Missense alleles of the HMW glutenin subunits Dx5 and Dy10 have small changes in function relative to missense changes in Puroindoline a and b

Abstract

Background and Objectives. The high molecular weight glutenin subunit genes (HMW-GS) in wheat (Triticum aestivum L.) play a key role in determining dough functionality. More specifically allelic variation for the Glu-D1 subunit loci is consistently associated with bread making quality. Since much of the hard wheat germplasm is fixed for the more favorable 1Dx5 + 1Dy10 haplotype, our goal was to identify allelic variation in 1Dx5 and 1Dy10 HMW-GS genes using an ethyl methanesulfonate (EMS) mutagenized population in the soft white spring wheat cultivar “Alpowa.” The same source population was previously used in screening for creating novel alleles in Puroindoline a (Pina) and Puroindoline b (Pinb) which are responsible for grain hardness variation. Direct sequencing of 384 M3 families identified 135 point mutations equally dispersed across 1Dx5 and 1Dy10. The mutation discovery rate was 1/12.7 kb of DNA, equal to that found in the Pin loci. Mutation carrying plants were crossed to the nonmutagenized Alpowa parent to create F2 populations segregating for the induced EMS HMW Glutenin mutations. Swelling index of gluten (SIG) was measured from seed from field grown plants to assess the impact of each EMS mutation upon Glutenin function. Findings. Nonsense mutations in both 1Dx5 and 1Dy10 reduced SIG values, indicating both 1Dx5 and 1Dy10 are needed for gluten functionality. Missense mutations did not alter SIG values compared to their wild type counterparts. In contrast missense mutations within the tryptophan-rich region of both PINS increased grain hardness pointing to the importance of this protein region. No such region in 1Dx5 or 1Dy10 was found indicating these proteins are structurally stable and tolerant of amino acid substitution. Conclusions. Results from the Glu-D1 subunit and Pin loci show that useful phenotypic variation can be created using EMS mutagenesis for genes where naturally occurring mutations occur from amino substitutions as is the case for the Pin loci. Significance and Novelty. EMS induced mutations are useful for creating new alleles in storage proteins with much greater impacts in genes that have a defined active site as for the Pin loci than in genes such as the HMW Glu-1 genes which have a long repetitive region.