Re-sequencing transgenic plants revealed rearrangements at T-DNA inserts, and integration of a short T-DNA fragment, but no increase of small mutations elsewhere

Henk J. Schouten*, Henri van de Geest, Sofia Papadimitriou, Marian Bemer, Jan G. Schaart, Rene Smulders, Gabino Sanchez Perez, Elio Schijlen

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

13 Citations (Scopus)

Abstract

Key message: Transformation resulted in deletions and translocations at T-DNA inserts, but not in genome-wide small mutations. A tiny T-DNA splinter was detected that probably would remain undetected by conventional techniques. Abstract: We investigated to which extent Agrobacterium tumefaciens-mediated transformation is mutagenic, on top of inserting T-DNA. To prevent mutations due to in vitro propagation, we applied floral dip transformation of Arabidopsis thaliana. We re-sequenced the genomes of five primary transformants, and compared these to genomic sequences derived from a pool of four wild-type plants. By genome-wide comparisons, we identified ten small mutations in the genomes of the five transgenic plants, not correlated to the positions or number of T-DNA inserts. This mutation frequency is within the range of spontaneous mutations occurring during seed propagation in A. thaliana, as determined earlier. In addition, we detected small as well as large deletions specifically at the T-DNA insert sites. Furthermore, we detected partial T-DNA inserts, one of these a tiny 50-bp fragment originating from a central part of the T-DNA construct used, inserted into the plant genome without flanking other T-DNA. Because of its small size, we named this fragment a T-DNA splinter. As far as we know this is the first report of such a small T-DNA fragment insert in absence of any T-DNA border sequence. Finally, we found evidence for translocations from other chromosomes, flanking T-DNA inserts. In this study, we showed that next-generation sequencing (NGS) is a highly sensitive approach to detect T-DNA inserts in transgenic plants.
Original languageEnglish
Pages (from-to)493-504
JournalPlant Cell Reports
Volume36
Issue number3
DOIs
Publication statusPublished - Mar 2017

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transgenic plants
mutation
DNA
genome
Arabidopsis thaliana
Agrobacterium radiobacter
chromosomes
genomics

Keywords

  • Agrobacterium tumefaciens-mediated transformation
  • Arabidopsis thaliana
  • Molecular characterization
  • Mutation frequency
  • Next-generation sequencing
  • Splinter

Cite this

@article{abb59dc0f1384116bc4d44f19a613760,
title = "Re-sequencing transgenic plants revealed rearrangements at T-DNA inserts, and integration of a short T-DNA fragment, but no increase of small mutations elsewhere",
abstract = "Key message: Transformation resulted in deletions and translocations at T-DNA inserts, but not in genome-wide small mutations. A tiny T-DNA splinter was detected that probably would remain undetected by conventional techniques. Abstract: We investigated to which extent Agrobacterium tumefaciens-mediated transformation is mutagenic, on top of inserting T-DNA. To prevent mutations due to in vitro propagation, we applied floral dip transformation of Arabidopsis thaliana. We re-sequenced the genomes of five primary transformants, and compared these to genomic sequences derived from a pool of four wild-type plants. By genome-wide comparisons, we identified ten small mutations in the genomes of the five transgenic plants, not correlated to the positions or number of T-DNA inserts. This mutation frequency is within the range of spontaneous mutations occurring during seed propagation in A. thaliana, as determined earlier. In addition, we detected small as well as large deletions specifically at the T-DNA insert sites. Furthermore, we detected partial T-DNA inserts, one of these a tiny 50-bp fragment originating from a central part of the T-DNA construct used, inserted into the plant genome without flanking other T-DNA. Because of its small size, we named this fragment a T-DNA splinter. As far as we know this is the first report of such a small T-DNA fragment insert in absence of any T-DNA border sequence. Finally, we found evidence for translocations from other chromosomes, flanking T-DNA inserts. In this study, we showed that next-generation sequencing (NGS) is a highly sensitive approach to detect T-DNA inserts in transgenic plants.",
keywords = "Agrobacterium tumefaciens-mediated transformation, Arabidopsis thaliana, Molecular characterization, Mutation frequency, Next-generation sequencing, Splinter",
author = "Schouten, {Henk J.} and {van de Geest}, Henri and Sofia Papadimitriou and Marian Bemer and Schaart, {Jan G.} and Rene Smulders and Perez, {Gabino Sanchez} and Elio Schijlen",
year = "2017",
month = "3",
doi = "10.1007/s00299-017-2098-z",
language = "English",
volume = "36",
pages = "493--504",
journal = "Plant Cell Reports",
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}

Re-sequencing transgenic plants revealed rearrangements at T-DNA inserts, and integration of a short T-DNA fragment, but no increase of small mutations elsewhere. / Schouten, Henk J.; van de Geest, Henri; Papadimitriou, Sofia; Bemer, Marian; Schaart, Jan G.; Smulders, Rene; Perez, Gabino Sanchez; Schijlen, Elio.

In: Plant Cell Reports, Vol. 36, No. 3, 03.2017, p. 493-504.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Re-sequencing transgenic plants revealed rearrangements at T-DNA inserts, and integration of a short T-DNA fragment, but no increase of small mutations elsewhere

AU - Schouten, Henk J.

AU - van de Geest, Henri

AU - Papadimitriou, Sofia

AU - Bemer, Marian

AU - Schaart, Jan G.

AU - Smulders, Rene

AU - Perez, Gabino Sanchez

AU - Schijlen, Elio

PY - 2017/3

Y1 - 2017/3

N2 - Key message: Transformation resulted in deletions and translocations at T-DNA inserts, but not in genome-wide small mutations. A tiny T-DNA splinter was detected that probably would remain undetected by conventional techniques. Abstract: We investigated to which extent Agrobacterium tumefaciens-mediated transformation is mutagenic, on top of inserting T-DNA. To prevent mutations due to in vitro propagation, we applied floral dip transformation of Arabidopsis thaliana. We re-sequenced the genomes of five primary transformants, and compared these to genomic sequences derived from a pool of four wild-type plants. By genome-wide comparisons, we identified ten small mutations in the genomes of the five transgenic plants, not correlated to the positions or number of T-DNA inserts. This mutation frequency is within the range of spontaneous mutations occurring during seed propagation in A. thaliana, as determined earlier. In addition, we detected small as well as large deletions specifically at the T-DNA insert sites. Furthermore, we detected partial T-DNA inserts, one of these a tiny 50-bp fragment originating from a central part of the T-DNA construct used, inserted into the plant genome without flanking other T-DNA. Because of its small size, we named this fragment a T-DNA splinter. As far as we know this is the first report of such a small T-DNA fragment insert in absence of any T-DNA border sequence. Finally, we found evidence for translocations from other chromosomes, flanking T-DNA inserts. In this study, we showed that next-generation sequencing (NGS) is a highly sensitive approach to detect T-DNA inserts in transgenic plants.

AB - Key message: Transformation resulted in deletions and translocations at T-DNA inserts, but not in genome-wide small mutations. A tiny T-DNA splinter was detected that probably would remain undetected by conventional techniques. Abstract: We investigated to which extent Agrobacterium tumefaciens-mediated transformation is mutagenic, on top of inserting T-DNA. To prevent mutations due to in vitro propagation, we applied floral dip transformation of Arabidopsis thaliana. We re-sequenced the genomes of five primary transformants, and compared these to genomic sequences derived from a pool of four wild-type plants. By genome-wide comparisons, we identified ten small mutations in the genomes of the five transgenic plants, not correlated to the positions or number of T-DNA inserts. This mutation frequency is within the range of spontaneous mutations occurring during seed propagation in A. thaliana, as determined earlier. In addition, we detected small as well as large deletions specifically at the T-DNA insert sites. Furthermore, we detected partial T-DNA inserts, one of these a tiny 50-bp fragment originating from a central part of the T-DNA construct used, inserted into the plant genome without flanking other T-DNA. Because of its small size, we named this fragment a T-DNA splinter. As far as we know this is the first report of such a small T-DNA fragment insert in absence of any T-DNA border sequence. Finally, we found evidence for translocations from other chromosomes, flanking T-DNA inserts. In this study, we showed that next-generation sequencing (NGS) is a highly sensitive approach to detect T-DNA inserts in transgenic plants.

KW - Agrobacterium tumefaciens-mediated transformation

KW - Arabidopsis thaliana

KW - Molecular characterization

KW - Mutation frequency

KW - Next-generation sequencing

KW - Splinter

U2 - 10.1007/s00299-017-2098-z

DO - 10.1007/s00299-017-2098-z

M3 - Article

VL - 36

SP - 493

EP - 504

JO - Plant Cell Reports

JF - Plant Cell Reports

SN - 0721-7714

IS - 3

ER -