Generation of a Selectable Marker Free, Highly Expressed Single Copy Locus as Landing Pad for Transgene Stacking in Sugarcane
CABBI Theme: Feedstock Production
Keywords: Biomass Analytics, Genomics
Zhao, Y., Kim, J.Y., Karan, R., Jung, J.H., Pathak, B., Williamson, B., Kannan, B., Wang, D., Fan, C., Yu, W., Dong, S., Srivastava, V., Altpeter, F. March 27, 2019. “Generation of a Selectable Marker Free, Highly Expressed Single Copy Locus as Landing Pad for Transgene Stacking in Sugarcane.” Plant Molecular Biology 99-509:1-17. DOI: 10.1007/s11103-019-00856-4.
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.
A1: Alignment of single-copy pJKIS lines sequencing data with plasmid sequence in the ubiquitin promoter, intron and lox76 region.
A2: Alignment of single-copy and two-copy pJKIe lines sequencing data with plasmid sequence in the excision region after heat treatment.
A3: Evaluation of the presence of FRT sites in Ins20 and Ins62 lines by Sanger sequencing of PCR amplicons.
A4: Genomic sequence flanking the transgene integration site in line FLP83e following Sanger sequencing of cloned Tail PCR amplicons.