ISSN 2167-0447
International Journal of Agricultural Sciences Vol. 1(1), pp. 020-024, December 2011, ISSN: 2167-0447 ©2011 International Scholars Journals
Full Length Research Paper
Vistra Green-Stained cDNA AFLP Technique for Identification of Transcript Profiles in Supernodulating Soybean
Puji Lestari1 and Suk Ha Lee2
1Indonesian Center for Agricultural Biotechnology and Genetic Reseources Research and Development, Jl.Tentara Pelajar No. 3 A Bogor 16111, Indonesia
2Seoul National University, Seoul, South Korea
*Corresponding Author's E-mail: [email protected]
Received December 5, 2011; Accepted December 22, 2011
Abstract
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 non-denaturing 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.
Keywords: biological nitrogen fixation, Bradyrhizobium japonicum, cDNA AFLP, soybean, supernodulation, transcript derived fragmen (TDF).