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Cereal Chem. 82(5):601-608The effects of varying the gluten composition at constant protein, protein content at constant composition, and glutenin-to-gliadin (glu/gli) ratio on durum semolina rheological properties and the quality of the spaghetti derived from these doughs was investigated using the reconstitution method. Reconstituted flours were built up from a common durum starch and water-soluble fraction but with varying gluten types from a range of wheats at both 12 and 9% total protein. A 10-g mixograph and microextensigraph properties were affected by the source of the gluten, which was related to glutenin composition and polymeric molecular weight distribution. Cooked pasta firmness was highly correlated to mixograph development time (MDDT). Furthermore, varying the protein content (9-20%) showed an increase in mixograph peak resistance (PR) with no effect on extensigraph Rmax. Pasta firmness increased and stickiness decreased with increasing protein content. In another experiment, the glutenin and gliadin fractions isolated from durum wheat were added to the respective base semolina to investigate the effect of varying the glu/gli ratio by 1.3-1.6 fold. Increasing the ratio increased MDDT but had no effect on PR and resistance breakdown. Variable effects were obtained for spaghetti firmness. The information obtained should prove useful to durum breeders by providing further evidence for the importance of protein to pasta quality.

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PCR was used to amplify low-molecular-weight (LMW) glutenin genes from the Glu-A3 loci of hexaploid wheat cultivars containing different Glu-A3 alleles. The complete coding sequence of one LMW glutenin gene was obtained for each of the seven alleles Glu-A3a to Glu-A3g. Chromosome assignment of PCR products using Chinese Spring nulli-tetrasomic lines confirmed the amplified products were from chromosome 1A. All sequences were classified as LMW-i-type genes based on the presence of an N-terminal isoleucine residue and eight cysteine residues located within the C-terminal domain of the predicted, mature amino acid sequence. All genes contained a single uninterrupted open reading frame, including the sequence from the Glu-A3e allele, for which no protein product has been identified. Comparison of LMW glutenin gene sequences obtained from different alleles showed a wide range of sequence identity between the genes, with between 1 and 37 single nucleotide polymorphisms and between one and five insertion/deletion events between genes from different alleles. Allele-specific PCR markers were designed based on the DNA polymorphisms identified between the LMW glutenin genes, and these markers were validated against a panel of cultivars containing different Glu-A3 alleles. This collection of markers represents a valuable resource for use in marker-assisted breeding to select for specific alleles of this important quality-determining locus in bread wheat.

Electrophoretic characterisation of fractions collected from gluten protein extracts subjected to size-exclusion high-performance liquid chromatographyThe electrophoretic analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; reduced and unreduced) of fractions, collected from a size exclusion-high performance liquid chromatography (SE-HPLC) separation of gluten proteins using a column with pore size of around 400A, showed clear resolution for the seven elution ranges studied in two Australian bread wheat lines. Polymeric proteins -high molecular weight (HMW) glutenin subunits, low molecular weight (LMW) glutenin subunits, HMW albumins and some modified m-gliadins -appeared exclusively in the region within the first peak of the chromatogram (fractions 1 to 5), the limit being a region that resolved as a small peak before the large peak of gliadins and where some o-gliadins eluted. A larger proportion of HMW glutenin subunits and B subunits contributed to polymer formation of higher molecular weight.The polymer size decreased as the proportion of the other protein components increased. IntroductionWheat endosperm proteins have been extensively studied due to their viscoelastic properties that give wheat flours unique baking characteristics. They are usually classified according to their solubility, amino acid composition, size or polymerising behaviour. Wheat proteins may be separated into albumin (soluble in water), globulin (soluble in salt solution), gliadin (70% ethanol soluble) and glutenin (insoluble in ethanol) [l]. From a functional point of view, they are also grouped into polymeric protein (mainly glutenin) held together by interchain disulphide bonds, and monomeric protein (gliadins and albumins/globulins) [2], in which the only disulphide bonds are intrachain. Statistical relationships between these groups (particularly glutenins) and breadmaking quality have been established and, as a general result, close correlations have been found [2,3]. To reach these conclusions, wheat proteins have been characterised in detail using a wide range of methodologies, particularly over the last twenty-five years.Electrophoretic techniques using SDS-PAGE have been essential in determining the importance of glutenin polypeptides in wheat quality [4], but prior to electrophoresis in an SDS-PAGE gel, the glutenin polymer needs to be broken into subunits by the reduction of the disulphide bonds that build the polymeric structure. Four different types of subunits are found: high molecular weight (HMW) glutenin subunits, low molecular weight (LMW) glutenin subunits, HMW albumin subunits (mostly

The effects of α‐ + β‐, γ‐, ω‐ and total gliadins on mixing, extension baking, and techno‐functional properties of doughs from hard and soft flours were measured using small‐scale techniques. The addition of all gliadin fractions resulted in decreased mixing time, peak resistance, maximum resistance to extension, and loaf height, and in increased resistance breakdown and extensibility. The various gliadin fractions showed differences in functional properties, with γ‐gliadin reducing the mixing time and maximum resistance to extension to the greatest extent, ω‐gliadin contributing to the greatest reduction in loaf height, and α‐ + β‐gliadins having the least effect on reducing loaf height. The effects of gliadin fractions on loaf height were correlated with molecular mass, and effects on mixing time, maximum resistance to extension, and extensibility were correlated with hydrophobicity.

The coding regions of 28 entries of hexaploid wheat gamma-gliadin genes, gene fragments or pseudogenes in GenBank were used for nucleotide alignment. These sequences could be divided into nine subgroups based on nucleotide variation. The chromosomal locations of five of the seven unassigned subgroups were identified through subgroup-specific polymerase chain reactions (PCR) using Chinese Spring group-1 nulli-tetrasomic lines. Multiple single nucleotide polymorphisms (SNPs) and small insertions/deletions were identified in each subgroup. With further mining from wheat expressed sequence tag databases and targeted DNA sequencing, two SNPs were confirmed and one SNP was discovered for genes at the Gli-A1, Gli-B1 and Gli-D1 loci. A modified allele-specific PCR procedure for assaying SNPs was used to generate dominant DNA markers based on these three SNPs. For each of these three SNPs, two allele-specific primer sets were used to test Chinese Spring and 52 commercial Australian wheat varieties representing a range of low-molecular-weight (LMW) alleles. PCR results indicated that all were positive with one of the primer sets and negative with the other, with the exception of three varieties containing the 1BL/1RS chromosomal translocation that were negative for both. Furthermore, markers GliA1.1, GliB1.1 and GliD1.1 were found to be correlated with Glu-A3 a, b or c, Glu-B3 b, c, d or e and Glu-D3 a, b or e LMW glutenin alleles, respectively. Markers GliA1.2, GliB1.2 and GliD1.2 were found to be correlated with the Glu-A3 d or e, Glu-B3 a, g or h and Glu-D3 c alleles, respectively. These results indicated that the gamma-gliadin SNP markers could be used for detecting linked LMW glutenin subunit alleles that are important in determining the quality attributes of wheat products.

Increased expression of the high molecular weight glutenin subunit (HMW-GS) Bx7 is associated with improved dough strength of wheat (Triticum aestivum L.) flour. Several cultivars and landraces of widely different genetic backgrounds from around the world have now been found to contain this so-called 'over-expressing' allelic form of the Bx7 subunit encoded by Glu-B1al. Using three methods of identification, SDS-PAGE, RP-HPLC and PCR marker analysis, as well as pedigree information, we have traced the distribution and source of this allele from a Uruguayan landrace, Americano 44D, in the mid-nineteenth century. Results are supported by knowledge of the movement of wheat lines with migrants. All cultivars possessing the Glu-B1al allele can be identified by the following attributes: (1) the elution of the By sub-unit peak before the Dx sub-unit peak by RP-HPLC, (2) high expression levels of Bx7 (>39% Mol% Bx), (3) a 43 bp insertion in the matrix-attachment region (MAR) upstream of the gene promoter relative to Bx7 and an 18 bp nucleotide duplication in the coding region of the gene. Evidence is presented indicating that these 18 and 43 bp sequence insertions are not causal for the high expression levels of Bx7 as they were also found to be present in a small number of hexaploid species, including Chinese Spring, and species expressing Glu-B1ak and Glu-B1a alleles. In addition, these sequence inserts were found in different isolates of the tetraploid wheat, T. turgidum, indicating that these insertion/deletion events occurred prior to hexaploidization.