Genetic analysis of opaque2 modifier loci in quality protein maize

Abstract

Quality protein maize (QPM) was created by selecting genetic modifiers that convert the starchy endosperm of an opaque2 (o2) mutant to a hard, vitreous phenotype. Genetic analysis has shown that there are multiple, unlinked o2 modifiers (Opm), but their identity and mode of action are unknown. Using two independently developed QPM lines, we mapped several major Opm QTLs to chromosomes 1, 7 and 9. A microarray hybridization performed with RNA obtained from true breeding o2 progeny with vitreous and opaque kernel phenotypes identified a small group of differentially expressed genes, some of which map at or near the Opm QTLs. Several of the genes are associated with ethylene and ABA signaling and suggest a potential linkage of o2 endosperm modification with programmed cell death.

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

“…5B), showing that the protein absence was not a regulatory effect but due rather to the loss of these genes. Given that the 27-and 50-kD g-zein genes are closely linked (being separated by approximately 27 kb; Holding et al, 2008), the most likely scenario was that a deletion covered both genes, which was confirmed by sequencing exoncaptured genomic DNA (below).…”

“…For identification of DNA sequence deletions in the induced mutants, we combined exon-capture DNA sequencing and RNA sequencing (RNA-seq). Among the opaque variants induced in QPM, one had a deletion that eliminates both the 27-and 50-kD g-zein loci, which are within a significant QPM QTL on chromosome 7 (Holding et al, 2008(Holding et al, , 2011. By demonstrating the dosage-dependent action of 27-kD g-zein in o2 endosperm modification, this mutant highlights the potential of this approach for identifying other o2 modifier genes as well as genes involved in a variety of aspects of endosperm development.…”

“…Although the genetic and biochemical mechanisms responsible for this increase are unknown, the degree of endosperm vitreousness in QPM correlates with the level of 27-kD g-zein protein . Furthermore, the 27-kD g-zein gene, along with the closely linked 50-kD g-zein, maps to the most significant quantitative trait loci (QTL) for endosperm modification located on chromosome 7 Holding et al, 2008Holding et al, , 2011. QPM endosperm accumulates larger numbers of small, g-zein-rich protein bodies.…”

“…The molecular characterization of opaque mutants has shown that vitreous endosperm formation depends on more than just correct abundance and spatial organization of zein proteins (Holding and Larkins, 2006;Holding et al, 2007Holding et al, , 2010Myers et al, 2011;Wang et al, 2012). Furthermore, mapping studies indicate there are several QTLs for endosperm modification in QPM Holding et al, 2008Holding et al, , 2011.…”

Nonredundant Function of Zeins and Their Correct Stoichiometric Ratio Drive Protein Body Formation in Maize Endosperm

“…For RNAi lines with dominant phenotypes, opaque T2 kernels were planted. RNAi and the pZ1C:27g plants were selected for the presence of the transgene using leaf genomic PCR using DNA extracted according to the urea method (Holding et al, 2008). For the 18-DAP stage, kernels were removed, without damaging the base of the kernels, using a razor blade and treated as follows.…”