Forrest.Wold-McGimsey@colostate.edu, Caitlynd.d@hotmail.com, Rocio.Alarcon-Reverte@rothamsted.ac.uk, Karl@tesorogenetics.com, Andrew.Katz@colostate.edu, John.Stromberger@colostate.edu, Esten.Mason@colostate.edu, Stephen.Pearce@rothamsted.ac.uk

In wheat (Triticum aestivum L.), polyphenol oxidase (PPO) enzymes released from the aleurone layer of the grain during milling results in the discoloration of flour, dough, and end-use products, reducing their value. Loss-of-function mutations in the PPO1 and PPO2 paralogous genes on homoeologous group 2 chromosomes confer reduced PPO activity in the wheat grain. However, limited natural variation and the proximity of these genes complicates the selection of extremely low-PPO wheat varieties by recombination.

A CRISPR/Cas9 construct with one single guide RNA targeting a conserved copper binding domain was used to edit all seven PPO1 and PPO2 genes in the spring wheat cultivar 'Fielder'. Five of the seven edited T1 lines exhibited significant reductions in PPO activity, and T2 lines had PPO activity up to 86.7% lower than wild-type.

The same construct was transformed into the elite winter wheat cultivars 'Guardian' and 'Steamboat', which have five PPO1 and PPO2 genes. In these varieties PPO activity was reduced by >90% in both T1 and T2 lines.

In all three varieties, dough samples from edited lines exhibited reduced browning. Using this method, we hope to develop elite wheat lines with near-zero PPO grain content with no loss to their favorable qualities.

This study demonstrates that multi-target editing at late stages of variety development could complement selection for beneficial alleles in crop breeding programs by inducing novel variation in loci inaccessible to recombination.