Abstract: The gene encoding betaine aldehyde dehydrogenase (BADH), an important osmoregulation gene, has been transformed into many crops, including wheat (Triticum aestivum L.), rice (Oryza sativa L.), maize (Zea maysL.), rape (Brassica campestris L.), and potato (Solanum tuberosumL.). Transgenic crops carring BADH gene enhances tolerance to salinity and drought stresses. Gene badh had been integrated into the most important forage crop alfalfa (Medicago sativa L.) in our previous work, and 42 transgenic plants with improved salt tolerance were obtained. Since they were derived from the same transformant vector, these plants were not able to be distinguished from each other by commonly used methods, such as screening detection, gene specific detection, and vector specific detection. To differentiate these transformants in molecular level, we performed the thermal asymmetric interlaced PCR (TAIL-PCR) to separate the T-DNA flanking sequences for identification of the transgenic plants in event specific detection. A total of six sequences flanking either the left or the right borders of the T-DNA were obtained, which included the flanking sequences at both left and right borders of plant B127, and the left border flanking sequences of plants B125, B138, B295, and B196. The left border sequence of T-DNA was completely deleted from the vector and was thus not integrated into the genome of alfalfa in the transgenic plant B196. Although the left border flanking sequence in the transgenic plant B127 was not changed, it was filled with a DNA sequence of unknown origin. The forward and backward primers for PCR were designed based on the characteristics of the flanking sequences originating from the vector sequence and the alfalfa genomic sequence adjacent to the integrated vector sequence, respectively. The results of amplification in 42 BADH-transgenic alfalfa plants showed that plants B106, B125, B127, B138, B157, B158, B289, B295, and B305 presented the same amplification banding patterns. Plants B196, B203, B220, and B223 produced the same banding pattern which was different from that in other plants. These results indicated that the plants with identical amplification banding pattern may come from the same transformation event. Based on the findings in the present study, we successfully used the flanking sequences separated by TAIL-PCR analysis in developing event specific detection method, which can be used not only to differentiate the origins of various transformants, but also to distinguish the transformants from each other. It is useful in protection of transgenic crops and labeling of transgenic products.

Key words: Transgenic alfalfa, BADH, T-DNA, Flanking sequence, Event specific detection