Abstract
Sugarcane is the source of 80% of the sugar and 26% of the bioethanol produced globally. However, its complex, highly polyploid genome (2n = 100 – 120) impedes crop improvement. Here, we report efficient and reproducible gene targeting (GT) in sugarcane, enabling precise co-editing of multiple alleles via template-mediated and homology-directed repair (HDR) of DNA double strand breaks induced by the programmable nuclease CRISPR/Cas9. The evaluation of 146 independently transformed plants from five independent experiments revealed a targeted nucleotide replacement that resulted in both targeted amino acid substitutions W574L and S653I in the acetolactate synthase (ALS) in 11 lines in addition to single, targeted amino acid substitutions W574L or S653I in 25 or 18 lines, respectively. Co-editing of up to three ALS copies/alleles that confer herbicide tolerance was confirmed by Sanger sequencing of cloned long polymerase chain reaction (PCR) amplicons. This work will enable crop improvement by conversion of inferior alleles to superior alleles through targeted nucleotide substitutions.
Highlights
Sugarcane (Saccharum spp. hybrid) is the source of 80% of the world’s sugar and 26% of its bioethanol, and it is an exceptionally productive crop due to its superior light conversion and waterand nitrogen-use efficiencies (Byrt et al, 2011)
We provide an evaluation of two alternative sgRNAs used alone or in combination for altering one or two codons (W574L and/or S653I) that are known to confer herbicide tolerance
The expression cassettes for two sgRNAs were designed and customsynthesized to induce double-strand breaks (DSBs) by Cas9 near these two codons
Summary
Sugarcane (Saccharum spp. hybrid) is the source of 80% of the world’s sugar and 26% of its bioethanol, and it is an exceptionally productive crop due to its superior light conversion and waterand nitrogen-use efficiencies (Byrt et al, 2011). The genome of sugarcane is the most complex of any domesticated agricultural species (2n = 100–120) (Piperidis and D’Hont, 2020). Modern sugarcane cultivars are derived from hybridization between Saccharum officinarum (2n = 80, x = 10) and Saccharum spontaneum (2n = 40–128, x = 8); these are responsible for high sugar content and stress tolerance or vigor, respectively (Piperidis and D’Hont, 2020). Most chromosomes are derived from S. officinarum, depending on the cultivar, with 10–20% originating from S. spontaneum and ∼10% from interspecific recombinants (D’Hont et al, 1996). Modern cultivars typically have 12 copies of each of the first four basic chromosomes, while parent species tend to differ in those basic chromosomes. One to four of these copies correspond to entire S. spontaneum chromosomes or interspecific recombinant chromosomes.
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