DRP005328
Efficient production of a triple recessive mutant with altered seed dormancy in bread wheat via CRISPR/Cas9 genome editing
Bread wheat has three sets of sub-genomes, making mutations difficult to observe, especially for traits controlled by recessive genes. Here, we produced hexaploid wheat (Triticum aestivum) cv. Fielder lines with loss-of-function of homoeoalleles of Qsd1, which controls seed dormancy in barley, by Agrobacterium-mediated CRISPR/Cas9. Of the eight transformed wheat events produced, three independent events carrying multiple mutations in wheat Qsd1 homoeoalleles were obtained. Notably, one line had mutations in every homoeoallele. We crossed this plant with wild-type cv. Fielder to generate a transgene-free triple recessive mutant, as revealed by Mendelian segregation. The mutant showed a significantly longer seed dormancy period than wild type, which may result in reduced pre-harvest sprouting of grains on spikes. PCR, Southern blotting and whole-genome shotgun sequencing revealed that this segregant lacked transgenes including Cas9 in its genomic sequence. The process of genome editing, genotyping and selection was completed in only 14 months, as accelerated generation advancement was performed using embryo culture. This strategy provides a model for precisely targeted Agrobacterium-mediated CRISPR/Cas9 genome editing to develop multiple recessive mutants in hexaploid wheat.