A Y‐linked A‐class MADS ‐box gene as a maleness‐determining candidate in the hardy‐rubber tree ( Eucommia ulmoides )

Eucommia ulmoides is a dioecious plant (with separate genetically male and female individuals) important in medicine and rubber production in the order Garryales. Flowers develop unisexually from inception (termed type II unisexual flowers). We used PacBio HiFi and Hi-C sequencing to assemble a high-quality haplotype-resolved male genome of E. ulmoides, and identified this species' sex-linked region (SLR). We aimed to find sex-determining candidate genes, and test the suggestion that sex in dioecious plants with type II unisexual flowers can be determined by MADS-box genes. Comparative genomic and population genetic analyses revealed a male heterogametic system (XY), with a roughly 32 Mb Y-linked region. An older evolutionary stratum (S1) includes a Y-specific A-class MADS-box gene, EuMADS65, a duplicate of EuMADS38, which is in the pseudoautosomal recombining region. Expression and functional experiments suggest neofunctionalization of EuMADS65 after the duplication, to act as the male-determining factor by regulating the expression of another sex-linked gene, EuAP3, a stamen organ identity B-class gene. As dioecy is prevalent among other species of Garryaceae, E. ulmoides probably did not evolve dioecy from a cosexual ancestor, and we evaluate the possibility that this duplication was involved in a sex chromosome turnover.