Vistra Green-Stained cDNA AFLP Technique for Identification of Transcript Profiles in Supernodulating Soybean

Abstract


Puji Lestari and Suk Ha Lee

To understand the molecular bases of symbiotic association, cDNA-AFLP technique was performed to identify differential transcripts between supernodulating soybean mutant, SS2-2, and its wild type, Sinpaldalkong 2. To determine optimum time point for identifying the transcripts, templates were prepared from soybean leaves depending on time after inoculation (TAI) of Bradyrhizobium japonicum on 3 soybean genotypes, SS2-2, Sinpaldalkong 2 and Jangyeobkong. While to identify the trasncripts associated in symbiotic interaction, cDNA-AFLP was carried out based on pre-determined time on 2 genotypes, SS2-2 and Sinpaldalkong 2. Gel-based vistra-green stained cDNA-AFLP system with nondenaturing polyacrylamide gel was established in this study. As the results, transcript-derived fragments (TDFs) showed the polymorphic banding patterns regardless of TAI. Using 22 primer combinations of EcoRI and MseI, polymorphism levels of the TDFs among 3 genotypes were found to be the highest on one week after inoculation (WAI), determining the optimum time point for isolating differential transcripts profiles. A total of 4000 amplicons were recognized, leading 147 differential fragments to be specific in the SS2-2 (61 fragments) and 86 TDFs specific to the wild type. Approximately 80.3% of fragments were successfully reamplified, and used as candidates for differentially expressed genes in the symbiotic association specifically in supernodulating and normal soybeans.

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