Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing

• DOI: 10.1186/s12864-015-2079-y
• Publication Date: 2015-11-26T08:14:54Z
• Authors: Mai Tsuda; Akito Kaga; Toyoaki Anai; Takehiko Shimizu; Takashi Sayama; Kyoko Takagi; Kayo Machita; Satoshi Watanabe; Minoru Nishimura; Naohiro Yamada; Satomi Mori; Harumi Sasaki; Hiroyuki Kanamori; Yuichi Katayose; Masao Ishimoto
• Keywords: Sanger sequencing; Ethyl methanesulfonate; Amplicon; Nonsense mutation

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 identification novel alleles useful breeding. Development cost-effective high-throughput protocols using next generation sequencing (NGS) technologies is expected to simplify retrieval mutants mutations interest. To increase density, seeds Japanese elite cultivar Enrei were treated chemical mutagen ethyl methanesulfonate (EMS); M2 produced by M1 plants EMS once again. The resultant library, which consisted DNA from 1536 plants, revealed large morphological physiological variations. Based on whole-genome re-sequencing analysis 12 lines, average number base changes was 12,796 per line. On average, 691 35 line missense nonsense mutations, respectively. Two screening strategies resolution melting (HRM) indexed amplicon designed retrieve mutants; confirmed Sanger as final step. In comparison HRM several genes, allows one scan longer sequence range skip steps know information because it uses systematic pooling index NGS reads, simplifies discovery amino acid substitutions. developed. (1 mutation/74 kb) achieved repeating treatment. our sufficiently obtain plant gene mutated. Thus, will potential yield