For each accession, genomic DNA was extracted from the same plant used to increase the seeds evaluated in this study. DNA extractions were performed at the USDA-ARS Small Grains and Potato Germplasm Research Unit using the CTAB protocol (Stewart and Via 1993 (link)). The DNA was precipitated by adding isopropanol, followed by washing of the pellet with ice-cold 70% ethanol, and resuspension in 200 µL of Tris HCl ethylenediaminetetraacetic acid (pH 8.0).
Genotyping was carried out at the USDA-ARS genotyping laboratory at Fargo, North Dakota, using the Infinium wheat SNP 9K iSelect assay from the Illumina platform (Illumina Inc., San Diego, CA) developed by the International Wheat SNP Consortium (Cavanagh et al. 2013 (link)). The raw Illumina SNP data were processed with the GenomeStudio v2011.1 software (Illumina). The array yielded 5234 scorable SNP markers. The polymorphic SNPs were ordered according to the scaled map positions of the hexaploid wheat 9K SNP consensus map (Cavanagh et al. 2013 (link)). The arm orientation of chromosomes 4A, 5A, and 5B presented here is in opposite orientation to the published consensus map (Cavanagh et al. 2013 (link)).
The dataset was filtered using a 10% cutoff for missing data in either loci or accessions (23 accessions were eliminated). On the basis of the filtered SNP data, a triangular identity-by-state genetic similarity matrix (Kang et al. 2008 (link)) was then obtained for all possible pairs of accessions. For groups of accessions with ≥0.99 genetic similarity, only one representative accession (the one with the lowest number of missing data) was retained per group. After applying these filtering criteria, a total of 875 accessions were retained for the GWAS. Only SNPs with minor allele frequency (MAF) ≥0.10 (i.e., minor allele present in at least 87 accessions) were considered for GWAS. Of the 4585 SNPs that satisfied this criterion, 4374 were positioned on the consensus map. Low-frequency SNPs were discarded to focus on SNPs with greater statistical power (Turner et al. 2011 ). The downside of this approach is the potential loss of true resistance loci present at low frequency (increase in false negatives). In this study we prioritized the reliability of the detected QTL over the sensitivity of the analyses.
Molecular markers tightly associated to two well-characterized loci conferring resistance to multiple pathogens were included as internal controls. The diagnostic KaspLr34 assay (http://www.cerealsdb.uk.net/cerealgenomics/CerealsDB/SNPs/Documents/MAS , = wMAS000003) was designed around a 3-bp indel in exon 11 of the Lr34/Yr18 gene (Lagudah et al. 2009 (link)). Marker csSNP856 (=Kasp856) is tightly linked to the Lr67/Yr46 locus but is not diagnostic for the resistance gene (Forrest et al. 2014 ). Data for these two control markers are summarized in File S3 .
Genotyping was carried out at the USDA-ARS genotyping laboratory at Fargo, North Dakota, using the Infinium wheat SNP 9K iSelect assay from the Illumina platform (Illumina Inc., San Diego, CA) developed by the International Wheat SNP Consortium (Cavanagh et al. 2013 (link)). The raw Illumina SNP data were processed with the GenomeStudio v2011.1 software (Illumina). The array yielded 5234 scorable SNP markers. The polymorphic SNPs were ordered according to the scaled map positions of the hexaploid wheat 9K SNP consensus map (Cavanagh et al. 2013 (link)). The arm orientation of chromosomes 4A, 5A, and 5B presented here is in opposite orientation to the published consensus map (Cavanagh et al. 2013 (link)).
The dataset was filtered using a 10% cutoff for missing data in either loci or accessions (23 accessions were eliminated). On the basis of the filtered SNP data, a triangular identity-by-state genetic similarity matrix (Kang et al. 2008 (link)) was then obtained for all possible pairs of accessions. For groups of accessions with ≥0.99 genetic similarity, only one representative accession (the one with the lowest number of missing data) was retained per group. After applying these filtering criteria, a total of 875 accessions were retained for the GWAS. Only SNPs with minor allele frequency (MAF) ≥0.10 (i.e., minor allele present in at least 87 accessions) were considered for GWAS. Of the 4585 SNPs that satisfied this criterion, 4374 were positioned on the consensus map. Low-frequency SNPs were discarded to focus on SNPs with greater statistical power (Turner et al. 2011 ). The downside of this approach is the potential loss of true resistance loci present at low frequency (increase in false negatives). In this study we prioritized the reliability of the detected QTL over the sensitivity of the analyses.
Molecular markers tightly associated to two well-characterized loci conferring resistance to multiple pathogens were included as internal controls. The diagnostic KaspLr34 assay (
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