Timing of post‐fertilization pressure shock treatment for the production of mitotic gynogens in six salmonid species

Doubled haploid gynogens are individuals whose genetic material consists of two identical maternal chromosome sets and who lack paternal genetic contributions. These individuals can be useful in whole-genome sequence assembly to eliminate allelic variation in an individual that otherwise complicates the discrimination of SNPs and paralogs in regular diploids. This is particularly important in salmonid species, which have extensive remnants of an ancestral whole-genome duplication. Further, doubled haploid individuals are fully homozygous and can be used to generate clonal lines. Here, successful timing was determined for late pressure shocking for producing doubled haploid gynogens in five Oncorhynchus species (Chinook Salmon O. tshawytscha, Coho Salmon O. kisutch, Chum Salmon O. keta, Pink Salmon O. gorbuscha, and Sockeye Salmon O. nerka) and one Salvelinus species (domesticated Artic Char Salvelinus alpinus). For this study, sperm was treated with UV irradiation to inactivate the paternal nuclear DNA and used to fertilize eggs. The resulting zygotes were pressure shocked at various times following fertilization to form doubled haploid embryos via inhibition of the first cell division (mitotic gynogenesis). At an incubation temperature of 10.5 degrees C, successful postfertilization pressure shock times for maximal survival of confirmed gynogens were 2.41-2.83 accumulated thermal units (ATUs) for Coho Salmon, 2.63-2.84 ATUs for Chum Salmon, 2.84-3.06 ATUs for Pink Salmon, 2.19 ATUs for Sockeye Salmon, and 2.63-3.06 ATUs for Chinook Salmon, whereas for Arctic Char a shock time of 2.29-2.54 ATUs at 4 degrees C incubation was successful. Survival to fry stage ranged from less than 1% to 11.7%. Survivors were genotyped using microsatellite markers to assess nonpaternity and maternal homozygosity and were found to be 92.5% gynogens when averaged across all treatments and species. Mitotic gynogenetic individuals were produced in all six salmonid species and were used in subsequent studies to generate whole-genome sequences for Chinook, Coho, Chum, and Sockeye salmons.