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

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. 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:
 [1];  [2];  [1];  [3];  [1];  [1];  [4];  [4];  [5];  [5];  [5];  [6]; ORCiD logo [4]
  1. Univ. of Florida, Gainesville, FL (United States). Agronomy Dept. Plant Molecular and Cellular Biology Program. Genetics Inst.
  2. 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
  3. 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
  4. 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
  5. Syngenta Crop Protection, LLC, Research Triangle Park, NC (United States)
  6. Univ. of Arkansas, Fayetteville, AR (United States). Crop, Soil and Environmental Sciences
Publication Date:
Research Org.:
Univ. of Florida, Gainesville, FL (United States); Syngenta Crop Protection, LLC, Research Triangle Park, NC (United States); Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1505510
Grant/Contract Number:  
SC0018420
Resource Type:
Accepted Manuscript
Journal Name:
Plant Molecular Biology
Additional Journal Information:
Journal Volume: 100; Journal Issue: 3; Journal ID: ISSN 0167-4412
Publisher:
Springer
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; sugarcane; selection marker removal; site-specific recombination; insulator; FLP; FLPe

Citation Formats

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., 2019. Web. doi:10.1007/s11103-019-00856-4.
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.
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. Wed . "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. https://www.osti.gov/servlets/purl/1505510.
@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 = 3,
volume = 100,
place = {United States},
year = {2019},
month = {3}
}

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