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Generation of a selectable marker free, highly expressed single copy locus as landing pad for transgene stacking in sugarcane

  • Yang Zhao
  • Jae Y. Kim
  • Ratna Karan
  • Je H. Jung
  • Bhuvan Pathak
  • Bruce Williamson
  • Baskaran Kannan
  • Duoduo Wang
  • Chunyang Fan
  • Wenjin Yu
  • Shujie Dong
  • Vibha Srivastava
  • Fredy AltpeterEmail author
Article
  • 141 Downloads

Abstract

Key message

A selectable marker free, highly expressed single copy locus flanked by insulators was created as landing pad for transgene stacking in sugarcane. These events displayed superior transgene expression compared to single-copy transgenic lines lacking insulators. Excision of the selectable marker gene from transgenic sugarcane lines was supported by FLPe/FRT site-specific recombination.

Abstract

Sugarcane, a tropical C4 grass in the genus Saccharum (Poaceae), accounts for nearly 80% of sugar produced worldwide and is also an important feedstock for biofuel production. Generating transgenic sugarcane with predictable and stable transgene expression is critical for crop improvement. In this study, we generated a highly expressed single copy locus as landing pad for transgene stacking. Transgenic sugarcane lines with stable integration of a single copy nptII expression cassette flanked by insulators supported higher transgene expression along with reduced line to line variation when compared to single copy events without insulators by NPTII ELISA analysis. Subsequently, the nptII selectable marker gene was efficiently excised from the sugarcane genome by the FLPe/FRT site-specific recombination system to create selectable marker free plants. This study provides valuable resources for future gene stacking using site-specific recombination or genome editing tools.

Keywords

Sugarcane Selection marker removal Site-specific recombination Insulator FLP FLPe 

Notes

Acknowledgements

The authors are thankful to Syngenta Crop Protection, LLC. and CPBR for financial support. This work was also co-funded by the DOE Center for Advanced Bioenergy and Bioproducts Innovation (U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under Award Number DE-SC0018420). Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the U.S. Department of Energy. The authors would like to thank Dr. Hardev Sandhu (Everglades Research and Educational Center, UF-IFAS, Belle Glade, FL) for providing tops of sugarcane cultivar CP 88-1762 and Sun Gro Horticulture, Apopka, FL, for donation of the Fafard #2 potting mix.

Author contributions

F.A. and V.S. conceived and designed the experiments; Y.Z., J.Y.K. and J.H.J. constructed the plasmids; Y.Z., B.P. and B.W. generated the transgenic plants; Y.Z., B.K. and D.W. analyzed the transgenic plants; C.F., W.Y. and S.D. performed the TaqMan® qPCR assay; R.K. and Y.Z. carried out Southern Blot hybridization; Y.Z. and F.A. wrote the manuscript. All authors read and approved the final manuscript.

Supplementary material

11103_2019_856_MOESM1_ESM.docx (58 kb)
Supplementary material 1 (DOCX 58 KB)
11103_2019_856_MOESM2_ESM.xlsx (19 kb)
Supplementary material 2 (XLSX 19 KB)

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Yang Zhao
    • 1
  • Jae Y. Kim
    • 1
    • 5
  • Ratna Karan
    • 1
  • Je H. Jung
    • 1
    • 6
  • Bhuvan Pathak
    • 1
  • Bruce Williamson
    • 1
  • Baskaran Kannan
    • 1
    • 4
  • Duoduo Wang
    • 1
    • 4
  • Chunyang Fan
    • 2
  • Wenjin Yu
    • 2
  • Shujie Dong
    • 2
  • Vibha Srivastava
    • 3
  • Fredy Altpeter
    • 1
    • 4
    Email author
  1. 1.Agronomy Department, Plant Molecular and Cellular Biology Program, Genetics InstituteUniversity of Florida - IFASGainesvilleUSA
  2. 2.Syngenta Crop Protection, LLCResearch Triangle ParkUSA
  3. 3.Crop, Soil and Environmental SciencesUniversity of ArkansasFayettevilleUSA
  4. 4.DOE Center for Advanced Bioenergy and Bioproducts InnovationUniversity of Florida - IFASGainesvilleUSA
  5. 5.Department of Plant Resources, College of Industrial ScienceKongju National UniversityYesanRepublic of Korea
  6. 6.Smart Farm Research Center, Institute of Natural ProductsKorea Institute of Science and Technology (KIST)Gangwon-doRepublic of Korea

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