Commercial production of spinach (Spinacia oleracea L.) is centered in California and Arizona in the US, where downy mildew caused by Peronospora effusa is the most destructive disease. Nineteen typical races of P. effusa have been reported to infect spinach, with 16 identified after 1990. The regular appearance of new pathogen races breaks the resistance gene introgressed in spinach. We attempted to map and delineate the RPF2 locus at a finer resolution, identify linked single nucleotide polymorphism (SNP) markers, and report candidate downy mildew resistance (R) genes. Progeny populations segregating for RPF2 locus derived from resistant differential cultivar Lazio were infected using race 5 of P. effusa and were used to study for genetic transmission and mapping analysis in this study. Association analysis performed with low coverage whole genome resequencing-generated SNP markers mapped the RPF2 locus between 0.47 to 1.46 Mb of chromosome 3 with peak SNP (Chr3_1, 221, 009) showing a LOD value of 61.6 in the GLM model in TASSEL, which was within 1.08 Kb from Spo12821, a gene that encodes CC-NBS-LRR plant disease resistance protein. In addition, a combined analysis of progeny panels of Lazio and Whale segregating for RPF2 and RPF3 loci delineated the resistance section in chromosome 3 between 1.18–1.23 and 1.75–1.76 Mb. This study provides valuable information on the RPF2 resistance region in the spinach cultivar Lazio compared to RPF3 loci in the cultivar Whale. The RPF2 and RPF3 specific SNP markers, plus the resistant genes reported here, could add value to breeding efforts to develop downy mildew resistant cultivars in the future.

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