Mapping and selection of downy mildew resistance in spinach cv. whale by low coverage whole genome sequencing

Abstract

Spinach (Spinacia oleracea) is a popular leafy vegetable crop and commercial production is centered in California and Arizona in the US. The oomycete Peronospora effusa causes the most important disease in spinach, downy mildew. A total of nineteen races of P. effusa are known, with more than 15 documented in the last three decades, and the regular emergence of new races is continually overcoming the genetic resistance to the pathogen. This study aimed to finely map the downy mildew resistance locus RPF3 in spinach, identify single nucleotide polymorphism (SNP) markers associated with the resistance, refine the candidate genes responsible for the resistance, and evaluate the prediction performance using multiple machine learning genomic prediction (GP) methods. Segregating progeny population developed from a cross of resistant cultivar Whale and susceptible cultivar Viroflay to race 5 of P. effusa was inoculated under greenhouse conditions to determine downy mildew disease response across the panel. The progeny panel and the parents were resequenced at low coverage (1x) to identify genome wide SNP markers. Association analysis was performed using disease response phenotype data and SNP markers in TASSEL, GAPIT, and GENESIS programs and mapped the race 5 resistance loci (RPF3) to 1.25 and 2.73 Mb of Monoe-Viroflay chromosome 3 with the associated SNP in the 1.25 Mb region was 0.9 Kb from the NBS-LRR gene SOV3g001250. The RPF3 locus in the 1.22-1.23 Mb region of Sp75 chromosome 3 is 2.41-3.65 Kb from the gene Spo12821 annotated as NBS-LRR disease resistance protein. This study extended our understanding of the genetic basis of downy mildew resistance in spinach cultivar Whale and mapped the RPF3 resistance loci close to the NBS-LRR gene providing a target to pursue functional validation. Three SNP markers efficiently selected resistance based on multiple genomic selection (GS) models. The results from this study have added new genomic resources, generated an informed basis of the RPF3 locus resistant to spinach downy mildew pathogen, and developed markers and prediction methods to select resistant lines.

Skim resequencing finely maps the downy mildew resistance loci RPF2 and RPF3 in spinach cultivars whale and Lazio

“…For the Viroflay x Lazio panel, 192 seedlings (comprising 142 resistant and 48 susceptible seedlings and two parents) were sequenced to generate genotype datasets. In addition, 192 progeny panels segregating from Viroflay x Whale for race 5 of P. effusa reported earlier [ 16 ] were included to perform a meta-analysis.…”

“…Downy mildew pathogen resistance from field evaluations in the diverse germplasm panel showed R genes between 0.3 to 1.5 Mb of chromosome 3 in Monoe-Viroflay genome assembly [ 15 ]. The RPF3 locus in Whale was located at 1.25 Mb and at 2.73–2.74 Mb in Monoe-Viroflay genome assembly, and three regions of Sp75 genome assembly located at 1.19, 1.22–1.23, and 1.75–1.76 Mb [ 16 ]. The RPF2 locus was recently reported to be between 1.11 to 1.72 Mb on chromosome 3 of Sp75 genome assembly [ 17 ].…”

“…Genome wide association studies (GWAS) are commonly used to identify chromosomal regions associated with trait expression in self-pollinated and cross-pollinated crop species. GWAS have been used to map the disease resistance regulating genomic regions and have identified single nucleotide polymorphism (SNP) markers that are associated with many traits in spinach [ 13 , 15 , 16 , 18 , 19 ]. In this study, we employed the low coverage whole genome resequencing method, also known as skim resequencing, to generate SNP genotype datasets.…”

“…In this study, we employed the low coverage whole genome resequencing method, also known as skim resequencing, to generate SNP genotype datasets. We selected this method as a continuation of our previous report [ 16 ] because of its capability to offer complete genome coverage, which is advantageous for genotyping compared to the GBS method commonly used in spinach genotyping studies [ 13 , 14 , 20 ], which is limited by incomplete genome coverage. Skim resequencing has been extensively applied in various crop species for discovering genome-wide genetic variants, particularly SNPs, and in trait mapping studies in diverse and bi-parental populations [ 21–24 ].…”

Insights into spinach domestication from genomes sequences of two wild spinach progenitors,Spinacia turkestanicaandS. tetrandra

“…Notably, one region was identified at 0.5–1.5 Mb of the S. oleracea chromosome 1 (Fig. 5c), overlapping with the downy mildew (DM) resistance loci, RPF1 – RPF3 (Bhattarai et al, 2022; Gao et al, 2022; She et al, 2018), RPF11 (Dijkstra and Raedts, 2019), and RPF12 (Dijkstra and Raedts, 2019), which confer resistance against at least 17 downy mildew of the 19 races of this pathogen reported in spinach. A neighbor-joining tree using SVs within this introgression region relationship in the 94 Spinacia accessions suggested that the DM loci originated from both wild species as donors (Fig.…”