Recreating viable YY^h genotype uncovers the role of CpYYL underlying YY lethality in papaya

Papaya is a trioecious species with XX females, XY males, and XY^h hermaphrodites. Any combination of Y and Y^h chromosomes is lethal. The gene underlying YY lethality is pivotal in the transition from stage 2 to stage 3 in sex chromosome evolution. Here, we identify and functionally validate the YY lethality gene in papaya, CpYYL. Loss of CpYYL function accelerates glycolysis and reduces sucrose accumulation in embryo, causing embryo abortion. CpYYL interacts with CpAKRP and overexpression of both genes partially rescues seed abortion in the heterozygous emb506 and akrp mutants, indicating conserved role in plastid differentiation and embryo development. Viable YY^h and Y^hY^h plants are generated through genetic engineering and crossing, although both genotypes show high seedling mortality. Crossing YY^h male with XX female papaya shows 1:1 ratio of male to hermaphrodite plants without recombination between MSY and HSY. Identification of CpYYL fills a critical gap in papaya sex chromosome evolution, and provides new resources for genomic and epigenetic studies.