Mechanism analysis of alfalfa anther development based on single cell transcriptome

Anthers consist of various specialized cell types and play a significant role in plant reproduction. Although extensive research has been conducted on the molecular mechanisms underlying anther development and regulation, the single-cell transcriptional landscape and dynamic regulation during anther development in Medicago sativa (alfalfa) have largely remained unexplored. In this study, we constructed the first single-cell transcriptome atlas of M. sativa anthers, providing a comprehensive view of cell type-specific gene expression and epigenetic modifications.The reconstruction of developmental trajectories for tapetal cells and microspores led to the identification of novel genes and elucidated the regulatory networks involved in tapetum formation. Our findings revealed the rapid functional transitions of tapetal cells during the tetrad stage, including cell formation, specialization, and programmed cell death. Additionally, we analyzed the stages at which tapetal degradation and microspore shrinkage occurred in the sterile line.Overall, this study offers valuable insights into the molecular mechanisms of anther development in M. sativa. Specifically, we identified KIN14P as a regulator of microtubules and the cytoskeleton during mitosis, while transcription factors ERF3 and ERF025 were shown to influence anther development through ethylene response pathways. These findings provide essential theoretical foundations for the development of novel male-sterile lines and enhance the breeding capabilities of M. sativa.