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

Nature Protocols volume 4, pages 650–661 (2009)Cite this article

Abstract

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.

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Figure 1: Binary vector pVec8-GFP used for Agrobacterium-mediated transformation of Brachypodium. The vector pVec8-GFP16 (14,558 nt) contains two expression units within the T-DNA (5,663 nt).
Figure 2: Examples of regions flanking the right or left border of the T-DNA for different types of inserts into the plant genome.
Figure 3: Analysis of flanking regions adjacent to the left or right border of the T-DNA.
Figure 4: PCR amplification of the sequences flanking the left border of the T-DNA(s) inserted into 58 independently transformed Bd21 plant lines.
Figure 5: Example of a raw PCR2 product sequence retrieved from the transgenic line BdAA1.
Figure 6: Analysis of flanking regions adjacent to the right border of two T-DNA inserts (genetically linked or unlinked) into the plant genome.

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Acknowledgements

This study was supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC) and through a Short-Term Marie Curie EST fellowship.

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Authors and Affiliations

  1. Department of Crop Genetics, John Innes Centre, Norwich Research Park, Colney Lane, NR4 7UH, Norwich, UK

    Vera Thole, Sílvia C Alves, Barbara Worland & Philippe Vain

  2. Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park, Colney Lane, NR4 7UH, Norwich, UK

    Michael W Bevan

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  1. Vera Thole
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  2. Sílvia C Alves
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Correspondence to Philippe Vain.

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Thole, V., Alves, S., Worland, B. et al. A protocol for efficiently retrieving and characterizing flanking sequence tags (FSTs) in Brachypodium distachyon T-DNA insertional mutants. Nat Protoc 4, 650–661 (2009). https://doi.org/10.1038/nprot.2009.32

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