Development of a high-throughput method for the screening of a maize mutant collection

Exploiting genetic variability is at the heart of breeding activities. More than a decade ago, Biogemma has contributed to the creation of a maize mutant collection produced by insertion of Mutator transposon: “the Gene Machine”. After years of recurrent gene-per-gene screenings, we devised a procedure that combines molecular approaches described in the literature and specific in-house optimizations. Hence we improved the preparation of NGS library and the PCRs for the selective enrichment of library fragments encompassing Mutator sequences. Furthermore, we developed custom primers for Illumina sequencing in order to optimize the fraction of informative reads. We also completely automated the analysis with a bioinformatic pipeline that manages every analysis step from raw data to the identification of individual plant carrying the putative germinal insertions. Following the initial method development, a large-scale validation step has been carried out on part of the mutant collection and has proven the reliability of the method. The comprehensive characterization of the “Gene Machine” resource once for all will greatly ease and speed up our reverse genetics studies.