LAUSR

Summary Transcriptional gene silencing (TGS) can offer a straightforward tool for functional analysis of plant genes, particularly in polyploid species such as wheat, where genetic redundancy poses a challenge in applying mutagenesis approaches, including CRISPR gene editing. In this study, we demonstrate efficient TGS in wheat, mediated by constitutive RNA expression of a promoter inverted repeat (pIR). pIR‐mediated TGS of two anther‐specific genes, TaMs45 and TaMs1, abolished their function resulting in male sterility. Whilst TGS of TaMs45 required transcriptional silencing of all three homoeologs, a B‐genome‐specific pIR for TaMs1 was sufficient to confer male sterility. We further show that the pIRs effect TGS of TaMs45 gene through DNA methylation of homologous promoter sequence, successfully suppressing transcription of all three homoeologs. Applying pIR‐mediated TGS in wheat, we have generated a dominant male fertility system for production of hybrid seed and demonstrated the efficacy of this system under greenhouse and field conditions. This report describes the first successful TGS in wheat, whilst providing a dominant negative approach as alternative to gene knockout strategies for hybrid wheat breeding and seed production.