GENOME EDITING AND MARKER-ASSISTED SELECTION:A PATHWAY TO SUSTAINABLE RICE BREEDING

Rice (Oryza sativa L.) is the staple food for half of the world’s population. Italy represents the largest cultivation area in Europe characterised by irrigated systems and temperate japonica elite cultivars tailored to specific market classes. Among biotic stresses, rice blast caused by the fungus Mag-naporthe oryzae (anamorph: Pyricularia oryzae) remains the most destructive rice disease worldwide and a growing concern under climate variability. While extensive genetic knowledge exists on blast resistance, ranging from major NLR resistance (R) genes to quantitative loci and susceptibility (S) genes, its routine exploitation in Italian rice breeding programs is still limited. This PhD aimed to translate blast-resistance genetic knowledge into practical, breeding-oriented solutions for Italian elite germplasm, while also advancing our understanding of O. sativa-M. oryzae interaction. Two complementary approaches were pursued. First, CRISPR/Cas9 was used to generate targeted knockouts of candidate S genes, belonging to the Heavy-metal Isoprenylated Plant Protein (HIPP) family in elite backgrounds, focusing on Pi21 and the AVR-Pik host targets OsHIPP19 and OsHIPP20. A yeast two-hybrid screening against a broad effector panel identified a possible candi-date Pi21-interacting effector. Edited lines were produced in multiple Italian genetic backgrounds and advanced to stable, transgene-free loss-of-function lines. Although field disease pressure in 2025 was insufficient to quantify blast responses, controlled inoculation assays with a compatible isolate indi-cated that editing of S genes resulted in reduced lesion development, consistent with quantitatively decreased susceptibility; Unexpectedly, HIPP-edited lines developed spontaneous necrotic lesions reminiscent of an immune response, even in the absence of the pathogen. Second, two marker-assisted backcrossing (MABC) pipelines were initiated to introgress and stack resistance determinants from donor accessions into Italian elite backgrounds. In the Teqing (Donor)/ Laser (Recipient) program, PCR markers were developed to track Pi-b, the linked Pid2/3 interval, and bsr-d1. In the Gigante Vercelli (Donor)/Zar (Recipient) program, markers were designed to fol-low structural and allelic variation at the Pish cluster and a complex resistance hotspot on chromo-some 4. In MABC populations, a 2025 field trial in a RCBD showed that Pi-b tracked robust panicle blast reduction in Laser/Teqing BC2F2 families, whereas Zar/Gigante Vercelli BC3F2 families shifted toward lower incidence mainly due to the Pish cluster and, to a lesser extent, QTL4. Overall, this work provides both applied and mechanistic advances for blast resistance in Italian rice, combining S-gene editing and marker-assisted introgression with host–pathogen interaction analyses, and identifying Pi-b and the Pish cluster as key drivers of reduced disease incidence under local pathogen populations, with QTL4 supporting resistance.