A population of deletion mutants and an integrated mapping and exome-seq pipeline for gene discovery in maize

To better understand maize endosperm filling and maturation, we used gamma irradiation of the B73 maize reference line to generate mutants with opaque endosperm and reduced kernel fill phenotypes, and created a population of 1788 lines including 47 Mo17 × F2s showing stable, segregating and viable kernel phenotypes. For molecular characterization of the mutants, we developed a novel functional genomics platform that combined Bulked Segregant RNA and Exome sequencing (BSREx-seq) to map causative mutations and identify candidate genes within mapping intervals. To exemplify the utility of the mutants and provide proof-ofconcept for the bioinformatics platform, we present detailed characterization of line 937; an opaque mutant harboring a 6203-bp in-frame deletion covering six exons within the Opaque-1 gene. In addition, we describe mutant line 146 which contains a 4.8 Kb intra-gene deletion within the Sugary-1 gene and line 916 in which an 8.6 Kb deletion knocks out a Cyclin A2 gene. The publically available algorithm developed in this work improves the identification of causative deletions and its corresponding gaps within mapping peaks. This study demonstrates the utility of gamma irradiation for forward genetics in large non-dense genomes such as maize since deletions often affect single genes. Furthermore, we show how this classical mutagenesis method becomes applicable for functional genomics when combined with state-of-the-art genomics tools