Fhb7 is an FHB resistance gene derived from chromosome 7E of tall wheatgrass species Thinopyrum ponticum (2n = 10x = 70) and Th. elongatum (2n = 2x = 14). Fhb7^Thp was the first allele of Fhb7 cloned from Th. ponticum, which encodes a glutathione S-transferase (GST) and confers FHB resistance through detoxifying trichothecene. This allele has been integrated into wheat chromosome 7D by wide crosses and homoeologous recombination. More recently, a novel allele (Fhb7^The2) of Fhb7 from Th. elongatum has been transferred to wheat chromosome 7B by translocation, which can be used for FHB resistance improvement in both common and durum wheat. To verify the function of GST in the Fhb7 resistance, we conducted targeted mutagenesis of the GST-encoding alleles through wide hybridization between wheat lines carrying Fhb7 and transgenic maize expressing Cas9 and single guide RNA (sgRNA). We generated transgenic maize plants by Agrobacterium-mediated transformation using a binary vector expressing Cas9 and sgRNA for targeting the Fhb7 alleles. Three Fhb7-carrying wheat lines, named CS-Fhb7 with Fhb7^Thp and PI 702949 with Fhb7^The2 both in Chinese Spring background, and FW23-09-80 with Fhb7^The2 in elite hard red spring wheat (HRSW), were used as female parents for pollination with the transgenic maize plants and a total of 170 haploid plants were generated through embryo rescue. PCR amplification and Sanger sequencing indicated that 22%, 24% and 13% of the haploid plants derived from CS-Fhb7 (Fhb7^Thp), PI 702949 (Fhb7^The2), and FW23-09-80 (Fhb7^The2), respectively, had mutations at the target sites. Doubled haploid (DH) mutant lines were produced by colchicine treatment of the haploid plants and evaluated for FHB resistance in greenhouse. The results indicated that DH lines with Fhb7 alleles mutated were significantly more susceptible to FHB compared to their wild types carrying the original Fhb7 alleles. Our study not only confirmed the important role of GST in FHB resistance for both Fhb7^Thp and Fhb7^The2 alleles, but also demonstrated the power of the wheat × maize hybridization combined with genome editing technology in functional characterization of genes in wheat.

ACKNOWLEDGEMENT AND DISCLAIMER

This material is based upon work supported by North Dakota Wheat Commission and the U.S. Department of Agriculture under Agreement No. 59-0206-2-156. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative (USWBSI). 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.