Unique gliadin patterns in Chinese winter wheat cultivars

Cultivated Chinese wheat germplasm has been a valuable genetic resource in international plant breeding. Patterns of gliadin among cultivated Chinese accessions are unknown, despite the proven value and potential novelty. The objective of this work was to analyse the diversity within improved Chinese wheat germplasm. The electrophoretic banding patterns of gliadin in winter wheat cultivars and advanced lines were determined by acid-polyacrylamide gel electrophoresis. For 148 leading commercial cultivars and promising advanced lines used in this study, 48 patterns were identified, 29 corresponding to ω -gliadin, nine to γ -gliadin, five to β -gliadin and five to α -gliadin. The most frequent patterns were A6 in ω ; B in γ ; B in β and A in the region of α . A total of 116 band types appeared in the 148 samples: 94 accessions had unique gliadin types, and 22 gliadin, types while not unique, were found in 54 accessions. The gliadin patterns of Chinese wheat cultivars and lines greatly differed from the patterns of wheat lines from other countries. Three patterns, E, J, H, M, N and O in the ω -zone had not been reported previously. Three wheat zones of China, the Northern Winter Wheat Region, the Yellow and Huai Valley River valleys Winter Wheat Region and the Southwestern Winter Wheat Region, showed different frequencies in their gliadin patterns. This information can be used to monitor genetic diversity with Chinese wheat germplasm.     

Polyacrylamide gel electrophoresis of wheat gliadins: the use of a moving boundary for improved resolution

Summary The process of the separation of gliadin proteins of wheat, using polyacrylamide gel electrophoresis, was studied in detail. It was shown that electrophoresis is a dynamic process during which the pH of the gel changes together with the potassium ion concentration. The most positive effect on the separation of the gliadins was generated by a moving front, which is a boundary between regions with a low concentration of K⁺ ions and a low pH, and region with a high concentration of K⁺ ions and a high pH, after optimization of the concentrations of the cations and anions in the electrode solutions. The finding was exploited for the development of an extremely simple electrophoresis system, in which buffers were not needed for obtaining a high resolution. The system was further improved by applying a stacking gel. The advantages of this system are discussed. This new approach may be useful for improving electrophoresis systems for other applications.     

Disulphide structure of high-molecular-weight (HMW-) gliadins as affected by terminators

This study aimed at elucidating SS-bonds of HMW-gliadins (HGL) from wheat with the focus on terminators of glutenin polymerisation. HGL from wheat flour extracts non-treated or treated with the S-alkylation reagent N-ethylmaleinimide (NEMI) were compared. HGL from wheat flour Akteur were isolated, hydrolysed with thermolysin and the resulting peptides pre-separated by gel permeation chromatography and analysed by liquid chromatography/mass-spectrometry using alternating electron transfer dissociation/collision-induced dissociation. Altogether, 22 and 28 SS-peptides from samples without and with NEMI treatment, respectively, were identified. Twenty-six peptides included standard SS-bonds of α- and γ-gliadins, high-molecular-weight and low-molecular-weight glutenin subunits. Eleven SS-bonds were identified for the first time. Fifteen peptides unique to HGL contained cysteine residues from gliadins with an odd number of cysteines (ω5-, α- and γ-gliadins). Thus, gliadins with an odd number of cysteines, glutathione and cysteine had acted as terminators of glutenin polymerisation. Decisive differences between samples without and with NEMI treatment were not obvious showing that the termination of polymerisation was already completed in the flour. The two HGL samples, however, were different in the majority of ten peptides that included disulphide-linked low-molecular-weight (LMW) thiols such as glutathione and cysteine with the former being enriched in the non-treated HGL-sample.     

Atomic Force Microscopy of A-Gliadin Fibrils and in situ Degradation

Atomic force microscopy (AFM) has been used in air and in aqueous buffer to study the structure of fibrils formed by the self-assembly of A-gliadin protein molecules. The images showed fibrils with a diameter of between 15 and 30 nm and lengths ranging from about 100 nm to 2 μm. No branched fibrils were observed, and there was no indication of a strong lateral inter-fibril interaction that would result in side-by-side association. Disassembly of the fibrils occurred when the pH of the aqueous buffer was reduced. In contrast the reverse process of fibril assembly and adsorption to the mica surface was less readily observed in situ. Some short fibrils were observed to assemble, but the lengths and densities were considerably less than those obtained by external deposition and drying.     

An assessment of cultivated emmer germplasm for gluten proteins

The storage protein composition of 61 accessions of Triticum dicoccum was analyzed by SDS-PAGE (HMW- and LMW-glutenin subunits) and Acid-PAGE (gliadins). In the HMW-glutenin subunits, four allelic variants at the Glu-A1 and eight at the Glu-B1 locus were detected resulting in a total of 17 patterns. The Glu-B1 locus was found to be more polymorphic than the Glu-A1 locus. Interestingly, the presence of HMW subunits like 13+16, 2^∗ and 1 associated with good quality was observed. Three accessions were null for both the Glu-A1 and Glu-B1 loci. There was less variation for gliadins. Three different gamma gliadin fractions coded by Gli-B1 locus were detected and there were eight different LMW-B glutenin patterns at the Glu-3 loci. The variability can be used to improve the utility of this crop.     

A proposed method of polyacrylamide gel electrophoresis in acid environments applied to gliadins of wheat grains

Summary In this paper we describe the method of wheat gliadin electrophoresis in use at RIVRO, Wageningen. It differs from other techniques mainly by the application of an alternative buffer system, making it possible to polymerise the gels in a buffered alkaline environment and to perform the run at pH 3.1 without extensive buffer changing steps. Advantages are a greater gel reproducibility and the ease of gel handling. Furthermore, a rationalised protein extraction procedure, a cheap shaking system for staining baths and a better (slower moving) tracking dye are described.     

Genetic polymorphisms at Gli-Dt gliadin loci in Aegilops tauschii as revealed by acid polyacrylamide gel and capillary electrophoresis

Gliadin variation at Gli‐D^t1 and Gli‐D^t2 loci in 198 Aegilops tauschii accessions was studied by acid polyacrylamide gel electrophoresis (A‐PAGE) and capillary electrophoresis (CE). High genetic polymorphisms were found at both gliadin coding loci, revealing a total of 184 and 169 gliadin variants at the Gli‐D^t1 and Gli‐D^t2 loci, respectively. In particular, 12 gliadin blocks encoded by different alleles were apparently expressed and readily identified in six synthetic hexaploids produced by hybridization between Triticum durum and Ae. tauschii accessions. Compared with Ae. tauschii ssp. eusquarrosa, the gliadin profile of the D genome in Ae. tauschii ssp. strangulata more resembles that of T. aestivum, supporting the view that the subspecies strangulata is the most likely progenitor of bread wheat. Capillary electrophoresis analysis showed that the method is capable of separating and characterizing gliadins with speed, in high resolution using small sample amounts, and is well‐suited to detect protein alleles and to identify desirable genotypes in wheat quality improvement.     

Complexity of the Gli-A3 locus in bread wheat

Two bread wheat cultivars, ‘Ariana 8’ and ‘Cajeme 71’, and 129 F₂, grains from the cross between them were analysed for gliadin composition. Two monodimensional (A-PAGE and SDS-PAGE) and two different two-dimensional (SDS-PAGE x SDS-PAGE and A-PAGE x SDS-PAGE) electrophoretic methods were used. Parents differed at the Gli-Al locus, detected by A-PAGE. The SDS-PAGE of the aqueous ethanol extractable protein under nonreduced conditions showed two bands of ‘Ariana 8’ and one of ‘Cajeme 71’, encoded by genes located 22 cM from the Gli-Al locus, and therefore, located at the Gli-A3 locus. This locus has been considered to contain genes coding for ω-gliadins alone. The two-dimensional maps of the parents showed that one band from ‘Ariana 8’ was an ω-gliadin, but the other two bands, one from each parent, were γ-gliadins. Results obtained indicated that GH-A3, like Gli-Al, is a complex locus coding for both ω-and γ-gliadins.     

The genetics of gliadin and glutenin,the major storage proteins of the wheat endosperm

This paper reviews our present knowledge of the chromosomal location of the genes that control the synthesis of gliadins and glutenins, the two major groups of storage protein in the endosperm of wheat (Triticum aestivum). Allelic relationships and genetic linkage between genes are also discussed. The areas that require futher investigation are identified.     

Genetic Diversity of Recurrent Selection Populations with Ms2 Gene Assessed by Gliadins in Common Wheat （Triticum aestivum L.）

The male-sterile lines with Ms2 gene were highly evaluated in recurrent selection in wheat （Triticum aestivum L.）. Three populations C6 （population after six cycles of selection）, C7 （population after seven cycles of selection）, and C8 （population after eight cycles of selection） were constructed through recurrent selection with 12 parental materials （P）. Acid polyacrymide gel electrophoresis （A-PAGE） analysis was used to identify gliadin patterns and evaluate the genetic diversity in 12 parents and three populations. A total of 63 bands were identified, of which 17 polymorphic bands and 7 unique bands were present in populations and seven polymorphic bands and four unique bands were present in parents. The number of polymorphic and unique bands decreased gradually from C6 to C8, especially for to- and y-gliadins. The genetic distances in C6, C7, and C8 were calculated. The distributions of genetic distance were different in three recurrent selection populations. From C6 to C8, the genetic distance was 0.2687, 0.2652 and 0.1987, respectively. Statistically significant differences were detected between C7 and C8 with the T value of 37.9718. The result of cluster analysis based on genetic similarity matrix of three populations fitted well to those of principle coordinates analysis （PCoA）. Compared with 12 parents, almost all individuals of three populations are new genotypes. Most of the individuals from C6 and C7 could be divided into two groups, while most individuals of C8 were in one cluster. In conclusion, the results indicated that the genetic diversity was decreased severely according to the information revealed by A-PAGE, although some variations could be created in the recurrent selection. It was necessary to introduce diverse germplasm based on the genetic database of recurrent population to maintain and improve the breeding efficiency in the further program.