Haplotype-resolved powdery mildew resistance loci reveal the impact of heterozygous structural variation on NLR genes in Muscadinia rotundifolia

Muscadinia rotundifolia cv. Trayshed is a valuable source of resistance to grape powdery mildew. It carries two powdery mildew resistance-associated genetic loci, Run1.2 on chromosome 12 and Run2.2 on chromosome 18. The purpose of this study was to identify candidate resistance genes associated with each haplotype of the two loci. Both haplotypes of each resistance-associated locus were identified, phased, and reconstructed. Haplotype phasing allowed the identification of several structural variation events between haplotypes of both loci. Combined with a manual refinement of the gene models, we found that the heterozygous structural variants affected the gene content, with some resulting in duplicated or hemizygous nucleotide-binding leucine-rich repeat (NLR) genes. Heterozygous structural variations were also found to impact the domain composition of some NLRs. By comparing the NLRs at Run1.2 and Run2.2 loci, we discovered that the two loci include different numbers and classes of NLR genes. To identify powdery mildew resistance-associated genes, we performed a gene expression profiling of the NLR genes at Run1.2b and Run2.2 loci with or without powdery mildew present. Several NLR genes were constitutively expressed, suggesting a role in powdery mildew resistance. These first complete, haplotype-resolved resistance-associated loci and the candidate NLR genes identified by this study are new resources that can aid the development of powdery mildew-resistant grape cultivars.