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CRISPR/Cas9-mediated restoration of Tamyb10 to create pre-harvest sprouting-resistant red wheat

Yiwang Zhu1,†, Yarong Lin2,†, Yujin Fan1,3,4, Yiwei Wang1,5, Pengfeng Li1, Jiang Xiong1, Yuhan He1,Shifeng Cheng1, Xingguo Ye6, Feng Wang2, Justin Goodrich7, Jian-Kang Zhu8, Ke Wang6,*andCui-Jun Zhang1,*



Wheat pre-harvest sprouting (PHS) reduces yield and grain quality and occurs in almost every wheat-growing region around the world. The R2R3-MYB transcription factor Tamyb10 of hexaploid wheat activates flavonoid biosynthesis genes to specify red grain colour and influences PHS. In most white wheat varieties, the Tamyb10-A1a, Tamyb10-B1a, and Tamyb10-D1a genes have large insertions or deletions, which disrupt the IRTKAL/IRC motif and regulatory function. Among the Tamyb10 genes, the Tamyb10-B1a allele has a 19-bp deletion in nearly 88.6% of bread wheat lines; this deletion causes a frameshift in the open-reading frame and disrupts the resulting protein. In this study, the 1-bp insertions restored the reading frame by converting the frameshift mutation in the Tamyb10-B1a allele (19-bp deletion) to in-frame mutation (18-bp deletion). We successfully converted a white wheat variety into a red one and improved its PHS tolerance through CRISPR/Cas9-mediated functional restoration of the Tamyb10-B1a allele. The present study also presents an alternative strategy for crop improvement by editing genes that were lost through domestication via the restoration of reading frames.