Identification of the T-DNA Flanking Sequences and Event-Specific PCR Analysis of High-Content Oleic Acid Transgenic Soybean Based on Genome Re-sequencing
Abstract：The specific detection method based on the specificity of the connection area between external T-DNA and plant genome has very high specificity and accuracy, which is an important technical means to effectively supervise and manage the transgenic event and its derivatives, and ensure the healthy development of the transgenic industry. In the previous study, 2 transgenic soybean (Glycine max) events were obtained with >78% oleic acid content. To isolate the flanking sequences of T-DNAs with endogenous genes and promote the safety assessment and application of these 2 transgenic events, genome re-sequencing combined with PCR was carried out to analyze the flanking sequences of T-DNAs of these 2 transgenic events. There were 1 and 2 copies T-DNAs in these 2 transgenic events by Southern blot analysis, respectively. Using genome re-sequencing compared to reference genome information based on BWA-Burrows-Wheeler Alignment tool method (http://bio-bwa.sourceforge.net/), flanking sequences of T-DNAs in the transgenic soybean 'E2D9050' and 'EB8072' genomes were isolated. The T-DNA of 'E2D9050' was integrated into the position 51410941 on Chr04 with one copy. The T-DNA of 'EB8072' was integrated into soybean genome with 2 copies in position 38147218 on Chr19 and the pattern of integration was inverted insertion with right borders of these copies interfaced. These results were in accord with the results of Southern blot analysis. According to the flanking sequences of these positions, primers were designed and PCR was amplified to verify these flanking sequences of insertion sites. Event-specific primers were designed based on these fragments and the sensitivities of these pairs of primer were both 0.1%. It provides an accurate and fast method for identification of these kinds of transgenic event.
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