Characterisation of Wheat Disease Resistance Genes Through the Application of Molecular Genetics and Deep Sequencing Technology

Bread wheat (Triticum aestivum) is a vital crop that supplies ~20% of globally consumed calories. However, fungal pathogens can devastate production in wheat growing regions and pose a major threat to food security. Pathogens can rapidly evolve to overcome genetically resistant varieties so there is much focus on the study of disease resistance genes (R genes) to combat this threat. Cloning specific R genes has been a major effort as the immense size and complexity of the wheat genome means it can take a decade or more to isolate a single R gene using classic map-based methods. However, the advancement of next-generation DNA sequencing (NGS) has significantly reduced the time and resources required for R gene cloning, allowing more research into their biology and evolution. Most R genes encode immune receptor proteins known as nucleotide-binding leucine rich repeats (NLRs). The technique known as mutagenesis and R gene enrichment sequencing (MutRenSeq) targets NLRs and was used to clone three distinct R genes in wheat, Pm1 conferring resistance to powdery mildew (Blumeria graminis), Lr13, conferring resistance to leaf rust (Puccinia triticana), and Sr6, conferring resistance to stem rust (P. graminis). These genes reside in regions of the genome that did not permit map-based cloning methods. A bioinformatic protocol was developed to enhance the outcomes of target identification from sequencing data. Candidates were identified for each gene and genetic validation was performed to confirm their identity. Further work explored the unique aspects of these genes which are associated with hybrid necrosis (Lr13), chromosomal rearrangement (Pm1) and temperature sensitivity (Sr6). This work highlights the functional and genetic diversity of NLRs and demonstrates cloning of R genes from complex genomes is no longer a major bottleneck in pursuing novel sources of disease resistance and a deeper understanding of host-pathogen interactions