Analysis of common wheat varieties and near-isogenic lines by PCR with allele-specific primers for Gli-1 and Glu-3 loci

The allelic characteristics of the Gli-A1, Gli-B1, Gli-D1 and Glu-A3 loci of 14 bread wheat varieties and 6 near-isogenic lines derived from Bezostaya 1 have been detected by PCR analysis. The conformity of molecular-genetic data and electrophoresis of storage proteins has been determined: the allelic variants of gliadins Gli-A1o and Gi-A1m correspond to the PCR-allele GliA1.2, the gliadin variants Gli-A1f, Gli-A1b, Gli-A1c correspond to the PCR-allele GliA 1.1, the allelic variants Gli-B1b, Gli-B1d--to the PCR-allele GliB1.1 and the variants Gli-B1e, Gli-B1g, Gli-B1c-to the PCR-allele GliB1.2. A new PCR-allele at the GliB) locus in the line Gli-B1-12 (with the gliadin block Gli-B1o from Levent) was identified.     

Comparison of alleles at the <Emphasis Type="Italic">Gli-1</Emphasis> loci of common wheat by means of two-dimensional electrophoresis of gliadin and RFLP analysis

Allelic variants of the Gli-1 locus is known to control groups (blocks) of gliadin polypeptides (gliadins). Some allelic variants of blocks that differ in the electrophoretic (acid gel) mobility (EM) of only one gliadin of the block were compared using two-dimensional electrophoresis (SDS-PAGE) and the RFLP procedure. It was found that, in these pairs of similar alleles (Gli-B1f, Gli-B1s, and Gli-D1a as compared with Gli-B1e, Gli-B1n, and Gli-D1c, respectively), faster γ-gliadin had smaller molecular weight (MW). Alleles at the Gli-A1 locus (Gli-A1j, Gli-A1i, Gli-A1a, Gli-A1k, and Gli-A1f) differ in the EM of the γ-gliadin so that Gli-A1j controls the slowest γ-gliadin and Gli-A1f controls the fastest one. We found that, in this order of alleles, faster γ-gliadin always had smaller MW. It was suggested that similar alleles might arise from one another by spontaneous mutations changing the number of repeating sequences or length of the polyglutamine domain present in the γ-gliadin gene thereby influencing MW and EM of encoding polypeptide. Other mechanisms of the mutational appearance of new alleles were found earlier by comparison of allele pairs: Gli-D1a and Gli-D1k (gene silencing) and Gli-D1b and Gli-D1d (gene amplification). We discovered contrasting families of alleles at the Gli-B1 and at the Gli-D1 loci and also two variants of apparently the same allele Gli-D1a that differed in the number of encoded ω-gliadins. Families of alleles at one locus of T. aestivum might inherit from different genotypes of corresponding diploid donor, as we suggested earlier.     

Comparison of Alleles at <Emphasis Type="Italic">Gli-2</Emphasis> Loci of Common Wheat by Means of Two-Dimensional Electrophoresis of Gliadin

Electrophoretic mobility (EM) and molecular weight (MW) of some allelic variants of α- and β-gliadins contrlled by Gli-2 loci were compared by means of two-dimensional (APAGE × SDS) electrophoresis. Comparison of α-gliadins of the alleles Gli-A2b and Gli-A2p, of β-gliadins of the Gli-B2b and Gli-B2c, and of β-gliadins of the Gli-D2b, Gli-D2c, Gli-D2j, and Gli-D2r indicated that a gliadin with lower EM had, as a rule, bigger MW which is known to depend on the length of the polyglutamine domain of gliadin of α-type. However, allelic variants of the α-gliadin encoded by Gli-D2b and Gli-D2e differ in EM but not in apparent MW. It might be caused by a substitution of some charged/uncharged aminoacids in the polypeptide of gliadin. Allele Gli-B2o which is very frequent in up-to-date common wheat germplasm originated probably by means of unequal crossingover. Some alleles at Gli-A2 is found to control completely different blocks of gliadins and therefore might come to common wheat from different genotypes of the polymorphic diploid donor of the A genome. The results indicate that the reason of the known more vast polymorphism of gliadins controlled by Gli-2 loci as compared with Gli-1 loci is the considerable difference of the structure, first, of Gli-1 and Gli-2 loci (Gli-2 loci have more expressed genes per locus) and, second, of genes encoding gliadins of α- and γ-types (α-gliadins are shown to contain a long polyglutamine sequences highly variable in their length).     

Allelic diversity at gliadin-coding gene loci in cultivars of spring durum wheat (<Emphasis Type="Italic">Triticum durum</Emphasis> Desf.) Bred in Russia and former Soviet republics in the 20th century

Allelic diversity at five gliadin-coding gene loci has been studied in the most important spring durum wheat cultivars released in Russia and former Soviet republics in the 20th century (66 cultivars). Seven, 5, 8, 13, and 2 allelic variants of blocks of gliadin components controlled by the loci Gli-A1 ^d , Gli-B1 ^d , Gli-A2 ^d , Gli-B2 ^d , and Gli-B5 ^d , respectively, have been identified. The allelic diversity did not exhibit a consistent trend during the period studied. Nei’s diversity index (H) was 0.68 in the period from 1929 to 1950, increased to 0.70 in 1951–1980, and decreased to 0.58 after the year 1981. It has been found that the most frequent alleles in this collection are relatively rare in other regions of the world, which suggests unique ways of the formation of the diversity of durum wheat cultivars in the former Soviet Union. The efficiency of electrophoresis of storage proteins as a method for identification of durum wheat cultivars by the gliadin electrophoretic pattern has been estimated.     

Catalogue of alleles of gliadin-coding loci in durum wheat (Triticum durum Desf.)

Gliadins are seed storage proteins which are characterized by high intervarietal polymorphism and can be used as genetic markers. As a result of our work, a considerably extended catalogue of allelic variants of gliadin component blocks was compiled for durum wheat; 74 allelic variants for four gliadin-coding loci were identified for the first time. The extended catalogue includes a total of 131 allelic variants: 16 for locus Gli-A1(d), 19 for locus Gli-B1(d), 41 for locus Gli-A2(d), and 55 for locus Gli-B2(d). The electrophoretic pattern of the standard cultivar and a diagram are provided for every block identified. The number of alleles per family is quite small for loci Gli-A1(d) and Gli-B1(d) of durum wheat, as contrasted to loci Gli-A2(d) and Gli-B2(d) that are characterized by large families including many alleles. The presence of large block families determines a higher diversity of durum wheat for loci Gli-A2(d) and Gli-B2(d) as compared to Gli-A1(d) and Gli-B1(d). The catalogue of allelic variants of gliadin component blocks can be used by seed farmers to identify durum wheat cultivars and evaluate their purity; by breeders, to obtain homogenous cultivars and control the initial stages of selection; by gene bank experts, to preserve native varieties and the original biotypic composition of cultivars.     

Analysis of Inheritance of Morphological and Biochemical Characters Introgressed into Common Wheat from <Emphasis Type="Italic">Aegilops speltoides</Emphasis> Tausch.

Genetic control of some morphological traits and the gliadin composition were examined in plants of two lines of common wheat carrying genes introgressed from the wild diploid cereal Aegilops speltoides. Leaf hairiness was shown to be controlled by a single introgressed dominant gene that was not allelic to the known common wheat gene Hl1. Waxlessness of the whole plant is controlled by the introgressed from Ae. speltoides inhibitor gene allelic to gene W1 ^I located on chromosome 2B. This gene was epistatic to the introgressed gene controlling spike waxlessness. The introgressed gene of spike color was shown to be allelic to Rg1 located on chromosome 1B of common wheat. However, the former gene proved to be linked to an allele of the Gli-B1 locus other than in wheat.__________Translated from Genetika, Vol. 41, No. 6, 2005, pp. 793–799.Original Russian Text Copyright © 2005 by Pshenichnikova, Lapochkina, Shchukina, Berezovskaya, Trufanov.     

Genealogical and statistical analyses of the inheritance of gliadin-coding alleles in a model set of common wheat <Emphasis Type="Italic">Triticum aestivum</Emphasis> L. cultivars

Allelic diversity of the gliadin-coding loci Gli-1 and Gli-2 was compared with the genealogical profiles of common wheat cultivars developed in Saratov. Allele tracking through their pedigrees and hierarchic cluster analysis associated 31 Gli alleles with groups of original ancestors. The cultivars Poltavka (12 alleles of six loci) and Selivanovskii Rusak (six alleles of six loci) were identified as sources of the majority of alleles. The results of the cluster analysis fully coincided with the results of allele tracking for alleles occurring at high frequencies. For rare alleles, the resolution of the cluster analysis was somewhat lower and depended on the similarity/distance measure. Thus, it proved possible to indirectly identify the donors of gene alleles by multidimensional statistics even when data on alleles identified in ancestors are unavailable. This approach to the analysis of inheritance has two limitations: detailed pedigree data should be known, and relatively high frequencies (no less than 15–20%) should be observed for the alleles in a sample under study. Cluster analysis was used to study the association of gliadin alleles with commercial quality classes. The most important gliadin-coding alleles, which mark strong cultivars, were identified. In the Saratov cultivars, such alleles include Gli-A1f, GliB1e, Gli-D1a, Gli-A2q, Gli-B2s, and Gli-D2e, which were inherited from the landrace Poltavka, and Gli-A1i, Gli-A2s, and Gli-B2q, which were inherited from the landrace Selivanovskii Rusak.     

Genetic diversity of local spring bread wheats (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) of West and East Siberia in gliadin genes

A considerable polymorphism in gliadin genes was detected in the wheat landraces of West Siberia (Altai krai, Omsk oblast, and Kurgan oblast) and the local cultivars characteristic of several East Siberian regions (Krasnoyarsk krai, Irkutsk oblast, Tuva, and Yakutia), and the genetic formulas were determined. The common alleles characteristic of the wheats of both regions were detected, namely, Gli-A1f, Gli-A1j, Gli-A1i, Gli-A1m, Gli-B1e, Gli-B1m, Gli-D1a, Gli-A2q, Gli-A2k, Gli-A2u, Gli-D2a, and Gli-D2q, as well as 14 novel alleles unknown earlier. It was demonstrated that several genotypes had formed in Siberia. Of them, the genotypes Gli-A1f_Gli-B1e_Gli-D1a and Gli-A1j_Gli-B1e_Gli-D1a occur both in West and East Siberia, whereas the genotypes Gli-A1i_Gli-B1m_Gli-D1a_Gli-A2new10, Gli-A1m_Gli-B1b_Gli-D1a_Gli-A2f, and Gli-A1m_Gli-B1m_Gli-D1a_Gli-A2u are found only in East Siberia.     

Molecular genetic characteristics of the <Emphasis Type="Italic">Wx-B1e</Emphasis> allele from common wheat and applicability of the DNA markers for its identification

Molecular genetic characterization of the Wx-B1e allele identified by the authors of the study in the common wheat cultivar Korotyshka was performed. The 804-bp Wx-B1e fragment was cloned and sequenced. Comparison of the sequence obtained with that for the wild-type allele of common wheat (Wx-B1a) demonstrated that Wx-B1e carried the 34-bp insertion, 8-bp deletion, and 23 nucleotide substitutions. BLAST analysis revealed the highest homology with the nucleotide sequences of Wx genes from Triticum spelta and Triticum durum. The amplification variants of four Wx-B1 molecular markers, applied worldwide for testing the collections for different Wx allelic variants, are demonstrated.     

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.     

Identification of SNPs and development of allele-specific PCR markers for γ-gliadin alleles in Triticum aestivum

The coding regions of 28 entries of hexaploid wheat gamma-gliadin genes, gene fragments or pseudogenes in GenBank were used for nucleotide alignment. These sequences could be divided into nine subgroups based on nucleotide variation. The chromosomal locations of five of the seven unassigned subgroups were identified through subgroup-specific polymerase chain reactions (PCR) using Chinese Spring group-1 nulli-tetrasomic lines. Multiple single nucleotide polymorphisms (SNPs) and small insertions/deletions were identified in each subgroup. With further mining from wheat expressed sequence tag databases and targeted DNA sequencing, two SNPs were confirmed and one SNP was discovered for genes at the Gli-A1, Gli-B1 and Gli-D1 loci. A modified allele-specific PCR procedure for assaying SNPs was used to generate dominant DNA markers based on these three SNPs. For each of these three SNPs, two allele-specific primer sets were used to test Chinese Spring and 52 commercial Australian wheat varieties representing a range of low-molecular-weight (LMW) alleles. PCR results indicated that all were positive with one of the primer sets and negative with the other, with the exception of three varieties containing the 1BL/1RS chromosomal translocation that were negative for both. Furthermore, markers GliA1.1, GliB1.1 and GliD1.1 were found to be correlated with Glu-A3 a, b or c, Glu-B3 b, c, d or e and Glu-D3 a, b or e LMW glutenin alleles, respectively. Markers GliA1.2, GliB1.2 and GliD1.2 were found to be correlated with the Glu-A3 d or e, Glu-B3 a, g or h and Glu-D3 c alleles, respectively. These results indicated that the gamma-gliadin SNP markers could be used for detecting linked LMW glutenin subunit alleles that are important in determining the quality attributes of wheat products.     

Allelic variation at the <Emphasis Type="Italic">VRN-1</Emphasis> promoter region in polyploid wheat

Vernalization, the requirement of a long exposure to low temperatures to induce flowering, is an essential adaptation of plants to cold winters. We have shown recently that the vernalization gene VRN-1 from diploid wheat Triticum monococcum is the meristem identity gene APETALA1, and that deletions in its promoter were associated with spring growth habit. In this study, we characterized the allelic variation at the VRN-1 promoter region in polyploid wheat. The Vrn-A1a allele has a duplication including the promoter region. Each copy has similar foldback elements inserted at the same location and is flanked by identical host direct duplications (HDD). This allele was found in more than half of the hexaploid varieties but not among the tetraploid lines analyzed here. The Vrn-A1b allele has two mutations in the HDD region and a 20-bp deletion in the 5 UTR compared with the winter allele. The Vrn-A1b allele was found in both tetraploid and hexaploid accessions but at a relatively low frequency. Among the tetraploid wheat accessions, we found two additional alleles with 32 bp and 54 bp deletions that included the HDD region. We found no size polymorphisms in the promoter region among the winter wheat varieties. The dominant Vrn-A1 allele from two spring varieties from Afghanistan and Egypt (Vrn-A1c allele) and all the dominant Vrn-B1 and Vrn-D1 alleles included in this study showed no differences from their respective recessive alleles in promoter sequences. Based on these results, we concluded that the VRN-1 genes should have additional regulatory sites outside the promoter region studied here.     

Variation at storage protein loci in winter common wheat cultivars of the central forest-steppe of Ukraine

Genotypes at the gliadin loci Gli-A1, Gli-B1, Gli-D1 and the high-molecular-weight glutenin subunit loci Glu-A1, Glu-B1, Glu-D1 were identified in 77 winter common wheat cultivars developed in the Central Forest Steppe of Ukraine in different periods of time. The highest level of variation was observed at the Gli-A1 locus. Predominant alleles (one or two per locus) were revealed. The comparison of allele frequencies in groups of cultivars developed in different periods of time (before 1996 and in 1996–2007) has demonstrated appearance of new alleles and change of frequencies of existing alleles at the storage protein loci. The high frequency of cultivars with the wheat-rye 1BL/1RS translocation was detected (about 40%). The wheat rye 1AL/1RS translocation was identified in six cultivars developed in the last decade. Four gliadin alleles, Gli-A1w (a marker for the 1AL/1RS translocation), Gli-A1x, Gli-A1y and Gli-B1x, were proposed for cataloging. The article is published in the original.     

Cytogenetic analysis of hybrids derived from wheat and <Emphasis Type="Italic">Tritipyrum</Emphasis> using conventional staining and genomic <Emphasis Type="Italic">in situ</Emphasis> hybridization

The new salt tolerant cereal, Tritipyrum (2n=6x=42, AABBE^bE^b) offers potential to introduce desirable characters for wheat improvements. This study was aimed to generate a segregating population from Iranian local wheat cultivars (2n=6x=42, AABBDD) and Tritipyrum crosses, study of the meiotic behaviour in F₂ hybrids and identification of E^b chromosomes in F₃ individuals. Results showed meiotic abnormalities in F₂ plants and different pairing frequency in the meiosis among F₂ plants. Genomic in situ hybridization revealed that total and E^b chromosome number of F₃ seeds ranged from 39 to 45 and 0 to 10, respectively. A significant prevalence of hyper-aneuploidy was observed among F₃ genotypes. C-banding patterns identified E^b chromosomes in Tritipyrum, indicating that it also can be useful to study wheat-Tritipyrum derivatives.     

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.     

Effect of an introgression from <Emphasis Type="Italic">Aegilops cylindrica</Emphasis> host on manifestation of productivity traits in winter common wheat F<Subscript>2</Subscript> plants

The effect of introgression of a chromosome 1D segment from Aegilops cylindrica to winter common wheat on productivity traits in F₂ plants was studied using storage protein loci as genetic markers. An allele of the gliadin-coding Gli-D1 locus served as a marker of the introgression. Using of two- and three-locus interaction models, it was shown that the introgression tagged with Gli-D1 affected the manifestation of productivity traits (productive tillering, grain weight per plant and grain number per plant) through interaction with other marker storage protein loci: Glu-B1, Glu-D1, and Gli-B2.Translated from Genetika, Vol. 40, No. 12, 2004, pp. 1662–1667.Original Russian Text Copyright © 2004 by Kozub, I. Sozinov, A. Sozinov.     

Susceptibility to <Emphasis Type="Italic">Fusarium </Emphasis>head blight is associated with the <Emphasis Type="Italic">Rht-D1b</Emphasis> semi-dwarfing allele in wheat

Fusarium head blight (FHB) is an important disease of wheat worldwide. The cultivar Spark is more resistant than most other UK winter wheat varieties but the genetic basis for this is not known. A mapping population from a cross between Spark and the FHB susceptible variety Rialto was used to identify quantitative trait loci (QTL) associated with resistance. QTL analysis across environments revealed nine QTL for FHB resistance and four QTL for plant height (PH). One FHB QTL was coincident with the Rht-1D locus and accounted for up to 51% of the phenotypic variance. The enhanced FHB susceptibility associated with Rht-D1b is not an effect of PH per se as other QTL for height segregating in this population have no influence on susceptibility. Experiments with near-isogenic lines supported the association between susceptibility and the Rht-D1b allele conferring the semi-dwarf habit. Our results demonstrate that lines carrying the Rht-1Db semi-dwarfing allele are compromised in resistance to initial infection (type I resistance) while being unaffected in resistance to spread within the spike (type II resistance).     

Genetic control of gliadin components in wheat <Emphasis Type="Italic">Triticum spelta</Emphasis> L.

The componental composition of electrophoretic spectra of gliadin in Triticum spelta L. was studied. By analogy with common wheat T. aestivum L., it was established that genes controlling gliadin components in spelt are also located in short arms of chromosomes of homeological groups 1 and 6. Analysis of gliadin spectra in F₂ grains from the crosses k-20539 × Ershovskaya 32 and k-20558 × Ershovskaya 32 revealed linkage of some components and their grouping into blocks (alleles) of coinherited gliadin components. Alleles of gliadin-coding loci identical to alleles of common wheat and new alleles earlier unknown for wheat populations have been identified.     

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.     

Frequencies of the <Emphasis Type="Italic">DRB1</Emphasis>, <Emphasis Type="Italic">DQA1</Emphasis>, <Emphasis Type="Italic">DQB1</Emphasis> and <Emphasis Type="Italic">TNFA</Emphasis> alleles in immigrant population of west Siberia

Based on population analysis of the DRB1, DQA1, DQB1 and TNFA allele frequency distribution patterns, regional features of immunogenetic structure of the population of West Siberia were investigated. Statistically significant linkage disequilibrium within the HLA class II region, as well as between the TNFA and DRB1, DQA1, and DQB1 was demonstrated. Population frequency distribution patterns of two- and multilocus haplotypes were examined.