M. C. Gianibelli

Biochemical, Genetic, and Molecular Characterization of Wheat Glutenin and Its Component Subunits

Of all the cereal grains, wheat is unique because wheat flour alone has the ability to form a dough that exhibits the rheological properties required for the production of leavened bread and for the wider diversity of foods that have been developed to take advantage of these attributes. The unique properties of the wheat grain reside primarily in the gluten-forming storage proteins of its endosperm. It is these dough-forming properties that are responsible for wheat being the most important source of protein in the human diet. The bread and durum wheats are polyploid species containing three (AABBDD) and two (AABB) related genomes, respectively. The genetic constitution of wheat is important because all quality traits result from the expression of genes and their interaction with the environment. The full spectrum of wheat-endosperm proteins has been exhibited in proteome studies involving the two-dimensional fractionation of the polypeptides (after disulfide-bond rupture), followed by dissection of the individual components for identification. This display (Fig. 1) shows that there are at least 1,300 polypeptides, over 300 of which have been identified by N-terminal amino

Characterisation and marker development for low molecular weight glutenin genes from Glu-A3 alleles of bread wheat (Triticum aestivum. L)

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.

Identification of SNPs and development of allele-specific PCR markers for γ-gliadin alleles in Triticum aestivum

The coding regions of 28 entries of hexaploid wheat γ-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 γ-gliadin SNP markers could be used for detecting linked LMW glutenin subunit alleles that are important in determining the quality attributes of wheat products.

Effect of source and proportion of waxy starches on pasta cooking quality

ABSTRACT Starches from the endosperm of three types of total-waxy cereals (bread wheat, maize, and barley) were used in reconstitution studies of durum wheat semolinas to investigate the effect of waxy starch on pasta cooking quality. The chemical composition and the pasting and gelatinization properties of the starches used in this study were evaluated to define the functional properties of each waxy starch. The rheological properties of dough semolinas were evaluated by small-scale mixograph. Spaghetti was prepared using a small-scale pasta extruder and its cooking quality was assessed using a texture analyzer. Cooked pasta firmness, resilience, and stickiness were measured. The substitution of semolina starch with waxy starches from different sources changed the functional properties of dough and their pasta quality. A decrease in firmness was detected in all the semolinas reconstituted with waxy starches. An increase in stickiness was found when semolinas with waxy starch from wheat were evaluated. No...

The characterisation and mapping of a family of LMW-gliadin genes: effects on dough properties and bread volume

Abstract.Analysis of a cDNA library from wheat cv Wyuna endosperm, indicated a significant size and sequence variation among seed-endosperm protein genes. In this study, a family of low-molecular-weight seed protein genes are analysed that are related to the gliadins and the low-molecular-weight glutenin subunits. Sequence analysis and comparison of these proteins showed that they are closely related to a 17-kDa protein from barley, ε hordein, which plays a role in beer foam stability in the brewing industry. Mapping of these genes in wheat shows that they are located on group 7 and 4 chromosomes, as opposed to a group 1 and 6 location for the glutenins and gliadins. It is possible that this family of proteins forms a new class of seed-endosperm proteins important in defining the quality characteristics of wheat flour. Therefore, a representative gene from this family was expressed in Escherichia coli and the purified protein was supplemented into a base wheat flour. Rheological analysis showed that the protein effected dough strength and resistance break down during mixing of the dough, and provided a 20% increase in loaf height after baking.

Multiple isoforms of starch branching enzyme-I in wheat: lack of the major SBE-I isoform does not alter starch phenotype

The role of starch branching enzyme-I (SBE-I) in determining starch structure in the endosperm has been investigated. Null mutations of SBE-I at the A, B and D genomes of wheat were identified in Australian wheat varieties by immunoblotting. By combining individual null mutations at the B and D genomes through hybridisation, a double-null mutant wheat, which lacks the B and D isoforms of SBE-I, was developed. Wheat mutants lacking all the three isoforms of SBE-I were generated from a doubled haploid progeny of a cross between the BD double-null mutant line and a Chinese Spring (CS) deletion line lacking the A genome isoform. Comparison of starch from this mutant wheat to that from wild type revealed no substantial alteration in any of the structural or functional properties analysed. Further analysis of this triple-null mutant line revealed the presence of another residual peak of SBE-I activity, referred to as SBE-Ir, in wheat endosperm representing < 3% of the activity of SBE-I in wild type endosperm.

Small-Scale Reconstitution of Durum Semolina Components

ABSTRACT Pasta prepared by extrusion from 25 g of semolina has been compared with that made from a standard laboratory extruder and found to have similar quality. Durum semolina was fractionated into its starch, gluten, water soluble, and residue fractions. The freeze-dried components were reconstituted and the properties of the reconstituted semolina (ReSem) have been measured. Examination using a 2 g-mixograph and micro-extension tester has shown that ReSem behaves similarly to the original semolina. ReSem and semolina were made into pasta using a small-scale pasta extruder and were of comparable cooking quality. The fractionation and reconstitution of durum semolina on this scale is a useful technique to evaluate the contribution of semolina components to pasta quality.