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.
Highlights
Cultivated potato (Solanum tuberosum L.) is the third most important food crop after rice and wheat (Devaux et al, 2014) and plays an essential role in human nutrition as a primary source of carbohydrates
We found that the DM S-RNase is highly expressed in mature flowers [245.5 Fragments Per Kilobase of exon model per Million mapped reads (FPKM)] compared with no expression in leaves or tubers in DM
High S-RNase expression levels were detected in carpels (4342.7 FKPM) consistent with its role in preventing self compatibility (Kao and Tsukamoto, 2004)
Summary
Cultivated potato (Solanum tuberosum L.) is the third most important food crop after rice and wheat (Devaux et al, 2014) and plays an essential role in human nutrition as a primary source of carbohydrates. While improvement of cultivated potatoes (2n = 4x = 48) relies on the discovery and introgression of genes from wild species for traits such as disease resistance, the polyploid nature of cultivated tetraploid potato hampers the fixation of desirable alleles in new cultivars. The introgression of critical dominant alleles such as the potato virus Y (PVY) disease-resistance gene in a triplex or quadruplex allelic configuration can take up to 15 years (Mendoza et al, 1996). Re-inventing potato as a diploid inbred/F1 hybrid variety (2n = 2x = 24) would allow the application of efficient breeding methods (Jansky et al, 2016) as inbred potatoes would. S-RNase Knockout in Diploid Potatoes accelerate the generation of new varieties with favorable allelic combinations targeting yield, tuber quality, and resistance traits. A significant barrier to this approach is the occurrence of gametophytic self-incompatibility (SI) in a majority of the diploid potato germplasm, thereby preventing the ability to generate diploid homozygous lines
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