A new two-gene system of sex determination in a Salicaceae.

In angiosperms, dioecy has evolved thousands of times, and the pathways underlying the required floral changes are therefore expected to exhibit diversity as well as parallelism. Here we investigate Itoa orientalis, a dioecious Salicaceae in which immature flowers are bisexual, but the stamens or pistils then abort. This contrasts with the floral development in dioecious species of Populus and Salix, which lack any morphologically bisexual stage. A haplotype-resolved Itoa genome assembly revealed an XY system of sex determination with a sex-determining region (SDR) spanning ∼6 Mb and encompassing the centromere. In females, the SDR contains an 11-bp deletion in the TAPETAL DEVELOPMENT and FUNCTION 1 (TDF1) gene that results in multiple premature stop codons. Experimental silencing of TDF1 in males led to defective stamens, providing direct evidence that TDF1 is a regulator of male function as it is in the phylogenetically distant dioecious Asparagus officinalis. A candidate gene for suppression of female function is the MINI ZINC FINGER 2 (MIF2) gene. These findings reveal that the Salicaceae family has both an ARR17-based one-gene sex-determining system in Populus and Salix, and a two-gene system in Itoa.