New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesis
- Equal contributors
1 Graduate School of Agricultural and Life Sciences, The University of Tokyo, 2971-4 Bentenjima, Maisaka, Hamamatsu, Shizuoka 431-0214, Japan
2 Yashima station, Stock Enhancement and Management Division, National Research Institute of Fisheries and Enhancement of Inland Sea, Fisheries Research Agency, 243 Yashima-higashi, Takamatsu, Kagawa 761-0111, Japan
3 Aquatic Animal Health Division, National Research Institute of Aquaculture, Fisheries Research Agency, 224-1 Hiruta, Tamaki, Mie 519-0423, Japan
4 Faculty of Agriculture, Kyusyu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
5 Aquaculture Technology Division, National Research Institute of Aquaculture, Fisheries Research Agency, 422-1 Nakatsuhamaura, Minami-ise, Mie 516-0193, Japan
BMC Genomics 2013, 14:786 doi:10.1186/1471-2164-14-786Published: 13 November 2013
In fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (
In this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297 kb, which is similar to values calculated for zebrafish and medaka TILLING libraries.
These results demonstrate that the TILLING method in fugu was established. We anticipate that this TILLING approach can be used to generate a wide range of mutant alleles, and be applicable to many farmed fish that can be chemically mutagenized.