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PLoS One. 2015 Oct 7;10(10):e0139672. doi: 10.1371/journal.pone.0139672. eCollection 2015.

High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture.

Author information

1
Plant Biology Department and Genome Center, University of California Davis, Davis, California, United States of America; Department of Integrative Genetics, National Institute of Genetics, Mishima, Japan.
2
Plant Biology Department and Genome Center, University of California Davis, Davis, California, United States of America.

Abstract

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA-genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.

PMID:
26445462
PMCID:
PMC4596565
DOI:
10.1371/journal.pone.0139672
[Indexed for MEDLINE]
Free PMC Article

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