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

Han, Hongjiang; Shen, Guoan; An, Tianyue; Song, Bo; Zhao, Suzhen; Qi, Xiaoquan

doi:10.1007/s11816-018-0489-4

Cited by 3 publications

(1 citation statement)

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“…Information on insertion sites forms an important molecular data for the safety assessment of transgenic organisms in the world 17 and requires a precise and efficient method for the isolation of flanking sequences. Currently, TAIL-PCR and ligation-mediated PCR are used for the identification of flanking sequences in complex genomes [17][18][19][20][21] , and the application of reverse PCR has become less frequent. Despite several improvements and optimizations, TAIL-PCR still has a lower success rate in practical application due to the non-specific products or short product fragments.…”

Section: Discussionmentioning

confidence: 99%

Cyclic Digestion and Ligation-Mediated PCR Used for Flanking Sequence Walking

Zhou

Sun

et al. 2020

Sci Rep

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Ligation-mediated PCR (LM-PCR) is a classical method for isolating flanking sequences; however, it has a common limitation of reduced success rate owing to the circularization or multimerization of target restriction fragments including the known sequence. To address this limitation, we developed a novel LM-PCR method, termed Cyclic Digestion and Ligation-Mediated PCR (CDL-PCR). The novelty of this approach involves the design of new adapters that cannot be digested after being ligated with the restriction fragment, and cyclic digestion and ligation may be manipulated to block the circularization or multimerization of the target restriction fragments. Moreover, to improve the generality and flexibility of CDL-PCR, an adapter precursor sequence was designed, which could be digested to prepare 12 different adapters at low cost. Using this method, the flanking sequences of T-DNA insertions were obtained from transgenic rice and Arabidopsis thaliana. The experimental results demonstrated that CDL-PCR is an efficient and flexible method for identifying the flanking sequences in transgenic rice and Arabidopsis thaliana.

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Section: Discussionmentioning

confidence: 99%

Cyclic Digestion and Ligation-Mediated PCR Used for Flanking Sequence Walking

Zhou

Sun

et al. 2020

Sci Rep

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EstablishingAc/DsStarter Lines for Large-Scale Transposon-Tagged Mutagenesis in Tomato

Kumari

Ponukumatla

Pandey

et al. 2023

Preprint

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Tomato (Solanum lycopersicum), a model system for ripening of fleshy fruits, has ~40,000 genes predicted by in silico homology-based annotation. However, functional validation is lacking for most annotated tomato genes. Among the strategies for functional annotations, transposon-tagged mutagenesis is the most powerful approach. Transposon-tagged genes can be functionally validated by phenotyping and activation tagging. However, the lack of a robust in planta transformation system precludes large-scale transposon-mutagenesis of tomato. To overcome this limitation, we developed two sets of starter lines in tomato, each carrying maize transposon elements (Ds) and transposases (Ac), respectively. The Ds and Ac lines were crossed to allow the Ac-mediated transposition of the Ds in the F1 generation. In the F2 generation, the location of excised Ds at new sites in the tomato genome was monitored. The Ds transposition was interspersed on different chromosomes of the tomato, indicating unlinked transposition of the Ds. The analysis of DNA sequences flanking Ds showed random integration of Ds in intergenic regions, genes, and the promoter region of the genome. Our study paves the way for the generation of large-scale transposon-tagged tomato lines using Ac/Ds starter lines and provides a potential tool for the functional validation of genes in tomato.

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