A Multi-Tiered Haplotype Strategy to Enhance Phased Assembly and Fine-Mapping of a Disease Resistance Locus.

Fine mapping of quantitative trait loci (QTL) to dissect the genetic basis of traits of interest is essential to modern breeding practice. Here, we employed a multi-tiered haplotypic marker system to increase fine mapping accuracy by constructing a chromosome-level, haplotype-resolved parental genome, accurate detection of recombination sites, and allele-specific characterization of the transcriptome. In the first tier of this system, we applied the preexisting panel of 2000 rhAmpSeq core genome markers that is transferable across the entire Vitis genus and provides a genomic resolution of 200 kb to 1 Mb. The second tier consisted of high-density haplotypic markers generated from Illumina skim sequencing data for samples enriched for relevant recombinations, increasing the potential resolution to hundreds of base pairs. We used this approach to dissect a novel Resistance to Plasmopara viticola-33 (RPV33) locus conferring resistance to grapevine downy mildew, narrowing the candidate region to only 0.46 Mb. In the third tier, we used allele-specific RNA-seq analysis to identify a cluster of three putative disease resistance RPP13-like protein 2 genes located tandemly in a non-syntenic insertion as candidates for the disease resistance trait. In addition, combining the rhAmpSeq core genome haplotype markers and skim sequencing-derived high-density haplotype markers enabled chromosomal-level scaffolding and phasing of the grape Vitis × doaniana 'PI 588149' assembly, initially built solely from PacBio HiFi reads, leading to the correction of 16 large-scale phasing errors. Our mapping strategy integrates high-density, phased genetic information with individual reference genomes to pinpoint the genetic basis of QTLs, and will likely be widely adopted in highly heterozygous species.