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

  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},
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
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).
Distribution and characterization of more than 1000 T-DNA tags in the genome of Brachypodium distachyon community standard line Bd21.
A collection of 4117 fertile T-DNA lines has been generated by Agrobacterium-mediated transformation of the diploid community standard line Bd21 of Brachypodium distachyon by the BrachyTAG programme, and more than half of the T-DNAs inserted in genic regions.
Using Combined Methods of Genetic Mapping and Nanopore-Based Sequencing Technology to Analyze the Insertion Positions of G10evo-EPSPS and Cry1Ab/Cry2Aj Transgenes in Maize
The combined method of genetic mapping and the nanopore-based sequencing technology will be a useful approach for identifying the insertion positions of transgenic sequences in other GM plants with relatively large and complex genomes.
A new method to identify flanking sequence tags in chlamydomonas using 3’-RACE
It is proposed that the 3’-RACE FST method can be used to build large scale FST libraries in Chlamydomonas and other transformable organisms.
TADEA-PCR is a highly efficient method of amplifying unknown flanking fragments of T-DNA transformants.
Forward genetic analysis, widely used to find new gene functions, benefits from the availability of mutants. At present, based on Agrobacterium-mediated plant transformation technology, many transfer
Investigating transgene integration and organization in cotton (Gossypium hirsutum L.) genome.
This chapter uses conventional PCR and genomic Southern blot hybridization to characterize integration of T-DNA components and vector backbone fragments and surveys of genomic structure changes brought by transgene integration by comparing a pre-insertion site with corresponding transGene/plant junctions.
T-DNA mutagenesis in Brachypodium distachyon.
The utility of these mutant collections is illustrated with some examples from the BrachyTAG collection at the John Innes Centre-such as those in the eukaryotic initiation factor 4A (eIF4A) and brassinosteroid insensitive-1 (BRI1) genes.
Brachypodium distachyon T-DNA insertion lines: a model pathosystem to study nonhost resistance to wheat stripe rust
The phenotypic evaluation showed that the response of Brachypodium distachyon to PST was nonhost resistance (NHR), which allowed this plant-pathogen system as a model to explore the immune response and the molecular mechanism underlying wheat and PST.
A TILLING Platform for Functional Genomics in Brachypodium distachyon
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.


Generation of a flanking sequence-tag database for activation-tagging lines in japonica rice.
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
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.
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.
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
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.
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
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
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.
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
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.