Open Access Methodology article

New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesis

Miwa Kuroyanagi1, Takashi Katayama2, Tadashi Imai2, Yoshihisa Yamamoto2, Shin-ichi Chisada3, Yasutoshi Yoshiura3, Tomokazu Ushijima4, Tomonao Matsushita4, Masashi Fujita1, Aoi Nozawa1, Yuzuru Suzuki1, Kiyoshi Kikuchi1* and Hiroyuki Okamoto5*

Author Affiliations

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

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BMC Genomics 2013, 14:786  doi:10.1186/1471-2164-14-786

Published: 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 (

enomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported.


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.

TILLING; Fugu; ENU; HRM; Myostatin; Mutagenesis; Fish breeding