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.

中文翻译：

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面包小麦 (Triticum aestivum L.) 中的转录基因沉默及其在杂交种子生产中调节雄性生育力中的应用

转录基因沉默 (TGS) 可以为植物基因的功能分析提供直接的工具，特别是在小麦等多倍体物种中，其中遗传冗余对应用诱变方法（包括 CRISPR 基因编辑）提出了挑战。在这项研究中，我们证明了小麦中有效的 TGS，由启动子反向重复 (pIR) 的组成型 RNA 表达介导。pIR 介导的两个花药特异性基因TaMs45和TaMs1的 TGS消除了它们的功能，导致雄性不育。虽然TaMs45的 TGS需要所有三个同源物的转录沉默，但 TaMs1 的 B 基因组特异性 pIR足以赋予雄性不育。我们进一步表明 pIRs 影响 TaMs45 的TGS基因通过同源启动子序列的DNA甲基化，成功地抑制了所有三个同源物的转录。在小麦中应用 pIR 介导的 TGS，我们已经产生了用于生产杂交种子的显性雄性生育系统，并证明了该系统在温室和田间条件下的功效。本报告描述了小麦中第一个成功的 TGS，同时提供了一种显性阴性方法作为杂交小麦育种和种子生产的基因敲除策略的替代方案。