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

@article{Thole2009APF,
  title={A protocol for efficiently retrieving and characterizing flanking sequence tags (FSTs) in Brachypodium distachyon T-DNA insertional mutants},
  author={Vera Thole and Silvia Cristina Batezati Alves and Barbara Worland and Michael Webster Bevan and Philippe Vain},
  journal={Nature Protocols},
  year={2009},
  volume={4},
  pages={650-661}
}
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… 
Highly efficient generation of T-DNA insertion lines and isolation of flanking sequence tags (FSTs) of Brachypodium distachyon
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In this study, Agrobacterium-mediated transformation system was optimized to generate T-DNA mutants and a simple and highly efficient method was developed to isolate T- DNA flanking sequence tags (FSTs).
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TLDR
The new model plant for temperate grasses, Brachypodium distachyon, offers great potential as a tool for functional genomics, and a sodium azide-induced mutant collection and the TILLING platform should greatly facilitatefunctional genomics approaches in this model organism.
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References

SHOWING 1-10 OF 25 REFERENCES
Generation of a flanking sequence-tag database for activation-tagging lines in japonica rice.
TLDR
Mapping of the FSTs on chromosomes revealed that T- DNA integration frequency was generally proportional to chromosome size, however, T-DNA insertions were non-uniformly distributed on each chromosome: higher at the distal ends and lower in regions close to the centromeres.
An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome
TLDR
This protocol describes an adapter ligation-mediated PCR method that has been used to screen a mutant library and identify over 150,000 T-DNA insertional mutants; the method can also be applied to map individual mutants.
High-throughput generation of sequence indexes from T-DNA mutagenized Arabidopsis thaliana lines.
TLDR
The pipeline included robotized extraction of genomic DNA in a 96-well format, an adapter-ligation PCR method for amplification of plant sequences adjacent to T-DNA borders, automated purification and sequencing of PCR products, and computational trimming of the resulting sequence files.
Agrobacterium-mediated transformation of the temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional mutagenesis.
TLDR
A facile, efficient and rapid transformation system for Bd21 is described using Agrobacterium-mediated transformation of compact embryogenic calli derived from immature embryos to facilitate large-scale functional genomics research in this model system.
T-DNA insertional mutagenesis for functional genomics in rice.
  • J. Jeon, S. Lee, G. An
  • Biology
    The Plant journal : for cell and molecular biology
  • 2000
TLDR
The large population of T-DNA-tagged lines will be useful for identifying insertional mutants in various genes and for discovering new genes in rice.
Screening of transgenic plants by amplification of unknown genomic DNA flanking T-DNA.
TLDR
The PCR patterns obtained were specific and reproducible for different plants from a given transgenic line and the number of PCR products obtained could be considered a good estimation of the T-DNA copy number.
Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics
TLDR
A highly efficient seed-embryo callus transformation procedure of japonica rice is established that results both in a high frequency (75–95%) of co-cultured calli yielding resistant cell lines and the generation of multipleresistant cell lines per co- Cultured callus.
A large-scale study of rice plants transformed with different T-DNAs provides new insights into locus composition and T-DNA linkage configurations
TLDR
Comparison of transGene presence and expression in progeny plants showed that segregation of the transgene phenotype was not a reliable indicator of either transgenes inheritance or T-DNA linkage, as only 60–80% of thetransgenic loci were detected by the expression study.
On Defining T-DNA.
TLDR
Examination of transferred DNA in several hundred plants independently transformed using an Agrobacterium-mediated binary vector system reveals a high frequency of “beyond the border” DNA transfer, which may contribute to the design of ongoing investigations into the molecular mechanism of T-DNA transfer.
T-DNA–mediated transfer of Agrobacterium tumefaciens chromosomal DNA into plants
TLDR
It is demonstrated that large (up to ∼18 kb) gene-bearing fragments of Agrobacterium chromosomal DNA (AchrDNA) can be integrated into Arabidopsis thaliana genomic DNA during transformation.
...
...