Detection of induced mutations in Ca FAD 2 genes by next‐generation sequencing leading to the production of improved oil composition in Crambe abyssinica

Summary Crambe abyssinica is a hexaploid oil crop for industrial applications. An increase of erucic acid (C22:1) and reduction of polyunsaturated fatty acid ( PUFA ) contents in crambe oil is a valuable improvement. An increase in oleic acid (C18:1), a reduction in PUFA and possibly an increase in C22:1 can be obtained by down‐regulating the expression of fatty acid desaturase2 genes ( Ca FAD 2 ), which code for the enzyme that converts C18:1 into C18:2. We conducted EMS ‐mutagenesis in crambe, followed by Illumina sequencing, to screen mutations in three expressed Ca FAD 2 genes. Two novel analysis strategies were used to detect mutation sites. In the first strategy, mutation detection targeted specific sequence motifs. In the second strategy, every nucleotide position in a Ca FAD 2 fragment was tested for the presence of mutations. Seventeen novel mutations were detected in 1100 one‐dimensional pools (11 000 individuals) in three expressed Ca FAD 2 genes, including non‐sense mutations and mis‐sense mutations in CaFAD2‐C1 , ‐ C2 and ‐ C3 . The homozygous non‐sense mutants for Ca FAD 2‐C3 resulted in a 25% higher content of C18:1 and 25% lower content of PUFA compared to the wild type. The mis‐sense mutations only led to small changes in oil composition. Concluding, targeted mutation detection using NGS in a polyploid was successfully applied and it was found that a non‐sense mutation in even a single Ca FAD 2 gene can lead to changes in crambe oil composition. Stacking the mutations in different Ca FAD 2 may gain additional changes in C18:1 and PUFA contents.