EMS was commonly used to induce mutations for various organisms, causing nucleotides to mispair with their complementary bases. So, chemical mutagenesis has become the best method for inducing mutations in genetic studies. Simple PCR-based detection and high-throughput technologies helped to screen and identify mutations. Degenerate oligonucleotide primed PCR (DOP-PCR) became getting attention for mutation survey because the requirement of sequence information and high cost for designing primers could be diminished. Also, high-throughput sequencing instruments, such as GS-FLX, allowed characterization of nucleic acids and massive mutant analysis. A total of 6,696 aligned pairs for Sinpaldalkong 2 vs. SS2-2 and 6,935 for Sinpaldalkong 2 vs. 25-1-1 were formed for mutation detection. A mutation every 437 bp in SS2-2 and every 402 bp in 25-1-1 was observed. About 2/3 of a total of mutations were single base variation in both comparisons. Mutated and non-mutated fragments from SS2-2 and 25-1-1 were distributed on all LGs. The 25-1-1 had more mutations than SS2-2 compared with their wild type, Sinpaldalkong 2. Local compositional bias was also observed around the mutated G. Our modified DOP-PCR primers were successfully amplified and their amplicons were located on randomly but somewhat targeted regions of soybean genome.

Expressed sequence taqs (ESTs) have accepted to be a valuable tool for discovering single nucleotide polymorphism (SNP) in many species. For detection putative SNPs in soybean genome, approximately 90,000 EST sequences from genotype Williams 82 were downloaded from NCBI. The paralog sequences of these ESTs were distinguished by TGI clustering tools (TGICL) performing megablast and EST cluster analysis, and the EST clusters were used as reference sequences for detection putative SNPs by in silico. The EST clusters were aligned with EST sequence from other cultivars of soybean by Polybase (computer software). The results revealed that putative SNPs were distributed in 5,677 clusters with frequency of 1 SNP per 333 nucleotide sites. For SNP validation, 43 primer pairs were designed from EST clusters containing putative SNPs for sequencing genomic DNA of Williams 82, Harosoy, Peking, Pureunkong, Jinpumkong 2, Hwangkeumkong and IT182932. From results of sequencing PCR, we totally found 99 SNPs from 33 primer pairs. Twenty-three and 47 out of 99 SNPs showed polymorphisms between Pureunkong and Jinpumkong 2, and Hwangkeumkong and IT182932, respectively. The SNPs discovered from this study can be used for genetic mapping in the four genotypes of soybean.