Identification and Biological Characterization of Candidate Genes for Resistance to Soil-Borne Cereal Mosaic Virus in Durum Wheat using CRISPR-Cas9 Genome Editing Systems

Soil-Borne Cereal Mosaic Virus (SBCMV) significantly harms wheat yields and grain quality. To sustainably meet rising food demands, genetic resistance of plants to the virus is the most effective plant protection strategy, relying on New Genomics Techniques due to the low rate of natural beneficial mutations. This research aims to identify and biologically characterize SBCMV resistance genes in durum wheat. Sbm2, a major Quantitative Trait Locus (QTL) located on chromosomal arm 2BS, is supposedly involved in SBCMV resistance. A GWAS analysis on the genotypes of the UNIBO Durum Panel narrowed the most probable causal region of Sbm2 to a 2.661 Mb region (0.2 cM), encompassing 93 genes. An RNA-Seq analysis within this region identified ten differentially expressed genes comparing gene expression levels from virus-resistant and virus-susceptible cultivars; from the identified set, three genes were prioritized as the strongest candidates for conferring virus resistance, based on their expression levels and gene ontology. To validate the biological function of these candidate genes by inducing knockout mutations, plasmid vectors harboring multiple gRNAs for CRISPR-Cas9 genome editing were assembled via Golden Gate assembly. The efficiency of the designed gRNA/Cas9 systems was subsequently validated in leaf protoplasts isolated from virus-resistant Svevo cultivar plants and analyzed through NGS amplicon sequencing of the target sites. Currently, high-efficiency editing plasmids are being used to transform Svevo embryos via Agrobacterium tumefaciens-mediated transformation, aiming to prove, through phenotypic analysis of the in vitro-regenerated plants, the biological function of these three candidate genes and their effective involvement in conferring SBCMV resistance.