Abstract
Genome editing has emerged as a technology with a potential to revolutionize plant breeding. In this study, we report on generating, in less than ten months, Tomelo, a non-transgenic tomato variety resistant to the powdery mildew fungal pathogen using the CRISPR/Cas9 technology. We used whole-genome sequencing to show that Tomelo does not carry any foreign DNA sequences but only carries a deletion that is indistinguishable from naturally occurring mutations. We also present evidence for CRISPR/Cas9 being a highly precise tool, as we did not detect off-target mutations in Tomelo. Using our pipeline, mutations can be readily introduced into elite or locally adapted tomato varieties in less than a year with relatively minimal effort and investment.
Highlights
As the world population grows, there is an increasing demand for food
In this study we report on generating a tomato variety resistant to the powdery mildew fungal pathogen Oidium neolycopersici using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas[9] technology, which is based on the Cas[9] DNA nuclease guided to a specific DNA target by a single guide RNA
We aimed at determining the speed and efficiency at which we could generate a transgene-free genetically edited slmlo[1] tomato variety using the CRISPR/Cas[9] system
Summary
The plasmid pAGM4723::Cas9_sgRNA1_sgRNA2 (Supplementary sequence file 1). GCR7589, a derivative of tomato cultivar Moneymaker, was transformed with the pAGM4723::Cas9_sgRNA1_sgRNA2 construct as previously described[12]. The DNA was extracted from the plant tissue using 500 ul of CTAB buffer (2% CTAB, 1.42 M NaCl, 20 mM EDTA and 100 mM Tris pH 8) for 30 min at 60 °C. Detection of Cas9-induced deletions and T-DNA in plant genomic DNA using PCR genotyping. The libraries were sequenced in 2 lanes paired end with 151 bp read length and 8 bp index read using 300cycle HiSEQ3000 SBS kits (Cat No: FC-410-1003). Each tomato line was analyzed separately with the SHORE pipeline[13], using BWA aln (v0.6.2)[14] with option “-n 0.05” to map the reads to the Solanum lycopersicum (version SL2.40.26; ftp://ftp.ensemblgenomes.org/pub/release-26/plants/fasta/solanum_lycopersicum/dna/) reference genome. The statistics was done using the algorithm described[15]
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