title: Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing.
abstract: BACKGROUND
  Functions of most genes predicted in the soybean genome have not been clarified. A mutant library with a high mutation density would be helpful for functional studies and for identification of novel alleles useful for breeding. Development of cost-effective and high-throughput protocols using next generation sequencing (NGS) technologies is expected to simplify the retrieval of mutants with mutations in genes of interest.
  
  
  RESULTS
  To increase the mutation density, seeds of the Japanese elite soybean cultivar Enrei were treated with the chemical mutagen ethyl methanesulfonate (EMS); M2 seeds produced by M1 plants were treated with EMS once again. The resultant library, which consisted of DNA and seeds from 1536 plants, revealed large morphological and physiological variations. Based on whole-genome re-sequencing analysis of 12 mutant lines, the average number of base changes was 12,796 per line. On average, 691 and 35 per line were missense and nonsense mutations, respectively. Two screening strategies for high resolution melting (HRM) analysis and indexed amplicon sequencing were designed to retrieve the mutants; the mutations were confirmed by Sanger sequencing as the final step. In comparison with HRM screening of several genes, indexed amplicon sequencing allows one to scan a longer sequence range and skip screening steps and to know the sequence information of mutation because it uses systematic DNA pooling and the index of NGS reads, which simplifies the discovery of mutants with amino acid substitutions.
  
  
  CONCLUSIONS
  A soybean mutant library with a high mutation density was developed. A high mutation density (1 mutation/74 kb) was achieved by repeating the EMS treatment. The mutation density of our library is sufficiently high to obtain a plant in which a gene is nonsense mutated. Thus, our mutant library and the indexed amplicon sequencing will be useful for functional studies of soybean genes and have a potential to yield useful mutant alleles for soybean breeding.
authors.affiliation: Agronomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. tsuda.mai.fu@u.tsukuba.ac.jp.|Present address: Gene Research Center, Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1, Ten-nodai, Tsukuba, Ibaraki, 305-8574, Japan. tsuda.mai.fu@u.tsukuba.ac.jp.
authors.forename: Mai
authors.initials: M
authors.lastname: Tsuda
authors.affiliation: Agronomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. kaga@affrc.go.jp.
authors.forename: Akito
authors.initials: A
authors.lastname: Kaga
authors.affiliation: Faculty of Agriculture, Saga University, 1 Honjo-machi, Saga, 840-8502, Japan. anai@cc.saga-u.ac.jp.
authors.forename: Toyoaki
authors.initials: T
authors.lastname: Anai
authors.affiliation: Agronomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. tshimiz@affrc.go.jp.
authors.forename: Takehiko
authors.initials: T
authors.lastname: Shimizu
authors.affiliation: Agronomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. tsayama@affrc.go.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Sayama
authors.affiliation: Agronomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. kytakagi@affrc.go.jp.|Present Address: Soil Science and Plant Nutrition Division, National Agriculture and Food Research Organization Agricultural Research Center, 3-1-1, Kannondai, Tsukuba, Ibaraki, 305-8666, Japan. kytakagi@affrc.go.jp.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Takagi
authors.affiliation: Agronomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. kay0machk0@gmail.com.
authors.forename: Kayo
authors.initials: K
authors.lastname: Machita
authors.affiliation: Agronomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. nabemame@cc.saga-u.ac.jp.|Faculty of Agriculture, Saga University, 1 Honjo-machi, Saga, 840-8502, Japan. nabemame@cc.saga-u.ac.jp.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Agronomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. nisimura@agr.niigata-u.ac.jp.|Present Address: Faculty of Agriculture, Niigata University, 8050, Ikarashi 2-no-cho, Nishi-ku, Niigata, 950-2181, Japan. nisimura@agr.niigata-u.ac.jp.
authors.forename: Minoru
authors.initials: M
authors.lastname: Nishimura
authors.affiliation: Nagano Vegetable and Ornamental Crops Experiment Station, 1066-1, Soga, Shiojiri, Nagano, 399-6461, Japan. nyamada@chushin-exp.pref.nagano.jp.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Yamada
authors.affiliation: Agronomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. morisatomi@affrc.go.jp.
authors.forename: Satomi
authors.initials: S
authors.lastname: Mori
authors.affiliation: Agronomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. sasaharu@affrc.go.jp.
authors.forename: Harumi
authors.initials: H
authors.lastname: Sasaki
authors.affiliation: Agronomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. kanamo@affrc.go.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kanamori
authors.affiliation: Agronomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. katayose@affrc.go.jp.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Katayose
authors.affiliation: Agronomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. ishimoto@affrc.go.jp.
authors.forename: Masao
authors.initials: M
authors.lastname: Ishimoto
mesh_terms: D059014:High-Throughput Nucleotide Sequencing; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics; D013025:Glycine max / Q000235:genetics*