Creation and functional analysis of new Puroindoline alleles in Triticum aestivum

The Hardness ( Ha) locus controls grain texture and affects many end-use properties of wheat ( Triticum aestivum L.). The Ha locus is functionally comprised of the Puroindoline a and b genes, Pina and Pinb, respectively. The lack of Pin allelic diversity is a major factor limiting Ha functional analyses and wheat quality improvement. In order to create new Ha alleles, a 630 member M-2 population was produced in the soft white spring cultivar Alpowa using ethylmethane sulfonate mutagenesis. The M-2 population was screened to identify new alleles of Pina and Pinb. Eighteen new Pin alleles, including eight missense alleles, were identified. F-2 populations for four of the new Pin alleles were developed after crossing each back to non-mutant Alpowa. Grain hardness was then measured on F-2:3 seeds and the impact of each allele on grain hardness was quantified. The tested mutations were responsible for between 28 and 94% of the grain hardness variation and seed weight and vigor of all mutation lines was restored among the F-2 populations. Selection of new Pin alleles following direct phenotyping or direct sequencing is a successful approach to identify new Ha alleles useful in improving wheat product quality and understanding Ha locus function.