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International audience; 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.

Simple Summary The selection of varieties more resilient to disease and climate change requires generating new genetic diversity for breeding. The main mechanism for reshuffling genetic information is through the recombination of chromosomes during meiosis. We showed in oilseed rape (Brassica napus, AACC, 2n = 4x = 38), which is a natural hybrid formed from a cross between turnip (B. rapa, AA, 2n = 2x = 20) and cabbage (B. oleracea, CC, 2n = 2x = 18), that there is significantly more crossovers occurring along the entire A chromosomes in allotriploid AAC (crossbetween B. napus and B. rapa) than in diploid AA or allotetraploid AACC hybrids. We demonstrated that these allotriploid AAC hybrids are highly efficient to introduce new variability within oilseed rape varieties, notably by enabling the introduction of small genomic regions carrying genes controlling agronomically interesting traits. Abstract 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.

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.

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.

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.

support from the 'Plate-forme Génomique Environnementale et Fonctionnelle' (OSUR: INEE-CNRS) and the Genouest Bioinformatic Plateform (University of Rennes 1); International audience; † Background and Aims To date chloroplast genomes are available only for members of the non-protein amino acidaccumulating clade (NPAAA) Papilionoid lineages in the legume family (i.e. Millettioids, Robinoids and the 'inverted repeat-lacking clade', IRLC). It is thus very important to sequence plastomes from other lineages in order to better understand the unusual evolution observed in this model flowering plant family. To this end, the plastome of a lupine species, Lupinus luteus, was sequenced to represent the Genistoid lineage, a noteworthy but poorly studied legume group. † Methods The plastome of L. luteus was reconstructed using Roche-454 and Illumina next-generation sequencing. Its structure, repetitive sequences, gene content and sequence divergence were compared with those of other Fabaceae plastomes. PCR screening and sequencing were performed in other allied legumes in order to determine the origin of a large inversion identified in L. luteus. † Key Results The first sequenced Genistoid plastome (L. luteus: 155 894 bp) resulted in the discovery of a 36-kb inversion, embedded within the already known 50-kb inversion in the large single-copy (LSC) region of the Papilionoideae. This inversion occurs at the base or soon after the Genistoid emergence, and most probably resulted from a flip-flop recombination between identical 29-bp inverted repeats within two trnS genes. Comparative analyses of the chloroplast gene content of L. luteus vs. Fabaceae and extra-Fabales plastomes revealed the loss of the plastid rpl22 gene, and its functional relocation to the nucleus was verified using lupine transcriptomic data. An investigation into the evolutionary rate of coding and non-coding sequencesamong legume plastomes resulted in the identification of remarkably variable regions. †Conclusions This study resulted in the discovery of a novel, major 36-kb inversion, specific to the Genistoids. Chloroplast mutational hotspots were also identified, which contain novel and potentially informative regions for molecular evolutionary studies at various taxonomic levels in the legumes. Taken together, the results provide new insights into the evolutionary landscape of the legume plastome.

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.

DNA of plastid (chloroplast) origin comprises between 1% and 10% of the mitochondrial genomes of higher plants, but functions are currently considered to be limited to rare instances where plastid tRNA genes have replaced their mitochondrial counterparts, where short patches of mitochondrial genes evolved using their homologous plastidic copies by gene conversion or where a new promoter region is created. Here, we show that, in some angiosperms, plastid-derived DNA in mitochondrial genomes (also called mtpt for mitochondrial plastid DNA) contributes codons to unrelated mitochondrial protein-coding sequences and may also have a role in posttranscriptional RNA processing. We determined that these transfers of plastid DNA occurred a few to 150 Ma and that mtpts can sometimes remain dormant many millions of years before contributing to the mitochondrial proteome.

With 1 figure and 3 tables Abstract The Rosodae supertribe is composed of economically important species (strawberry, rose and raspberry). In order to extend the repertoire of markers for optimizing genetic mapping and synteny within this supertribe, we developed 20 functional markers from two Fragaria cDNA libraries enriched in AG/AC microsatellite motifs. We tested their transferability within the Rosodae supertribe, including two reference diploid and octoploid strawberry segregating populations. Out of these 20 functional markers, 70% revealed polymorphism in both diploid and octoploid Fragaria segregating populations and were mapped on the diploid genetic map to provide useful information for further marker-assisted selection. These 20 EST-SSRs were also tested in 17 wild Fragaria species and displayed a high cross-species transferability (mean of 95%), providing new markers for further diversity studies within the genus. These markers were also transferable to the phylogenetically close Potentilla genus, whereas their transferability was lower for the polyploid Duchesnea indica species and for the more distant Rosa genus. Their use for genetic mapping within the Rosodae will further permit a comparison of genetic information among the different genera.

Gene and whole-genome duplications are widespread in plant nuclear genomes, resulting in sequence heterogeneity. Identification of duplicated genes may be particularly challenging in highly redundant genomes, especially when there are no diploid parents as a reference. Here, we developed a pipeline to detect the different copies in the ribosomal RNA gene family in the hexaploid grass Spartina maritima from next-generation sequencing (Roche-454) reads. The heterogeneity of the different domains of the highly repeated 45S unit was explored by identifying single nucleotide polymorphisms (SNPs) and assembling reads based on shared polymorphisms. SNPs were validated using comparisons with Illumina sequence data sets and by cloning and Sanger (re)sequencing. Using this approach, 29 validated polymorphisms and 11 validated haplotypes were reported (out of 34 and 20, respectively, that were initially predicted by our program). The rDNA domains of S. maritima have similar lengths as those found in other Poaceae, apart from the 5′-ETS, which is approximately two-times longer in S. maritima. Sequence homogeneity was encountered in coding regions and both internal transcribed spacers (ITS), whereas high intragenomic variability was detected in the intergenic spacer (IGS) and the external transcribed spacer (ETS). Molecular cytogenetic analysis by fluorescent in situ hybridization (FISH) revealed the presence of one pair of 45S rDNA signals on the chromosomes of S. maritima instead of three expected pairs for a hexaploid genome, indicating loss of duplicated homeologous loci through the diploidization process. The procedure developed here may be used at any ploidy level and using different sequencing technologies.

Nuclear genomes of eukaryotes are bombarded by a continuous deluge of organellar DNA which contributes significantly to eukaryote evolution. Here, we present a new PCR-based method that allows the specific amplification of nuclear integrants of organellar DNA (norgs) by exploiting recent deletions present in organellar genome sequences. We have used this method to amplify nuclear integrants of plastid DNA (nupts) from the nuclear genomes of several nicotiana species and to study the evolutionary forces acting upon these sequences. The role of nupts in endosymbiotic evolution and the different genetic factors influencing the time available for a chloroplastic gene to be functionally relocated in the nucleus are discussed.

International audience

International audience