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Summary  Identifying genic male‐sterility (GMS) genes and elucidating their roles are important to unveil plant male reproduction and promote their application in crop breeding. However, compared with  Arabidopsis  and rice, relatively fewer maize GMS genes have been discovered and little is known about their regulatory pathways underlying anther and pollen development. Here, by sequencing and analysing anther transcriptomes at 11 developmental stages in maize B73, Zheng58 and M6007 inbred lines, 1100 transcription factor (TF) genes were identified to be stably differentially expressed among different developmental stages. Among them, 14 maize TF genes (9 types belonging to five TF families) were selected and performed CRISPR/Cas9‐mediated gene mutagenesis, and then, 12 genes in eight types, including  ZmbHLH51 ,  ZmbHLH122 ,  ZmTGA9‐1/‐2/‐3 ,  ZmTGA10 ,  ZmMYB84 ,  ZmMYB33‐1/‐2 ,  ZmPHD11  and  ZmLBD10/27 , were identified as maize new GMS genes by using DNA sequencing, phenotypic and cytological analyses. Notably,  ZmTGA9‐1/‐2/‐3  triple‐gene mutants and  ZmMYB33‐1/‐2  double‐gene mutants displayed complete male sterility, but their double‐ or single‐gene mutants showed male fertility. Similarly,  ZmLBD10/27  double‐gene mutant displayed partial male sterility with 32.18% of aborted pollen grains. In addition,  ZmbHLH51  was transcriptionally activated by ZmbHLH122 and their proteins were physically interacted. Molecular markers co‐segregating with these GMS mutations were developed to facilitate their application in maize breeding. Finally, all 14‐type maize GMS TF genes identified here and reported previously were compared on functional conservation and diversification among maize, rice and  Arabidopsis . These findings enrich GMS gene and mutant resources for deeply understanding the regulatory network underlying male fertility and for creating male‐sterility lines in maize.

CRISPR/Cas9‐based discovery of maize transcription factors regulating male sterility and their functional conservation in plants