Cold-shock induced androgenesis without egg irradiation and subsequent production of doubled haploids and a clonal line in Japanese flounder, Paralichthys olivaceus

Abstract

Androgenesis is a useful manipulation to fix male-specific genetic traits as well as to restore the genotypes of cryopreserved sperm, and it has been induced by the genetic inactivation of the egg nucleus with gamma-, X- or UV-ray irradiation before fertilization. Recently, a technique for cold-shock induced androgenesis was developed in a freshwater species, the loach, Misgurnus anguillicaudatus, in which viable androgenetic diploids and doubled haploids (DHs) were successfully produced by the fertilization of diploid sperm and chromosome doubling by heat-shock treatment, respectively. This technique was immediately applied for cloning in the zebrafish, Danio rerio. Here, we reported the first successful induction of androgenetic development by means of cold-shock treatment (at 0°C for 15min) just after the fertilization (within 10s) of eggs in a marine aquaculture fish species, the Japanese flounder, Paralichthys olivaceus. Then, androgenetic embryos thus generated were subjected to hydrostatic pressure treatment (650kg/cm2, 6min) after incubation at 17°C so as to produce DHs by chromosome doubling. The yield rate of putative DHs, which was estimated as the frequency of diploid larvae at the first feeding stage in relation to the total eggs used, ranged from 0.81% to 1.79%. The complete homozygosity of these putative DHs was genetically verified using 36 microsatellite markers that covered 24 linkage groups of Japanese flounder. Thus, an androgenetic clonal line was produced with the sperm of a mito-gynogenetic DH male by using cold-shock and subsequent hydrostatic pressure-shock treatments. Statement of relevanceThe results of our manuscript confirm the broad applicability of the cold-shock induced androgenesis method in commercially important marine fish species.