Highly efficient generation of T-DNA insertion lines and isolation of flanking sequence tags (FSTs) of Brachypodium distachyon

Brachypodium distachyon (Brachypodium) has been developed as a model system for the temperate grasses. Hence, establishing a large insertion mutant population and identifying T-DNA insertion sites are imperative for functional genomics studies. Currently, Brachypodium has an established T-DNA collection resource, but it is far less than needed. In addition, the conventional methods for the isolation of flanking sequence tags (FSTs) characterizing the T-DNA inserts, such as TAIL-PCR, adapter ligation-mediated PCR, and inverse PCR, are time-consuming, laborious, and expensive. In this study, Agrobacterium-mediated transformation system was optimized to generate T-DNA mutants. Approximately 7000 T-DNA insertion lines were obtained. Furthermore, a simple and highly efficient method was developed to isolate T-DNA flanking sequence tags (FSTs). The procedures simplified the multi-step reaction that is required for the conventional inverse PCR and several reactions were conducted within a microscale reaction system in one tube. It is flexible to isolate FSTs for either individual or large numbers of T-DNA lines. To rapidly process the large-scale sequence data, a serial of Perl scripts were developed using the Perl Programming Language (http://www.perl.org). Using these methods, a total of 794 flanking sequences were isolated and analyzed in detail. Although the methods were developed in the process of isolating and analyzing T-DNA flanking sequences of Brachypodium, it can be applied to other plants.