Functional analysis of Puroindoline genes in Wheat (Triticum aestivum)
Grain hardness variation has large effects upon many different end-use properties of wheat (Triticum aestivum). The Hardness (Ha) locus consisting of the Puroindoline a and b genes (Pina and Pinb) controls the majority of grain hardness variation. Starch production is a growing end-use of wheat. The first objective of this study was to estimate the differences in starch yield due to natural and transgenically conditioned grain hardness differences. To accomplish this goal, a small scale wet milling protocol was used to characterize the wet milling properties of two independent groups of isogenic materials varying in grain hardness and in Pin expression level. The results of the first study demonstrate that the Ha locus and puroindoline expression are both linked to wet-milling starch yield and that selection for increased Ha function increases starch yield via enhanced separation of starch granules and the protein matrix during wet milling. The lack of Pin allelic diversity is a major factor limiting Ha functional analyses and wheat quality improvement. So the second objective of this study was to create new Ha alleles in the soft white spring cultivar Alpowa using ethylmethane sulfonate (EMS) mutagenesis. The M 2 population was screened to identify new alleles of Pina and Pinb. One hundred and forty eight new Pin alleles, including 68 missense alleles, were identified. F 2 populations for 49 of the new Pin alleles including 43 unique missense ones 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 comprised a range of functionality from neutral to function abolishing mutations. Seed weight and vigor of all mutation lines was restored among all of the F 2 populations. The new alleles have the potential to improve end use properties of soft and hard wheats.