Gene editing of shelf-life related genes in strawberry

In crop plants, most traits are determined by dominant alleles. Such traits can easily be selected for in breeding programs because of their dominant phenotypes, also when in a heterozygous state. Several important traits are however determined by recessive alleles, which are only visible in a homozygous state. Studies in autogamous crops show the value of recessive alleles for important agronomic traits, such as disease resistance (S-genes), fruit ripening (ethylene biosynthesis), and shelf-life (cell wall degrading enzymes). Such alleles may be loss-of-function variants but may also represent favorable rare alleles with altered functionality or with a specific expression pattern. In polyploid crops, functional analysis and application of recessive alleles is difficult, because for this they should be fixed in a fully homozygous state, which is very difficult or virtually impossible to achieve in many polyploids. Moreover, selfing in polyploid crops is often hampered by self-incompatibility and inbreeding depression, making it even more complex. For this reason, many recessive alleles remain undiscovered in polyploids and hence their application in breeding is very limited if at all. The aim of this project is to develop a way to explore and utilise the hidden potential of recessive genes. The advent of genome editing technology, e.g. using CRISPR-Cas, may change the discovery of recessive alleles in polyploid species. Using CRISPR-Cas, mutations can be targeted to a specific region of a gene, and in polyploid genomes this frequently results in multiallelic mutagenesis. In the first step of our gene editing strategy, we will create mutant lines in which all alleles of a gene are knocked out. This gene editing at the gene level enables us to determine which candidate genes are most relevant for our traits of interest. The second gene editing step is performed at the allele level: for selected candidate genes we will make allelic series in which most alleles are knocked out but leaving one or a few specific alleles untargeted. This enables functional analysis of the different individual alleles and allows the discovery of favorable rare alleles in polyploid crops. Research in the proposed project will aim at the discovery of recessive alleles of shelf-life related genes in the allo-octoploid crop strawberry.