Identifying and Isolating Meiotic Mutants in a Polyploid Brassica Crop
This chapter provides a detailed description of TILLING and CRISPR-Cas9 approaches for the purpose of studying genes/factors involved in meiotic recombination in the polyploid species B. napus. The TILLING approach involves the screening and identification of EMS-mutagenized M2 B. napus plants. The strategy for high-throughput plant pooling, the set up for microfluidic PCR and sequencing is provided and the parameters for the analysis of sequence results and the detection of mutants are explained. The CRISPR-Cas system relies on the optimal design of guide RNAs and their efficient expression. The procedure for the generation and detection of knockout mutants is described with the aims to simultaneously target homologous genes.
Key wordsBrassica napus Gene editing Meiotic recombination Microfluidic PCR Polyploidy Reverse genetics Targeting induced local lesions in genomes
This work has benefited from a French State grant (reference ANR-10-LABX-0040-SPS) managed by the French National Research Agency under an Investments for the Future program (reference n°ANR-11-IDEX-0003-02). It has also been funded through the ANR project ANR-14-CE19-0004—CROC and with the support of INRA BAP division (Appel à Manifestation d’intérêt 2012; HyperRec). We also want to acknowledge CEA-IbFJ-Genoscope that gave EPGV group access to their Illumina sequencing resources as well as Isabelle Le Clainche, Aurélie Chauveau, Elodie Marquand and Aurélie Canaguier. A.B. was funded by a “Young Scientist Contracts” (CJS) from INRA. A.G. was funded by the Marie-Curie “COMREC” network FP7 ITN-606956.
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