Sequence-specific nucleases have been applied to engineer targeted modifications in polyploid genomes , but simultaneous modification of multiple homoeoalleles has not been reported. Here we use transcription activator–like effector nuclease (TALEN) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 (refs. 4 , 5 ) technologies in hexaploid bread wheat to introduce targeted mutations in the three homoeoalleles that encode MILDEW-RESISTANCE LOCUS (MLO) proteins . Genetic redundancy has prevented evaluation of whether mutation of all three MLO alleles in bread wheat might confer resistance to powdery mildew, a trait not found in natural populations . We show that TALEN-induced mutation of all three TaMLO homoeologs in the same plant confers heritable broad-spectrum resistance to powdery mildew. We further use CRISPR-Cas9 technology to generate transgenic wheat plants that carry mutations in the TaMLO-A1 allele. We also demonstrate the feasibility of engineering targeted DNA insertion in bread wheat through nonhomologous end joining of the double-strand breaks caused by TALENs. Our findings provide a methodological framework to improve polyploid crops. Bread wheat ( Triticum aestivum L., 2 n = 42, AABBDD) is a major staple crop worldwide. It provides approximately 20% of all calories consumed by humans ( http://faostat.fao.org/site/339/default.aspx ). Given the
Nature Biotechnology – Nature Publishing Group (NPG)
Published: Jul 20, 2014
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