Detection and characterization of a glutenin subunit with unusual high Mr at the Glu-A1 locus in hexaploid wheat

A hexaploid wheat landrace collected from the Baluchistan province of Pakistan was found to possess a novel high-molecular-weight glutenin subunit (HMW-GS). The subunit has a very slow electrophoretic mobility as revealed by SDS-PAGE, and its molecular weight is comparable to that of the highest molecular weight glutenin subunit (2.2 encoded in the D-genome) reported so far in hexaploid wheat varieties and landraces of Japanese origin. Evidence obtained from (PCR) gene amplification studies using the primers specific for Glu-1 loci proved that the gene coding for this novel subunit belongs to the Glu-A1 locus located on the long arm of chromosome 1A. Digestion of the amplified gene (PCR product) with restriction enzymes indicated that the novel gene differs from prevailing Glu-A1 alleles (null, 1 and 2^*) by an extra DNA fragment of approximately 600 base pairs. The results also indicated that the novel subunit is most probably a derivative of subunit 2^* that has very likely incorporated the 600-bp fragment following a process of unequal crossing over. The present findings were further substantiated by reserved phase high performance liquid chromatography (RP-HPLC) analysis.     

Gene-assisted selection for high molecular weight glutenin subunits in wheat doubled haploid breeding programs

Molecular markers based on DNA sequence variations of the coding and/or promoter regions of the wheat (Triticum aestivum L.) HMW glutenin genes located at the Glu-1 loci were developed. Markers characteristic of alleles Glu-A1-1a (encoding Ax1 subunit) and Glu-A1-1c (encoding Ax2* subunit) at the Glu-A1 locus, alleles Glu-B1ak (encoding Bx7* subunit) and Glu-B1al for overexpressed Bx7 subunit at the Glu-B1 locus and alleles Glu-D1-1a (encoding Dx2 subunit) and Glu-D1-1d (encoding Dx5 subunit) at the Glu-D1 locus were tested using genomic DNA of haploid leaf tissue. A method for simultaneously extracting DNA from 96 haploid leaf tissue pieces is described. Two of the developed markers were dominant and two were co-dominant. A F₁-derived population segregating for all HMW glutenin genes was used to test the validity of the markers and their usefulness in doubled haploid breeding programs. SDS-PAGE analysis of seed storage protein was performed on seeds from the doubled haploid lines. A total of 299 lines were tested with the DNA markers on the haploid tissue and validated by protein analysis of the corresponding DH seeds. PCR markers and SDS-PAGE analysis showed between 2 and 8.5% discrepancies depending on the marker. Applications of DNA markers for gene-assisted-selection of haploid tissue and use in breeding programs are discussed. Advantages and disadvantages of dominant and co-dominant markers are outlined.     

Atomic force microscopy of a hybrid high-molecular-weight glutenin subunit from a transgenic hexaploid wheat

The high-molecular-weight glutenin subunits (HMW-GS) of wheat gluten in their native form are incorporated into an intermolecularly disulfide-linked, polymeric system that gives rise to the elasticity of wheat flour doughs. These protein subunits range in molecular weight from about 70 K-90 K and are made up of small N-terminal and C-terminal domains and a large central domain that consists of repeating sequences rich in glutamine, proline, and glycine. The cysteines involved in forming intra- and intermolecular disulfide bonds are found in, or close to, the N- and C-terminal domains. A model has been proposed in which the repeating sequence domain of the HMW-GS forms a rod-like beta-spiral with length near 50 nm and diameter near 2 nm. We have sought to examine this model by using noncontact atomic force microscopy (NCAFM) to image a hybrid HMW-GS in which the N-terminal domain of subunit Dy10 has replaced the N-terminal domain of subunit Dx5. This hybrid subunit, coded by a transgene overexpressed in transgenic wheat, has the unusual characteristic of forming, in vivo, not only polymeric forms, but also a monomer in which a single disulfide bond links the C-terminal domain to the N-terminal domain, replacing the two intermolecular disulfide bonds normally formed by the corresponding cysteine side chains. No such monomeric subunits have been observed in normal wheat lines, only polymeric forms. NCAFM of the native, unreduced 93 K monomer showed fibrils of varying lengths but a length of about 110 nm was particularly noticeable whereas the reduced form showed rod-like structures with a length of about 300 nm or greater. The 110 nm fibrils may represent the length of the disulfide-linked monomer, in which case they would not be in accord with the beta-spiral model, but would favor a more extended conformation for the polypeptide chain, possibly polyproline II.     

Use of recombinant inbred lines of wheat for study of associations of high-molecular-weight glutenin subunit alleles to quantitative traits

Summary Recombinant inbred lines (RILs) derived by single plant descent to F₈ from a hybrid of Anza, a low-quality cultivar, and Cajeme 71, a high-quality cultivar, differed in alleles at three high-molecular-weight glutenin (HMW-glu) seed storage protein loci. The 48 RILs were classified by SDS-PAGE for the Anza alleles Glu-Alc (null), Glu-B1b (subunits 7 + 8), and Glu-D1a (subunits 2 + 12) and for Cajeme 71 alleles Glu-A1a (sub-unit 1), Glu-B1I (subunits 17 + 18), and Glu-D1d (subunits 5 + 10). All RILs and parents were grown in a replicated field trial with three levels of nitrogen (N) fertilization. Additive and additive x additive gene effects for the three loci were detected by orthogonal comparisons of means for each of six wheat end-use quality traits. Each HMW-glu genotype was represented by three to ten RILs so that variability among RILs within each HMW-glu genotype could be examined. N effects were consistently small. All traits except flour yield were highly correlated with predictor traits studied earlier. Flour protein content, baking water absorption, dough mixing time, bread loaf volume, and bread loaf crumb score were all correlated, suggesting similar gene control for these traits; however, specific additive locus contributions were evident: _B for flour yield; _B and _D for flour protein; and _B for absorption, but differing in sign; all three loci for mixing time, but _B was negative; and all three loci were positively associated with loaf volume. Digenic epistatic effects were significant for flour yield (_AD), flour protein (_AB), and absorption and mixing time (_AD, _BD). Only flour yield showed a trigenic epistatic effect. Six of seven epistatic effects were negative, thus showing how progress in breeding for high quality may be impeded by interaction of genes which, by themselves, have strong positive additive effects. Considerable genetic variance among RILs within a HMW-glu genotype was detected for all traits, and the summation of effects accounted for a mean of 13% of the parental differences for the six traits examined in this study. Clearly, further resolution of the genetics of wheat quality would be desirable from a plant breeding point of view.     

The characterization and comparative analysis of high-molecular-weight glutenin genes from genomes A and B of a hexaploid bread wheat

Summary Two high-molecular-weight subunit (HMWS) glutenin genes from the A and B genomes of the hexaploid bread wheat Triticum aestivum L. cv Cheyenne have been isolated and sequenced. Both of these genes are of the high M_r class (x-type) of HMW glutenins, and have not been previously reported. The entire set of six HMW genes from cultivar Cheyenne have now been isolated and characterized. An analysis of the Ax and Bx sequences shows that the Ax sequence is similar to the homoeologous gene from the D genome, while the Bx repeat structure is significantly different. The repetitive region of these proteins can be modelled as a series of interspersed copies of repeat modifs of 6, 9, and 15 amino acid residues. The evolution of these genes includes single-base substitutions over the entire coding region, plus insertion/deletions of single or blocks of repeats in the central repetitive domain.     

Use of recombinant inbred lines of wheat for study of associations of high-molecular-weight glutenin subunit alleles to quantitative traits

Summary The high-molecular-weight glutenin subunits (HMW glutenin), encoded by alleles at homoeologous lociGlu-A1,Glu-B1, andGlu-D1 on the long arms of chromosomes1A,1B, and1D of a set of F₈ random recombinant inbred lines (RIL) derived from the bread wheat cross Anza × Cajeme 71, were classified by SDS-PAGE. Anza has poor breadmaking quality and HMW-glutenin subunits (Payne numbers) null (Glu-A1c), 7+8 (Glu-B1b), and 2+12 (Glu-D1a); Cajeme 71 has good quality and 1 (Glu-A1a), 17+18 (Glu-B1i), and 5+10 (Glu-D1d). The combinations of these alleles in the RIL were examined for associations with grain yield and four indicators of grain quality — protein content, yellowberry, pearling index, and SDS sedimentation volume. Data were obtained from a field experiment with three nitrogen fertilization treatments on 48 RIL and the parents. Orthogonal partitioning of the genetic variance associated with the three HMW glutenin subunit loci into additive and epistatic (digenic and trigenic) effects showed strong associations of these loci with grain yield and the indicators of quality; however, the associations accounted for no more than 25% of the differences between the parents. Genetic variance was detected among the RIL, which had the same HMW glutenin genotype for all traits. Epistatic effects were absent for grain yield and yellowberry, but were substantial for grain protein content, pearling index, and SDS sedimentation volume. All three loci had large single-locus additive effects for grain yield, protein, and SDS sedimentation volume. Yellowberry was largely influenced byGlu-B1 andGlu-D1, whereas pearling index was associated withGlu-A1 andGlu-B1. Even though the observed associations-of effects of HMW glutenin loci with the quantitative characters were small relative to the total genetic variability, they are of considerable importance in understanding the genetics of wheat quality, and are useful in the development of new wheat varieties with specific desired characteristics.     

Isolation and molecular characterization of high molecular weight glutenin subunit genes 1Bx13 and 1By16 from hexaploid wheat

The high molecular weight glutenin subunit （HMW-GS） pair 1Bx13＋1By16 are recognized to positively correlate with bread-making quality; however, their molecular data remain unknown. In order to reveal the mechanism by which 1By16 and 1Bx13 creates high quality, their open reading frames （ORFs） were amplified from common wheat Atlas66 and Jimai 20 using primers that were designed based on published sequences of HMW glutenin genes. The ORF of 1By16 was 2220bp, deduced into 738 amino acid residues with seven cysteines including 59 hexapeptides and 22 nanopeptides motifs. The ORF of 1Bx13 was 2385bp, deduced into 795 amino acid residues with four cysteines including 68 hexapeptides, 25 nanopeptides and six tripepUdes motifs. We found that 1By16 was the largest y-type HMW glutenin gene described to date in common wheat. The 1By16 had 36 amino acid residues inserted in the central repetitive domain compared with 1By15. Expression in bacteria and western-blot tests confirmed that the sequence cloned was the ORF of HMW-GS 1By16, and that 1Bx13 was one of the largest 1Bx genes that have been described so far in common wheat, exhibiting a hexapeptide （PGQGQQ） insertion in the end of central repetitive domain compared with 1Bx7. A phylogenetic tree based on the deduced full-length amino acid sequence alignment of the published HMW-GS genes showed that the 1By16 was clustered with Glu-1B-2, and that the 1Bx13 was clustered with Glu-1B-1 alleles.     

Characterization of two HMW glutenin subunit genes from Taenitherum Nevski

The compositions of high molecular weight (HMW) glutenin subunits from three species of Taenitherum Nevski (TaTa, 2n = 2x = 14), Ta. caput-medusae, Ta. crinitum and Ta. asperum, were investigated by SDS-PAGE analysis. The electrophoresis mobility of the x-type HMW glutenin subunits were slower or equal to that of wheat HMW glutenin subunit Dx2, and the electrophoresis mobility of the y-type subunits were faster than that of wheat HMW glutenin subunit Dy12. Two HMW glutenin genes, designated as Tax and Tay, were isolated from Ta. crinitum, and their complete nucleotide coding sequences were determined. Sequencing and multiple sequences alignment suggested that the HMW glutenin subunits derived from Ta. crinitum had the similar structures to the HMW glutenin subunits from wheat and related species with a signal peptide, and N- and C-conservative domains flanking by a repetitive domain consisted of the repeated short peptide motifs. However, the encoding sequences of Tax and Tay had some novel modification compared with the HMW glutenin genes reported so far: (1) A short peptide with the consensus sequences of KGGSFYP, which was observed in the N-terminal of all known HMW glutenin genes, was absent in Tax; (2) There is a specified short peptide tandem of tripeptide, hexapeptide and nonapeptide and three tandem of tripeptide in the repetitive domain of Tax; (3) The amino acid residues number is 105 (an extra Q presented) but not 104 in the N-terminal of Tay, which was similar to most of y-type HMW glutenin genes from Elytrigia elongata and Crithopsis delileana. Phylogenetic analysis indicated that Tax subunit was mostly related to Ax1, Cx, Ux and Dx5, and Tay was more related to Ay, Cy and Ry.     

Identification and localization of molecular markers linked to the Lr9 leaf rust resistance gene of wheat

Near-isogenic lines (NILs) for the leaf rust resistance gene Lr9 were screened for polymorphisms at the molecular level. RAPD (random amplified polymorphic DNA) primers as well as RFLP (restriction fragment length polymorphism) markers were used. Out of 395 RAPD primers tested, three showed polymorphisms between NILs, i.e., an additional band was found in resistant lines. One of these polymorphic bands was cloned and sequenced. Specific primers were synthesized, and after amplification only resistant lines showed an amplified product. Thus, these primers define a sequence-tagged site that is specific for the translocated fragment carrying the Lr9 gene. A cross between a resistant NIL and the spelt (Triticum spelta) variety Oberkulmer was made, and F₂ plants were analyzed for genetic linkage. All three polymorphisms detected by the PCR (polymerase chain reaction) and one RFLP marker (cMWG684) showed complete linkage to the Lr9 gene in 156 and 133 plants analyzed, respectively. A second RFLP marker (PSR546) was closely linked (8±2.4 cM) to the Lr9 gene and the other four DNA markers. As this marker maps to the distal part of the long arm of chromosome 6B of wheat, Lr9 and the other DNA markers also map to the distal region of 6BL. All three PCR markers detected the Lr9 gene in independently derived breeding lines and varieties, thus proving their general applicability in wheat breeding programs.     

Genetic control of the mitochondrial form of superoxide dismutase in hexaploid wheat

Extracts of mature grains of a large number of aneuploid derivatives of Triticum aestivum cv. Chinese Spring and of the members of five wheat-alien chromosome addition series were subjected to isoelectric focusing in polyacrylamide gels in order to study the genetic control of superoxide dismutase (SOD). Evidence was obtained that homologous structural genes for the mitochondrial form of SOD are located in the long arms of the homoeologous group 2 chromosomes of Chinese Spring and in chromosome 2R of Secale cereale cv. Imperial. The SOD gene loci located in chromosomes 2A, 2B, 2D, and 2R were designated Sod-A1, Sod-B1, Sod-D1, and Sod-R1, respectively. Chromosome-arm pairing data indicate that 2DL is not homoeologous to either 2AS or 2BL. The results of this study suggest, however, that 2BL is partially homoeologous to both 2AL and 2DL.Technical article No. 21074 of the Texas Agricultural Experiment Station. This work was supported by USDA Grant 83-CRCR-1-1322 to GEH.     

Classification of wheat low-molecular-weight glutenin subunit genes and its chromosome assignment by developing LMW-GS group-specific primers

On the basis of sequence analysis, 69 known low-molecular-weight glutenin subunit (LMW-GS) genes were experimentally classified into nine groups by the deduced amino acid sequence of the highly conserved N-terminal domain. To clarify the chromosomal locations of these groups, 11 specific primer sets were designed to carry out polymerase chain reactions (PCR) with the genomic DNA of group 1 ditelosomic lines of Chinese Spring, among which nine primer sets proved to be LMW-GS group-specific. Each group of LMW-GS genes was specifically assigned on a single chromosome arm and hence to a specific locus. Therefore, these results provided the possibility to predict the chromosome location of a new LMW-GS gene based on its deduced N-terminal sequence. The validity of the classification was confirmed by the amplifications in 27 diploid wheat and Aegilops accessions. The length polymorphisms of LMW-GS genes of groups 1 and 2, and groups 3 and 4.1 were detected in diploid A-genome and S-genome accessions, respectively. The diploid wheat and Aegilops species could be used as valuable resources of novel allele variations of LMW-GS gene in the improvement of wheat quality. The nine LMW-GS group-specific primer sets could be utilized to select specific allele variations of LMW-GS genes in the marker-assisted breeding. Electronic Supplementary Material Supplementary material is available for this article at Hai Long and Yu-Ming Wei are the two authors who have contributed equally to this paper     

Molecular discrimination of Bx7 alleles demonstrates that a highly expressed high-molecular-weight glutenin allele has a major impact on wheat flour dough strength

High-molecular-weight glutenin subunits (HMW-GS) are important determinants of wheat dough quality as they confer visco-elastic properties to the dough required for mixing and baking performance. With this important role, the HMW-GS alleles are key markers in breeding programs. In this work, we present the use of a PCR marker initially designed to discriminate Glu1 Bx7 and Glu1 Bx17 HMW-GS. It was discovered that this marker also differentiated two alleles, originally both scored as Glu1 Bx7, present in the wheat lines CD87 and Katepwa respectively, by a size polymorphism of 18 bp. The marker was scored across a segregating doubled-haploid (DH) population (CD87 × Katepwa) containing 156 individual lines and grown at two sites. Within this population, the marker differentiated lines showing the over-expression of the Glu1 Bx7 subunit (indicated by the larger PCR fragment), derived from the CD87 parent, relative to lines showing the normal expression of the Glu1 Bx7 subunit, derived from the Katepwa parent. DNA sequence analysis showed that the observed size polymorphism was due to an 18 bp insertion/deletion event at the C-terminal end of the central repetitive domain of the Glu1 Bx 7 coding sequence, which resulted in an extra copy of the hexapeptide sequence QPGQGQ in the deduced amino-acid sequence of Bx7 from CD87. When the DH population was analysed using this novel Bx7 PCR marker, SDS PAGE and RP HPLC, there was perfect correlation between the Bx7 PCR marker results and the expression level of Bx7. This differentiation of the population was confirmed by both SDS-PAGE and RP-HPLC. The functional significance of this marker was assessed by measuring key dough properties of the 156 DH lines. A strong association was shown between lines with an over expression of Bx7 and high dough strength. Furthermore, the data demonstrated that there was an additional impact of Glu-D1 alleles on dough properties, with lines containing both over-expressed Bx7 and Glu-D1 5+10 having the highest levels of dough strength. However, there was no statistically significant epistatic interaction between Glu-B1 and Glu-D1 loci.Communicated by J.W. Snape     

Genetic analysis of photosynthetic variation in hexaploid and tetraploid wheat and their interspecific hybrids

Intra- and inter-specific variation in CO₂ assimilation rate (A) in Triticum spp. is well documented for reproductive growth stages. Research was conducted to characterize early vegetative photosynthetic variation in a diverse set of cultivated hexaploid wheat (T. aestivum L.) germplasm and in wild tetraploid (T. dicoccoides Korn) and hexaploid x tetraploid populations. Choice of hexaploid genotypes was based on maximum genetic distance between cultivars within the HRW and SRW wheat classes of the USA. The tetraploid material was produced by hybridizing two accessions of T. dicoccoides previously shown to differ widely in A and A/Chl but with similar leaf morphology. Genetic variability in the HRW and SRW gene pools was attributed to more recently developed descendent lines and unrelated lines rather than parental lines. Phenotypic distributions for A, stomatal conductance (gs), and internal CO₂ concentration (Ci) in the F₂ tetraploid population were continuous and showed transgressive segregation, reflecting quantitative inheritance with intermediate heritability. Variability in A was not associated with chlorophyll content or CO₂ supply to the mesophyll measured as Ci. Genetic variability in A was also observed in the interspecific backcross population, 2*TAM W-101/PI 428109, thereby providing a germplasm pool to select for high A while restoring the D genome of hexaploid wheat. These results suggest that genetic improvement of vegetative assimilation rate is feasible in hexaploid wheat via homologous transfer from an alien source.Abbreviations HRW hard red winter - LA leaf area - r_G genotypic correlation - r_P phenotypic correlation - SRW soft red winter     

Production and characterization of a transgenic bread wheat line over-expressing a low-molecular-weight glutenin subunit gene

The end-use properties, and thus the value, of wheat flours are determined to a large extent by the proteins that make up the polymeric network called gluten. Low molecular weight glutenin subunits (LMW-GS) are important components of gluten structure. Their relative amounts and/or the presence of specific components can influence dough visco-elasticity, a property that is correlated with the end-use properties of wheat flour. For these reasons, manipulation of gluten dough strength and elasticity is important. We are pursuing this goal by transforming the bread wheat cultivar Bobwhite with a LMW-GS gene driven by its own promoter. Particle bombardment of immature embryos produced several transgenic lines, one of which over-expressed the LMW-GS transgene. Southern blots confirmed that the transgene was integrated into the wheat genome, although segregation analyses showed that its expression was sometimes poorly transmitted to progeny. We have determined that the transgene-encoded LMW-GS accumulates to very high levels in seeds of this line, and that it is incorporated into the glutenin polymer, nearly doubling its overall amount. However, SDS sedimentation test values were lower from the transgenic material compared to a non transgenic flour. These results suggest that the widely accepted correlation between the amount of the glutenin polymers and flour technological properties might not be valid, depending on the components of the polymer.     

Dosage effects of chromosomes of homoeologous groups 1 and 6 upon bread-making quality in hexaploid wheat

Summary The endosperm storage proteins, glutenin and gliadin, are major determinants of bread-making quality in hexaploid wheat. Genes encoding them are located on chromosomes of homoeologous groups 1 and 6. Aneuploid lines of these groups in spring wheat cultivar Chinese Spring have been used to investigate the effect of varying the dosage of chromosomes and chromosome arms upon bread-making quality, where quality has been assessed using the SDS-sedimentation test. Differences between the group 1 chromosomes for quality were greater than those between the group 6 chromosomes. The chromosomes were ranked within homoeologous groups for their effect on quality as follows (>=better quality): 1D>1B>1A and 6A>6B=6D. The relationship of chromosome dosage with quality was principally linear for four of the chromosomes, but not for 6B and 6D. Increases in the dosage of 1B, 6A and, especially, 1D, were associated with significant improvements in quality, whereas increases in the dosage of 1A were associated with reductions in quality. The effects of 1A and 1D were such that the best genotype for quality was nullisomic 1A-tetrasomic 1D. For group 1, effects of the long arm appeared in general to be more important than effects of the short arm. For group 6, effects were found associated with the long arms as well as with the short arms, a surprising result in view of the absence of genes encoding storage proteins on the long arms. Significant interactions were found between chromosomes and genetic backgrounds, and between individual chromosomes. Analysis of trials grown over two years demonstrated that, although additive environmental differences over years and genotype x years interaction were present, they were relatively small in magnitude compared with purely genetic differences.     

Homology modeling and molecular dynamics simulations of the N-terminal domain of wheat high molecular weight glutenin subunit 10

High molecular weight glutenin subunits (HMW-GS) are of a particular interest because of their biomechanical properties, which are important in many food systems such as breadmaking. Using fold-recognition techniques, we identified a fold compatible with the N-terminal domain of HMW-GS Dy10. This fold corresponds to the one adopted by proteins belonging to the cereal inhibitor family. Starting from three known protein structures of this family as templates, we built three models for the N-terminal domain of HMW-GS Dy10. We analyzed these models, and we propose a number of hypotheses regarding the N-terminal domain properties that can be tested experimentally. In particular, we discuss two possible ways of interaction between the N-terminal domains of the y-type HMW glutenin subunits. The first way consists in the creation of interchain disulfide bridges. According to our models, we propose two plausible scenarios: (1) the existence of an intrachain disulfide bridge between cysteines 22 and 44, leaving the three other cysteines free of engaging in intermolecular bonds; and (2) the creation of two intrachain disulfide bridges (involving cysteines 22-44 and cysteines 10-55), leaving a single cysteine (45) for creating an intermolecular disulfide bridge. We discuss these scenarios in relation to contradictory experimental results. The second way, although less likely, is nevertheless worth considering. There might exist a possibility for the N-terminal domain of Dy10, Nt-Dy10, to create oligomers, because homologous cereal inhibitor proteins are known to exist as monomers, homodimers, and heterooligomers. We also discuss, in relation to the function of the cereal inhibitor proteins, the possibility that this N-terminal domain has retained similar inhibitory functions.