RFLP patterns of gliadin alleles in Triticum aestivum L.: implications for analysis of the organization and evolution of complex loci.

A correspondence between RFLP patterns and gliadin alleles at the Gli-1 and Gli-2 loci was established in a set of 70 common wheat (T.aestivum L.) cultivars using γ-gliadin (K32) and α-gliadin (pTU1) specific probes. All Gli-B1 and Gli-D1 alleles which differed in encoded γ-gliadins showed definite RFLP patterns after hybridization with the K32 probe. Two groups of Gli-B1 alleles, Gli-B1b-like and Gli-B1e-like, were identified, and these could originate from distinct genotypes of the presumptive donor of the B-genome. Intralocus recombination and/or gene conversion as well as small deletions, gene silencing and gene amplification were assumed to be responsible for the origin of new gliadin alleles. Silent γ-gliadin sequences were shown to exist in all of the genotypes studied. K32 also differentiated Gli-A1a from all other Gli-A1 alleles as well as the Gli-B11 allele in cultivars carrying the 1B/1R (wheat/rye) translocation. PTU1 was shown to recognize several Gli-A2 alleles, but not the Gli-B2 or Gli-D2 alleles. Moreover, this probe hybridized to chromosome 1R sequences suggesting the existence of rye gene(s), probably silent, for α-gliadin-like proteins on chromosome 1R.

Genetic variation at storage protein-coding loci of common wheat (cv 'Chinese Spring') induced by nitrosoethylurea and by the cultivation of immature embryos in vitro.

Electrophoretic patterns of seed storage proteins, the high-molecular-weight glutenins and gliadins, were studied in 468 plants of the common wheat cultivar 'Chinese Spring' regenerated from callus culture of immature embryos, in 115 plants grown from seeds treated with nitrosoethylurea and in 260 control plants. From 5 to 21 single grains were analysed from each plant. In these three groups, the frequency of inherited mutations causing the loss of all proteins controlled by a locus (null-mutations, probably caused by a chromosomal deficiency) was 0.69%, 2.07%, and 0.05% per locus (the differences were statistically significant), respectively, while that of mutations causing the loss of a single protein band was 0.11%, 0.33%, and 0.05%, respectively. The loss of all of the gliadins controlled by Gli-B1 or GH-B2 (mutations were probably caused by a deletion of satellites of the corresponding chromosomes), was significantly higher than the loss of gliadins controlled by genomes A and D. Gene mutations altering the electrophoretic mobility of a single protein band in the pattern were found only in the second group of plants (0.44%). Therefore, chemical mutagenesis which produced not only more mutations than cultivation of immature wheat embryos in vitro, but also a higher ratio of mutations that altered DNA sequences, can be considered as an easier and comparatively more promising way for obtaining new improved variants of loci controlling biochemical characteristics in wheat. Somaclonal variation, on the other hand, was probably mainly caused by chromosomal abnormalities and could therefore hardly be considered as a useful tool in wheat breeding.

Production and genetic characterization of near-isogenic lines in the bread-wheat cultivar Alpe.

Two biotypes of the bread-wheat cultivar Alpe were shown to possess contrasting alleles at each of the glutenin (Glu-B1, Glu-D1, Glu-B3 and Glu-D3) and gliadin (Gli-B1 and Gli-D1) loci on chromosomes 1B and 1D. Fourteen near-isogenic lines (NILs) were produced by crossing these biotypes and used to determine the genetic control of both low-molecular-weight (LMW) glutenin subunits and gliadins by means of one-dimensional or two-dimensional electrophoresis. Genes coding for the B, C and D groups of EMW subunits were found to be inherited in clusters tightly linked with those controlling gliadins. Southern-blot analysis of total genomic DNAs hybridized to a γ-gliadin-specific cDNA clone revealed that seven NILs lack both the Gli-D1 and Glu-D3 loci on chromosome 1D. Segregation data indicated that these "null" alleles are normally inherited. Comparison of the "null" NILs with those possessing allele b at the Glu-D3 locus showed one B subunit, seven C subunits and two D subunits, as fractionated by two-dimensional A-PAGExSDS-PAGE, to be encoded by this allele. Alleles b and k at Glu-B3 were found to code for two C subunits plus eight and six B subunits respectively, whereas alleles b and k at Gli-B1 each controlled the synthesis of two ß-gliadins, one γ and two ω-gliadins. The novel Gli-B5 locus coding for two ω-gliadins was shown to recombine with the Gli-B1 locus on chromosome 1B. The two-dimensional map of glutenin subunits showed α-gliadins encoded at the Gli-A2 locus on chromosome 6A. The use of Alpe NILs in the study of the individual and combined effects of glutenin subunits on dough properties is discussed.

Gliadin alleles in Canadian western red spring wheat cultivars: use of two different procedures of acid polyacrylamide gel electrophoresis for gliadin separation.

Gliadin allele compositions of 21 Canadian spring common wheat cultivars, most of which belong to the Canada western red spring (CWRS) class, were studied and great similarity in their genotypes was confirmed. It was found that alleles frequent in the set of Canadian wheats (such as Gli-B1d, Gli-D1j, Gli-A2m, and Gli-D2h) are very rare or absent in common wheat cultivars from other regions and countries studied earlier, indicating that germplasm of CWRS cultivars is rather unique. It may be suggested that alleles frequent in Canadian cultivars relate to important technological characteristics of these wheats and may possibly serve as marker genes during selection for quality traits. Similarity of gliadin electrophoregrams obtained by two different acid polyacryl-amide gel electrophoretic procedures for the same genotype was established, and the component composition of allelic variants of blocks of gliadin components found in the set of Canadian cultivars and in standard cultivars Chinese Spring and Bezostaya 1 are described.

Recombination mapping of Gli-5, a new gliadin-coding locus on chromosomes 1A and 1B in common wheat.

Inheritance studies of gliadin loci on chromosomes 1A and 1B were carried out in the progeny from crosses between cv "Salmone" and six other common wheat varieties. The map distance between the Rg-1 locus for glume colour and the gliadin locus Gli-B1 on the satellite of chromosome 1B was calculated as 2.0±0.6 cM. An additional gliadin locus, Gli-B5, was mapped between Gli-B1 and Rg-1, 1.4 cM from the former. A genetic distance of 1.8±0.4 cM was obtained between the Hg-1 locus for hairy glumes and a gliadin locus that seems to be remote from Gli-A1 and homoeologous to Gli-B5. Statistically significant differences in recombination values were found in the six crosses, indicating the influence of genotype on the frequency of recombination. The similarity in chromosomal location of seed storage protein genes in wheat, barley and rye is discussed.

Genetics of gliadins coded by the group 1 chromosomes in the high-quality bread wheat cultivar Neepawa.

The inheritance and biochemical properties of gliadins controlled by the group 1 chromosomes of the high-quality bread wheat cultivar Neepawa were studied in the progeny of the cross Neepawa x Costantino by six different electrophoretic procedures. Chromosome 1B of Neepawa contains two gliadin loci, one (Gli-B1) coding for at least six ω- or γ-gliadins, the other (Gli-B3) controlling the synthesis of gliadin N6 only. The map distance between these loci was calculated as 22.1 cM. Amongst the chromosome 1A gliadins, three proteins are encoded at the Gli-A1 locus whereas polypeptides N14-N15-N16 are controlled by a remote locus which recombines with Gli-A1. Six other gliadins are controlled by a gene cluster at Gli-D1 on chromosome 1D. Canadian wheat cultivars sharing the Gli-B1 allele of Neepawa were found to differ in the presence or absence of gliadin N6. The electrophoretic mobilities of proteins N6 and N14-N15-N16 were unaffected by the addition of a reducing agent during two-dimensional sodium dodecyl sulphate polyacrylamid-gel electrophoresis, suggesting the absence of intra-chain disulphide bonds in their structure.

Polymorphism and inheritance of gliadin polypeptides in T. monococcum L.

More than 80 different gliadin electrophoretic patterns (spectra) have been found in 109 accessions of the diploid wheat Triticum monococcum. Each pattern consists of 15-20 gliadin bands. Some patterns are clearly related and might arise from one another through single mutations in the gliadin-coding loci. From the analysis of 15 grains of each, only 61 accessions were found to be uniform; others consisted of two or more grain variants differing in their gliadin spectrum. An analysis of F2 grains from three crosses between different accessions showed that groups (blocks) of components are jointly and codominantly inherited. Two independent major Gli loci were established. The close resemblance of the composition of some blocks of T. monococcum to some of those in polyploid wheats indicates that one locus in each T. monococcum genotype is located on chromosome 1A (Gli-A1) and the other on 6A (Gli-A2). However, the blocks of T. monococcum include more bands than corresponding (equivalent) blocks of polyploid wheats. Two out of 275 F2 grains of the cross k-14244 x k-20409 were found to have gliadin spectra which can be explained as a result of intralocus recombination. Also, a second gliadin-coding locus on chromosome 1A was found in the cross k-46140 x k-46753. This locus recombines with the main Gli-A1 locus with a frequency of about 22% and was clearly analogous to the additional Gli locus found earlier on chromosome 1A of certain polyploid wheats.

Chromosomal control of wheat gliadin protein epitopes: analysis with specific monoclonal antibodies.

The genetic relationships between small clusters of monomeric alcohol-soluble wheat (Triticum aestivum L.) grain storage proteins (gliadins) were studied using a panel of monoclonal antibodies and immunoblotting, ELISA, and RIA methods. Use of Chinese Spring nullisomic-tetrasomic lines showed that several narrow-specificity antibodies bound specifically to gliadins encoded by genes located on a single chromosome. In at least one case, antibodies bound to genetic "blocks" of gliadins, indicating that these block members have structural homology. However, often not all gliadins of a block were recognized by an antibody. For broad-specificity antibodies and some narrow-specificity antibodies, structural genes on several chromosomes were important. Studies with several primitive wheat species indicated that, while antibodies usually bound gliadins from the same genome in bread and primitive wheats, antibodies sometimes bound proteins of quite differing mobilities in the two wheat types. Use of antibodies to identify gliadin blocks is simpler than block analysis based on performing crosses, and should be of value in monitoring genotype/end-use quality relationships.

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.

Polymorphism and inheritance of gliadin components controlled by chromosome 6A of spring durum wheat.

The gliadin composition of 78 spring durum wheat varieties has been studied by one-dimensional (Al-lactate, pH 3.1) and two-dimensional (first dimension, Al-lactate, pH 3.1; second dimension, sodium dodecyl sulfate-polyacrylamide gel) electrophoresis. Analysis of hybrids has shown that all components of the alpha zone of gliadin spectra are inherited together as blocks and are, probably, coded for by a cluster of tightly linked genes located on chromosome 6A. Fourteen variants of gliadin blocks have been identified, which can be classified into five families on the basis of component composition. All families but one have analogues among chromosome 6A-controlled blocks of bread wheat. The results indicate that some of the genome A diploid genotypes that were ancestors of durum wheats were also ancestors of bread wheats and that polyploid wheats were produced by repeated allopolyploidization events, as has been suggested earlier.

Problems of interpreting results obtained in studies of somaclonal variation in gliadin proteins in wheat.

It is quite likely that somacloning increases the frequency of mutations: this has, in fact, been claimed for gliadin proteins. However, in such instances it is important to distinguish between true mutations and presumed changes in regenerants due to cross pollination or admixture. We present herein arguments that some so-claimed somaclonal variants in gliadincoding genes are unlikely to be due to mutations since they would have had to involve directed changes at several loci simultaneously.

Genetic analysis of gliadin-encoding genes reveals gene clusters as well as single remote genes.

Analysis of F2 grains from two different crosses has revealed a complex organization of the family of gliadin-coding genes located on chromosomes of the first homoeological group in hexaploid wheat. Chromosome 1A of variety 'Bezenchukskaya 98' was found to carry at least five gliadin-coding genes of which three genes form a cluster controlling the synthesis of the GLD1A1 block. Two additional genes are located on the both sides of this cluster and recombine with it at frequencies of 5±1.3% and 13±2.9%. Gliadinencoding genes recombining with the main clusters were also found on chromosomes 1B and 1A in the 'Bezenchukskaya 98' and 'Saratovskaya 210' varieties, respectively. In 'Chinese Spring', widely used in genetic studies, we discovered a recombination between genes located on chromosome 1A and controlling the synthesis of ω- and γ-gliadins. Varieties and biotypes of one variety may differ by the presence or absence of such "selfish" (not included in clusters) gliadin components. The similarity of organization of prolamine-coding genes on chromosomes in different cereals is considered.

Genetic analysis of gliadin components in winter wheat using two-dimensional polyacrylamide gel electrophoresis.

Blocks of gliadin components found both in a number of varieties and in single F2 grains of winter wheat intervarietal hybrids have been studied by two-dimensional electrophoresis combining electrophoresis in acidic aluminium-lactate buffer (pH3.1) and SDS-electrophoresis. Gliadin components (spots) have been shown to be inherited as linked groups (blocks), codominantly and in accordance with a gene dosage in triploid endosperm. Blocks include components differing in their electrophoretic mobility and molecular weight. Some allelic variants of blocks differ only in presence of few additional components or in the electrophoretic mobility of components with similar molecular weights; other variants may contain no similar components. Apparently, in the course of evolution, mutations in individual genes of gliadin-coding loci and processes changing the number of expressing genes and the sizes of their structural part occurred.

Blocks of gliadin components in winter wheat detected by one-dimensional polyacrylamide gel electrophoresis.

Inheritance of gliadin components in winter wheat has been studied by one-dimensional polyacrylamide gel electrophoresis. Single F2 grains from 36 intervarietal hybrid combinations have been analysed. The genetic analysis has revealed blocks, including 1-6 gliadin components, which are inherited as individual mendelian traits. About 80 variants of blocks have been detected. On the basis of the allelism test they are grouped into 6 series in accordance with the number of known gliadin-coding loci located on chromosomes of the homoeologous groups 1 and 6. Each series includes 8-18 blocks controlled by different alleles of one gliadin-coding locus. Blocks of components have been confirmed to be inherited codominantly in accordance to the gene dose in the triploid endosperm. The highest similarity between members of one series is observed in groups of blocks controlled by chromosomes ID and 6D. On the contrary, many blocks controlled by chromosomes 1A and 1B have no bands in common. The presented catalogue of blocks of components may be used to make up gliadin genetic formulae and to compare electrophoregrams obtained by different authors. Blocks of gliadin components are suitable genetic markers for use in revealing and studying heterogeneity of wheat varieties, in tracing their origin, in identifying recombinations, translocations and substitutions of the genetic material and in solving many other problems of the origin, evolution and selection of hexaploid wheat.