Development of Novel Monoclonal Antibodies to Wheat Alpha-Amylases Associated with Grain Quality Problems That Are Increasing with Climate Change.

Accurate, rapid testing platforms are essential for early detection and mitigation of late maturity α-amylase (LMA) and preharvest sprouting (PHS) in wheat. These conditions are characterized by elevated α-amylase levels and negatively impact flour quality, resulting in substantial economic losses. The Hagberg-Perten Falling Number (FN) method is the industry standard for measuring α-amylase activity in wheatmeal. However, FN does not directly detect α-amylase and has major limitations. Developing α-amylase immunoassays would potentially enable early, accurate detection regardless of testing environment. With this goal, we assessed an expression of α-amylase isoforms during seed development. Transcripts of three of the four isoforms were detected in developing and mature grain. These were cloned and used to develop E. coli expression lines expressing single isoforms. After assessing amino acid conservation between isoforms, we identified peptide sequences specific to a single isoform (TaAMY1) or that were conserved in all isoforms, to develop monoclonal antibodies with targeted specificities. Three monoclonal antibodies were developed, anti-TaAMY1-A, anti-TaAMY1-B, and anti-TaAMY1-C. All three detected endogenous α-amylase(s). Anti-TaAMY1-A was specific for TaAMY1, whereas anti-TaAMY1-C detected TaAMY1, 2, and 4. Thus, confirming that they possessed the intended specificities. All three antibodies were shown to be compatible for use with immuno-pulldown and immuno-assay applications.

Genome-wide associations for multiple pest resistances in a Northwestern United States elite spring wheat panel.

Northern areas of the western United States are one of the most productive wheat growing regions in the United States. Increasing productivity through breeding is hindered by several biotic stresses which slow and constrain targeted yield improvement. In order to understand genetic variation for stripe rust (Puccinia striiformis f. sp. tritici), Septoria tritici blotch (Mycosphaerella graminicola), and Hessian fly (Mayetiola destructor) in regional germplasm, a panel of 408 elite spring wheat lines was characterized and genotyped with an Illumina 9K wheat single nucleotide polymorphism (SNP) chip to enable genome-wide association study (GWAS) analyses. Significant marker-trait associations were identified for stripe rust (38 loci), Septoria tritici blotch (8) and Hessian fly (9) resistance. Many of the QTL corresponded with previously reported gene locations or QTL, but we also discovered new resistance loci for each trait. We validated one of the stripe rust resistance loci detected by GWAS in a bi-parental mapping population, which confirmed the detection of Yr15 in the panel. This study elucidated well-defined chromosome regions for multiple pest resistances in elite Northwest germplasm. Newly identified resistance loci, along with SNPs more tightly linked to previously reported genes or QTL will help future breeding and marker assisted selection efforts.