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Female germline cells in flowering plants differentiate from somatic cells to produce specialized reproductive organs, called ovules, embedded deep inside the flowers. We investigated the molecular basis of this distinctive developmental program by performing single-cell RNA sequencing (scRNA-seq) of 16,872 single cells of Arabidopsis thaliana ovule primordia at three developmental time points during female germline differentiation. This allowed us to identify the characteristic expression patterns of the main cell types, including the female germline and its surrounding nucellus. We then reconstructed the continuous trajectory of female germline differentiation and observed dynamic waves of gene expression along the developmental trajectory. A focused analysis revealed transcriptional cascades and identified key transcriptional factors that showed distinct expression patterns along the germline differentiation trajectory. Our study provides a valuable reference dataset of the transcriptional process during female germline differentiation at single-cell resolution, shedding light on the mechanisms underlying germline cell fate determination. Zhimin Hou, Yanhui Liu et al. used single cell RNA-seq to analyze the model organism, Arabidopsis thaliana, at three stages during female germline differentiation. They reconstructed the continuous trajectory of female germline differentiation, providing a valuable reference for future investigation of germline cell fate determination in A. thaliana. [ABSTRACT FROM AUTHOR]