Most vertebrate sex-determining genes (SDGs) emerge as neofunctionalized genes through duplication and/or mutation of ancestral genes that are involved with sexual differentiation. We previously demonstrated dm-W to be the SDG in the African clawed frog Xenopus laevis and found that a portion of this gene emerged from the masculinization gene dmrt1 after allotetraploidization by interspecific hybridization between two ancestral species around 17-18 million years ago. dm-W has four exons consisting of a noncoding exon 1, dmrt1-derived exons 2 and 3, and an orphan exon 4 (Ex4) of unknown origin that includes coding sequence. In this study, we searched for the origin of Ex4 and investigated the function of the coding sequence of this exon. We found that the Ex4-coding sequence (CDS) is derived from a non-coding portion of the hAT-10 family of DNA transposon. Evolutionary analysis of transposons and determination of the Ex4 sequences from three other species indicated that Ex4 was generated before the diversification of most or all extant allotetraploid species in subgenus Xenopus, during which time we hypothesize that transposase activity of this hAT superfamily was active. Using DNA-protein binding and transfection assays, we further demonstrate that the Ex4-encoded amino acid sequence increases the DNA-binding ability and transcriptional activity of DM-W. These findings suggest that the conversion of the noncoding transposon sequence to the CDS of dm-W contributed to neofunctionalization of a new chimeric SDG in the ancestor of the allotetraploid Xenopus species, offering new insights into de novo origin and functional evolution of chimerical genes.

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Interspecific hybridization between two closely related species sometimes resulted in a new species with allotetraploid genomes. Many clawed frog species belonging to the Xenopus genus have diverged from the allotetraploid ancestor created by the hybridization of two closely related species with the predicted L and S genomes. There are species-specific repeated sequences including transposable elements in each genome of organisms that reproduce sexually. To understand what happened on and after the hybridization of the two distinct systems consisting of repeated sequences and their corresponding piRNAs, we isolated small RNAs from ovaries and testes of three Xenopus species consisting of allotetraploid X. laevis and X. borealis and diploid X. tropicalis as controls. After a comprehensive sequencing and selection of piRNAs, comparison of their sequences showed that most piRNA sequences were different between the ovaries and testes in all three species. We compared piRNA and genome sequences and specified gene clusters for piRNA expression in each genome. The synteny and homology analyses showed many distinct piRNA clusters among the three species and even between the two L and/or S subgenomes, indicating that most clusters of the two allotetraploid species changed after hybridization. Moreover, evolutionary analysis showed that DNA transposons including Kolobok superfamily might get activated just after hybridization and then gradually inactivated. These findings suggest that some DNA transposons and their piRNAs might greatly influence allotetraploid genome evolution after hybridization.