Graduate Studies

 

First Advisor

David Holding

Degree Name

Doctor of Philosophy (Ph.D.)

Committee Members

Brandi Sigmon, James Schnable, Scott Sattler, Thomas Clemente

Department

Agronomy and Horticulture

Date of this Version

5-2025

Document Type

Dissertation

Citation

A dissertation presented to the faculty of the Graduate College at the University of Nebraska in partial fulfillment of requirements for the degree of Doctor of Philosophy

Major: Agronomy and Horticulture

Under the supervision of Professor David Holding

Lincoln, Nebraska, May 2025

Comments

Copyright 2025, Joseph Preston Hurst. Used by permission

Abstract

Maize grain is deficient in lysine. While the opaque2 mutation increases grain lysine, o2 is a transcription factor that regulates a wide network of genes beyond zeins, which leads to pleiotropic and often negative effects. Additionally, the drastic reduction in 19kDa and 22kDa alpha-zeins causes a floury kernel, unsuitable for agricultural use. Sorghum, closely related to maize, has a similar lack of lysine that can be corrected with prolamin alteration and also subject to the same deleterious kernel texture. CRISPR/cas9 gives researchers the ability to directly target genes of interest. In this research, gene editing was used to specifically target the 19kDa alpha zein gene family. This allows for proteome rebalancing to occur without an o2 mutation and without a total alpha-zein knockout. In addition, a previously gene edited high lysine sorghum line was observed more in depth. The results demonstrate the possibility of developing high lysine maize and sorghum with minimal penalty to kernel texture. Finally, the repetitive nature of the prolamins in these two species presents an interesting challenge in gene editing. We show examples of the effects of having many CRISPR targets in a local region, and the complex structural variations that may arise. In addition, our approaches give some examples on selecting targets and characterizing alleles in such situations.

Advisor: David Holding

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