Androgenetic doubled haploids induced without irradiation of eggs in loach (Misgurnus anguillicaudatus)

Androgenetic doubled haploids (DHs) were induced in the loach Misgurnus anguillicaudatus (Cobitidae) without irradiation of the eggs. The eggs of wild-type females were activated with the intact sperm of an orange-phenotype male, and treated (within 10 s of activation) at 3 ± 0.5 °C for 30 min, to eliminate the female nucleus. The eggs were then incubated in a water bath at 20 ± 0.5 °C for 35 min. Finally, diploidy was restored (65 min after activation) by heat-shock treatment at 42 ± 0.5 °C for 2 min. Under these conditions, the yield rate (mean ± SD) of putative DHs relative to the total number of eggs used was 10.43 ± 1.69%, which was significantly higher (P < 0.05) than the yield rates obtained under the remaining heat-shock initiation conditions (55 min, 60 min, and 70 min after activation). We analyzed the ploidy status of the putative DH by using flow cytometry. All-male inheritance was confirmed by the expression of the recessive orange body color trait and microsatellite genotypes. We detected no maternally derived alleles or heterozygous genotypes at any of the 28 loci (covering 27 linkage groups) of loach, indicating the exclusively paternal inheritance and homozygosity of the obtained androgenetic DHs.