Title: ZmMTOPVIB is essential for double-strand break formation and bipolar spindle

35 The programmed formation of DNA double-strand breaks (DSBs) during early 36 meiosis is catalyzed by SPO11, a conserved ortholog to the A subunit of an archaeal 37 topoisomerase VI (TopoVI) which functions as a hetero-tetramer comprising two A and 38 two B subunits. An essential role of the meiotic TopoVI B subunit (TopVIB) in DSB 39 formation has been reported in mouse, Arabidopsis and rice. Very recently, rice MTopVIB 40 was revealed to have an unexpected role in meiotic bipolar spindle assembly, highlighting 41 multiple functions of MTopVIB during rice meiosis. In this work, the meiotic TopVIB in 42 maize (ZmMTOPVIB) was characterized. The ZmmtopVIB mutant plants exhibited 43 normal vegetative growth but male and female sterility. DSB formation is abolished in 44 mutant meiocytes. Despite normal assembly of axial elements, synapsis was severely 45 3 affected and homologous pairing was disrupted in mutants. Importantly, we showed that 46 bipolar spindle assembly was also affected in ZmmtopVIB, resulting in triad and polyad 47 formation. Overall, our results demonstrate that ZmMTOPVIB plays critical roles in DSB 48 formation and homologous recombination. In addition, the newly-discovered function of 49 MTOPVIB in bipolar spindle assembly is likely conserved across different monocots. 50