Abstract
In many plant species, gene dosage is an important cause of phenotype variation. Engineering gene dosage, particularly in polyploid genomes, would provide an efficient tool for plant breeding. The hexaploid oilseed crop Camelina sativa, which has three closely related expressed subgenomes, is an ideal species for investigation of the possibility of creating a large collection of combinatorial mutants. Selective, targeted mutagenesis of the three delta‐12‐desaturase (FAD2) genes was achieved by CRISPR‐Cas9 gene editing, leading to reduced levels of polyunsaturated fatty acids and increased accumulation of oleic acid in the oil. Analysis of mutations over four generations demonstrated the presence of a large variety of heritable mutations in the three isologous CsFAD2 genes. The different combinations of single, double and triple mutants in the T3 generation were isolated, and the complete loss‐of‐function mutants revealed the importance of delta‐12‐desaturation for Camelina development. Combinatorial association of different alleles for the three FAD2 loci provided a large diversity of Camelina lines with various lipid profiles, ranging from 10% to 62% oleic acid accumulation in the oil. The different allelic combinations allowed an unbiased analysis of gene dosage and function in this hexaploid species, but also provided a unique source of genetic variability for plant breeding.
Highlights
Plant breeding is based on the recombination of a diversity of alleles to select the optimal combinations for a defined trait
The creation of specific DNA breaks can be obtained by the expression of nucleases such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and, more recently, the Cas9 protein associated with type II clustered regulatory interspaced short palindromic repeats (CRISPR) (Hsu et al, 2014)
Selective mutagenesis of CsFAD2 genes by CRISPR-Cas9 was highly effective in Camelina
Summary
Plant breeding is based on the recombination of a diversity of alleles to select the optimal combinations for a defined trait. Classical breeding relies on the availability of alleles in natural accessions, and in the different subgenomes in the case of polyploid crop species These limitations could be overcome by genetic modifications that allow the dominant expression of a transgene carrying the desired trait or allele, or alternatively the dominant down-regulation of endogenous genes by silencing. The specificity of the system relies on the perfect match of 8–12 nucleotides of the 50 end of the target, while some mismatches are tolerated at the 30 end (Cong et al, 2013) This system offers a simple, rapid and flexible method to induce single or even multiple mutations in the genome, defined by the specificity of the selected sgRNA sequence
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