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. 2018 Mar 27:9:380.
doi: 10.3389/fpls.2018.00380. eCollection 2018.

Genome-Wide Association Study for Identification and Validation of Novel SNP Markers for Sr6 Stem Rust Resistance Gene in Bread Wheat

Amira M I Mourad  1   2 , Ahmed Sallam  1   3 , Vikas Belamkar  1 , Stephen Wegulo  4 , Robert Bowden  5 , Yue Jin  6 , Ezzat Mahdy  2 , Bahy Bakheit  2 , Atif A El-Wafaa  2 , Jesse Poland  7 , Peter S Baenziger  1
Affiliations

Genome-Wide Association Study for Identification and Validation of Novel SNP Markers for Sr6 Stem Rust Resistance Gene in Bread Wheat

Amira M I Mourad et al. Front Plant Sci. .

Abstract

Stem rust (caused by Puccinia graminis f. sp. tritici Erikss. & E. Henn.), is a major disease in wheat (Triticum aestivium L.). However, in recent years it occurs rarely in Nebraska due to weather and the effective selection and gene pyramiding of resistance genes. To understand the genetic basis of stem rust resistance in Nebraska winter wheat, we applied genome-wide association study (GWAS) on a set of 270 winter wheat genotypes (A-set). Genotyping was carried out using genotyping-by-sequencing and ∼35,000 high-quality SNPs were identified. The tested genotypes were evaluated for their resistance to the common stem rust race in Nebraska (QFCSC) in two replications. Marker-trait association identified 32 SNP markers, which were significantly (Bonferroni corrected P < 0.05) associated with the resistance on chromosome 2D. The chromosomal location of the significant SNPs (chromosome 2D) matched the location of Sr6 gene which was expected in these genotypes based on pedigree information. A highly significant linkage disequilibrium (LD, r2 ) was found between the significant SNPs and the specific SSR marker for the Sr6 gene (Xcfd43). This suggests the significant SNP markers are tagging Sr6 gene. Out of the 32 significant SNPs, eight SNPs were in six genes that are annotated as being linked to disease resistance in the IWGSC RefSeq v1.0. The 32 significant SNP markers were located in nine haplotype blocks. All the 32 significant SNPs were validated in a set of 60 different genotypes (V-set) using single marker analysis. SNP markers identified in this study can be used in marker-assisted selection, genomic selection, and to develop KASP (Kompetitive Allele Specific PCR) marker for the Sr6 gene.

Highlights: Novel SNPs for Sr6 gene, an important stem rust resistant gene, were identified and validated in this study. These SNPs can be used to improve stem rust resistance in wheat.

Keywords: SNP validation; genome-wide association study; haplotypes; linkage disequilibrium; marker-assisted selection; single marker analysis.

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Figures

FIGURE 1
FIGURE 1
The infection response of the genotypes in the (A) association set (A-set, 270 genotypes) and (B) validation set (V-set, 60 genotypes) to infection with the common stem rust race in Nebraska, QFCSC using the linear scale of Kumssa et al. (2015).
FIGURE 2
FIGURE 2
Analysis of population structure using 35,216 SNP markers: (A) Estimated population structure of 259 winter wheat genotypes (k = 2). The y-axis is the sub-population membership, and the x-axis is the genotypes. (B) Delta-k for different numbers of sub-populations as determined by Evanno et al. (2005).
FIGURE 3
FIGURE 3
(A) Manhattan plot displaying SNP marker-trait association identified for stem rust resistance in GWAS using 259 winter wheat lines of the A-set. Redline is significance threshold of 5% Bonferroni correction. (B) Quantile-Quantile (QQ) plot used to evaluate the performance of the mixed linear model used for of GWAS for stem rust resistance using mixed linear model (MLM).
FIGURE 4
FIGURE 4
Linkage disequilibrium (LD) analysis in the association set (A-set): heatmap of LD between the significant SNPs detected by the mixed linear model (MLM) (at a significance level of 5% Bonferroni correction) and SSR marker (Xcfd43) that predicts Sr6.
FIGURE 5
FIGURE 5
Haplotype block analysis for chromosome 2D, (A) the 306 haplotype blocks on the whole chromosome, (B) the nine haplotype blocks containing the 32 significant SNPs associated with stem rust resistance. SNPs with red circles are significantly associated with stem rust resistance based on 5% Bonferroni correction.
FIGURE 6
FIGURE 6
Linkage disequilibrium (LD) analysis in the validation set (V-set): heatmap of LD between the significant SNPs detected by the mixed linear model (MLM) (at significance level of 5% Bonferroni correction) and SSR marker Xcfd43 that predicts Sr6.
FIGURE 7
FIGURE 7
Histogram of the percentage of missing loci for each significant SNP (identified in the association set) in the validation set.
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