Starch granule (SG)-associated proteins are involved in starch synthesis and the interaction between SGs and the endosperm protein matrix. In this study, SG proteins were sequentially extracted with the chaotropic reagent, urea from 1 M to 4 M, and then profiled using an integrated proteomic approach including one- and two-dimensional electrophoresis, mass spectrometry and antibody-based enzyme-linked immunosorbent assay (ELISA). The results demonstrated that the SG-associated proteins were dominated by granule-bound starch synthase (GBSS), gliadin. low molecular weight glutenin subunits (LMW-GS), serine protease inhibitors, alpha-amylase inhibitors and puroindolines. A protein with an apparent molecular mass of 50 kDa, expressed in cultivar hard wheat Kukri but not in soft wheat Triller was identified as a novel member of the 'S' group of LMW-GS, designated as LMW-GS-'S'. Further characterization using a broad wheat population revealed that LMW-GS-'S' was selectively expressed in hard wheat cultivars while deleted in all soft wheats tested. Its relationship with hardness was confirmed by its expression in tetraploid durum wheats, which are among the hardest wheats around the world. Monoclonal antibody (MAb) F8-14E6 against LMW-GS-'S' was developed and used in an ELISA to screen 90 Glu allele-defined doubled haploid Janz/Kukri wheat lines. The allele that encodes LMW-GS-'S' was mapped to GluB3h (p < 0.001). (C) 2011 Elsevier Ltd. All rights reserved.

Glutenin genes were known to influence maximum dough resistance (Rmax), dough extensibility (extensibility), and dough development time, whereas puroindoline genes were known to influence grain hardness, flour water absorption (water absorption), and milling yield. These are important determinants of grain quality of wheat in Australia. This study was conducted to investigate the combined effect of these genes on Rmax, extensibility, dough development time, water absorption, and milling yield in a large dataset assembled from the breeding programs based at Horsham, Victoria; Roseworthy, South Australia; and Wagga Wagga, New South Wales; for at least 10 seasons. The effect of the glutenin genes on Rmax, extensibility, and dough development time was confirmed, as was the effect of the puroindoline genes on water absorption and milling yield. In addition, puroindoline genes were shown to significantly affect extensibility and dough development time. The Pina-D1a/Pinb-D1b genotype increased extensibility, dough development time, and milling yield relative to the Pina-D1b/Pinb-D1a genotype. Both of these genotypes are present in cultivars classified as hard-grained in southern Australia. Therefore, the allelic composition of both glutenin and puroindoline genes is required to predict the grain quality of hard wheat in southern Australian breeding programs. The glutenin and puroindoline genes in combination accounted for more than 50% of the genotypic variance for these traits, except for milling yield, but a substantial proportion of the genotypic variation could not be attributed to these genes, indicating that other genes affecting the traits were present in the populations of these wheat-breeding programs.

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Glutenins are the major determinant of dough characteristics in wheat. These proteins are determined by genes at 6 loci, with multiple alleles present in southern Australian breeding programs. Previously, we estimated the effects of these genes on maximum dough resistance (Rmax), dough extensibility and dough development time. Subsequently, the allele previously classified as Glu-B1b was found to consist of 2 alleles, with one, now considered to be Glu-B1al, producing an overexpression of the Bx7 glutenin subunit. Therefore, there is a potential bias in our previous estimates. An extended dataset was analysed with the 2 alleles now separated. These analyses identified negligible biases in our previous estimates, probably due to a low frequency of Glu-B1al before 1999. However, Glu-B1al produced significantly higher Rmax, dough extensibility, and dough development time values than all other alleles at the Glu-B1 locus. Therefore, at intermediate allele frequencies, substantial bias in estimates of the effects of the Glu-B1 alleles can be expected without correct identification of Glu-B1al.

In Canada in 1993, a special market class of wheat, Canada Western Extra Strong (CWES), was established to segregate wheat varieties known to produce very strong and extensible doughs. These exceptional dough properties enable CWES cultivars to be blended with wheats of lesser quality as well as being suited to the manufacture of frozen dough products. The high molecular weight (HMW) glutenin allele (Glu-B1al) that confers these properties, particularly dough strength, has now been identified. Typically, the presence of the Glu-B1al (7+8*) allele is associated with the overexpression of HMW-GS 1Bx 7. RP-HPLC was used to quantify the proportion (% area) of individual HMW-GS relative to total HMW-GS in wheat varieties of different origin. The B genome contributed the highest percentage of HMW-GS, with the exception of Glu-B1d (6+8*) where the D genome contributed the most. Cultivars that possessed the Glu-B1al allele contained a significantly higher (P < 0.001) proportion of HMW-GS (56.80 ± 3.25%) encoded by the B genome. This suggests that the proportion of Glu-B1 subunits, relative to the total amount of HMW-GS expressed, has a major effect on dough strength. We also identified germplasm, of different origin, that contains the Glu-B1al allele and overexpresses subunit 7, including the most likely source of this allele in bread wheat cultivars. The Glu-B1al allele in the varieties identified in this paper could be traced, at least through one parent, to the Argentinean bread wheat cultivar Klein Universal II. RP-HPLC elution and expression profiles of various common HMW-GS are also discussed.

High-molecular-weight glutenin subunits (HMW-GS) are important determinants of wheat dough quality as they confer visco-elastic properties to the dough required for mixing and baking performance. With this important role, the HMW-GS alleles are key markers in breeding programs. In this work, we present the use of a PCR marker initially designed to discriminate Glu1 Bx7 and Glu1 Bx17 HMW-GS. It was discovered that this marker also differentiated two alleles, originally both scored as Glu1 Bx7, present in the wheat lines CD87 and Katepwa respectively, by a size polymorphism of 18 bp. The marker was scored across a segregating doubled-haploid (DH) population (CD87 × Katepwa) containing 156 individual lines and grown at two sites. Within this population, the marker differentiated lines showing the over-expression of the Glu1 Bx7 subunit (indicated by the larger PCR fragment), derived from the CD87 parent, relative to lines showing the normal expression of the Glu1 Bx7 subunit, derived from the Katepwa parent. DNA sequence analysis showed that the observed size polymorphism was due to an 18 bp insertion/deletion event at the C-terminal end of the central repetitive domain of the Glu1 Bx 7 coding sequence, which resulted in an extra copy of the hexapeptide sequence QPGQGQ in the deduced amino-acid sequence of Bx7 from CD87. When the DH population was analysed using this novel Bx7 PCR marker, SDS PAGE and RP HPLC, there was perfect correlation between the Bx7 PCR marker results and the expression level of Bx7. This differentiation of the population was confirmed by both SDS-PAGE and RP-HPLC. The functional significance of this marker was assessed by measuring key dough properties of the 156 DH lines. A strong association was shown between lines with an over expression of Bx7 and high dough strength. Furthermore, the data demonstrated that there was an additional impact of Glu-D1 alleles on dough properties, with lines containing both over-expressed Bx7 and Glu-D1 5+10 having the highest levels of dough strength. However, there was no statistically significant epistatic interaction between Glu-B1 and Glu-D1 loci.

Starch properties were measured on the doubled haploid progeny of 2 crosses, one between Cranbrook and Halberd and the other between CD87 and Katepwa. Properties studied included starch peak and final viscosity measured by Rapid Visco Analyser, starch granule size distribution measured by laser light scattering, starch gelatinisation temperature by differential scanning calorimetry, and flour swelling volume. In the Cranbrook Halberd cross (samples from 2 environments), a highly significant quantitative trait locus (QTL) was located on chromosome 4A for both starch peak viscosity and starch/flour swelling volume, centred around the Wx-B1 locus. In previous studies, this locus has been found to be linked to Japanese noodle quality. The increases in starch peak viscosity and flour swelling volume are derived from the Halberd parent, consistent with the fact that Halberd is null for the Wx-B1 locus on chromosome 4A and is missing the respective granule-bound starch synthase protein, whereas Cranbrook is a wheat line carrying the normal 3 Wx loci. The final starch viscosity also showed an association with the Wx-B1 locus. In the CD87 Katepwa cross, the progeny showed an association between peak viscosity and a marker on chromosome 7A. This appeared to be near the Wx-A1 locus. In some experiments, flour viscosity showed a highly significant QTL on chromosome 7B, apparently at the same locus as the late maturity - amylase derived from the Cranbrook parent. Starch gelatinisation onset temperature indicated a significant QTL on chromosomes 2B and 7A (LOD = 2.58 and 3.66, respectively, in interval analyses). Starch gelatinisation peak temperatures indicated a QTL on chromosome 7A, which, although not in the significant (P = 0.05) class based on regression analyses, indicated a LOD score of 3.06 in interval analyses. Heat of gelatinisation (H) indicated a suggestive QTL (LRS = 14.5 with a threshold of 14.7 for P < 0.05, LOD = 2.65 for interval analysis), on chromosome 4A, at the Wx-B1 locus with an increased effect coming from the Halberd parent. The A:B granule ratio analysis indicated a significant QTL on chromosome 4B, but this was not observed in all environments and may be due to the fact that the QTL corresponded to the position of a major QTL controlling plant growth.

The Cranbrook/Halberd doubled haploid population has provided a unique opportunity to examine in detail the contributions made by a number of different high molecular weight (HMW) and low molecular weight (LMW) glutenin alleles to the dough properties in a set of homogeneous lines of wheat. A range of different instruments was employed, including Farinograph, Extensograph, Do-Corder, Resistograph, and GRL/EasyMix, to study the dough rheology of the lines from 3 sites over 2 years. Correlation studies showed that 2 basic parameters (dough strength and extensibility) were measured by these different instruments. The results presented are mainly from the Extensograph, which is a major Australian standard for determining release and marketing classification of Australian wheats. Approaches to investigate the data include bulk segregant analysis, distribution of protein alleles in the population, and multiple linear regression. As expected, the HMW glutenin alleles made a major contribution to dough strength, with a minor, but not insignificant, contribution from the LMW glutenin alleles. From a knowledge of their glutenin alleles, a glutenin strength score (GSS) was devised to allow breeders to rank the dough strength of various lines. The GSS scoring system is based on both HMW and LMW glutenin alleles, adding to a total out of 10. Extensibility, on the other hand, was predominantly influenced by protein levels in the flour and environmental conditions such as site and season. However, the LMW glutenin alleles make a significant genetic contribution to the extensibility, which can be assessed by using a glutenin extensibility score. These two glutenin quality scores currently include only the alleles present in the parents, Cranbrook and Halberd, but this could be expanded to include a wider range of alleles by analysis of the quality data from other doubled haploid populations. These quality scores would then be an extremely useful tool for assessing the potential quality of parental and early generation germplasm in wheat breeding programs, by a knowledge of the allelic composition of their HMW and LMW glutenins.

Starch properties were measured on the doubled haploid progeny of 2 crosses, one between Cranbrook and Halberd and the other between CD87 and Katepwa. Properties studied included starch peak and final viscosity measured by Rapid Visco Analyser, starch granule size distribution measured by laser light scattering, starch gelatinisation temperature by differential scanning calorimetry, and flour swelling volume. In the Cranbrook Halberd cross (samples from 2 environments), a highly significant quantitative trait locus (QTL) was located on chromosome 4A for both starch peak viscosity and starch/flour swelling volume, centred around the Wx-B1 locus. In previous studies, this locus has been found to be linked to Japanese noodle quality. The increases in starch peak viscosity and flour swelling volume are derived from the Halberd parent, consistent with the fact that Halberd is null for the Wx-B1 locus on chromosome 4A and is missing the respective granule-bound starch synthase protein, whereas Cranbrook is a wheat line carrying the normal 3 Wx loci. The final starch viscosity also showed an association with the Wx-B1 locus. In the CD87 Katepwa cross, the progeny showed an association between peak viscosity and a marker on chromosome 7A. This appeared to be near the Wx-A1 locus. In some experiments, flour viscosity showed a highly significant QTL on chromosome 7B, apparently at the same locus as the late maturity - amylase derived from the Cranbrook parent. Starch gelatinisation onset temperature indicated a significant QTL on chromosomes 2B and 7A (LOD = 2.58 and 3.66, respectively, in interval analyses). Starch gelatinisation peak temperatures indicated a QTL on chromosome 7A, which, although not in the significant (P = 0.05) class based on regression analyses, indicated a LOD score of 3.06 in interval analyses. Heat of gelatinisation (H) indicated a suggestive QTL (LRS = 14.5 with a threshold of 14.7 for P < 0.05, LOD = 2.65 for interval analysis), on chromosome 4A, at the Wx-B1 locus with an increased effect coming from the Halberd parent. The A:B granule ratio analysis indicated a significant QTL on chromosome 4B, but this was not observed in all environments and may be due to the fact that the QTL corresponded to the position of a major QTL controlling plant growth.