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Application of the CRISPR/Cas9 system in Dioscorea zingiberensis

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Abstract

Dioscorea zingiberensis is a major pharmaceutical plant that produces diosgenin, an important starting material for steroidal hormones. To date, no genome editing approach in D. zingiberensis has been reported. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system has proven to be an efficient targeted genome modification tool and has been successfully applied in many plants, including rice, soybean, wheat, and Arabidopsis. Here, we report CRISPR/Cas9-mediated targeted mutagenesis in D. zingiberensis using an Agrobacterium tumefaciens-mediated transformation method. The target guide RNA was designed in the first exon of the farnesyl pyrophosphate synthase gene (Dzfps), which is a critical gene involved in the synthesis of secondary metabolites. The single guide RNA expression cassette was driven by the OsU3 promoter, and Cas9 was driven by the 35S promoter. High frequencies of mutants were detected in T0 plants. Among 15 transformed plants, nine mutants that contained five types of mutations at the predicted double-stranded break site were identified. The transcript levels of Dzfps and the content of squalene in isolated mutants were significantly decreased compared with those in wild-type plants. Overall, our research provides a rapid and efficient approach for targeted genome modification in D. zingiberensis.

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Abbreviations

CRISPR/Cas9:

The clustered regularly interspaced short palindromic repeats

Dzfps :

Farnesyl pyrophosphate synthase gene

sgRNA:

Single guide RNA

DSBs:

Double-strand breaks

NHEJ:

Non-homologous end joining

HDR:

Homology-directed repair

FPS:

Farnesyl pyrophosphate synthase

HygR:

Hygromycin resistance gene

AS:

Acetosyringone

qRT-PCR:

Real-time reverse transcriptase-polymerase chain reaction

PTCs:

Premature termination codons

PAM:

Protospacer adjacent motif

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Acknowledgements

This work was funded by the National Natural Science Foundation of China (No. 31270345).

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Authors and Affiliations

  1. State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, 430072, China

    Shan Feng, Wei Song, Ruirui Fu, Hong Zhang, Anran Xu & Jiaru Li

Authors
  1. Shan Feng
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  2. Wei Song
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  3. Ruirui Fu
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  4. Hong Zhang
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  5. Anran Xu
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  6. Jiaru Li
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Corresponding author

Correspondence to Jiaru Li.

Additional information

Communicated by Sergio J. Ochatt.

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Fig. S1

Generation of transgenic plants. (a) Callus induced from the stems of D. zingiberensis. (b) Generation of hygromycin-resistant callus. (c) Shoot elongation and root induction. (d) Acclimatisation of a transformed shoot. (TIF 9490 KB)

Fig. S2

PCR analysis of the HygR gene in transgenic lines. M, DNA marker; 1, Positive control; 2–7, Different transgenic lines; 8, Wild-type plant. Red arrowheads indicate the PCR product of the HygR gene, ~400 bp. (TIF 4247 KB)

Fig. S3

New mutation type in shoots generated from mutant #5 by rhizome breeding. (a) Sequences of wild-type and mutation induced at the target site; blue capital letters, protospacer adjacent motif; red capital letters, target sequence; dashes, deletions. (b) Sequence peaks of the wild type and mutation type at the target site. Red arrowheads indicate the locations of the mutations. #5-1 indicate the mutant generated from mutant #5. (TIF 1401 KB)

Table S1 (DOCX 15 KB)

Text S1

Full-length Dzfps (DOCX 14 KB)

Text S2

Premature termination codons (PTCs) and protein sequences generated by frameshift mutations of Dzfps. CDS, coding sequence; blue capital letters, target sequence; red capital letters, protospacer adjacent motif; dashes, deletions; yellow rectangle, termination codon. (DOCX 17 KB)

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Feng, S., Song, W., Fu, R. et al. Application of the CRISPR/Cas9 system in Dioscorea zingiberensis. Plant Cell Tiss Organ Cult 135, 133–141 (2018). https://doi.org/10.1007/s11240-018-1450-5

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  • DOI: https://doi.org/10.1007/s11240-018-1450-5

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