Overcoming Self-Incompatibility in Diploid Potato Using CRISPR-Cas9

Abstract

Potato breeding can be redirected to a diploid inbred/F1 hybrid variety breeding strategy if self-compatibility can be introduced into diploid germplasm. However, the majority of diploid potato clones (<i>Solanum</i> spp.) possess gametophytic self-incompatibility that is primarily controlled by a single multiallelic locus called the <i>S</i>-locus which is composed of tightly linked genes, <i>S-RNase</i> (<i>S</i>-locus RNase) and multiple <i>SLFs</i> (<i>S</i>-locus F-box proteins), which are expressed in the style and pollen, respectively. Using <i>S-RNase</i> genes known to function in the Solanaceae gametophytic SI mechanism, we identified <i>S-RNase</i> alleles with flower-specific expression in two diploid self-incompatible potato lines using genome resequencing data. Consistent with the location of the <i>S</i>-locus in potato, we genetically mapped the <i>S-RNase</i> gene using a segregating population to a region of low recombination within the pericentromere of chromosome 1. To generate self-compatible diploid potato lines, a dual single-guide RNA (sgRNA) strategy was used to target conserved exonic regions of the <i>S-RNase</i> gene and generate targeted knockouts (KOs) using a Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (Cas9) approach. Self-compatibility was achieved in nine <i>S-RNase</i> KO T₀ lines which contained bi-allelic and homozygous deletions/insertions in both genotypes, transmitting self compatibility to T₁ progeny. This study demonstrates an efficient approach to achieve stable, consistent self-compatibility through <i>S-RNase</i> KO for use in diploid potato breeding approaches.