Measuring Genotypic Variation in Wheat Seed Iron First Requires Stringent Protocols to Minimize Soil Iron Contamination

Measuring genotypic variation in Fe is an important task for plant breeders trying to biofortify wheat (Triticum aestivum L.), but levels of Fe are often enhanced in wheat seed due to soil derived contamination. Seed Al levels can be used to identify genotypes that are contaminated with Fe. The sources of contamination were identified and removed by milling the wheat grains in a modified Kett mill for 5 s before analysis. Thirty contaminated lines from Australia and 240 contaminated lines from Pakistan were cleaned for the validation of the 5 s milling method. Before cleaning, average Al concentrations were 10 mg kg^-1. After cleaning, 183 out of the 240, or 76%, had Al less than 5.0 mg kg^-1, and average Al levels were 4.2 mg kg^-1. Therefore, the cleaning procedure was highly successful in reducing Al levels in contaminated samples. The ratio between Al removed and Fe removed was fairly consistent and followed a 1:1 relationship across two environments. Twenty genotypes of a wheat population were evaluated for their Fe concentration before and after cleaning and ranking changed significantly and cleaning also improved the precision of quantitative trait loci (QTL) analysis, with QTL QGFe.ta-3B having a logarithm of the odds score of 1.1 before cleaning, which rose to 3.4 after cleaning. The method provides a valuable cleaning procedure for plant breeders wanting to analyze wheat seed and also provides evidence for reducing the influence of soil Fe contamination on seed by simply adjusting the Fe concentrations based on the level of Al in the grain.w