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Title: Deletion mutagenesis identifies a haploinsufficient role for gamma-zein in opaque-2 endosperm modification

item YUAN, LINGLING - University Of Nebraska
item DOU, YONGCHAO - University Of Nebraska
item Kianian, Shahryar
item ZHANG, CHI - University Of Nebraska
item HOLDING, DAVID - University Of Nebraska

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/24/2014
Publication Date: 1/24/2014
Citation: Yuan, L., Dou, Y., Kianian, S., Zhang, C., Holding, D.R. 2014. Deletion mutagenesis identifies a haploinsufficient role for gamma-zein in opaque-2 endosperm modification. Plant Physiology. 164:119-130.

Interpretive Summary: The methods we developed using gamma irradiation in generating mutants was applied to maize. The utility of this approach was tested by identifying mutants that modify quality protein maize (QPM). QPM is a variant of maize kernel with much higher lysine content being more beneficial to human consumption due to its more balanced amino acid levels. Analysis of mutants generated indicate that the 27-kD alpha zein is a modifier of this locus and has a strong influence on QPM production. The two major findings of this work are: 1) gamma irradiation can be broadly applied to many plant species to generate functional mutations for important traits and 2) this method in combination with modern sequencing methods provides an efficient and rapid procedure for analysis of genes underlying those traits.

Technical Abstract: Quality Protein Maize (QPM) is a hard kernel variant of the high-lysine mutant, opaque-2. Using gamma irradiation, we created opaque QPM variants to identify opaque-2 modifier genes and to investigate deletion mutagenesis combined with Illumina sequencing as a maize functional genomics tool. A K0326Y-QPM deletion mutant was null for the 27- and 50-kD alpha zeins and abolished vitreous endosperm formation. Illumina exon- and RNA-seq revealed a 1.2 Mb deletion encompassing the 27- and 50-kD alpha zein genes on chromosome 7, and a deletion of at least 232 Kbp on chromosome 9. Protein body number was reduced by over 90% while protein body size is similar to wild type. Kernels hemizygous for the alpha zein deletion had intermediate 27- and 50-kD alpha zein levels and were semi-vitreous, indicating haploinsufficiency of these gene products in opaque-2 endosperm modification. The alpha zein deletion further increases lysine in QPM in its homozygous and hemizygous states. This work identifies 27-kD alpha zein as an opaque-2¬ modifier gene within the largest QPM QTL, and suggests the 50-kD alpha zein also contributes to this QTL. It further demonstrates that genome-wide deletions in non-reference maize lines can be identified through a combination of assembly of Illumina reads against the B73 genome and integration of RNA-seq data.