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In vivo modification of a maize engineered minichromosome

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Abstract

Engineered minichromosomes provide efficient platforms for stacking transgenes in crop plants. Methods for modifying these chromosomes in vivo are essential for the development of customizable systems for the removal of selection genes or other sequences and for the addition of new genes. Previous studies have demonstrated that Cre, a site-specific recombinase, could be used to modify lox sites on transgenes on maize minichromosomes; however, these studies demonstrated somatic recombination only, and modified minichromosomes could not be recovered. We describe the recovery of an engineered chromosome composed of little more than a centromere plus transgene that was derived by telomere-mediated truncation. We used the fiber fluorescence in situ hybridization technique and detected a transgene on the minichromosome inserted among stretches of CentC centromere repeats, and this insertion was large enough to suggest a tandem insertion. By crossing the minichromosome to a plant expressing Cre-recombinase, the Bar selection gene was removed, leaving behind a single loxP site. This study demonstrates that engineered chromosomes can be modified in vivo using site-specific recombinases, a demonstration essential to the development of amendable chromosome platforms in plants.

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Acknowledgment

Research was supported by NSF grant DBI 0701297.

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Authors and Affiliations

  1. Division of Biological Sciences, University of Missouri, Columbia, Columbia, MO, 65211, USA

    Robert T. Gaeta, Rick E. Masonbrink, Changzeng Zhao, Abhijit Sanyal, Lakshminarasimhan Krishnaswamy & James A. Birchler

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  1. Robert T. Gaeta
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  2. Rick E. Masonbrink
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  3. Changzeng Zhao
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  4. Abhijit Sanyal
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  5. Lakshminarasimhan Krishnaswamy
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  6. James A. Birchler
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Corresponding author

Correspondence to James A. Birchler.

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ESM 1

Karyotype analysis of Cre 1.1. The karyotype of Cre 1.1 plants was determined using CentC (green), TAG (green), NOR (green), and transgene (pTF:UbiCre; red). Cre 1.1 was located on 9L (arrow). Scale bar 10μm (PDF 28.8 kb)

ESM 2

Sequence comparison of the fragments show in Fig. 7, lanes 1 and 3 revealed the expected rearrangement in mIGT-1 (underlined). The sequence in bold in both sequences is the 34bp loxP target sequence. The sequence upstream of loxP in both sequences is the 5' end of En TIR. The underline sequence in the IGT-1 sequenced fragment is the 3' end of the NOS terminator adjacent to the Bar gene, and the underlined sequence in the mIGT-1 sequenced fragment is the 3' end of the ubiquitin promoter fragment. The recombinant molecule sequenced in mIGT-1 would be expected if the Bar-NOS fragment was deleted by Cre-mediated excision (PDF 50.6 kb)

ESM 3

Sequence comparison of the recovered transgene and genomic fragment (top) to the En TIR sequence in the pGZ vector (bottom) (PDF 167 kb)

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Gaeta, R.T., Masonbrink, R.E., Zhao, C. et al. In vivo modification of a maize engineered minichromosome. Chromosoma 122, 221–232 (2013). https://doi.org/10.1007/s00412-013-0403-3

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  • DOI: https://doi.org/10.1007/s00412-013-0403-3

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