Analysis Of Gliadins Research Articles

The introgression of chromosome 6P specifying for increased numbers of florets and kernels from Agropyron cristatum into wheat

A wheat (Triticum aestivum L.) line 4844 with superior numbers of florets and grains per spike was derived from the cross between Fukohokomugi wheat and Agropyron cristatum (L.) Gaertn. In order to determine the genetic control of floret and kernel number per spike in this line, chromosome addition and substitution lines that were derived from line 4844 were characterized by means of in situ hybridization, microsatellite (SSR), and gliadin analyses. Genomic in situ hybridization analysis with biotinylated P genomic DNA of A. cristatum as a probe demonstrated that the increased number of florets and grains in a spike was associated with the introgression of an A. cristatum chromosome. Fluorescence in situ hybridization, using a repetitive sequence, pAs1, derived from Aegilops squarrosa L., indicated the replacement of chromosome 6D of wheat in the wheat-A. cristatum chromosome substitution lines. This was confirmed by microsatellite analyses with wheat SSR markers specific for chromosome 6D, suggesting that the A. cristatum chromosome was homoeologous to group 6 and was therefore designated as 6P. This conclvsion was further confirmed by amplification using EST-SSR markers and gliadin analysis. The increased number of florets and kernels within a spike of the wheat-A. cristatum hybrids thus was controlled by gene(s) located on A. cristatum chromosome 6P.

Genetic diversity and structure in a natural Hordeum chilense population based on gliadin analysis

Hordeum chilense Roem. et Schult. is a South American wild barley that occurs exclusively in Chile and Argentina, where it is a component of natural pastures. This species has been crossed with durum and bread wheats to obtain a new amphiploid, named tritordeum, which presents agronomic traits of a new crop. The knowledge of the reproductive system is very important for the management of any species with breeding purposes. In this work with this finality, two hundred seventy seeds of 32 original spikes from a natural population were analysed for the variation of gliadin by A-PAGE. The data suggested that 98% of the genetic diversity appeared between spikes whereas only the 2.0% was within spikes. Only four out of 32 analysed spikes showed certain level of heterozygosity for the ω-gliadins. The Wright's fixation index for this population was established in F = 0.993 and the cross-fertilisation rate was equal to 0.4%, which suggested that H. chilense is an autogamous species.

Analyses of Acid‐PAGE Gliadin Pattern of Indian Wheats (Triticum aestivum L.) Representing Different Environments and Periods

Acid‐PAGE analysis of gliadins from 159 Indian wheat (Triticum aestivum L.) cultivars developed during the last five decades was accomplished to identify gliadin band patterns as well as the extent of genetic diversity. Extensive polymorphism [genetic diversity index (H) = 0.875] in gliadin pattern was observed in the cultivars studied. A total of 147 band patterns were identified, of which 45 different mobility bands were in the region of ω gliadins, 42 in the region of γ gliadins, 30 in the region of β, and 29 in the region of α gliadins. Zone‐wise genetic diversity index was highest in North Western Plains Zone (H = 0.904) followed by North Eastern Plains Zone (H = 0.878), Central Zone (H = 0.864), Peninsular Zone (H = 0.844), and Northern Hills Zone (H = 0.836). The α gliadin pattern 1, 8, 11, and 20; β gliadin pattern 21; γ gliadin pattern 15 and 20; and ω gliadin pattern 4, 18, and 38 were specific to cultivars of Northern Zones and α gliadin 20 and β gliadin 22 to Central and Peninsular Zones. Period‐wise highest mean genetic diversity (H = 0.915) was observed in cultivars identified during 1971 through 1980 and lowest (H = 0.868) in cultivars developed after 1990. The reduction in genetic diversity during 1990 onward might be because of enhanced use of 1BL.1RS translocation. This information can be used in breeding programs to maintain genetic diversity within Indian wheat germplasm.

Development of a general procedure for complete extraction of gliadins for heat processed and unheated foods

In the past, one of the major problems in gluten analysis has been the unavailability of an efficient, universal, extraction procedure of gliadins - the alcohol-soluble proteins of gluten - from both heat processed and unprocessed products. This study was designed to develop a universal, extraction procedure capable of extracting the totality of gliadins from both unprocessed and heat processed foods for coeliac patients. A simple quantitative extraction solution containing 250 mM 2-mercaptoethanol and 2 M guanidine hydrochloride ('cocktail'), was developed to extract gliadins from heated foods. The diluted reducing and disaggregating agents reaching the micro plate at low concentration do not affect the ELISA system based on the R5 monoclonal antibody. The recovery of gliadins extracted by the cocktail from spiked samples was nearly complete, with an average mean value of 95.5%, which is clearly superior to 44.4% obtained with conventional 60% aqueous ethanol. The cocktail always yielded either slightly similar or higher values than 60% aqueous ethanol depending on the type of foods: 1.1-fold in unheated foods, 1.4-fold in wheat starches and 3.0-fold in heated foods. False positives or negatives were never observed using the cocktail solution. We present a general complete gliadin extraction procedure based on reducing and disaggregating agents for both heated and unheated foods as a crucial tool for gliadin analysis. The new extraction solution is used for corresponding proteins from rye (secalins) and barley (hordeins). The cocktail was employed as the extraction method in the international ring trial evaluation of sandwich R5-ELISA as proposed by the Codex Alimentarius and organized by the Working Group on Prolamin Analysis and Toxicity.

General experimental aspects for capillary electrophoretic analysis of gliadins in Triticum species

Capillary electrophoresis (CE) of gliadins from Triticum species in acidic or isoelectric buffers is currently used. The performance of five buffers are compared here. The highest resolving power and the shortest time of analysis resulted associated to the isoelectric buffers. The slight different performance of the iminodiacetic acid (IDA) and aspartic acid (Asp) buffer systems should be taken into consideration before to start CE analyses. IDA buffer should be preferred when screening large germplasm collections or when a detailed characterisation of ω-gliadins is required. Asp buffer proved to be most satisfactory for cultivar discrimination or varietal identification.

Assay of gliadin by real-time immunopolymerase chain reaction.

Patients with coeliac disease (gluten-sensitive enteropathy) are intolerant against gliadins from wheat and the respective proteins from related cereals and have to keep a lifelong gluten-free diet. For control of gliadin in gluten-free food sensitive assay techniques are necessary. We developed an immunopolymerase chain reaction (iPCR) assay for gliadin. In this technique immunological detection of gliadin by a monoclonal antibody R5 conjugated with an oligonucleotide is amplified by PCR. For quantification, iPCR was performed as real-time PCR (real-time iPCR) in one step. By means of real-time iPCR, the sensitivity of gliadin analysis was increased more than 30-fold above the level reached by enzyme immunoassay. Real time-iPCR using R5 directly conjugated with oligonucleotide was clearly more sensitive than real time-iPCR applying sequentially biotinylated R5, streptavidin, and biotinylated oligonucleotide. With directly conjugated R5 gliadin was detected at a concentration as low as 0.16 ng/mL corresponding to 16 microg gliadin/100 g food or 0.16 ppm (corresponding to 0.25 g of food extracted in 10 mL of solvent and 25-fold dilution of the extract prior to analysis). This is the first report applying the highly sensitive technique of iPCR for gliadin analysis. Furthermore, this is the first approach to perform real-time iPCR in one step without changing the reaction vessels after enzyme immunoassay for subsequent PCR analysis thus minimizing risks of contamination and loss of sensitivity.

Polymorphism, variation and genetic identity of Spanish common wheat germplasm based on gliadin alleles

Gliadin analysis has shown that Spanish common wheat germplasm is highly polymorphic and rather unique. In total, 81 different alleles, 70 of them catalogued, were identified in the 52 Spanish landraces studied, and 25 new gliadin alleles, not present in germplasm from other countries, were found. Eleven alleles were identified at the Gli-A1 locus, 14 at the Gli-B1, 9 at the Gli-D1, 17 at the Gli-A2, 14 at the Gli-B2 and 16 at the Gli-D2. Comparison of gliadin alleles among varieties released before and after 1966 revealed significant differences for 46 gliadin alleles. Forty-six alleles (36 of them catalogued) present in the material developed before 1966 were lost. In contrast, 25 alleles (23 of them catalogued) appear for the first time in the genepool bred after 1966. On the other hand, most of the Gli alleles frequent in recent Spanish cultivars were already present in Spanish landraces. In general, the results showed that Spanish varieties have suffered qualitative alterations of genetic variation during the last 35 years mainly due to changes in breeding goals and crosses with foreign germplasm. Seventy-five percent of the landraces analysed were shown to have maintained their genetic identity in the genebank although they have been regenerated several times during the last century. However, mistakes in three accessions modified 10 alleles (allele losses or presence of new alleles), and error in one suggests alteration of five alleles. These results demonstrate that great care during maintenance and handling of samples is an important requirement in conserving germplasm collections. Gliadin alleles allowed us to identify 48 different genotypes, some accession duplicates and one intermixing. Also, several mistakes could be resolved using gliadins as genetic markers.

Análise numérica dos eletroforegramas de gliadinas de cultivares de triticale

Buscando automatizar o processo de identificação de cultivares pelo método de eletroforese de proteínas, o presente trabalho teve por objetivo avaliar a viabilidade da utilização de valores de intensidade das bandas como dados adicionais de polimorfismos na discriminação de cultivares e comparar o comportamento de vários coeficientes de similaridade na análise dos eletroforegramas de gliadinas. Os eletroforegramas de gliadinas de quatro cultivares de triticale estreitamente relacionadas foram comparados pela análise computacional utilizando seis coeficientes de similaridade binários (presença/ausência) (Jaccard, Sorensen-Dice, Nei & Li, Simple Matching, Yule e Baroni-Urbani) e cinco quantitativos (Pearson product moment correlation, Spearman, Percent Similarity, Modified Morisita e Gower). As análises quantitativas dos eletroforegramas levaram em conta a intensidade das bandas, disposta em diferentes números de classes, possibilitando avaliar o efeito da variabilidade desse parâmetro. Foram utilizadas cerca de 60 amostras individuais de sementes de cada cultivar. Apesar do baixo polimorfismo intervarietal, os coeficientes médios de similaridade dentro das cultivares sempre foram maiores do que entre cultivares, o que indica o alto poder discriminatório dos testes. O estudo demonstra a viabilidade de identificar cultivares de triticale pela comparação numérica dos dados de eletroforese de gliadinas, obtidos das amostras, com uma biblioteca de eletroforegramas. Esse procedimento garante uma comparação mais objetiva dos eletroforegramas, em relação à análise visual. Foi constatado também, que a intensidade (densidade) das bandas nos eletroforegramas de gliadinas é muito variável, sendo portanto um parâmetro pouco confiável na análise de polimorfismos de gliadinas em triticale. Ainda com relação ao parâmetro intensidade, há uma perda progressiva na confiabilidade dos resultados de comparação com o aumento no número de classes de intensidade.

Sample Preparation Optimization for the Analysis of Gliadins in Food by Matrixassisted Laser DesorptionIonization Timeofflight Mass Spectrometry

Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) of wheat gliadin extracts from distinct types of wheat varieties displays a similar characteristic gliadin mass pattern within the 30–40 kDa range. Direct observation of this pattern in food samples was used to identify gluten gliadins in food rapidly and easily. A modified sample preparation for gliadin analysis by MALDI/TOF-MS has improved sensitivity of gliadin detection by a factor of more than 50. This procedure allows the direct detection of complex mixtures of gluten gliadins in food sample extracts down to a concentration of 0.01 mg ml-1, i.e. down to levels which are toxic for coeliac patients. This highly sensitive improved method has been also exemplified by the facile detection of individual proteins at low concentrations (100–400 pmol ml-1). © 1997 by John Wiley & Sons, Ltd.

Sample Preparation Optimization for the Analysis of Gliadins in Food by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) of wheat gliadin extracts from distinct types of wheat varieties displays a similar characteristic gliadin mass pattern within the 30–40 kDa range. Direct observation of this pattern in food samples was used to identify gluten gliadins in food rapidly and easily. A modified sample preparation for gliadin analysis by MALDI/TOF-MS has improved sensitivity of gliadin detection by a factor of more than 50. This procedure allows the direct detection of complex mixtures of gluten gliadins in food sample extracts down to a concentration of 0.01 mg ml-1, i.e. down to levels which are toxic for coeliac patients. This highly sensitive improved method has been also exemplified by the facile detection of individual proteins at low concentrations (100–400 pmol ml-1). © 1997 by John Wiley & Sons, Ltd.

Genetic characterization of storage proteins in a set of F1-derived haploid lines in bread wheat

Wheat storage proteins were evaluated by SDS-PAGE in a population of 206 doubled haploid (DH) lines, produced from a cross between bread wheat cvs Chinese Spring (CS) and Courtot (CT). The analysis of gliadins and high- and low-molecular-weight glutenins gave rise to 11 protein markers between parental varieties. Among these, one each was encoded at the Glu-A1, Gli-A1, Gli-A2, Gli-A5, Glu-B3, Gli-B1 and Gli-D1 loci and four were encoded at the Glu-D3 locus. Only the Gli-A2 marker showed a distorted segregation. A distance of 1.94 cM was evaluated between the Gli-A1 locus and the recently found Gli-A5 locus. Among the DH lines, only nine exhibited an unexpected pattern. The chromosome allocation was determined for almost all the LMW-GS and gliadin bands of CS using nullitetrasomic and ditelosomic lines. Two C LMW-GS were found to be coded by 6DS. Similarly, substitution lines into CT allowed the allelic determination of numerous LMW-GS and gliadin bands. A correspondence between gliadin markers separated in SDS-PAGE and in A-PAGE revealed that the common allele Gli-Aa between CS and CT determined in A-PAGE was able to be separated into two alleles when SDS-PAGE was used.

HMW-GS variation of somaclones and its variants in Triticum aestivum

Research on somacolonal variation in hexaploid wheat (Triticum aestivum L.) has been conducted a lot and morphological, cytological, and biochemical variations have been observed. Evidence for gene mutations has been obtained through the analysis of gliadin and glutenin in wheat grain, but loci of mutations have not been located. The aims of this study are: firstly, to detect mutation loci of specific gene (Glu-1) to provide exact evi-dence for gene mutation by analyzing changes of high-molecular-weight glutenin subunits

Capillary electrophoresis of wheat gliadin: Initial studies and application to varietal identification

Wheat gluten proteins have important functional properties, and are reliable indicators of genotype. They are also very heterogeneous, demanding use of powerful analytical techniques. Gel electrophoresis methods are widely used for gluten analysis, but they are labor-intensive, and their data are not easily quantified. Methods of capillary electrophoresis (CE) are now available that promise to overcome these disadvantages. We here report successful application and optimization of two CE procedures for gliadin analysis. The first procedure used 0.06 mol/L sodium borate buffer, pH 9.0, containing 200 mL/L acetonitrile + 10 g/L sodium dodecyl sulfate; separations were excellent, but reproducibility was difficult to maintain. The second buffer, 0.1 mol/L phosphate, pH 2.5, containing a linear hydrophilic polymer, overcame this problem, providing separations of gliadins that are rapid, high-resolution, automatic, and easily quantified. CE analyses of gliadin also reliably differentiate varieties of all U.S. wheat classes. CE is thus a valuable technique for gliadin analysis, complementary to other methods of electrophoresis and chromatography. CE has much potential to become a routine tool for wheat varietal identification and classification, and for prediction of quality.

Molecular analysis of gliadin and glutenin genes in T. durum cv. Lira. A model system to analyse the molecular bases of quality differences in durum wheat cultivars

As a model system to study the molecular bases of differences between high- and lowquality durum wheat cultivars, designated as biotypes γ-45 and γ-42, respectively, the cultivar Lira, which contains both biotypes, was examined. Polymerase chain reaction analysis of biotypes 42 and 45 was carried out by using oligonucleotide primers from flanking nucleotide sequences coding for α-, β- and γ-gliadin and for high molecular weight (HMW) and low molecular weight (LMW) glutenins. A different amplified band for γ-gliadin sequences was observed between the two biotypes. Southern blot analysis of the same biotypes showed different hybridization patterns for both γ-gliadin and LMW glutenin genes. The hybridization patterns of LMW glutenin sequences revealed the presence of additional bands in biotype 45, suggesting that the quantitative differences in LMW glutenins between biotypes 42 and 45, considered responsible for their quality differences, are most probably due to a higher number of LMW genes in biotype 45. This conclusion seems also to be supported by the results obtained in other durum wheat cultivars belonging to types 42 and 45.

Agarose gel isoelectric focusing of gliadins in the presence of ethanol

Conditions for the analysis of gliadins by isoelectric focusing in the presence of ethanol were worked out. Comparison of the electrophoretic band patterns obtained in aqueous and ethanolic media showed that the resolution was highly improved in the presence of ethanol. The gliadins were identified by immunological detection in nitrocellulose blots obtained from the agarose gels.

Analysis of phylogenetic relations of durum, carthlicum and common wheats by means of comparison of alleles of gliadin-coding loci

Polymorphism and inheritance of wheat storage protein, gliadin, of durum (macaroni) and carthlicum wheats have been studied. Analysis of gliadin in 78 cultivars and in F2 seeds of intercultivar crosses of durum wheat revealed three different chromosome 1A-encoded blocks of components similar to those found in common wheat (GLD1A2, GLD1A18, GLD1A19). Most of the durum cultivars studied had these three blocks; GLD1A2 was also frequent in common wheat. In contrast, all chromosome 1B-encoded blocks of durum clearly differed in component composition from those found in common wheat. Therefore, durum could not be an ancestor or a derivate of recent bread wheat. Analysis of gliadin in the collection of carthlicum wheat (14 accessions) revealed several suspected chromosome 1A, 1B, and 6A-controlled blocks, some of which were similar to those in common wheat, while others were different. Therefore, carthlicum is likely to be an ancestor or a derivate of some forms of bread wheat. There were also chromosome 1A and 6A-, but not 1B-encoded blocks which were identical in durum and carthlicum wheats. The results confirm that all three wheats share the same genome A, but emphasize the heterogeneity of genotypes among donors of this genome. Discovery of identical blocks in tetraploids and hexaploids indicates polyphyletic [from different genotypes of donor (s)] origin of these wheats.

Accumulation of Gliadin and Glutenin Polypeptides during Development of Normal and Sulphur-deficient Wheat Seed: Analysis using Specific Monoclonal Antibodies

Skerritt, J. H., Lew, P. Y. and Castle, S. L. 1988. Accumulation of gliadin and glutenin polypeptides during development of normal and sulphur-deficient wheat seed: analysis using specific monoclonal antibodies.—J. exp. Bot. 39: 723-737. A panel of monoclonal antibodies with various specificities for wheat (Triticum aestivum L.) gluten polypeptides has been used to analyse the accumulation of these polypeptides in the endosperm of developing wheat seeds grown under normal and sulphur-deficient conditions. Immunoblots of polypeptides fractionated by SDS PAGE allowed a qualitative analysis of gliadin and HMW glutenin accumulation for high- and low-sulphur seeds 8 d to 30 d after anthesis (d.a.a.). In addition, quantitative analysis of the deposition of various gluten polypeptides was performed, with a solid-phase radioimmunoassay on extracts of seeds harvested 4-36 d.a.a. The initial accumulation of HMW glutenin subunits was detectable at an earlier stage of development than that of gliadins for both normal and sulphur-deficient seeds. The initiation of detectable gliadin accumulation was asynchronous with an order of alpha-gliadins, beta-, gamma- and some omega-gliadins and finally the remaining omega-gliadins. In sulphur deficiency, all gliadins reached a constant proportion of the dry weight of the endosperm earlier than in normal wheat, while a more marked increase in the proportion of HMW glutenin occurred late in grain development. The protein blot studies also identified a putative omega-gliadin polypeptide which was detectable late in seed development and only in sulphur-deficient seeds.

Chromosomal control of wheat gliadin: analysis by reversed-phase high-performance liquid chromatography

Gliadin proteins of the hexaploid wheat variety 'Chinese Spring', and of its nullisomic-tetrasomic and ditelocentric aneuploid lines, were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC). Reversed-phase separations were carried out at 70°C on C8 and C18 columns using a gradient of increasing acetonitrile concentration in the presence of 0.1% trifluoroacetic acid. Thirty-five components were separated and all were found to be controlled by genes on the short arms of group 1 and group 6 chromosomes (the complex Gli-1 and Gli-2 loci). Results indicated that gluten polypeptides elute as groups in order of increasing hydrophobicity in the following approximate order: (1) albumins plus globulins, (2) ω-gliadins, (3) high molecular weight (MW) glutenin subunits, (4) α-type gliadins, (5) low MW glutenin subunits, and (6) γ-gliadins. The three distinct protein types coded by genes at the complex Gli-I loci (ω-gliadins, γ-gliadins, and low MW glutenin subunits) thus have uniquely different surface hydrophobicities. Similarly, gene locations for hexaploid 'Cheyenne' gliadins and durum gliadin proteins in the varieties 'Langdon', 'Edmore', and 'Kharkovskaya-5' were determined through RP-HPLC analysis of aneuploid lines. All results confirm known locations of genes for gliadin proteins, and demonstrate that RP-HPLC is a powerful new tool for analysis of gliadins in breeding and genetic studies.

High‐resolution proton, carbon‐13 and nitrogen‐15 nuclear magnetic resonance studies of wheat proteins at high magnetic fields: Spectral assignments, changes with concentration and heating treatments of flinor gliadins in solution—comparison with gluten spectra

AbstractThe first high‐field and high‐resolution proton, carbon‐13 and nitrogen‐15 nuclear magnetic resonance (n.m.r.) spectra of gliadin solutions are reported. Collective assignments are proposed based on ‘random coil’ chemical shifts and amino acid analysis of gliadins. Additional information from X‐ray diffraction, photoacoustic spectroscopy, dielectric relaxation and electrophoretic measurements is considered in the interpretation of the n.m.r. spectra. Marked changes in the n.m.r. spectra of gliadin solution were observed with increasing concentration or temperature. An interpretation of these effects in molecular terms is proposed and the practical implications of n.m.r. measurements for wheat variety identification and correlation with rheological testing indicated. The relevance of the results to studies of glutens is also noted.

Glutamic Acid of Proteins

RELEVANT to the discussion which has taken place recently between Chibnall1 and Kogl and Erxleben2,3 are some facts which I have accumulated in an analysis of gliadin. Kogl and Erxleben find that the glutamic acid derived from the proteins of malignant tissue is partially racemized and suggest that this might be a characteristic of such proteins. Chibnall found tumour proteins to contain the bulk of their glutamic acid in the ordinary active form with [α]D + 31·6° in 9 per cent hydrochloric acid, and he thought that any racemization was incidental to the methods employed in its isolation. The work described here indicates that a vegetable protein such as gliadin also contains part of its glutamic acid in a racemic form, and that it is probably a general characteristic of proteins and not of special significance for the proteins of tumours.