Glutenin Subunit Genes Research Articles

Genetic basis of an elite wheat cultivar Guinong 29 with harmonious improvement between multiple diseases resistance and other comprehensive traits

Fungal diseases, such as powdery mildew and rusts, significantly affect the quality and yield of wheat. Pyramiding diverse types of resistance genes into cultivars represents the preferred strategy to combat these diseases. Moreover, achieving collaborative improvement between diseases resistance, abiotic stress, quality, and agronomic and yield traits is difficult in genetic breeding. In this study, the wheat cultivar, Guinong 29 (GN29), showed high resistance to powdery mildew and stripe rust at both seedling and adult plant stages, and was susceptible to leaf rust at the seedling stage but slow resistance at the adult-plant stage. Meanwhile, it has elite agronomic and yield traits, indicating promising coordination ability among multiple diseases resistance and other key breeding traits. To determine the genetic basis of these elite traits, GN29 was tested with 113 molecular markers for 98 genes associated with diseases resistance, stress tolerance, quality, and adaptability. The results indicated that two powdery mildew resistance (Pm) genes, Pm2 and Pm21, confirmed the outstanding resistance to powdery mildew through genetic analysis, marker detection, genomic in situ hybridization (GISH), non-denaturing fluorescence in situ hybridization (ND-FISH), and homology-based cloning; the stripe rust resistance (Yr) gene Yr26 and leaf rust resistance (Lr) genes Lr1 and Lr46 conferred the stripe rust and slow leaf rust resistance in GN29, respectively. Meanwhile, GN29 carries dwarfing genes Rht-B1b and Rht-D1a, vernalization genes vrn-A1, vrn-B1, vrn-D1, and vrn-B3, which were consistent with the phenotypic traits in dwarf characteristic and semi-winter property; carries genes Dreb1 and Ta-CRT for stress tolerance to drought, salinity, low temperature, and abscisic acid (ABA), suggesting that GN29 may also have elite stress-tolerance ability; and carries two low-molecular-weight glutenin subunit genes Glu-B3b and Glu-B3bef which contributed to high baking quality. This study not only elucidated the genetic basis of the elite traits in GN29 but also verified the capability for harmonious improvement in both multiple diseases resistance and other comprehensive traits, offering valuable information for breeding breakthrough-resistant cultivars.

Molecular Characterization and Marker Development of the HMW-GS Gene from Thinopyrum elongatum for Improving Wheat Quality

The quality of wheat primarily depends on its storage protein quality, especially in regards to gluten content and high-molecular-weight glutenin subunits (HMW-GS). The number of HMW-GS alleles is limited in bread wheat (Triticum aestivum L.), whereas it is abundant in wheat relatives. Therefore, HMW-GS alleles from wheat relatives could provide a potential for improving quality in wheat breeding. Thinopyrum elongatum (EE) is one of the relatives of wheat. The E genome is closely related to the ABD genome in wheat; therefore, Th. elongatum is often used as an excellent exogenous gene donor for wheat genetic improvement. In this study, the high-molecular glutenin subunit gene was cloned and sequenced from Th. elongatum. A specific molecular marker for identifying the Glu-1Ey subunit gene was developed and applied to detected wheat-Th. elongatum alien introgression lines. Quality analysis indicated that the substitution and addition lines containing Th. elongatum alleles significantly (p < 0.05) increased grain protein content by 3.76% to 5.11%, wet-gluten content by 6.55% to 8.73%, flour 8-MW by 0.25% to 6.35%, and bread volume value by 33.77 mL to 246.50 mL, in comparing it with Chinese Spring. The GMP content and lactic acid SRC showed significant positive correlations with flour processing quality and might be used as indicators for wheat quality. The results were expected to provide a novel route for improving processing quality in wheat quality breeding.

BZIP transcription factors repress the expression of wheat (Triticum aestivum L.) high molecular weight glutenin subunit genes in vegetative tissues

High molecular weight glutenin subunits (HMW GS) represent an important fraction of endosperm-specific seed-storage proteins that provide elasticity to bread dough. Previously, the second cis-regulatory module (CRM2) was found to be one of the most conserved part of HMW GS promoters, which indicated its pre-eminent role in their gene regulation. Here, we observed that deletion of CRM2 from the promoters of the Bx7 and By8 HMW GS genes increased the leakage of their transient expression in wheat leaf tissue. The effect of a VP1, an Myb and an antisense bZIP transcription factor (TF)-binding site, potentially involved in endosperm-specific regulation within CRM2, was then studied. The activity of a Bx7 gene promoter containing a mutant CRM2 with altered VP1 and Myb TF-binding sites, but an intact bZIP TF-binding site, was similarly low to that of the wild type in leaves. Transactivation analysis and EMSA indicated the binding of TFs TabZIP34 and TabZIP115 to the Skn-1 motif GTCAT in CRM2 and the repression of Bx7 and By8 HMW GS gene promoter activity in leaves. TabZIP34 and TabZIP115 may be involved in the downregulation of HMW GS gene expression in vegetative tissues and early-stage endosperm as well its modulation during seed maturation.

Missense alleles of the HMW glutenin subunits Dx5 and Dy10 have small changes in function relative to missense changes in Puroindoline a and b

AbstractBackground and ObjectivesThe high molecular weight glutenin subunit genes (HMW‐GS) in wheat (Triticum aestivum L.) play a key role in determining dough functionality. More specifically allelic variation for the Glu‐D1 subunit loci is consistently associated with bread making quality. Since much of the hard wheat germplasm is fixed for the more favorable 1Dx5 + 1Dy10 haplotype, our goal was to identify allelic variation in 1Dx5 and 1Dy10 HMW‐GS genes using an ethyl methanesulfonate (EMS) mutagenized population in the soft white spring wheat cultivar “Alpowa.” The same source population was previously used in screening for creating novel alleles in Puroindoline a (Pina) and Puroindoline b (Pinb) which are responsible for grain hardness variation. Direct sequencing of 384 M3 families identified 135 point mutations equally dispersed across 1Dx5 and 1Dy10. The mutation discovery rate was 1/12.7 kb of DNA, equal to that found in the Pin loci. Mutation carrying plants were crossed to the nonmutagenized Alpowa parent to create F2 populations segregating for the induced EMS HMW Glutenin mutations. Swelling index of gluten (SIG) was measured from seed from field grown plants to assess the impact of each EMS mutation upon Glutenin function.FindingsNonsense mutations in both 1Dx5 and 1Dy10 reduced SIG values, indicating both 1Dx5 and 1Dy10 are needed for gluten functionality. Missense mutations did not alter SIG values compared to their wild type counterparts. In contrast missense mutations within the tryptophan‐rich region of both PINS increased grain hardness pointing to the importance of this protein region. No such region in 1Dx5 or 1Dy10 was found indicating these proteins are structurally stable and tolerant of amino acid substitution.ConclusionsResults from the Glu‐D1 subunit and Pin loci show that useful phenotypic variation can be created using EMS mutagenesis for genes where naturally occurring mutations occur from amino substitutions as is the case for the Pin loci.Significance and NoveltyEMS induced mutations are useful for creating new alleles in storage proteins with much greater impacts in genes that have a defined active site as for the Pin loci than in genes such as the HMW Glu‐1 genes which have a long repetitive region.

Trade-offs in the genetic control of functional and nutritional quality traits in UK winter wheat.

A complex network of trade-offs exists between wheat quality and nutritional traits. We investigated the correlated relationships among several milling and baking traits as well as mineral density in refined white and whole grain flour. Our aim was to determine their pleiotropic genetic control in a multi-parent population over two trial years with direct application to practical breeding. Co-location of major quantitative trait loci (QTL) and principal component based multi-trait QTL mapping increased the power to detect QTL and revealed pleiotropic effects explaining many complementary and antagonistic trait relationships. High molecular weight glutenin subunit genes explained much of the heritable variation in important dough rheology traits, although additional QTL were detected. Several QTL, including one linked to the TaGW2 gene, controlled grain size and increased flour extraction rate. The semi-dwarf Rht-D1b allele had a positive effect on Hagberg falling number, but reduced grain size, specific weight, grain protein content and flour water absorption. Mineral nutrient concentrations were lower in Rht-D1b lines for many elements, in wholemeal and white flour, but potassium concentration was higher in Rht-D1b lines. The presence of awns increased calcium content without decreasing extraction rate, despite the negative correlation between these traits. QTL were also found that affect the relative concentrations of key mineral nutrients compared to phosphorus which may help increase bioavailability without associated anti-nutritional effects of phytic acid. Taken together these results demonstrate the potential for marker-based selection to optimise trait trade-offs and enhance wheat nutritional value by considering pleiotropic genetic effects across multiple traits.

Characterization of novel low-molecular-weight glutenin subunit genes from the diploid wild wheat relative Aegilops umbellulata

AbstractLow molecular weight glutenin subunits (LWM-GSs) play a crucial role in determining wheat flour processing quality. In this work, 35 novel LMW-GS genes (32 active and three pseudogenes) from three Aegilops umbellulata (2n = 2x = 14, UU) accessions were amplified by allelic-specific PCR. We found that all LMW-GS genes had the same primary structure shared by other known LMW-GSs. Thirty-two active genes encode 31 typical LMW-m-type subunits. The MZ424050 possessed nine cysteine residues with an extra cysteine residue located in the last amino acid residue of the conserved C-terminal III, which could benefit the formation of larger glutenin polymers, and therefore may have positive effects on dough properties. We have found extensive variations which were mainly resulted from single-nucleotide polymorphisms (SNPs) and insertions and deletions (InDels) among the LMW-GS genes in Ae. umbellulata. Our results demonstrated that Ae. umbellulata is an important source of LMW-GS variants and the potential value of the novel LMW-GS alleles for wheat quality improvement.

Genome-wide identification, characteristics and expression of the prolamin genes in Thinopyrum elongatum

BackgroundProlamins, unique to Gramineae (grasses), play a key role in the human diet. Thinopyrum elongatum (syn. Agropyron elongatum or Lophopyrum elongatum), a grass of the Triticeae family with a diploid E genome (2n = 2x = 14), is genetically well-characterized, but little is known about its prolamin genes and the relationships with homologous loci in the Triticeae species.ResultsIn this study, a total of 19 α-gliadin, 9 γ-gliadin, 19 ω-gliadin, 2 high-molecular-weight glutenin subunit (HMW-GS), and 5 low-molecular-weight glutenin subunit (LMW-GS) genes were identified in the Th. elongatum genome. Micro-synteny and phylogenetic analysis revealed dynamic changes of prolamin gene regions and genetic affinities among Th. elongatum, Triticum aestivum, T. urartu and Aegilops tauschii. The Th. elongatum genome, like the B subgenome of T. aestivum, only contained celiac disease epitope DQ8-glia-α1/DQ8.5-glia-α1, which provided a theoretical basis for the low gluten toxicity wheat breeding. The transcriptome data of Th. elongatum exhibited differential expression in quantity and pattern in the same subfamily or different subfamilies. Dough rheological properties of T. aestivum-Th. elongatum disomic substitution (DS) line 1E(1D) showed higher peak height values than that of their parents, and DS6E(6D) exhibited fewer α-gliadins, which indicates the potential usage for wheat quality breeding.ConclusionsOverall, this study provided a comprehensive overview of the prolamin gene family in Th. elongatum, and suggested a promising use of this species in the generation of improved wheat breeds intended for the human diet.

Seed-specific expression of TaYUC10 significantly increases auxin and protein content in wheat seeds.

Present study revealed that specific expression of TaYUC10.3 in wheat young seeds could increase the content of auxin, and protein. Auxin is a vital endogenous hormone in plants, which is involved in the regulation of various physiological and biochemical processes in plants. The flavin-containing monooxygenase encoded by the YUCCA gene is a rate-limiting enzyme in the tryptophan-dependent pathway of auxin synthesis. TaYUC10.3 was identified, cloned and found that it was abundantly expressed in wheat young seeds. In this study, a seed-specific expression vector of TaYUC10.3 was constructed with the promoter of 1Bx17 glutenin subunit gene and transformed wheat using the particle bombardment method. The quantitative RT-PCR showed that TaYUC10.3 was expressed in a large amount in young seeds of the transgenic lines. Plant hormone-targeted metabolomics showed that the auxin content of the transgenic lines was significantly increased compared with controls. The GC / MS non-targeted metabolite multiple statistical analyses showed that the variable importance in projection (VIP) of tryptophan reduced in the transgenic lines. Simultaneously, the VIP of indole acetic acid increased. The precursor amino acids for synthesizing some proteins and carbohydrates were upregulated in the transgenic lines. Subsequently, it was found that the protein content of the seeds of the transgenic TaYUC10.3 wheat was significantly higher than that of the control. The wet gluten content and sedimentation value of the transgenic TaYUC10.3 wheat were also high. This result indicated that TaYUC10.3 might participate in auxin synthesis and affects the protein content of wheat seeds.

Cloning and characterization of special HMW glutenin subunit genes from Aegilops longissima L. and their potential for wheat quality improvement.

Identification and cloning of new glutenin genes from wheat-related species can provide candidate gene resources for quality improvement of wheat. In this study, ten special high-molecular-weight glutenin subunits (HMW-GS), including five x-type (1Sl2x, 1Sl16x, 1Sl17x, 1Sl23x, and 1Sl25x) and five y-type (1Sl2y, 1Sl6y1, 1Sl16y, 1Sl17y, and 1Sl23y) from Aegilops longissima L. (SlSl, 2n = 2x = 14) were identified, and their complete encoding genes were cloned by allelic-specific polymerase chain reaction (AS-PCR). The deduced amino acid (aa) residues of the x-type subunit genes ranged from 821 aa (2469bp) to 941 aa (2829bp), while those of the y-type subunit genes varied from 749 aa (2250bp) to 771 aa (2361bp). These special HMW-GS had a longer central repetitive domain with more glutamine repeats and glutamine residues compared to the previously characterized HMW-GS in common wheat, which provided a structural basis for superior gluten quality formation. The authenticity of the four cloned genes were verified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Abundant single-nucleotide polymorphism (SNP) and insertion/deletion (InDel) variations among these genes were identified, which would benefit for developing specific molecular markers used for wheat gluten quality improvement. Phylogenetic analysis revealed that the 1Sl-encoded HMW-GS had close relationships with those from bread wheat, which were divergent from Triticum species at 2.10-10.00 million years ago. Our results indicate that the 1Sl genome contains superior candidate glutenin genes that have potential application values for the improvement of wheat bread making quality.

Molecular characterization and phylogenetic analysis of active y-type high molecular weight glutenin subunit genes at Glu-A1 locus in wheat

An increased number of high molecular weight glutenin subunits (HMW-GSs) is advantageous to the formation of glutenin macropolymer, the major determinant of end-product quality. Out of the six HMW-GS genes, Glu-1Ay is typically inactive in most hexaploid wheat cultivars. In the current study, we characterized two active Glu-1Ay genes Glu-1AyN11 and Glu-1AyC422 from a hexaploid wheat cultivar N11 and a durum wheat cultivar C422, respectively. The molecular masses of the 1Ay subunit of N11 (Glu-1AyN11) and C422 (Glu-1AyC422) were 61.1 kDa and 63.9 kDa, respectively, measured by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Molecular characterization of Glu-1AyN11 and Glu-1AyC422 showed their complete coding regions were 1764 bp and 1827 bp in length, respectively. The deduced amino acid sequence of both Glu-1AyN11 and Glu-1AyC422 has a similar typical primary structure as the other y-type HMW-GSs, but in comparison to Glu-1By and Glu-1Dy subunits, the Glu-1Ay subunit has one less conserved cysteine residue in the repetitive domain. Phylogenetic analysis revealed that the inactive Glu-1Ay had a closer evolutionary relationship with Glu-1AyC422 than with Glu-1AyN11. The divergence time among Glu-1AyN11, Glu-1AyC422, and other y-type subunit genes was also estimated.

Genomics Approach to Identify the Cause of the Missing Omega-5 Gliadin Protein in O-Free Wheat

A previous work developed and identified a new omega-5 gliadin deficient wheat line named O-free by crossing Keumkang and Olgeuru, which is nutritionally quite meaningful in that omega-5 gliadin is one of the known wheat allergens. To verify the characteristics of the O-free, we performed RNA sequencing (RNAseq) analysis of the O-free and the two parent lines (Keumkang and Olgeuru). The results of the similarity analysis with the ESTs for gliadins and glutenins showed that the O-free ESTs had no similarity with the omega-5 gliadin sequences but had similarity to other gliadins and glutenins. Furthermore, mapping results between the raw RNAseq data from the O-free and the omega-5 gliadin sequence showed a clear deletion of the N-terminal sequences which are an important signature of omega-5 gliadin. We also designed specific PCR primers that could identify omega-5 gliadin in the genomic DNA. The results showed that no omega-5 gliadin fragments were detected in the O-free. According to these results, we confirmed that the deficiency of omega-5 gliadin in the O-free is not caused by post-transcriptional or post-translational regulations such as epigenetic phenomena but by a simple deletion in the chromosome. Furthermore, we showed that the low-molecular weight glutenin subunit (LMW-GS) gene in the O-free had a single nucleotide polymorphism (SNP) causing a premature stop codon, resulting in a truncated polypeptide. We expect that the O-free line may serve as an excellent source of wheat that could prevail in the hypo-allergen wheat market, which has recently gained interest world-wide.

Cloning and characterization of a novel low-molecular-weight glutenin subunit gene with an unusual molecular structure of Aegilops uniaristata

Low-molecular-weight glutenin subunits (LMW-GSs) are one of the important factors for the dough processing quality. In this study, a novel LMW-GS, designated LMW-N13, from the wheat relative species Aegilops uniaristata PI 554421 was cloned and characterized. Unlike previously published LMW-GSs, LMW-N13 has a large molecular weight and is the largest LMW-GS published thus far. Sequence alignments demonstrated that LMW-N13 is a LMW-i-type subunit but contains nine cysteine residues which is one more than typical LMW-i-type subunits. In addition, four insertions are present in the repetitive domain that resulted in the large molecular weight. In vitro analysis showed that LMW-N13 could improve the dough quality of different base flours.

Low molecular weight glutenin subunit gene composition at Glu-D3 loci of Aegilops tauschii and common wheat and a further view of wheat evolution.

A comprehensive comparison of LMW-GS genes between Ae. tauschii and its progeny common wheat. Low molecular weight glutenin subunits (LMW-GSs) are determinant of wheat flour processing quality. However, the LMW-GS gene composition in Aegilops tauschii, the wheat D genome progenitor, has not been comprehensively elucidated and the impact of allohexaploidization on the Glu-D3 locus remains elusive. In this work, using the LMW-GS gene molecular marker system and the full-length gene-cloning method, LMW-GS genes at the Glu-D3 loci of 218 Ae. tauschii and 173 common wheat (Triticum aestivum L.) were characterized. Each Ae. tauschii contained 11 LMW-GS genes, and the whole collection was divided into 25 haplotypes (AeH01-AeH25). The Glu-D3 locus in common wheat lacked the LMW-GS genes D3-417, D3-507 and D3-552, but shared eight genes of identical open reading frame (ORF) sequences when compared to that of Ae. tauschii. Therefore, the allohexaploidization induces deletions, but exerts no influence on LMW-GS gene coding sequences at the Glu-D3 locus. 92.17% Ae. tauschii had 7-9 LMW-GSs, more than the six subunits in common wheat. The haplotypes AeH16, AeH20 and AeH23 of Ae. tauschii ssp. strangulate distributed in southeastern Caspian Iran were the main putative D genome donor of common wheat. These results facilitate the utilization of the Ae. tauschii glutenin gene resources and the understanding of wheat evolution.

Characterization of novel LMW glutenin subunit genes at the Glu-M3 locus from Aegilops comosa.

We report the characterization of seven novel low-molecular-weight glutenin subunit (LMW-GS) genes from Aegilops comosa (2n = 2x = 14, MM). We found that all seven LMW-GS genes possessed the same primary structure shared by other known LMW-GSs. Three genes (comosa-M1, comosa-M2, and comosa-M3) encode LMW-m-type subunits, two (comosa-I1 and comosa-I2) encode LWM-i-type subunits, and two (comosa-L1 and comosa-L2) encode LWM-l-type subunits. The comosa-M1 possessed seven cysteine residues, which resulted from a single-nucleotide polymorphism (SNP) of the G-A transition in the fifth position of the N-terminal sequence. Two l-type subunits, comosa-L1 and comosa-L2, contained nine cysteine residues with an extra cysteine residue located in the signal peptide and repetitive domain. In addition, a long insertion of 13 residues (LGQQPQ8/LVQQPQ8) was present in the end of the C-terminal II. Phylogenetic analysis implied that the comosa-L2 may be the intermediate type during the evolution of LMW-l and LMW-i-type genes. Our results demonstrated that the novel LMW-GSs, such as comosa-M1, comosa-L1, and comosa-L2, may have positive effects on dough properties.

Mechanisms, origin and heredity of Glu-1Ay silencing in wheat evolution and domestication.

Allotetraploidization drives Glu-1Ay silencing in polyploid wheat. The high-molecular-weight glutenin subunit gene, Glu-1Ay, is always silenced in common wheat via elusive mechanisms. To investigate its silencing and heredity during wheat polyploidization and domestication, the Glu-1Ay gene was characterized in 1246 accessions containing diploid and polyploid wheat worldwide. Eight expressed Glu-1Ay alleles (in 71.81% accessions) and five silenced alleles with a premature termination codon (PTC) were identified in Triticum urartu; 4 expressed alleles (in 41.21% accessions), 13 alleles with PTCs and 1 allele with a WIS 2-1A retrotransposon were present in wild tetraploid wheat; and only silenced alleles with PTC or WIS 2-1A were in cultivated tetra- and hexaploid wheat. Both the PTC number and position in T. urartu Glu-1Ay alleles (one in the N-terminal region) differed from its progeny wild tetraploid wheat (1-5 PTCs mainly in the repetitive domain). The WIS 2-1A insertion occurred ~ 0.13 million years ago in wild tetraploid wheat, much later than the allotetraploidization event. The Glu-1Ay alleles with PTCs or WIS 2-1A that arose in wild tetraploid wheat were fully succeeded to cultivated tetraploid and hexaploid wheat. In addition, the Glu-1Ay gene in wild einkorn inherited to cultivated einkorn. Our data demonstrated that the silencing of Glu-1Ay in tetraploid and hexaploid wheat was attributed to the new PTCs and WIS 2-1A insertion in wild tetraploid wheat, and most silenced alleles were delivered to the cultivated tetraploid and hexaploid wheat, providing a clear evolutionary history of the Glu-1Ay gene in the wheat polyploidization and domestication processes.

Molecular Cytogenetic Characterization of New Wheat-Dasypyrum breviaristatum Introgression Lines for Improving Grain Quality of Wheat.

As an important relative of wheat (Triticum aestivum L), Dasypyrum breviaristatum contains novel high molecular weight glutenin subunits (HMW-GSs) encoded by Glu-1Vb genes. We identified new wheat—D. breviaristatum chromosome introgression lines including chromosomes 1Vb and 1VbL.5VbL by fluorescence in situ hybridization (FISH) combined with molecular markers. We found that chromosome changes occurred in the wheat—D. breviaristatum introgression lines and particularly induced the deletion of 5BS terminal repeats and formation of a new type of 5B-7B reciprocal translocation. The results imply that the D. breviaristatum chromosome 1Vb may contain genes which induce chromosomal recombination in wheat background. Ten putative high molecular weight glutenin subunit (HMW-GS) genes from D. breviaristatum and wheat—D. breviaristatum introgression lines were isolated. The lengths of the HMW-GS genes in Dasypyrum were significantly shorter than typical HMW-GS of common wheat. A new y-type HMW-GS gene, named Glu-Vb1y, was characterized in wheat—D. breviaristatum 1Vb introgression lines. The new wheat—D. breviaristatum germplasm displayed reduced plant height, increased tillers and superior grain protein and gluten contents, improved gluten performance index. The results showed considerable potential for utilization of D. breviaristatum chromosome 1Vb segments in future wheat improvement.

Modification of a novel x-type high-molecular-weight glutenin subunit gene from Aegilops markgrafii to improve dough strength of wheat flour

High-molecular-weight glutenin subunits (HMW-GS) in bread wheat are major determinants of dough viscoelastic properties and the end-use quality of wheat flour. Cysteine residues, which form intermolecular disulphide bonds in HMW-GS, could improve the strength of gluten. To our knowledge, the number and position of cysteine residues in HMW-GS are conserved between wheat (Triticum aestivum) and Aegilops markgrafii. In the present study, we modified a gene (1Cx1.1) from Ae. markgrafii for an HMW-GS that possessed the typical structure and conserved number of cysteines. Site-directed mutagenesis was carried out in 1Cx1.1 to investigate how the position of cysteine residues in HMW-GS affects the mixing properties of dough. Six HMW-GS containing an extra cysteine residue were expressed in Escherichia coli, and the proteins were purified at sufficient scale for incorporation into flour to test dough quality. There were large differences in dough property among samples containing different modified subunits. Cysteine substituting in the N-terminal or repetitive-domain of HMW-GS could significantly improve dough quality. The results showed that the strategy was useful for providing genetic resources for gene engineering, and hence could be valuable for improving the processing quality of wheat.

Allelic variation of high-molecular-weight glutenin genes in bread wheat

The composition and quantity of high-molecular-weight glutenin subunits plays an important role in determining the bread-making quality of wheat. Molecular-genetic analysis of allelic composition of high-molecular-weight glutenin genes in 102 bread wheat cultivars and lines from different geographical regions was conducted. Three alleles at the Glu-A1 locus, nine alleles at the Glu-B1 locus, and two alleles at the Glu-D1 locus were identified. Among the investigated cultivars and lines, 21 were characterized by intracultivar polymorphism. High allelic variation of high-molecular-weight glutenin subunit genes was shown for the collection: 21 and 9 combinations were defined in monomorphic and polymorphic cultivars and lines, respectively. However, the major part of the collection (66.7%) contained four allelic combinations: Glu-A1b Glu-B1c Glu-D1d, Glu-A1b Glu-B1c Glu-D1-2a, Glu-A1a Glu-B1c Glu-D1d, and Glu-A1b Glu-B1c Glu-D1d/Glu-D1-2a. Fourteen cultivars of bread wheat were selected, and they were characterized by a favorable allelic composition of Glu-1 loci.

Allelic variation at high-molecular weight and low-molecular weight glutenin subunit genes in Moroccan bread wheat and durum wheat cultivars

Glutenin is a major protein fraction contributing to the functional properties of gluten and dough. The glutenin constitutes 30–40% of the protein in wheat flour and about half of that in gluten. It is essential to identify correct glutenin alleles and to improve wheat quality by selecting alleles that exert favorable effects. Moroccan wheat cultivars are unique in West Asia and North Africa region, since many of them possess resistance to Hessian fly, a pest, which is becoming important in other countries in the region. Hence, these cultivars are being used as donor for the resistance in the breeding program. Here, we determine the allelic variation in high-molecular weight glutenin subunits (HMW-GS) and low-molecular weight glutenin subunits (LMW-GS) in Moroccan cultivars of bread and durum wheat using the gene-specific PCR markers. In 20 cultivars of bread wheat, 9 different allele variants were detected at HMW-GS and 13 different allele variants were detected at LMW-GS, in which the alleles Glu-A1b (2*), Glu-B1i (17 + 18), Glu-B1c (7*/7 + 9), Glu-D1d (5 + 10), Glu-A3c, Glu-B3 h, and Glu-D3b were the most frequents. In 26 cultivars of durum wheat, less allelic variation was found: seven different allele variants at HMW-GS and six different allele variants at LMW-GS were identified, in which the major alleles were Glu-A1c (null), Glu-B1b (7 + 8), Glu-B1e (20), Glu-A3c, and Glu-B3d. The mean value of the genetic diversity for the glutenin loci was 0.502 in bread wheat and 0.449 in durum wheat. Most of the glutenin alleles carried by Moroccan bread wheat cultivars impart good bread-making quality. Most of the durum wheat glutenin alleles were related to low strength dough or poor quality and need to be improved. To improve quality of Moroccan durum wheat, essentially, Glu-A1c and Glu-B3d alleles of the genes should be replaced with the better alleles through breeding.

Molecular cloning and functional characterization of two novel high molecular weight glutenin subunit genes in Aegilops markgrafii.

The high molecular weight glutenin subunits (HMW-GS) in bread wheat are major determinants of the viscoelastic properties of dough and the end-use quality of wheat flour. Two novel HMW-GSs, 1Cx1.1 and 1Cy9.1, from the diploid species Aegilops markgrafii (CC) were identified in the present study. The corresponding open-reading frames of the genes of 1Cx1.1 and 1Cy9.1 were isolated and sequenced using allele-specific polymerase chain reaction. Sequence comparison demonstrated that the HMW-GSs from Ae. markgrafii possess a similar primary structure to the homologous proteins in wheat and related species. A tandem tripeptide exists in the central repetitive domain of 1Cx1.1, and this unique structure is very rare in the HMW-GSs of other genomes. To confirm the authenticity of these isolated endogenous HMW-GS, the heterologous proteins produced by removing the signal peptides expressed by E. coli exhibited the same electrophoretic mobility as the native proteins. Subsequently, the single protein was purified at a sufficient scale for incorporation into flour to performsodium dodecyl sulphate (SDS) sedimentation testing. Notably, the SDS sedimentation volume was less with the addition of 1Cx1.1 than it was with 1Cy9.1.