Deletion Mutagenesis Identifies a Haploinsufficient Role for gamma-Zein in opaque2 Endosperm Modification

Quality Protein Maize (QPM) is a hard kernel variant of the high-lysine mutant opaque2. Using gamma-irradiation, we created opaque QPM variants to identify opaque2 modifier genes and to investigate deletion mutagenesis combined with Illumina sequencing as a maize (Zea mays) functional genomics tool. A K0326Y QPM deletion mutant was null for the 27-and 50-kD gamma-zeins and abolished vitreous endosperm formation. Illumina exon and RNA sequencing revealed a 1.2-megabase pair deletion encompassing the 27-and 50-kD gamma-zein genes on chromosome 7 and a deletion of at least 232 kb on chromosome 9. Protein body number was reduced by over 90%, while protein body size is similar to the wild type. Kernels hemizygous for the gamma-zein deletion had intermediate 27-and 50-kD gamma-zein levels and were semivitreous, indicating haploinsufficiency of these gene products in opaque2 endosperm modification. The gamma-zein deletion further increased lysine in QPM in its homozygous and hemizygous states. This work identifies 27-kD gamma-zein as an opaque2 modifier gene within the largest QPM quantitative trait locus and may suggest the 50-kD gamma-zein also contributes to this quantitative trait locus. It further demonstrates that genome-wide deletions in nonreference maize lines can be identified through a combination of assembly of Illumina reads against the B73 genome and integration of RNA sequencing data.