganucleases, I-SceI and a customized meganuclease, in tomato and oilseed rape. Different strategies were explored for the use of these meganucleases. The activity of a customized and a I-SceI meganucleases was first estimated by the use of a reporter construct GFFP with the target sequences and enabled to demonstrate that both meganucleases can generate double-strand break and HDR mediated recombination in a reporter gene. Interestingly, I-SceI seems to have a higher DSB efficiency than the customized meganuclease: up to 62.5% in tomato and 44.8% in oilseed rape. Secondly, the same exogenous landing pad was introduced in both species. Despite being less efficient compared to I-SceI, the customized meganuclease was able to generate the excision of an exogenous transgene (large deletion of up to 3316 bp) present in tomato. In this paper, we also present some pitfalls to be considered before using meganucleases (e.g., potential toxicity) for plant genome editing.
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Edição de Genes , Solanum lycopersicum , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Genes Reporter , Solanum lycopersicum/genética , TransgenesRESUMO
With the rise of long-read sequencers and long-range technologies, delivering high-quality plant genome assemblies is no longer reserved to large consortia. Not only sequencing techniques, but also computer algorithms have reached a point where the reconstruction of assemblies at the chromosome scale is now feasible at the laboratory scale. Current technologies, in particular long-range technologies, are numerous, and selecting the most promising one for the genome of interest is crucial to obtain optimal results. In this study, we resequenced the genome of the yellow sarson, Brassica rapa cv. Z1, using the Oxford Nanopore PromethION sequencer and assembled the sequenced data using current assemblers. To reconstruct complete chromosomes, we used and compared three long-range scaffolding techniques, optical mapping, Omni-C, and Pore-C sequencing libraries, commercialized by Bionano Genomics, Dovetail Genomics, and Oxford Nanopore Technologies, respectively, or a combination of the three, in order to evaluate the capability of each technology.
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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.
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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.
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Brassica napus , Brassica , Aneuploidia , Brassica/genética , Brassica napus/genética , Genoma de Planta/genética , PoliploidiaRESUMO
BACKGROUND: The combination of long reads and long-range information to produce genome assemblies is now accepted as a common standard. This strategy not only allows access to the gene catalogue of a given species but also reveals the architecture and organization of chromosomes, including complex regions such as telomeres and centromeres. The Brassica genus is not exempt, and many assemblies based on long reads are now available. The reference genome for Brassica napus, Darmor-bzh, which was published in 2014, was produced using short reads and its contiguity was extremely low compared with current assemblies of the Brassica genus. FINDINGS: Herein, we report the new long-read assembly of Darmor-bzh genome (Brassica napus) generated by combining long-read sequencing data and optical and genetic maps. Using the PromethION device and 6 flowcells, we generated â¼16 million long reads representing 93× coverage and, more importantly, 6× with reads longer than 100 kb. This ultralong-read dataset allows us to generate one of the most contiguous and complete assemblies of a Brassica genome to date (contig N50 > 10 Mb). In addition, we exploited all the advantages of the nanopore technology to detect modified bases and sequence transcriptomic data using direct RNA to annotate the genome and focus on resistance genes. CONCLUSION: Using these cutting-edge technologies, and in particular by relying on all the advantages of the nanopore technology, we provide the most contiguous Brassica napus assembly, a resource that will be valuable to the Brassica community for crop improvement and will facilitate the rapid selection of agronomically important traits.
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Brassica napus , Nanoporos , Brassica napus/genética , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , FenótipoRESUMO
[This corrects the article DOI: 10.3389/fpls.2018.00368.].
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Traditionally, reference genomes in crop species rely on the assembly of one accession, thus occulting most of intraspecific diversity. However, rearrangements, gene duplications, and transposable element content may have a large impact on the genomic structure, which could generate new phenotypic traits. Comparing two Brassica rapa genomes recently sequenced and assembled using long-read technology and optical mapping, we investigated structural variants and repetitive content between the two accessions and genome size variation among a core collection. We explored the structural consequences of the presence of large repeated sequences in B. rapa 'Z1' genome vs. the B. rapa 'Chiifu' genome, using comparative genomics and cytogenetic approaches. First, we showed that large genomic variants on chromosomes A05, A06, A09, and A10 are due to large insertions and inversions when comparing B. rapa 'Z1' and B. rapa 'Chiifu' at the origin of important length differences in some chromosomes. For instance, lengths of 'Z1' and 'Chiifu' A06 chromosomes were estimated in silico to be 55 and 29 Mb, respectively. To validate these observations, we compared using fluorescent in situ hybridization (FISH) the two A06 chromosomes present in an F1 hybrid produced by crossing these two varieties. We confirmed a length difference of 17.6% between the A06 chromosomes of 'Z1' compared to 'Chiifu.' Alternatively, using a copy number variation approach, we were able to quantify the presence of a higher number of rDNA and gypsy elements in 'Z1' genome compared to 'Chiifu' on different chromosomes including A06. Using flow cytometry, the total genome size of 12 Brassica accessions corresponding to a B. rapa available core collection was estimated and revealed a genome size variation of up to 16% between these accessions as well as some shared inversions. This study revealed the contribution of long-read sequencing of new accessions belonging to different cultigroups of B. rapa and highlighted the potential impact of differential insertion of repeat elements and inversions of large genomic regions in genome size intraspecific variability.
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Polyploidy or whole genome duplication is a frequent and recurrent phenomenon in flowering plants that has played a major role in their diversification, adaptation and speciation. The adaptive success of polyploids relates to the different evolutionary fates of duplicated genes. In this study, we explored the impact of the whole genome triplication (WGT) event in the Brassiceae tribe on the genes involved in the self-incompatibility (SI) signalling pathway, a mechanism allowing recognition and rejection of self-pollen in hermaphrodite plants. By taking advantage of the knowledge acquired on this pathway as well as of several reference genomes in Brassicaceae species, we determined copy number of the different genes involved in this pathway and investigated their structural and functional evolutionary dynamics. We could infer that whereas most genes involved in the SI signalling returned to single copies after the WGT event (i.e. ARC1, JDP1, THL1, THL2, Exo70A01) in diploid Brassica species, a few were retained in duplicated (GLO1 and PLDα) or triplicated copies (MLPK). We also carefully studied the gene structure of these latter duplicated genes (including the conservation of functional domains and active sites) and tested their transcription in the stigma to identify which copies seem to be involved in the SI signalling pathway. By taking advantage of these analyses, we then explored the putative origin of a contrasted SI phenotype between two Brassica rapa varieties that have been fully sequenced and shared the same S-allele (S60).
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Evolução Biológica , Brassica , Genoma de Planta , Transdução de Sinais , Brassica/genética , Genoma de Planta/genética , Pólen , Transdução de Sinais/genéticaRESUMO
The large French research project GENIUS (2012-2019, https://www6.inra.genius-project_eng/ ) provides a good showcase of current genome editing techniques applied to crop plants. It addresses a large variety of agricultural species (rice, wheat, maize, tomato, potato, oilseed rape, poplar, apple and rose) together with some models (Arabidopsis, Brachypodium, Physcomitrella). Using targeted mutagenesis as its work horse, the project is limited to proof of concept under confined conditions. It mainly covers traits linked to crop culture, such as disease resistance to viruses and fungi, flowering time, plant architecture, tolerance to salinity and plant reproduction but also addresses traits improving the quality of agricultural products for industrial purposes. Examples include virus resistant tomato, early flowering apple and low-amylose starch potato. The wide range of traits illustrates the potential of genome editing towards a more sustainable agriculture through the reduction of pesticides and to the emergence of innovative bio-economy sectors based on custom tailored quality traits.
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Agricultura/tendências , Sistemas CRISPR-Cas/genética , Produtos Agrícolas/genética , Edição de Genes/métodos , Animais , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Brachypodium/genética , Brachypodium/crescimento & desenvolvimento , Bryopsida/genética , Bryopsida/crescimento & desenvolvimento , Produtos Agrícolas/crescimento & desenvolvimento , Genoma de Planta/genética , Mutagênese/genética , FenótipoRESUMO
Several plastid macromolecular protein complexes are encoded by both nuclear and plastid genes. Therefore, cytonuclear interactions are held in place to prevent genomic conflicts that may lead to incompatibilities. Allopolyploidy resulting from hybridization and genome doubling of two divergent species can disrupt these fine-tuned interactions, as newly formed allopolyploid species confront biparental nuclear chromosomes with a uniparentally inherited plastid genome. To avoid any deleterious effects of unequal genome inheritance, preferential transcription of the plastid donor over the other donor has been hypothesized to occur in allopolyploids. We used Brassica as a model to study the effects of paleopolyploidy in diploid parental species, as well as the effects of recent and ancient allopolyploidy in Brassica napus, on genes implicated in plastid protein complexes. We first identified redundant nuclear copies involved in those complexes. Compared with cytosolic protein complexes and with genome-wide retention rates, genes involved in plastid protein complexes show a higher retention of genes in duplicated and triplicated copies. Those redundant copies are functional and are undergoing strong purifying selection. We then compared transcription patterns and sequences of those redundant gene copies between resynthesized allopolyploids and their diploid parents. The neopolyploids showed no biased subgenome expression or maternal homogenization via gene conversion, despite the presence of some non-synonymous substitutions between plastid genomes of parental progenitors. Instead, subgenome dominance was observed regardless of the maternal progenitor. Our results provide new insights on the evolution of plastid protein complexes that could be tested and generalized in other allopolyploid species.
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Brassica/genética , Cloroplastos/genética , Duplicação Gênica/genética , Genomas de Plastídeos/genética , Brassica napus/genética , Evolução Molecular , PoliploidiaRESUMO
Allopolyploidy, combining interspecific hybridization with whole genome duplication, has had significant impact on plant evolution. Its evolutionary success is related to the rapid and profound genome reorganizations that allow neoallopolyploids to form and adapt. Nevertheless, how neoallopolyploid genomes adapt to regulate their expression remains poorly understood. The hypothesis of a major role for small noncoding RNAs (sRNAs) in mediating the transcriptional response of neoallopolyploid genomes has progressively emerged. Generally, 21-nt sRNAs mediate posttranscriptional gene silencing by mRNA cleavage, whereas 24-nt sRNAs repress transcription (transcriptional gene silencing) through epigenetic modifications. Here, we characterize the global response of sRNAs to allopolyploidy in Brassica, using three independently resynthesized Brassica napus allotetraploids originating from crosses between diploid Brassica oleracea and Brassica rapa accessions, surveyed at two different generations in comparison with their diploid progenitors. Our results suggest an immediate but transient response of specific sRNA populations to allopolyploidy. These sRNA populations mainly target noncoding components of the genome but also target the transcriptional regulation of genes involved in response to stresses and in metabolism; this suggests a broad role in adapting to allopolyploidy. We finally identify the early accumulation of both 21- and 24-nt sRNAs involved in regulating the same targets, supporting a posttranscriptional gene silencing to transcriptional gene silencing shift at the first stages of the neoallopolyploid formation. We propose that reorganization of sRNA production is an early response to allopolyploidy in order to control the transcriptional reactivation of various noncoding elements and stress-related genes, thus ensuring genome stability during the first steps of neoallopolyploid formation.
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Brassica napus/genética , Especiação Genética , Pequeno RNA não Traduzido/metabolismo , Tetraploidia , Brassica napus/metabolismo , Elementos de DNA TransponíveisRESUMO
Plant genomes are often characterized by a high level of repetitiveness and polyploid nature. Consequently, creating genome assemblies for plant genomes is challenging. The introduction of short-read technologies 10 years ago substantially increased the number of available plant genomes. Generally, these assemblies are incomplete and fragmented, and only a few are at the chromosome scale. Recently, Pacific Biosciences and Oxford Nanopore sequencing technologies were commercialized that can sequence long DNA fragments (kilobases to megabase) and, using efficient algorithms, provide high-quality assemblies in terms of contiguity and completeness of repetitive regions1-4. However, even though genome assemblies based on long reads exhibit high contig N50s (>1 Mb), these methods are still insufficient to decipher genome organization at the chromosome level. Here, we describe a strategy based on long reads (MinION or PromethION sequencers) and optical maps (Saphyr system) that can produce chromosome-level assemblies and demonstrate applicability by generating high-quality genome sequences for two new dicotyledon morphotypes, Brassica rapa Z1 (yellow sarson) and Brassica oleracea HDEM (broccoli), and one new monocotyledon, Musa schizocarpa (banana). All three assemblies show contig N50s of >5 Mb and contain scaffolds that represent entire chromosomes or chromosome arms.
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Brassica rapa/genética , Brassica/genética , Mapeamento Cromossômico/métodos , Cromossomos de Plantas/genética , Genoma de Planta/genética , Nanoporos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Óptica e Fotônica/métodos , Sequências Repetitivas de Ácido Nucleico/genéticaRESUMO
Polyploidization is a widespread phenomenon, especially in flowering plants that have all undergone at least one event of whole genome duplication during their evolutionary history. Consequently, a large range of plants, including many of the world's crops, combines more than two sets of chromosomes originating from the same (autopolyploids) or related species (allopolyploids). Depending on the polyploid formation pathway, different patterns of recombination will be promoted, conditioning the level of heterozygosity. A polyploid population harboring a high level of heterozygosity will produce more genetically diverse progenies. Some of these individuals may show a better adaptability to different ecological niches, increasing their chance for successful establishment through natural selection. Another condition for young polyploids to survive corresponds to the formation of well-balanced gametes, assuring a sufficient level of fertility. In this review, we discuss the consequences of polyploid formation pathways, meiotic behavior and recombination regulation on the speciation success and maintenance of polyploid species.
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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.
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Leptosphaeria maculans, the causal agent of blackleg disease, interacts with Brassica napus (oilseed rape, canola) and other Brassica hosts in a gene-for-gene manner. The avirulence gene AvrLmJ1 has been cloned previously and shown to interact with an unidentified Brassica juncea resistance gene. In this study, we show that the AvrLmJ1 gene maps to the same position as the AvrLm5 locus. Furthermore, isolates complemented with the AvrLmJ1 locus confer avirulence towards B. juncea genotypes harbouring Rlm5. These findings demonstrate that AvrLmJ1 is AvrLm5 and highlight the need for shared resources to characterize accurately avirulence and/or resistance genes.
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Ascomicetos/genética , Ascomicetos/patogenicidade , Brassica napus/microbiologia , Resistência à Doença/genética , Doenças das Plantas/microbiologia , Genes Fúngicos/genética , VirulênciaRESUMO
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.
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Brassica/genética , Troca Genética , Variação Genética , Meiose/genética , Brassica/crescimento & desenvolvimento , Genoma de Planta , Polimorfismo de Nucleotídeo Único , Poliploidia , Recombinação GenéticaRESUMO
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 â¼70% 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.