Unlocking wheat fertility: map-based cloning spotlights anther-specific TaAGO5c as a master modulator.

The development of reproductive organs plays a vital role in the production of gametes. However, the regulators of this process are poorly understood in wheat. Here, we identified a wheat sterile mutant ste with indehiscent anthers and shrunken pollen grains. Cytological analysis suggested that the tapetum cells degenerated earlier; Ubisch bodies exhibited abnormal shapes, while the microspore mother cells displayed meiosis defects, leading to the failure of tetrads formation in the ste. Bulked segregant analysis (BSA) and map-based cloning indicated that the TaAGO5c encoding an Argonaute protein is responsible for the sterility in ste. Gene editing-mediated knockout of three TaAGO5c homeologous genes resulted in a severely decreased self-seeding rate, confirming its function in reproductive development. TaAGO5c was predominantly expressed in the young spikes and anthers from the unicellular microspore stage to mature pollen stage. Subcellular localization in wheat protoplasts suggested that TaAGO5c is localized in the cytoplasm. Transcriptome sequencing analysis suggested that DEGs between the ste and control in the 30-40 mm and 60-70 mm young spikes were significantly enriched in several pathways associated with tapetum development and meiosis. Furthermore, some key genes related to these processes were downregulated in the ste mutant, which may be responsible for the abnormal development of tapetum and microspore. These results collectively suggest that TaAGO5c is a crucial regulator in reproductive development of wheat. This study also provides new insights into the genetic regulation of tapetum cells and Ubisch bodies development in wheat.