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Simultaneous site-directed mutagenesis of duplicated loci in soybean using a single guide RNA

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Using a gRNA and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two GmPPD loci in soybean. Mutations in GmPPD loci were confirmed in at least 33% of T2 seeds.

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is a powerful tool for site-directed mutagenesis in crops. Using a single guide RNA (gRNA) and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two homoeologous loci in soybean (Glycine max), GmPPD1 and GmPPD2, which encode the orthologs of Arabidopsis thaliana PEAPOD (PPD). Most of the T1 plants had heterozygous and/or chimeric mutations for the targeted loci. The sequencing analysis of T1 and T2 generations indicates that putative mutation induced in the T0 plant is transmitted to the T1 generation. The inheritable mutation induced in the T1 plant was also detected. This result indicates that continuous induction of mutations during T1 plant development increases the occurrence of mutations in germ cells, which ensures the transmission of mutations to the next generation. Simultaneous site-directed mutagenesis in both GmPPD loci was confirmed in at least 33% of T2 seeds examined. Approximately 19% of double mutants did not contain the Cas9/gRNA expression construct. Double mutants with frameshift mutations in both GmPPD1 and GmPPD2 had dome-shaped trifoliate leaves, extremely twisted pods, and produced few seeds. Taken together, our data indicate that continuous induction of mutations in the whole plant and advancing generations of transgenic plants enable efficient simultaneous site-directed mutagenesis in duplicated loci in soybean.

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Acknowledgements

We thank Professor Holger Puchta (University of Karlsruhe) for permission to use plasmid DNAs of pEn-Chimera and pDe-CAS9, and M. Suzuki and Y. Kitsui for general technical assistance. This work was supported by the Cabinet Office, Government of Japan [the Cross-ministerial Strategic Innovation Promotion Program (SIP) for TY].

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Author notes

  1. Yuhei Kanazashi and Aya Hirose have contributed equally to this work.

Authors and Affiliations

  1. Graduate School of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo, Hokkaido, 060-8589, Japan

    Yuhei Kanazashi, Aya Hirose, Ippei Takahashi, Jun Abe & Tetsuya Yamada

  2. Plant Genome Engineering Research Unit, Institute of Agrobiological Sciences, National Agricultural and Food Research Organization, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan

    Masafumi Mikami, Masaki Endo, Sakiko Hirose & Seiichi Toki

  3. Graduate School of Nanobioscience, Yokohama City University, 22-2, Seto, Kanazawa-ku, Yokohama, Kanagawa, 236-0027, Japan

    Masafumi Mikami & Seiichi Toki

  4. Kihara Institute for Biological Research, Yokohama City University, 641-12 Maioka-cho, Yokohama, Kanagawa, 244-0813, Japan

    Seiichi Toki

  5. Soybean and Field Crop Applied Genomics Research Unit, Institute of Crop Science, National Agricultural and Food Research Organization, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8518, Japan

    Akito Kaga

  6. Plant Diversity Research Team, Genetic Resources Center, National Agricultural and Food Research Organization, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8518, Japan

    Ken Naito

  7. Division of Basic Research, Institute of Crop Science, National Agricultural and Food Research Organization, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8518, Japan

    Masao Ishimoto

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  1. Yuhei Kanazashi
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  2. Aya Hirose
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  3. Ippei Takahashi
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  4. Masafumi Mikami
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  6. Sakiko Hirose
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  7. Seiichi Toki
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  8. Akito Kaga
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  9. Ken Naito
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  11. Jun Abe
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  12. Tetsuya Yamada
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Corresponding author

Correspondence to Tetsuya Yamada.

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Communicated by Kinya Toriyama.

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Kanazashi, Y., Hirose, A., Takahashi, I. et al. Simultaneous site-directed mutagenesis of duplicated loci in soybean using a single guide RNA. Plant Cell Rep 37, 553–563 (2018). https://doi.org/10.1007/s00299-018-2251-3

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  • DOI: https://doi.org/10.1007/s00299-018-2251-3

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