A protocol for efficiently retrieving and characterizing flanking sequence tags (FSTs) in Brachypodium distachyon T-DNA insertional mutants

• Published in: Nature protocols Vol. 4; no. 5; pp. 650 - 661
• Main Authors: Vain, Philippe, Thole, Vera, Alves, Sílvia C, Worland, Barbara, Bevan, Michael W
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.04.2009; Nature Publishing Group
• Subjects: Analytical Chemistry; Barley; Base Sequence; Biological Techniques; Biomedical and Life Sciences; Cereal crops; Computational Biology/Bioinformatics; Deoxyribonucleic acid; DNA; DNA, Bacterial; DNA, Plant - chemistry; Genes; Genetic recombination; Genetic Vectors - chemistry; Genome, Plant; Genomes; Genomics; Grasses; Life Sciences; Methods; Microarrays; Molecular Sequence Data; Mutagenesis; Mutagenesis, Insertional; Organic Chemistry; Plant genetic engineering; Poaceae - genetics; Polymerase chain reaction; Polymerase Chain Reaction - methods; Protocol; Research parks; Rice; Sequence Analysis, DNA; Sequence Tagged Sites; Wheat; United Kingdom; United Kingdom > UK
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2009.32

Brachypodium distachyon is emerging as a new model system for bridging research into temperate cereal crops, such as wheat and barley, and for promoting research in novel biomass grasses. Here, we provide an adapter ligation PCR protocol that allows the large-scale characterization of T-DNA insertions into the genome of Brachypodium . The procedure enables the retrieval and mapping of the regions flanking the right and left borders (RB and LB) of the T-DNA inserts and consists of five steps: extraction and restriction digest of genomic DNA; ligation of an adapter to the genomic DNA; PCR amplification of the regions flanking the T-DNA insert(s) using primers specific to the adapter and the T-DNA; sequencing of the PCR products; and identification of the flanking sequence tags (FSTs) characterizing the T-DNA inserts. Analyzing the regions flanking both the LB and RB of the T-DNA inserts significantly improves FST retrieval and the frequency of mutant lines for which at least one FST can be identified. It takes approximately 16 or 10 d for a single person to analyze 96 T-DNA lines using individual or batch procedures, respectively.