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Title: High-throughput analysis of T-DNA location and structure using sequence capture

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 usemore » as commercial products. As a result, our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.« less
Authors:
 [1] ;  [2] ;  [2] ;  [2]
  1. Univ. of California, Davis, CA (United States); National Institute of Genetics, Mishima (Japan)
  2. Univ. of California, Davis, CA (United States)
Publication Date:
OSTI Identifier:
1239138
Grant/Contract Number:
SC0007183
Type:
Accepted Manuscript
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 10; Journal Issue: 10; Related Information: Data Availability: All short read sequence files are available under BioProject ID PRJNA287142 and SRA ID: SRP059868.; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Research Org:
Univ. of California, Davis, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
sequence alignment; plant genomics; Arabidopsis thaliana; genomic library construction; polymerase chain reaction; probe hybridization; repeated sequences; sequence assembly tools