Identification of novel putative alleles related to important agronomic traits of wheat using robust strategies in GWAS

Principal component analysis (PCA) is widely used in various genetics studies. In this study, the role of classical PCA (cPCA) and robust PCA (rPCA) was evaluated explicitly in genome-wide association studies (GWAS). We evaluated 294 wheat genotypes under well-watered and rain-fed, focusing on spike traits. First, we showed that some phenotypic and genotypic observations could be outliers based on cPCA and different rPCA algorithms (Proj, Grid, Hubert, and Locantore). Hubert’s method provided a better approach to identifying outliers, which helped to understand the nature of these samples. These outliers led to the deviation of the heritability of traits from the actual value. Then, we performed GWAS with 36,000 single nucleotide polymorphisms (SNPs) based on the traditional approach and two robust strategies. In the conventional approach and using the first three components of cPCA as population structure, 184 and 139 marker-trait associations (MTAs) were identified for five traits in well-watered and rain-fed environments, respectively. In the first robust strategy and when rPCA was used as population structure in GWAS, we observed that the Hubert and Grid methods identified new MTAs, especially for yield and spike weight on chromosomes 7A and 6B. In the second strategy, we followed the classical and robust principal component-based GWAS, where the first two PCs obtained from phenotypic variables were used instead of traits. In the recent strategy, despite the similarity between the methods, some new MTAs were identified that can be considered pleiotropic. Hubert's method provided a better linear combination of traits because it had the most MTAs in common with the traditional approach. Newly identified SNPs, including rs19833 (5B) and rs48316 (2B), were annotated with important genes with vital biological processes and molecular functions. The approaches presented in this study can reduce the misleading GWAS results caused by the adverse effect of outlier observations.