Cyril Falentin

Early allopolyploid evolution in the post-neolithic Brassica napus oilseed genome

The genomic origins of rape oilseed Many domesticated plants arose through the meeting of multiple genomes through hybridization and genome doubling, known as polyploidy. Chalhoub et al. sequenced the polyploid genome of Brassica napus, which originated from a recent combination of two distinct genomes approximately 7500 years ago and gave rise to the crops of rape oilseed (canola), kale, and rutabaga. B. napus has undergone multiple events affecting differently sized genetic regions where a gene from one progenitor species has been converted to the copy from a second progenitor species. Some of these gene conversion events appear to have been selected by humans as part of the process of domestication and crop improvement. Science, this issue p. 950 The polyploid genome of oilseed rape exhibits evolution through homologous gene conversion. Oilseed rape (Brassica napus L.) was formed ~7500 years ago by hybridization between B. rapa and B. oleracea, followed by chromosome doubling, a process known as allopolyploidy. Together with more ancient polyploidizations, this conferred an aggregate 72× genome multiplication since the origin of angiosperms and high gene content. We examined the B. napus genome and the consequences of its recent duplication. The constituent An and Cn subgenomes are engaged in subtle structural, functional, and epigenetic cross-talk, with abundant homeologous exchanges. Incipient gene loss and expression divergence have begun. Selection in B. napus oilseed types has accelerated the loss of glucosinolate genes, while preserving expansion of oil biosynthesis genes. These processes provide insights into allopolyploid evolution and its relationship with crop domestication and improvement.

Identification of the fertility restoration locus, Rfo, in radish, as a member of the pentatricopeptide‐repeat protein family

Ogura cytoplasmic male sterility (CMS) in radish (Raphanus sativus) is caused by an aberrant mitochondrial gene, Orf138, that prevents the production of functional pollen without affecting female fertility. Rfo, a nuclear gene that restores male fertility, alters the expression of Orf138 at the post‐transcriptional level. The Ogura CMS/Rfo two‐component system is a useful model for investigating nuclear–cytoplasmic interactions, as well as the physiological basis of fertility restoration. Using a combination of positional cloning and microsynteny analysis of Arabidopsis thaliana and radish, we genetically and physically delimited the Rfo locus to a 15‐kb DNA segment. Analysis of this segment shows that Rfo is a member of the pentatricopeptide repeat (PPR) family. In Arabidopsis, this family contains more than 450 members of unknown function, although most of them are predicted to be targeted to mitochondria and chloroplasts and are thought to have roles in organellar gene expression.

Integration of linkage maps for the Amphidiploid Brassica napus and comparative mapping with Arabidopsis and Brassica rapa

BackgroundThe large number of genetic linkage maps representing Brassica chromosomes constitute a potential platform for studying crop traits and genome evolution within Brassicaceae. However, the alignment of existing maps remains a major challenge. The integration of these genetic maps will enhance genetic resolution, and provide a means to navigate between sequence-tagged loci, and with contiguous genome sequences as these become available.ResultsWe report the first genome-wide integration of Brassica maps based on an automated pipeline which involved collation of genome-wide genotype data for sequence-tagged markers scored on three extensively used amphidiploid Brassica napus (2n = 38) populations. Representative markers were selected from consolidated maps for each population, and skeleton bin maps were generated. The skeleton maps for the three populations were then combined to generate an integrated map for each LG, comparing two different approaches, one encapsulated in JoinMap and the other in MergeMap. The BnaWAIT_01_2010a integrated genetic map was generated using JoinMap, and includes 5,162 genetic markers mapped onto 2,196 loci, with a total genetic length of 1,792 cM. The map density of one locus every 0.82 cM, corresponding to 515 Kbp, increases by at least three-fold the locus and marker density within the original maps. Within the B. napus integrated map we identified 103 conserved collinearity blocks relative to Arabidopsis, including five previously unreported blocks. The BnaWAIT_01_2010a map was used to investigate the integrity and conservation of order proposed for genome sequence scaffolds generated from the constituent A genome of Brassica rapa.ConclusionsOur results provide a comprehensive genetic integration of the B. napus genome from a range of sources, which we anticipate will provide valuable information for rapeseed and Canola research.

Molecular and phenotypic characterization of near isogenic lines at QTL for quantitative resistance to Leptosphaeria maculans in oilseed rape (Brassica napus L.).

The most common and effective way to control phoma stem canker (blackleg) caused by Leptosphaeria maculans in oilseed rape (Brassica napus) is by breeding resistant cultivars. Specific resistance genes have been identified in B. napus and related species but in some B. napus cultivars resistance is polygenic [mediated by quantitative trait loci (QTL)], postulated to be race non-specific and durable. The genetic basis of quantitative resistance in the French winter oilseed rape ‘Darmor’, which was derived from ‘Jet Neuf’, was previously examined in two genetic backgrounds. Stable QTL involved in blackleg resistance across year and genetic backgrounds were identified. In this study, near isogenic lines (NILs) were produced in the susceptible background ‘Yudal’ for four of these QTL using marker-assisted selection. Various strategies were used to develop new molecular markers, which were mapped in these QTL regions. These were used to characterize the length and homozygosity of the ‘Darmor-bzh’ introgressed segment in the NILs. Individuals from each NIL were evaluated in blackleg disease field trials and assessed for their level of stem canker in comparison to the recurrent line ‘Yudal’. The effect of QTL LmA2 was clearly validated and to a lesser extent, QTL LmA9 also showed an effect on the disease level. This work provides valuable material that can be used to study the mode of action of genetic factors involved in L. maculans quantitative resistance.

Homoeologous duplicated regions are involved in quantitative resistance of Brassica napus to stem canker

BackgroundSeveral major crop species are current or ancient polyploids. To better describe the genetic factors controlling traits of agronomic interest (QTL), it is necessary to understand the structural and functional organisation of these QTL regions in relation to genome duplication. We investigated quantitative resistance to the fungal disease stem canker in Brassica napus, a highly duplicated amphidiploid species, to assess the proportion of resistance QTL located at duplicated positions.ResultsGenome-wide association analysis on a panel of 116 oilseed rape varieties genotyped with 3228 SNP indicated that 321 markers, corresponding to 64 genomic regions, are associated with resistance to stem canker. These genomic regions are relatively equally distributed on the A (53%) and C (47%) genomes of B. napus. Overall, 44% of these regions (28/64) are duplicated homoeologous regions. They are located in duplications of six (E, J, R, T, U and W) of the 24 ancestral blocks that constitute the B. napus genome. Overall, these six ancestral blocks have 34 duplicated copies in the B.napus genome. Almost all of the duplicated copies (82% of the 34 regions) harboured resistance associated markers for stem canker resistance, which suggests structural and functional conservation of genetic factors involved in this trait in B. napus.ConclusionsOur study provides information on the involvement of duplicated loci in the control of stem canker resistance in B. napus. Further investigation of the similarity/divergence in sequence and gene content of these duplicated regions will provide insight into the conservation and allelic diversity of the underlying genes.

A Dominant Point Mutation in a RINGv E3 Ubiquitin Ligase Homoeologous Gene Leads to Cleistogamy in Brassica napus

This work examines a Brassica napus mutant with a petal-closed phenotype, finding that it is caused by a dominant mutation in a RINGv E3 ubiquitin ligase homoeologous gene that is highly conserved across eukaryotes. In the allopolyploid Brassica napus, we obtained a petal-closed flower mutation by ethyl methanesulfonate mutagenesis. Here, we report cloning and characterization of the Bn-CLG1A (CLG for cleistogamy) gene and the Bn-clg1A-1D mutant allele responsible for the cleistogamy phenotype. Bn-CLG1A encodes a RINGv E3 ubiquitin ligase that is highly conserved across eukaryotes. In the Bn-clg1A-1D mutant allele, a C-to-T transition converts a Pro at position 325 to a Leu (P325L), causing a dominant mutation leading to cleistogamy. B. napus and Arabidopsis thaliana plants transformed with a Bn-clg1A-1D allele show cleistogamous flowers, and characterization of these flowers suggests that the Bn-clg1A-1D mutation causes a pronounced negative regulation of cutin biosynthesis or loading and affects elongation or differentiation of petal and sepal cells. This results in an inhibition or a delay of petal development, leading to folded petals. A homoeologous gene (Bn-CLG1C), which shows 99.5% amino acid identity and is also constitutively and equally expressed to the wild-type Bn-CLG1A gene, was also identified. We showed that P325L is not a loss-of-function mutation and did not affect expression of Bn-clg1A-1D or Bn-CLG1C. Our findings suggest that P325L is a gain-of-function semidominant mutation, which led to either hyper- or neofunctionalization of a redundant homoeologous gene.

The Impact of Open Pollination on the Structural Evolutionary Dynamics, Meiotic Behavior and Fertility of Resynthesized Allotetraploid Brassica napus L.

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.

Comparative genomic analysis of duplicated homoeologous regions involved in the resistance of Brassica napus to stem canker.

All crop species are current or ancient polyploids. Following whole genome duplication, structural and functional modifications result in differential gene content or regulation in the duplicated regions, which can play a fundamental role in the diversification of genes underlying complex traits. We have investigated this issue in Brassica napus, a species with a highly duplicated genome, with the aim of studying the structural and functional organization of duplicated regions involved in quantitative resistance to stem canker, a disease caused by the fungal pathogen Leptosphaeria maculans. Genome-wide association analysis on two oilseed rape panels confirmed that duplicated regions of ancestral blocks E, J, R, U, and W were involved in resistance to stem canker. The structural analysis of the duplicated genomic regions showed a higher gene density on the A genome than on the C genome and a better collinearity between homoeologous regions than paralogous regions, as overall in the whole B. napus genome. The three ancestral sub-genomes were involved in the resistance to stem canker and the fractionation profile of the duplicated regions corresponded to what was expected from results on the B. napus progenitors. About 60% of the genes identified in these duplicated regions were single-copy genes while less than 5% were retained in all the duplicated copies of a given ancestral block. Genes retained in several copies were mainly involved in response to stress, signaling, or transcription regulation. Genes with resistance-associated markers were mainly retained in more than two copies. These results suggested that some genes underlying quantitative resistance to stem canker might be duplicated genes. Genes with a hydrolase activity that were retained in one copy or R-like genes might also account for resistance in some regions. Further analyses need to be conducted to indicate to what extent duplicated genes contribute to the expression of the resistance phenotype.

Chromosome-scale assemblies of plant genomes using nanopore long reads and optical maps

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.Assembling genomes to chromosome scale remains a challenge. Now, a study reports a strategy based on nanopore long reads and optical maps and uses it to produce high-quality chromosome-scale assemblies for the genomes of yellow sarson, broccoli and banana.