Flow cytometry is widely used to estimate ploidy levels and genome sizes in plants and animals. It is also a common tool for determining the reproductive mode in flowering plants through flow cytometric seed screening (FCSS), which can differentiate sexual and apomictic pathways. However, FCSS has limitations, as it is not universally applicable to all apomictic taxa and cannot resolve certain reproductive pathways. These challenges can be addressed by germinating seeds and genotyping seedlings and their parents. However, this approach can be time- and space-intensive and carry other drawbacks (e.g., the ploidy level of the endosperm remains undetected). Here, we present a streamlined method that combines flow cytometry with genotyping of the same seeds. Applying both methods to a single seed enables comprehensive insights into reproductive modes, detecting various forms of apomixis and automixis, assessment of the level of selfing, regardless of the mode of reproduction mode, and identifying pollen donors in the endosperm of apomictic seeds. Each protocol-FCSS and seed genotyping-can also be used independently to determine basic reproductive modes.

Faculty of Science Department of Botany and Zoology Masaryk University Brno Czech Republic

Faculty of Science Department of Botany Palacký University in Olomouc Olomouc Holice Czech Republic

Freie Universität Berlin ZE Botanischer Garten und Botanisches Museum Berlin Berlin Germany

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