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. 2017 Mar 28;7(1):482.
doi: 10.1038/s41598-017-00578-x.

Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion

Vladimir Nekrasov  1   2 , Congmao Wang  3 , Joe Win  1 , Christa Lanz  4 , Detlef Weigel  5 , Sophien Kamoun  6
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Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion

Vladimir Nekrasov et al. Sci Rep. .
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Abstract

Genome editing has emerged as a technology with a potential to revolutionize plant breeding. In this study, we report on generating, in less than ten months, Tomelo, a non-transgenic tomato variety resistant to the powdery mildew fungal pathogen using the CRISPR/Cas9 technology. We used whole-genome sequencing to show that Tomelo does not carry any foreign DNA sequences but only carries a deletion that is indistinguishable from naturally occurring mutations. We also present evidence for CRISPR/Cas9 being a highly precise tool, as we did not detect off-target mutations in Tomelo. Using our pipeline, mutations can be readily introduced into elite or locally adapted tomato varieties in less than a year with relatively minimal effort and investment.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Generating non-transgenic slmlo1 tomato lines resistant to powdery mildew. (a) The SlMlo1 locus was targeted by two sgRNAs; (b) T0 tomato transformants were tested for the presence of deletions using the PCR band shift assay; (c) Selected T0 transformants genotyped using the PCR band shift assay alongside wild type (WT); (d) SlMlo1 sequencing reads from selected T0 transformants; (e) Leaves of tomato plants inoculated with Oidium neolycopersici (5 weeks post inoculation); (f) PCR genotyping of the T1 generation for the presence T-DNA and the slmlo1 mutation. The agarose gels presented in panels (b and c) were cropped.
Figure 2
Illumina sequencing data. (a) Quantification of Illumina sequencing reads matching the T-DNA or vector backbone in wild type and slmlo1 T1 progeny lines; (b) Coverage of the T-DNA by Illumina reads; (c) Coverage of the SlMlo1 locus by Illumina reads.
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References

    1. Schiml S, Puchta H. Revolutionizing plant biology: multiple ways of genome engineering by CRISPR/Cas. Plant methods. 2016;12:8. doi: 10.1186/s13007-016-0103-0. - DOI - PMC - PubMed
    1. Waltz E. CRISPR-edited crops free to enter market, skip regulation. Nat Biotechnol. 2016;34:582. doi: 10.1038/nbt0616-582. - DOI - PubMed
    1. Waltz E. Gene-edited CRISPR mushroom escapes US regulation. Nature. 2016;532:293. doi: 10.1038/nature.2016.19754. - DOI - PubMed
    1. Huang S, Weigel D, Beachy RN, Li J. A proposed regulatory framework for genome-edited crops. Nature genetics. 2016;48:109–111. doi: 10.1038/ng.3484. - DOI - PubMed
    1. Acevedo-Garcia J, Kusch S, Panstruga R. Magical mystery tour: MLO proteins in plant immunity and beyond. The New phytologist. 2014;204:273–281. doi: 10.1111/nph.12889. - DOI - PubMed

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