DNA Sequence Analysis of Induced Mutants in Soybean

Chemical mutagens, such as ethylmethanesulfonate (EMS), that cause point mutations were commonly used to induce mutations in both plants and animals due to the high frequency of heritable mutations. Saturated mutagenized populations could be generated with relatively few individuals. High-throughput genome sequencing is now available using the GS-20 or GS-FLX machines from Roche/454 Life Sciences, which are based on the advantages of emulsion PCR and pyrosequencing. Because of the wide availability of several high throughput genotyping methods, single nucleotide polymorphisms (SNPs) have drawn the attention of researchers. Degenerate oligonucleotide primed PCR (DOP-PCR) has been used for SNP genotyping in various organisms. In this study, we report the massive DNA sequence analysis of soybean mutants induced by EMS mutagenesis. Three soybean genotypes were used: 'Sinpaldalkong 2,' 'SS2-2' and '25-1-1.' Sinpaldalkong 2 is a recommended soybean variety in Korea and SS2-2 was generated by EMS mutagenesis of Sinpaldalkong 2. Genotype 25-1-1 is a regenerated M3 plant from EMS-treated immature embryo culture of Sinpaldalkong 2. After genomic DNA from the three soybean genotypes was amplified with modified DOP-PCR, nucleotide sequences were determined using the GS-FLX. Assuming only orthologues show 100% identity in BLASTN, an average of 1,100 contigs and 7,000 singlets were formed in each soybean genotype. A total of 1,187 SNPs were detected, with a frequency as 1 SNP per 272bp, using POLYBAYES to survey sequence polymorphisms. (author)