Tetraploidization overcomes male hybrid sterility in dojo loach Misgurnus anguillicaudatus

Hybridization often causes alterations in gametogenesis and meiosis, resulting in gonadal, gametic, or zygotic sterility and asexuality, including the production of unreduced gametes. The dojo loach, Misgurnus anguillicaudatus , comprises two genetically divergent groups (A and B) in Japan. In artificially produced intergroup diploid hybrids, males exhibited sterility and sperm dysfunction. Here, inter-group diploid hybrid(s) (genomic constitution: AB, 2n-Hyb(s)) were produced via artificial fertilization between eggs from group B and sperm from group A. Subsequently, the tetraploid hybrid(s) (allotetraploid; genomic constitution: AABB, 4n-Hyb(s)) via inhibition of a mitotic division were successfully obtained by heat-shock treatment of the fertilized eggs at 41.4 °C for 2 min duration, starting at 22 min post fertilization. Mature 4n-Hyb males produced diploid sperm, whereas haploid, diploid, and tetraploid sperm were observed in the semen of 2n-Hyb males. The diploid sperm of the 4n-Hybs exhibited a normal external appearance, whereas the sperm of the 2n-Hybs displayed varying head sizes and multiple flagella. The fertilization of wild-type eggs with diploid sperm from 4n-Hybs mainly produces triploid progeny. Androgenesis via fertilization of UV-irradiated eggs with diploid sperm from 4n-Hybs successfully produced viable diploid progeny. The androgenetic progeny of the 4n-Hybs received no genetic contribution from the mother and were almost genetically identical. In conclusion, the intergroup hybrids recovered their fertility after tetraploidization. Diploid sperm of 4n-Hybs were likely produced by meiosis, ensuring bivalent formation between duplicated homologous chromosomes belonging to the same genetic group, which arises from tetraploidization. • Allotetraploids could be induced by heat-shock treatment in dojo loach. • Allotetraploids produced fertile diploid sperm. • Androgenetic diploids could be induced using fertile diploid sperm. • The fertile diploid sperm were genetically almost identical.