Deletion mutagenesis identifies a haploinsufficient role for γ-zein in opaque2 endosperm modification

Abstract

Quality Protein Maize (QPM) is a hard kernel variant of the high-lysine mutant opaque2. Using γ-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 γ-zeins and abolished vitreous endosperm formation. Illumina exon and RNA sequencing revealed a 1.2-megabase pair deletion encompassing the 27- and 50-kD γ-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 γ-zein deletion had intermediate 27- and 50-kD γ-zein levels and were semivitreous, indicating haploinsufficiency of these gene products in opaque2 endosperm modification. The γ-zein deletion further increased lysine in QPM in its homozygous and hemizygous states. This work identifies 27-kD γ-zein as an opaque2 modifier gene within the largest QPM quantitative trait locus and may suggest the 50-kD γ-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.

Figure 1.

M3 ear phenotypes of K0326Y QPM control ear and segregating and homozygous K0326Y deletion line 107. Inserts in A and C show light box phenotypes.

Figure 2.

A, SDS-PAGE analysis of zein proteins from mature kernel of deletion line 107 compared with K0326Y QPM and the W64A wild type and W64Ao2. B, Western analysis of zein proteins loaded at 1:1,000 dilution of gel shown in A, probed with total α-zein antiserum (1:10,000) and anti-27-kD γ-zein antiserum (1:2,000). WT, Wild type. [See online article for color version of this figure.]

Figure 3.

Low-magnification TEM images from immunogold labeling showing distribution of protein bodies in representative 18-DAP endosperm of the wild type, K0326Y QPM, and QPM deletion line 107. Subaleurone cell layers are labeled with 2nd, 3rd, and 4th. Cell walls are labeled with cw>, starch grains are labeled with S, and selected protein bodies are labeled with vertical arrows. Depicted protein bodies in C show connections to the endoplasmic reticulum. Bar = 5 μm. WT, Wild type.

Figure 4.

Immunogold TEM analysis showing α- and γ-zein distribution in protein bodies of fourth subaleurone cell layer of 18-DAP endosperm of the wild type, K0326Y QPM, and QPM deletion line 107. Bar = 1 μm. WT, Wild type.

Figure 5.

A, RT-PCR showing γ-zein transcript accumulation. B, Genomic PCR analysis of γ-zein genes.

Figure 6.

SDS-PAGE and kernel opacity analysis showing haploinsufficiency of γ-zein for endosperm modification. Selected kernels from a segregating ear are shown. [See online article for color version of this figure.]

Figure 7.

Exon-seq and genomic and RT-PCR verification of deletion on chromosome 7.02. Illumina reads column and PCR columns show nonmutagenized K0326Y versus line 107. Most gene identifications show absence of Illumina reads in more than one exon (denoted by multiple sets of coordinates). GRMZM2G117230 and 429842 are the 5′ and 3′ flanking intact genes. Genes with an asterisk are either low-abundance transcripts, endosperm nonexpressed genes, or pseudogenes.

Figure 8.

Exon-seq and genomic and RT-PCR verification of deletion on chromosome 9.03. Illumina reads column and PCR columns show nonmutagenized K0326Y versus line 107. Most gene identifications show absence of Illumina reads in more than one exon (denoted by multiple sets of coordinates). GRMZM2G398628 and 435380 are the two nondeleted genes flanking the 5′ end of the deletion, and GRMZM2G499214 and 108302 are the two nearest nondeleted genes flanking the 3′ end of the deletion. Genes with an asterisk are either low-abundance transcripts, endosperm nonexpressed genes, or pseudogenes.