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

BMC Genomics

Methodology Article

Hiroyuki Okamoto¹  Shin-ichi Chisada²  Yasutoshi Yoshiura²  Tomonao Matsushita³  Tomokazu Ushijima³  Kiyoshi Kikuchi⁴  Aoi Nozawa⁴  Yuzuru Suzuki⁴  Miwa Kuroyanagi⁴  Masashi Fujita⁴  Yoshihisa Yamamoto⁵  Takashi Katayama⁵  Tadashi Imai⁵ 

[1] Aquaculture Technology Division, National Research Institute of Aquaculture, Fisheries Research Agency, 422-1 Nakatsuhamaura, 516-0193, Minami-ise, Mie, Japan;Aquatic Animal Health Division, National Research Institute of Aquaculture, Fisheries Research Agency, 224-1 Hiruta, 519-0423, Tamaki, Mie, Japan;Faculty of Agriculture, Kyusyu University, 6-10-1 Hakozaki, 812-8581, Higashi-ku, Fukuoka, Japan;Graduate School of Agricultural and Life Sciences, The University of Tokyo, 2971-4 Bentenjima, Maisaka, 431-0214, Hamamatsu, Shizuoka, Japan;Yashima station, Stock Enhancement and Management Division, National Research Institute of Fisheries and Enhancement of Inland Sea, Fisheries Research Agency, 243 Yashima-higashi, 761-0111, Takamatsu, Kagawa, Japan;

关键词: TILLING;    Fugu;    ENU;    HRM;    Myostatin;    Mutagenesis;    Fish breeding;   

DOI  :  10.1186/1471-2164-14-786

 received in 2013-02-08, accepted in 2013-11-04,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundIn 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 (Targeting Induced Local Lesions INGenomes) 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.ResultsIn 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.ConclusionsThese 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.

【 授权许可】

Unknown   
© Kuroyanagi et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.