GENERATION OF HT-B AND HT-B PLUS S-RNASE KNOCKOUT LINES TO UNDERSTAND SELF-COMPATIBILITY IN DIPLOID POTATO

Domesticated potato (Solanum tuberosum L.) is the world’s third most important food crop and is a food security crop according to the Food and Agriculture Organization of the United Nations. Currently, commercial potatoes are autotetraploid and mainly produced via asexual clonal propagation. The autotetraploid nature of most cultivated potatoes in combination with acute inbreeding depression when self-fertilized over multiple generations cause challenges in making advances with traditional breeding schemes. The benefits of moving potato to a diploid breeding model include a simplified breeding scheme, easier fixation of desirable alleles, and generation of inbred lines that may be used to generate F1 hybrids with heterotic potential. A major hinderance to self-compatibility originates from the gametophytic self-incompatibility (GSI) system in which the S-RNase and HT-B genes play a critical role. Utilizing CRISPR-Cas9 gene editing, HT-B and HT-B + S-RNase knockout (KO) lines were produced. HT-B KOs produced parthenocarpic fruit but remained self-incompatible. However, the S-RNase and HT-B double KOs were self-compatible. Self-compatibility was measured quantitatively using fruit set, fruit weight, and seed count. Fruit set varied across both self-incompatible and self-compatible lines, with no clear trend in statistical significance. Double KO lines consistently displayed higher fruit weight than incompatible lines. Seed count served as the best measure of self-compatibility, with S-RNase and HT-B double KO lines producing up to three times mean seed per fruit when compared to S-RNase only KOs from prior studies. The lines with the highest levels of self-compatibility will serve as useful additions in advancing potato breeding to the diploid level.