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CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in diploid and octoploid strawberry

CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in diploid and octoploid strawberry

Wilson, Fiona M. ORCID: 0000-0002-2073-7400, Harrison, Kate, Armitage, Andrew D. ORCID: 0000-0002-0610-763X, Simkin, Andrew J. and Harrison, Richard J. (2019) CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in diploid and octoploid strawberry. Plant Methods, 15:45. ISSN 1746-4811 (doi:https://doi.org/10.1186/s13007-019-0428-6)

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Abstract

Background: Gene editing using CRISPR/Cas9 is a simple and powerful tool for elucidating genetic controls and for crop improvement and its use has been reported in a growing number of important food crops, including recently Fragaria. In order to inform application of the technology in Fragaria, we targeted the visible endogenous marker gene PDS (phytoene desaturase) in diploid Fragaria vesca ssp. vesca ‘Hawaii 4’ and octoploid F. × ananassa ‘Calypso’.

Results: Agrobacterium-mediated transformation of leaf and petiole explants was used for efficient stable integration of constructs expressing plant codon-optimised Cas9 and single guide sequences under control of the Arabidopsis U6-26 consensus promoter and terminator or Fragaria vesca U6III regulatory sequences. More than 80% (‘Hawaii 4’) and 50% (‘Calypso’) putative transgenic shoot lines (multiple shoots derived from a single callus) exhibited mutant phenotypes. Of mutant shoot lines selected for molecular analysis, approximately 75% (‘Hawaii 4’) and 55% (‘Calypso’) included albino regenerants with bi-allelic target sequence variants. Our results indicate the PDS gene is functionally diploid in ‘Calypso’.

Conclusion: We demonstrate that CRISPR/Cas9 may be used to generate biallelic mutants at high frequency within the genomes of diploid and octoploid strawberry. The methodology, observations and comprehensive data set pre- sented will facilitate routine application of this technology in Fragaria to single and multiple gene copy targets where mutant phenotypes cannot be identified visually.

Item Type: Article
Uncontrolled Keywords: fragaria, strawberry, diploid, polyploid, CRISPR/Cas9, phytoene desaturase
Subjects: Q Science > QH Natural history > QH301 Biology
Faculty / Department / Research Group: Faculty of Engineering & Science
Faculty of Engineering & Science > Natural Resources Institute
Faculty of Engineering & Science > Natural Resources Institute > Agriculture, Health & Environment Department
Last Modified: 03 Mar 2020 15:05
Selected for GREAT 2016: None
Selected for GREAT 2017: None
Selected for GREAT 2018: None
Selected for GREAT 2019: None
URI: http://gala.gre.ac.uk/id/eprint/27120

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