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

Abstract

BackgroundFunctions 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.ResultsTo 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.ConclusionsA 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2079-y) contains supplementary material, which is available to authorized users.

The Analysis of Physiological Variations in M2 Generation of Solanum melongena L. Mutagenized by Ethyl Methane Sulfonate

“…The plant height phenotype showed the highest mutation frequency, whereas the flower phenotype showed the lowest mutation frequency. A similar phenomenon was reported by Saito et al (2011) and Tsuda et al (2015). This phenomenon may be explained by the fact that the higher mutation frequency phenotypes were regulated by the more number of structure or regulatory genes.…”

“…A light-green exocarp mutant was discovered from the EMS-mutagenized cucumber line 406 with dark-green exocarp (Zhou et al, 2015). In soybean, the protein, oil, and sugar contents of the mutants are abundant (Tsuda et al, 2015). Although the EMS-induced mutation library shows abundant variations in the phenotypic trait and metabolic product content (Saito et al, 2011; Hwang et al, 2014; Arisha et al, 2015; Shikata et al, 2016), only the dominant mutation phenotypic traits are visualized in the M 1 generation.…”

“…The types of EMS-induced mutation include SNV (base transition, base insertion, and base deletion), CNV, and indel section. The C/G to T/A transitions are the predominant mutations in EMS mutants (Uchida et al, 2011; Henry et al, 2014; Tsuda et al, 2015; Shirasawa et al, 2016). These mutations affect the protein synthesis or structure, thereby leading to phenotypic change.…”

Mutant Resources and Mutagenomics in crop plants

“…Such mutant resource offers as an excellent material for understanding the radiation damage, physiological basis, ultrastructural changes and plant metabolic pathways, besides for gene mapping and is valuable for functional genomics (Lundquist, 2009;Mirajkar et al, 2016). Both functional genomics and mutant gene detection studies will require a mutant library having a high mutation density (Tsuda et al, 2015). For example, TOMATOMA (http://tomatoma.…”

Frequent generation of mutants with coincidental changes in multiple traits via ion-beam irradiation in soybean

“…Subsequently, plants have been produced with desirable traits in terms of seed components (Takagi et al, 1990;Fehr et al, 1991;Hajika et al, 1991;Kitamura, 1991;Takahashi et al, 1994;Hayashi et al, 1998), nodule formation (Carroll et al, 1985) or herbicide tolerance (Sebastian et al, 1989) via mutagenesis using X-ray or gamma-ray irradiation or chemicals. Recently, mutants have been generated using fast neutrons (Bolon et al, 2011) or ethyl methanesulfonate (Tsuda et al, 2015) in combination with a high-throughput next-generation sequencing approach, indicating the feasibility of these new methods to obtain mutants.…”