AUTHOR=Enciso-Rodriguez Felix , Manrique-Carpintero Norma C. , Nadakuduti Satya Swathi , Buell C. Robin , Zarka Daniel , Douches David TITLE=Overcoming Self-Incompatibility in Diploid Potato Using CRISPR-Cas9 JOURNAL=Frontiers in Plant Science VOLUME=10 YEAR=2019 URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2019.00376 DOI=10.3389/fpls.2019.00376 ISSN=1664-462X 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 (Solanum spp.) possess gametophytic self-incompatibility that is primarily controlled by a single multiallelic locus called the S-locus which is composed of tightly linked genes, S-RNase (S-locus RNase) and multiple SLFs (S-locus F-box proteins), which are expressed in the style and pollen, respectively. Using S-RNase genes known to function in the Solanaceae gametophytic SI mechanism, we identified S-RNase alleles with flower-specific expression in two diploid self-incompatible potato lines using genome resequencing data. Consistent with the location of the S-locus in potato, we genetically mapped the S-RNase 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 S-RNase 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 S-RNase 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 S-RNase KO for use in diploid potato breeding approaches.