Construction and Evaluation of an EMS-Induced Mutant Library of Japanese Soybean Cultivar Enrei

A mutant library with a high mutation frequency was developed to a high quality Japanese soybean cultivar Enrei to identify useful, novel alleles for breeding. The seeds were treated with the chemical mutagen 0.35% ethyl methanesulfonate (EMS). To increase the mutation frequency, bulk M₂ seeds from ~2,000 M₁ plants were also treated in a similar way and used to grow M₁′ plants of the almost same population size as in the M₁ generation (The generation after second mutagen treatment was called M₁′ to discriminate it from the initial M₁ generation). The resultant library consisted of DNA derived from 1,536 M₂′ plants; 1,437 of them were able to produce seeds. DNA from each M₂′ plant was quantified; equivalent amounts of DNA from four individual plants were pooled into each well of a 384-well plate and mutations were detected by high-resolution melting (HRM) analysis. Mutation was also screened based on next generation sequencing (NGS) analysis. Twenty four plants with base substitution mutations in a 1030 bp region of a certain gene were identified in the library. Among the detected mutations, 16 substitutions (8 non-synonymous and 8 synonymous) occurred at different positions. A total of 17.1 million mutations was estimated to be distributed over the 1.1-Gb soybean genome. The mutation frequency in the library (~1 substitution per 100 kbp) is comparable to those reported for other high-mutation-frequency libraries. Thus, the present library has a potential to yield useful mutant alleles for soybean breeding.