Analysis of EMS Mutagenized Soybean by Combination of DOP-PCR and GS-FLX

Ethylmethanesulfonate (EMS) has been commonly used to induce mutations for various organisms because it led to irreversible mutations with a high level of frequency. With relatively few individuals, saturated mutagenized populations could be generated by chemical mutagens. Since high-throughput sequencing instruments, such as GS-20 or GS-FLX from Roche/454 Life Sciences are now available, characterization of nucleic acids and massive mutant analysis are more feasible. Due to the requirement of sequence information and high cost for designing primers, degenerate oligonucleotide primed PCR (DOP-PCR) instead of direct sequencing was used for single nucleotide polymorphisms (SNPs) survey. In this study, we screen point mutation in soybean mutants generated by EMS mutagenesis using a combination of DOP-PCR methodology and GS-FLX. Four different modified DOP-PCR primers were used for amplifying genomic DNA of three soybean genotypes, Sinpaldalkong 2, SS2-2 and 25-1-1. Then, nucleotide sequences of these amplified PCR products were analyzed by GS-FLX. Different number and length of contigs and singlets were constructed depending on soybean genotypes and nucleotide identity, after sequences were trimmed and aligned. With 100% in identity, average 1,100 contigs and 7,000 singlets were formed in each soybean genotype. In order to survey sequence polymorphisms, POLYBAYES was used with base quality consideration. A total of 1,187 putative SNPs were detected, and these polymorphisms should be reconfirmed by direct sequencing after a homology search against GenBank databases. (author)