A novel amphibian sex determination candidate gene, evolved by structural variation in the regulatory region between X and Y

Most vertebrates develop distinct females and males, where sex is determined by repeatedly-evolved environmental or genetic triggers. Undifferentiated sex chromosomes and large genomes have caused major knowledge gaps in amphibians. Only a single master gene is known in >8650 species, the dmrt1 -paralogue ( dm-w ) of female-heterogametic clawed frogs ( Xenopus ; ZW♀/ZZ♂). Combining a chromosome-scale genome of a non-model amphibian, the European green toad, Bufo ( tes ) viridis , with ddRAD- and whole genome pool-sequencing revealed a novel candidate master gene ( bod1l ), governing a male-heterogametic system (XX♀/XY♂). Targeted sequencing across multiple taxa and a male long-read assembly uncovered structural X/Y-variation in the 5’-regulatory region, where a Y-specific non-coding RNA, only expressed in males, suggests regional enhancer-properties. Developmental transcriptomes and RNA in-situ hybridization provide evidence for timely and spatially relevant, sex-specific bod1l -gene expression in primordial gonads with coinciding differential H3K4me -methylation in pre-granulosa/pre-Sertoli cells, pointing to a novel mechanism of amphibian sex determination.