Mutagenesis in Sperm of Fugu (Takifugu rubripes) and Amago Salmon (Oncorhynchus masou ishikawae) using N-Ethyl-N-Nitrosourea (ENU) for Developing a New Aquaculture Breeding Technique of TILLING

TILLING (Targeting Induced Local Lesions IN Genomes) is a technique utilized for agricultural breeding, which is used to genetically improve crop strains for a commercial trait, by selecting single base mutations in a target gene. Recently, we have established how to mutagenize the genome of aquaculture fish species safely and stably. To mutagenize the DNA in sperm, it is useful to inject the alkylating agent, N-ethyl-N-nitrosourea (ENU), into the abdominal cavity of maturing male fish in the spawning season. We have tested this method using fugu (pufferfish, Takifugu rubripes) and amago salmon (Oncorhynchus masou ishikawae). To mutagenize the genome of fish species having an annual spawning season, there are several key considerations regarding treatment with the agent. (1) The timing of the ENU treatment during the season. (2) The reproductive period and harvest timing of the sperm after the treatment. (3) The period of the longevity after sexually maturation. To develop the TILLING system for aquaculture fish, it is also an important point how to detect the ratio of mutation in the sperm genome. As most aquaculture fish species do not have a recessive-trait strain such as albino, we adopted the method of High Resolution of Melting analysis (HRM) in fugu. We detected the mutation ratio as 1/360 kb in the test of fugu. We also detected the ratio with 0.4% of emerging rate of the recessive albino in amago salmon. In these results, we show a useful method to make mutations for practical genomic breeding as TILLING in aquaculture fish species.