Fusarium head blight (FHB) infection significantly reduces wheat (Triticum aestivum) productivity and causes mycotoxin contamination in harvested grain. Growing resistant wheat cultivars is one of the most promising approaches to minimize FHB damage. Fhb1 is the most stable quantitative trait locus (QTL) with a major effect on FHB resistance in wheat. Recently, three groups reported cloning of Fhb1. One group reported pore-forming toxin (TaPFT) as the candidate, and the other two reported histidine rich calcium binding protein (TaHRC) as the candidate, but they proposed different mechanisms of TaHRC in regulating Fhb1 resistance. Therefore, the causal gene and mechanism for Fhb1 remain to be determined. CRISPR/Cas9 genome editing technology can knock out a gene to determine its function. However, conventional gene editing is conducted using gene transformation, and most wheat cultivars have extremely low transformation rates. Wheat cultivars Bobwhite and Fielder is frequently used for wheat transformation, but none of them carries Fhb1. We developed an gene editing method that generates mutations in the targeted wheat genes without transformation. To edit a FHB resistant wheat accession Ning7840 carrying the Fhb1 resistance allele, Cas9-overexpressed Bobwhite plants were crossed to Ning7840 and the leaf tissues of selected F₂ plants with both Cas9 and the target genes were inoculated with Barley stripe mosaic virus (BSMV) carrying the integrated guide RNA (gRNA). The progeny of BSMV infected plants were screened for the mutations using PCR cloning and sequencing. After sequencing 291 M₁ plants, we found two substitution mutations that alter amino acid sequences in TaHRC, and one deletion mutation and 1 substitution which caused a frameshift in TaPFT. Some of the homozygous M₂ mutant plants are being phenotyped for FHB resistance this fall to validate their functions on FHB resistance. Therefore, the new genome editing system successfully produced targeted mutations and can be used to validate gene functions in wheat.

ACKNOWLEDGEMENT AND DISCLAIMER

This material is based upon work supported by the U.S. Department of Agriculture. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture.