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

doi:10.1007/s11103-019-00856-4

This content will become publicly available on March 27, 2020

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

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. 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.

Authors:

Zhao, Yang ^[1] ; Kim, Jae Y. ^[2] ; Karan, Ratna ^[1] ; Jung, Je H. ^[3] ; Pathak, Bhuvan ^[1] ; Williamson, Bruce ^[1] ; Kannan, Baskaran ^[4] ; Wang, Duoduo ^[4] ; Fan, Chunyang ^[5] ; Yu, Wenjin ^[5] ; Dong, Shujie ^[5] ; Srivastava, Vibha ^[6] ;

Univ. of Florida, Gainesville, FL (United States). Agronomy Dept. Plant Molecular and Cellular Biology Program. Genetics Inst.
Univ. of Florida, Gainesville, FL (United States). Agronomy Dept. Plant Molecular and Cellular Biology Program. Genetics Inst.; Kongju National Univ., Yesan (Korea, Republic of). Dept. of Plant Resources. College of Industrial Science
Univ. of Florida, Gainesville, FL (United States). Agronomy Dept. Plant Molecular and Cellular Biology Program. Genetics Inst.; Korea Inst. of Science and Technology (KIST), Gangwon (Korea, Republic of). Smart Farm Research Center. Inst. of Natural Products
Univ. of Florida, Gainesville, FL (United States). Agronomy Dept. Plant Molecular and Cellular Biology Program. Genetics Inst. DOE Center for Advanced Bioenergy and Bioproducts Innovation
Syngenta Crop Protection, LLC, Research Triangle Park, NC (United States)
Univ. of Arkansas, Fayetteville, AR (United States). Crop, Soil and Environmental Sciences

Publication Date:: 2019-03-27

Grant/Contract Number:: SC0018420

Type:: Accepted Manuscript

Journal Name:: Plant molecular biology

Additional Journal Information:: Journal Name: Plant molecular biology; Journal ID: ISSN 0167-4412

Publisher:: Springer

Research Org:: Univ. of Florida, Gainesville, FL (United States); Syngenta Crop Protection, LLC, Research Triangle Park, NC (United States)

Sponsoring Org:: USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); Syngenta Crop Protection, LLC (United States)

Country of Publication:: United States

Language:: English

Subject:: sugarcane; selection marker removal; site-specific recombination; insulator; FLP; FLPe

OSTI Identifier:: 1505510


                    Zhao, Yang, Kim, Jae Y., Karan, Ratna, Jung, Je H., Pathak, Bhuvan, Williamson, Bruce, Kannan, Baskaran, Wang, Duoduo, Fan, Chunyang, Yu, Wenjin, Dong, Shujie, Srivastava, Vibha, and Altpeter, Fredy. Generation of a selectable marker free, highly expressed single copy locus as landing pad for transgene stacking in sugarcane.  United States: N. p.,  
        Web.  doi:10.1007/s11103-019-00856-4.

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                    Zhao, Yang, Kim, Jae Y., Karan, Ratna, Jung, Je H., Pathak, Bhuvan, Williamson, Bruce, Kannan, Baskaran, Wang, Duoduo, Fan, Chunyang, Yu, Wenjin, Dong, Shujie, Srivastava, Vibha, & Altpeter, Fredy. Generation of a selectable marker free, highly expressed single copy locus as landing pad for transgene stacking in sugarcane.  United States.  doi:10.1007/s11103-019-00856-4.

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                    Zhao, Yang, Kim, Jae Y., Karan, Ratna, Jung, Je H., Pathak, Bhuvan, Williamson, Bruce, Kannan, Baskaran, Wang, Duoduo, Fan, Chunyang, Yu, Wenjin, Dong, Shujie, Srivastava, Vibha, and Altpeter, Fredy. 2019.  
        "Generation of a selectable marker free, highly expressed single copy locus as landing pad for transgene stacking in sugarcane".  United States.  
        doi:10.1007/s11103-019-00856-4.

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@article{osti_1505510,

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

  author       = {Zhao, Yang and Kim, Jae Y. and Karan, Ratna and Jung, Je H. and Pathak, Bhuvan and Williamson, Bruce and Kannan, Baskaran and Wang, Duoduo and Fan, Chunyang and Yu, Wenjin and Dong, Shujie and Srivastava, Vibha and Altpeter, Fredy},

  abstractNote = {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. 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.},

  doi          = {10.1007/s11103-019-00856-4},

  journal      = {Plant molecular biology},

  number       = ,

  volume       = ,

  place        = {United States},

  year         = {2019},

  month        = {3}

}

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Free Publicly Accessible Full Text
This content will become publicly available on March 27, 2020
Publisher's Version of Record
10.1007/s11103-019-00856-4
https://doi.org/10.1007/s11103-019-00856-4

Works referenced in this record:

Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome
journal, April 1995

Albert, Henrik; Dale, Emily C.; Lee, Elsa
The Plant Journal, Vol. 7, Issue 4, p. 649-659
DOI: 10.1046/j.1365-313X.1995.7040649.x

Site-Specific Integration of Transgenes in Soybean via Recombinase-Mediated DNA Cassette Exchange
journal, May 2009

Li, Z.; Xing, A.; Moon, B. P.
Plant Physiology, Vol. 151, Issue 3, p. 1087-1095
DOI: 10.1104/pp.109.137612

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

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