Brassica genomics (Genome assembly) and new sequencing technologies

Coordinato: Cyril Falentin et Mathieu Rousseau-Gueutin

Contacts : Cyril.Falentin@inrae.fr etMathieu.Rousseau-Gueutin@inrae.fr

Recherch

Context and Issues

Plant genomes are characterized by a high level of repetitiveness and polyploid nature, Consequently, creating genome assemblies for plant genomes is difficult using short-read technologies: these assemblies are generally incomplete and fragmented. With the recent advent of new sequencing technologies that can sequence long DNA fragments as well as the use of optical maps, it is now possible to produce chromosome-scale assemblies (with a high contiguity and completeness of repetitive regions) in a relatively short amount of time and with low funds. In the last years, in tight collaboration with the Bioinformatics RD and Sequencing Team from Genoscope (Evry, France), we developed in-house protocols for high-molecular weight DNA extraction and used a strategy based on long reads (MinION or PromethION sequencers, Oxford Nanopore Technologies) and optical maps (Saphyr system, Bionano Genomics) to produce chromosome-level assemblies of different Brassica varieties, containing highly repeated and complex regions such as centromeres or telomeres. Such high-quality assemblies are particularly important for the identification and fine understanding of the regulation of genes involved in important agronomic traits. In addition to Brassica genome assemblies, we also develop or adapt protocols to produce other types of NGS data (such as HiC, BS-Seq, ChIP-Seq, Direct RNA or Small RNA Seq) in order to better understand the evolutionary dynamic of duplicated genes and genomes and its role in modifying important traits, such as meiotic recombination.

 Methodology 

• Extraction of high molecular-weight genomic DNA
• Use of various NGS Sequencing technologies (Illumina, Oxford Nanopore Technologies)
• Optical mapping (Bionano Genomics)
• Genotyping (Illumina SNP array)

Main Results

Since a few years, we dedicated important amount of time and energy to develop or adapt protocols for new sequencing technologies. From this work, mainly developed through a tight collaboration with Bioinformatics RD and Sequencing Team from Genocope (Evry, France), we produced chromosome scale assemblies of various Brassica genotypes using long reads sequencing and optical maps (Belser et al. 2018; Rousseau-Gueutin et al. 2020; Istace et al. 2021). Compared to previous assemblies obtained using short-reads sequencing, these assemblies are far more complete and contiguous, even in highly repeated regions such as centromeres. As an example, the comparison of the last B. napus cv Darmor-bzh assembly based on these new technologies with the previous one based on short reads, we observed on average an increase of sequence assembly and content in pericentromeric regions by 80 and 24 folds, respectively. The high improvement of the genome assembly will definitely facilitate the identification of candidate genes involved in traits of interests. In addition, as we could observe that there are high intraspecific variations (in terms of genome structure and gene content, especially for copy number variation: Boutte et al. 2020), it is crucial to produce several high-quality reference assemblies for a given species. For that reason, we are currently assembling genomes of various Brassica genotypes that are important in various projects developed in our research unit, such as those related to recombination or the resistance to biotic or abiotic stresses. These different assemblies will definitely fasten the identification of the genes (alleles) involved in these important agronomic traits.

Partners

• CEA, Genoscope, Evry, France (teams ‘Research and Development Bioinformatics and Sequencing’)

Funding and Support (last 5 years)

• France Génomique ‘Polysuccess’ (2014-2020). How a polyploid becomes a new species: Brassica model (P.I.: A.M. Chèvre)

• Istace B., Belser C., Falentin C., Labadie K., Boideau F., Deniot G., Maillet L., Cruaud C., Bertrand L., Chèvre A.M., Wincker P., Rousseau-Gueutin M., Aury J.M. (2021) Sequencing and Chromosome-Scale Assembly of Plant Genomes, Brassica rapa as a Use Case. Biology 10(8), 732
• Rousseau-Gueutin M; Belser C; Da Silva C; Richard G; Istace B; Cruaud C; Falentin C; Boideau F; Boutte J; Delourme R; Deniot G; Engelen S; de Carvalho JF; Lemainque A; Maillet L; Morice J; Wincker P; Denoeud F; Chèvre A; Aury JM (2020): "Long-reads assembly
  of the Brassica napus reference genome, Darmor-bzh" GigaScience 9 (12) giaa137
• Boutte J, Maillet L, Chaussepied T, Letort S, Aury JM, Belser C, Boideau F, Brunet A, Coriton O, Deniot G, Falentin C, Huteau V, Lodé-Taburel M, Morice J, Trotoux G, Chèvre AM, Rousseau-Gueutin M, Ferreira de Carvalho (2020)  Genome Size Variation and Comparative Genomics Reveal Intraspecific Diversity in Brassica rapa. Front Plant Sci 11: 577536.
• Belser C., Istace B., Denis E., Dubarry M., Baurens F.-C., Falentin C., Genete M., Berrabah W., Chèvre A. M., Delourme R., Deniot G., Denoeud F., Duffé P., Engelen S., Lemainque A., Manzanares-Dauleux M. J., Martin G., Morice J., Noel B., Vekemans X., D’Hont A., Rousseau-Gueutin M., Barbe V., Cruaud C., Wincker P. & Aury J.-M. (2018). Chromosome-scale assemblies of plant genomes using nanopore long reads and optical maps. Nature Plants, 4(11), 879-887.  https://doi.org/10.1038/s41477-018-0289-4

Book Chapters 

• Falentin C. (2018). Séquençage d'ADN grande longueur par la technique Nanopore. In D. Tagu, S. Jaubert-Possamai, & A. Méreau (Eds.), Principes des techniques de biologie moléculaire et génomique (pp. 193-196): Quae.
• Falentin C. (2018). Cartographie optique de génomes (Optical Mapping). In D. Tagu, S. Jaubert-Possamai, & A. Méreau (Eds.), Principes des techniques de biologie moléculaire et génomique (pp. 204-207): Quae.
• Falentin C. (2018). Séquençage d'ADN grande longueur "synthétique" par la technique 10X Genomics. In D. Tagu, S. Jaubert-Possamai, & A. Méreau (Eds.), Principes des techniques de biologie moléculaire et génomique (pp. 201-203): Quae.
• Falentin C. (2018). Séquençage d'ADN par la technique Illumina. In D. Tagu, S. Jaubert-Possamai, & A. Méreau (Eds.), Principes des techniques de biologie moléculaire et génomique (pp. 186-189): Quae.
• Falentin C. (2018). Séquençage d'ADN par la technique Ion Torrent^TM. In D. Tagu, S. Jaubert-Possamai, & A. Méreau (Eds.), Principes des techniques de biologie moléculaire et génomique (pp. 197-200): Quae.
• Falentin C. (2018). Séquençage d'ADN par la technique Single Molecule real Time (SMRT) : PacBio. In D. Tagu, S. Jaubert-Possamai, & A. Méreau (Eds.), Principes des techniques de biologie moléculaire et génomique (pp. 190-192): Quae.