Comparative genomics of <i>Rpv3</i>, a multiallelic downy mildew resistance locus in grapevine (<i>Vitis</i> sp.)

Grapevine downy mildew, caused by the oomycete pathogen Plasmopara viticola, can lead to economically significant losses in humid climates. An ever-growing catalog of loci for resistance to P. viticola is available to breeders, including Rpv3 on the lower arm of chromosome 18. Widely used in French-American cultivars, Rpv3 is a complex TIR-NBS-LRR locus for which associated SSR markers have provided evidence of multiple alleles with varying degrees of resistance. However, SSRs lack the resolution to detect nuances between alleles and fully characterize the locus. PacBio long-read sequencing enables phased assembly of highly repetitive gene cluster regions, allowing for high resolution comparison among predicted alleles. Rpv3 haplotypes of eight Vitis genomes (‘Catawba’, ‘Chambourcin’, ‘Concord’, ‘Horizon’, MN1264, ‘Norton’, NY84.0101.03, and PN40024) were compared to identify differences in gene structure among Rpv3.1, Rpv3.2, Rpv3.3, co-located Rpv27, and susceptible SSR haplotypes. This region was extracted from each haplotype as delimited by their flanking SSR markers and ranged in length from 0.8 to 1.7 Mb. While there was strong consistency in gene structure within Rpv3.1, both Rpv3.2 and Rpv3.3 showed evidence of divergence between haplotypes. In addition to local alignments, the candidate genes identified in Rpv3.1 were tested for copy number and functional variation across haplotypes. The candidate gene region ranged in length from 85.3–220 kb from SSR UDV737. While the structure of this region and the TNL gene sequences were largely consistent within Rpv3.1 and Rpv3.2, the structure was variable among Rpv3.3 individuals. In spite of this structural variation among the Rpv3.3 SSR haplotype, their TNL gene sequences had strong similarity to Rpv27. The variation in gene structure shown in this study underscores the need for the refinement of allele naming, high-quality genome assemblies, as well as more informative, higher resolution marker systems for marker-assisted selection to improve resistance to grapevine downy mildew.