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ARS Home » Midwest Area » Ames, Iowa » Plant Introduction Research » Research » Research Project #438401

Research Project: Evaluation and Association Analysis of Resistance to Pythium on USDA Spinach Germplasm

Location: Plant Introduction Research

Project Number: 5030-21000-064-009-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Sep 1, 2020
End Date: Aug 31, 2022

To utilize the wordwide USDA germplasm collection of spinach to identify variation for tolerance to infection by Pythium species, which cause damping-off and root rot disease, and to identify tolerant and/or resistant lines for use in breeding programs or for cultivar release.

Using a whole genome resequencing (WGR) approach, the investigators will discover DNA markers such as single nucleotide polymorphisms (SNPs), insertion or deletion of bases (Indels), copy number variations (CNVs), and genomic variants by conducting association mapping of Pythium resistance in spinach. First, 25 days old plants of spinach accessions will be screened for reaction to Pythium infection under greenhouse conditions using a published protocol for foliar blight and pre- and postemergence damping-off, grown in pots in greenhouse conditions. The 400 USDA spinach accessions, 50 commercial spinach hybrids, and 30 Arkansas spinach lines will be screened, using an artificial inoculation of culture Pythium isolates mixed in the soil substrate, and pots with uninoculated substrate for controls. Following incubation and evaluation, disease severity data will be analyzed. Genomic DNA from the 480 spinach genotypes was provided to a contractor for whole genome resequencing (WGR) at 10X coverage (not part of this agreement). The WGR data will be used in conjunction with the results of disease screening trials to perform genome wide association analysis (GWAS). Ridge regression best linear unbiased prediction analysis (RR-BLUP) will be used to predict genomic estimated breeding value (GEBV) for genomic selection (GS). Training population subsets will be randomly selected from the association panel of the 480 spinach genotypes (100 times), with 384 in the training set and the remaining 96 accessions as the validation set. SNP markers associated with each trait will be used to predict the GEBV, and prediction accuracy estimates calculated with each reiteration of the process. Those SNP markers with high effect, as identified from GWAS, will be validated though kompetitive allele specific polymerase chain reaction (KASP) SNP genotyping in each of the 480 spinach genotypes. Spinach genotypes showing high resistance to Pythium will be selected, potentially for release as germplasm, and for use as parents in spinach breeding programs to develop superior spinach cultivars with high levels of resistance to Pythium infection.