Assessment of allergenicity of diploid and hexaploid wheat genotypes: identification of allergens in the albumin/globulin fraction.

Wheat is an important part of the daily diet of millions of people. However, this staple food is also responsible for food allergies. Ancient cultivars of wheat are gaining interest today but nothing is known about their allergenicity. Many wheat proteins have been reported as causative food allergens, including some prolamin-type gluten proteins, and salt soluble proteins of the albumin/globulin (A/G) type. The objective of this work is to obtain information about the allergenicity of the salt soluble A/G fraction of an ancient diploid cultivar compared with a standard hexaploid bread wheat cultivar using 20 sera from patients with wheat allergy. Differences in the IgE reactivity of sera towards the two genotypes were quantified by ELISA. Qualitative differences in IgE-binding proteins were searched after 1D or 2D electrophoresis. For most of the sera, the concentration in A/G specific IgE was higher for the hexaploid T. aestivum (cv Récital) than for the diploid T. monococcum (cv Engrain). The analysis of 2D spots revealed by immunoblotting leads to the identification by mass spectrometry of 39 IgE-binding proteins, some of them unknown until now as wheat allergens. Numerous allergens were identified, differences observed between Engrain and Récital will be discussed.

Development of isohomoeoallelic lines within the wheat cv. Courtot for high molecular weight glutenin subunits: transfer of the Glu-D1 locus to chromosome 1A.

Wheat quality depends on protein composition and grain protein content. High molecular weight glutenin subunits (HMW-GS) play an important role in determining the viscoelastic properties of gluten. In an attempt to improve the bread-making quality of hexaploid wheat by elaborating novel HMW-GS combinations, a fragment of wheat chromosome 1D containing the Glu-D1 locus encoding the Dx2+Dy12 subunits was translocated to the long arm of chromosome 1A using the ph1b mutation. The partially isohomoeoallelic line selected was characterized using cytogenetical and molecular approaches to assess the amount of chromatin introgressed in the translocated 1A chromosome. Triple-target genomic in situ hybridization indicated that the translocated 1A chromosome had a terminal 1D segment representing 25% of the length of the recombinant long arm. The translocation was also identified on the long arm using molecular markers, and its length was estimated with a minimum of 91 cM. Proteome analysis was performed on total endosperm proteins. Out of the 152 major spots detected, 9 spots were up-regulated and 4 spots were down-regulated. Most of these proteins were identified as alpha-, beta-, gamma-gliadins assigned to the chromosomes of homoeologous groups 1 and 6. Quantitative variations in the HMW-GS were only observed in subunit Dy12 in response to duplication of the Glu-D1 locus.

Genetic differences in omega-gliadins involved in two different immediate food hypersensitivities to wheat.

BACKGROUND: Anti-gliadin IgE are expressed in patients with food allergy associated to skin immediate hypersensitivity to hydrolyzed wheat proteins (IHHWP). It is not known if they react with omega5-gliadins, the major allergens in wheat dependant exercise-induced food anaphylaxis (WDEIA), encoded on wheat chromosomes 1B. METHODS: Unmodified gliadins from 14 wheat varieties expressing most of the 1B omega-gliadin alleles, were immunoprobed after SDS-PAGE and blotting, with four sera from patients with IHHWP, and two with WDEIA. Gliadins reacting with IgE were visualized using chemiluminescence and identified according to their mobility and typical SDS-PAGE pattern. The resulting signal was also measured to compare their IgE reactivity. RESULTS: IHHWP and WDEIA sera exhibited distinct patterns of reactivity. IgE of patients with IHHWP reacted mainly with all omega-gliadins alleles and one gamma-gliadin encoded respectively on chromosomes 1D and 1B, but not with any omega5-gliadins alleles as for WDEIA. A few other reactive alleles of omega-gliadins were encoded on chromosomes 1A. Unassigned additional bands of the whole gliadin pattern were also reactive. The four patients with IHHWP exhibited almost the same pattern of reactivity. Main differences concerned band reactivity which modulated the overall reactivity of each wheat variety. CONCLUSIONS: The IgE epitopes involved in IHHWP and WDEIA are different. This suggests that the protein state and the route of exposure to very similar gluten structures, probably orientate the pattern of epitope reactivity and the wheat food allergy manifestations.

Genetic analysis of dry matter and nitrogen accumulation and protein composition in wheat kernels.

The maximum rate and duration for grain dry matter (DM) and nitrogen (N) accumulation were evaluated in 194 recombinant inbred lines (RILs) from a cross between the two French wheat cultivars Récital and Renan. These cultivars were previously identified as having contrasting kinetics of grain DM and N accumulation. Grain protein composition was analysed by capillary electrophoresis (CE), which enabled quantification of the different storage protein fractions (alphabetagamma-gliadins, omega-gliadins, LMW glutenins, HMW glutenins, and each of their subunits). Correlation analyses revealed that DM and N accumulation rates were closely correlated and repeatable over several years, which was not the case for DM and N accumulation durations, and that protein composition was primarily influenced by the N accumulation rate. This was particularly true for the LMW-glutenins and the alphabetagamma-gliadins, the most abundant protein fractions. A genetic map of 254 molecular markers covering nearly 80% of the wheat genome was used for quantitative trait loci (QTL) analysis. A total of seven QTLs were found. Five QTLs were significantly associated with the kinetics of DM and N accumulation, and two of them also influenced protein composition. Two QTLs affected only the protein composition. One major QTL explained more than 70% of the total variation in HMW-GS Glu1B-x content.

Chromosome mapping and identification of amphiphilic proteins of hexaploid wheat kernels.

Amphiphilic proteomic analysis was carried out on the ITMI (International Triticae Mapping Population) population resulting from a cross between "Synthetic", i.e.: "W7984" and "Opata". Out of a total of 446 spots, 170 were specific to either of the two parents, and 276 were common to both. Preliminary analysis, which was performed on 80 progenies (Amiour et al. 2002a), was completed here using a total of 101 selfed lines. Seventy two Loci of amphiphilic spots placed at LOD = 5 were conclusively assigned to 15 chromosomes. Some spots mapped during the first analysis were eliminated because of the significant distortion segregation observed in the second analysis. Group-1 chromosomes had by far the greatest number of mapped spots (51). Using the Quantitative Trait Loci (QTLs) approach, analysis of the quantitative variation of each spot revealed that 96 spots out of the 170 specific ones showed at least one Protein Quantity Locus (PQL). These PQLs were distributed throughout the genome. With Matrix Laser Desorption Ionisation Time Of Flight (MALDI-TOF) spectrometry and Database interrogation, a total of 93 specific and 41 common spots were identified. This enabled us to show that the majority of these proteins are associated with membranes and/or play a role in plant defence against external invasions. Using multiple-regression analysis, other amphiphilic proteins, in addition to puroindolines, were shown to be involved in variation in kernel hardness in the ITMI population.

Mapping QTLs for grain hardness and puroindoline content in wheat ( Triticum aestivum L.).

Genes for puroindoline-a (Pin-a), puroindoline-b (Pin-b) and grain-softness proteins (GSP) have been shown to be linked to the dominant Ha locus responsible for the soft texture of the grain. Though linkage has been demonstrated of the puroindoline genes to the Ha locus, there is no clear evidence that puroindoline content is the product of the gene Ha. A segregating population of 115 recombinant inbred lines (RILs) originating from a cross between the hexaploid Synthetic wheat ( Triticum durum x Aegilops tauschii, W 7984) and the cultivar 'Opata' (M 85) was studied in two different experimental years to detect Quantitative Trait Loci (QTLs) for three traits: grain hardness (Hard), puroindoline-a (Pin-a) and puroindoline-b (Pin-b) contents. The detection of QTLs was performed using marker linear regression. Negative correlation coefficients (-0.86 and -0.80) were identified between grain hardness and puroindoline content (a and b, respectively) on data obtained in 1996. Results obtained in 1999 confirmed the negative correlation between Hard and Pin-a (-0.73); however a positive correlation coefficient was found with Pin-b content (0.41). Total phenotypic variation explained by each QTL was calculated (R2). For each of the Hard, Pin-a and Pin-b traits one major QTL was detected on the short arm of chromosome 5D, located close to the mta9 allele (puroindoline-a). For the first year (1996) the QTL in this region explained around 63% of the phenotypic variability in grain hardness, 77% in Pin-a and 45% in Pin-b contents. These values were confirmed in trials carried out in 1999 with a R2 value of 0.71, 0.72 and 0.25 for Hard, Pin-a and Pin-b, respectively. In 1996 and 1999 a second major QTL was detected for grain hardness on the long arm of the same chromosome. Present results indicate that it cannot be definitely concluded that puroindoline content represents a linear explanation for variations in grain hardness.

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.

Visualization of barley beta-glucan degrading isozymes after gel isoelectric focusing.

A simple method to study the polymorphism of barley (Hordeum vulgare L.) beta-(1-3,1-4)-glucanases (specific enzymes of barley beta-glucan hydrolysis) is described. Proteins of a crude extract of germinated barley kernels were separated in an immobilized pH gradient in two pH ranges (pH 3-10.5 and pH 4-7). beta-glucanases were visualized by contact printing with a polyacrylamide gel containing beta-glucan or lichenan. Patterns of beta-glucanases were revealed by staining with Congo Red with resultant clear zones on a stained background. Various conditions of germination, extraction and visualization were investigated. New isozyme bands could be detected and their nature and origin discussed.

Polymorphism of omega-gliadins in durum wheat as revealed by the two-step APAGE/SDS-PAGE technique.

Polymorphism of omega-gliadins was studied in 243 durum wheats from 27 countries using the two-step one-dimensional APAGE/SDS-PAGE technique. A total of 12 bands of different mobility were observed, and four of them were found to be different from those previously detected by Khelifi et al. (1992) in bread wheat. Fifteen alleles, six coded by the Gli-A1 locus and nine coded by the Gli-B1 locus, were identified, accounting for 19 different electrophoretic patterns. Seven new alleles were detected: two at the Gli-A1 locus and five at the Gli-B1 locus. The polymorphism found at the Gli-A1 and Gli-B1 loci was slightly greater than that found in bread wheat. Allelic differences between both species were higher at the Gli-B1 locus. A comparison of the frequencies of alleles in both species was carried out. The null allele, Gli-A1e, was more common in durum wheat than in bread wheat. The Gli-B1b allele, present in 60% of the bread wheats, was found in only 2% of the durum wheats and Gli-B1e, very common in durum wheat (45%), was rare in bread wheat (4%). The Gli-B1IV allele, common in durum wheat (28%), was not detected in bread wheat.

Selection indices for quality evaluation in wheat breeding.

From multilocation trials involving 125 cultivars of wheat of mainly French and European origin four tests - protein content, Pelshenke, modified Zeleny and the mixograph - were used to establish six selection indices. Three of these indices - IW1, IW2 and IW3 - were calculated in order to evaluate the genetic potentiality of the lines for dough strength as given by the Chopin alveograph. The indices IV1, IV2 and IV3 were established to evaluate loaf volume as measured by the French bread-making standard. A quality index IQ was calculated from the allelic effects of the high-molecular-weight (HMW) subunits of glutenin from 195 cultivars assessed by the Chopin alveograph and the Pelshenke test. Comparison of the relative efficiency of each of the six indices to the individual tests revealed the superiority of the indices over one or several technological parameters. The six selection indices and the quality index were compared using 30 very diverse F4 lines. Their ability to retain the good quality lines is discussed in particular.

Electrophoresis of gliadins in long acrylamide gels: method and nomenclature.

Gliadins, defined as wheat kernel storage proteins soluble in 70% ethanol, possess an electrophoretic diversity permitting us to identify different varieties. Because of genetic proximity, however, it sometimes proves impossible to distinguish between different varieties using standard methods. An electrophoresis method, utilizing a discontinuous buffer system with aluminum lactate and potassium lactate in the gel and sodium lactate in the electrode vessels, is described for two types of gels 18 cm and 32 cm in length. The number of bands is higher than with standard methods, and is significantly increased with long gels, facilitating the distinction between varieties. A nomenclature for the new bands is presented for several widely different cultivars.

High molecular weight glutenin subunit in durum wheat (T. durum).

The diversity of high molecular weight (HMW) glutenin subunits of 502 varieties of durum wheat (Triticum durum) from 23 countries was studied using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Twenty-nine types of patterns were observed with 18 mobility bands. A total of 18 alleles were identified by comparing the mobilities of their subunits to those previously found in hexaploid wheat (T. aestivum) and in Triticum turgidum var. dicoccum. Five new alleles were detected: two on the Glu A1 and three on the Glu B1 locus. Comparison of the frequency of alleles in the three species T. aestivum, T. dicoccum and T. durum was investigated. Significant differences exist between each of these species on the basis of the frequency distributions of their three and four common alleles at the Glu A1 and Glu B1 locus, respectively. The Glu B1c allele occuring very frequently in hexaploid wheats was not found in the two tetraploid species. More than 83% of the T. durum analysed were found to have the Glu A1c (null) allele.

A comparison of androgenetic doubled-haploid, and single seed descent lines in Triticale.

Sixty single seed descent (SSD) lines and about 25 anther-derived doubled-haploid (DH) lines were obtained from two triticale crosses. The frequency distributions of 10 quantitative agronomic traits were compared using parametric and non-parametric tests. A multivariate discriminant analysis was subsequently carried out. Gliadin patterns obtained from each line by polyacrylamide gel electrophoresis were used to calculate intra- and inter-population diversities from relative dissimilarity indices. It was found that DH and SSD lines show significant differences in frequency distributions of 1000 grain weight in both crosses, of heading date for one cross, and of lodging susceptibility for the other cross. The results of intra- and inter-population gliadin diversity indicate that although the SSD method theoretically provides more opportunity for recombination to occur than the DH method, it did not produce a greater range of recombinants. Since there is no significant difference between SSD- and DH-line distributions for grain yield, anther culture appears to be an efficient method for producing high yielding homozygous lines from F1 hybrids of triticale in a relatively short time.

Study of genetic determination of 20 gliadin bands.

Study of the genetic determination of the gliadins of two F2 progenies from bread wheat has enabled (1) confirmation of the co-dominant heredity of the presence of these bands in the F1, and (2) determination of the transmission of the presence/absence character for 20 bands. 10 bands are monogenically controlled; 10 others are coded by two pairs of alleles. Among the latter 2 bands split into two proteins in 2 dimensional electrophoresis. Analysis of the segregations, not taking into consideration the presence/absence character but only the concentrations of certain bands, led to the formulation of the hypothesis of regulator genes controlling the expression of structural genes.

Correlation between gliadin bands.

Starch gel electrophoresis of gliadins was carried out for 37 bread wheat cultivars chosen for their distant relationships. Simple correlations were calculated between each of the 41 bands (variates) observed with these wheats. It was found that a band is usually negatively correlated with the two neighbouring mobility bands. The number of bands positively or negatively correlated with a given band varies from 2 to 8. Taking the bands significantly positively correlated with each-other 32 groups were constituted. It appears likely that these correlations result from the fact that the genes determining the bands of a given group are situated on one chromosome. Study of the distribution of 15 gliadin bands from 312 F2 grains indicated that 2/3 of the correlations calculated for this progeny were due to linkages.