Introgression of useful genes from Thinopyrum intermedium to wheat for improvement of bread‐making quality

To study the influence of genes from Thinopyrum intermedium on traits affecting the bread‐making quality of wheat, two derivatives from a putative disomic addition line in cultivar ‘Vilmorin 27’ were used in cytological, biochemical and molecular characterization. Cytological analysis suggested that one of the derivatives (Line‐1) had a terminal deletion involving the long arm of chromosome 1D (2n = 42, Del‐1DL”), and the other (Line‐2) was a conventional addition line, but also carried the same deletion on chromosome 1D (2n = 44, Thi”+Del‐1DL”). Amplification and sequencing of high‐molecular‐weight glutenin subunit (HMW‐GS) genes coded by the Th. intermedium chromosome in Line‐2 indicated the presence of one x‐type with an extra cysteine and four (rather than one) unique y‐type genes. Rheological studies of Line‐1 showed significantly lower dough strength compared to ‘Vilmorin 27’, confirming the recognized role of Glu‐1D coded HMW‐GSs. Line‐2 showed significantly higher dough strength compared to the background cultivar, indicating a significant potential of Th. intermedium for improvement of bread‐making quality in wheat.     

Molecular structure of a novel y-type HMW glutenin subunit gene present in Triticum tauschii

An unusually small y-type high molecular weight (HMW) glutenin subunit gene from Triticum tauschii was sequenced. This gene, encoded at the Glu-D^t1 locus was designated 12.4^t and is the smallest HMW glutenin subunit gene described so far in Triticum species. Oligonucleotide primers based on published sequences of HMW glutenin genes were designed to amplify the encoding region and the central repetitive domain of the gene, which produced fragments of 1.4 and 0.85 kb, respectively. PCR products were cloned and sequenced. The derived amino acid sequence was compared with the amino acid sequences of the HMW glutenin subunits Dy12^t, from T. tauschii, and subunits Dy10 and Dy12 of T. aestivum. The amino acid sequence deduced from the nucleotide sequence demonstrated that deletions of hexapeptides and nonapeptides were responsible for the reduction in the size of this HMW glutenin subunit. The estimated molecular weight of the Dy12.4^t subunit, calculated on the basis of the deduced amino acid sequence, was 45,228 Daltons. There were also single amino acid differences in the N-, C-terminal and central repetitive domains of this gene in comparison to the three other y-type subunits encoded at the Glu-D1 locus. The Dy12.4^t subunit showed the highest similarity to the Dy12 subunit present in the hexaploid wheat Chinese Spring.     

Molecular cloning and characterization of six novel HMW‐GS genes from Aegilops speltoides and Aegilops kotschyi

Six novel high molecular weight glutenin subunits (HMW‐GS) from Aegilops speltoides (SS, 2n = 2x = 14) and Aegilops kotschyi (UUSS, 2n = 4x = 28) were identified by SDS‐PAGE and designated as ASy15*, AKx1*, AKx3*, AKx2.3, AKy20* and AKy8*, respectively. Their complete open reading frames (ORFs) were cloned and sequenced by allele‐specific PCR (AS‐PCR). Sequence comparison demonstrated that these novel genes displayed high single‐nucleotide polymorphisms (SNP) and InDel variations. In particular, AKy8* had an extra cysteine residue at position 140 in the central repetitive domain, while AKx2.3 had an unusually long repetitive domain (816 aa), which might have positive effects on gluten quality. Phylogenetic analysis showed that AKx1* and AKx3* belonged to the 1S genome, AKx2.3 to the 1U genome, and AKy20* and AKy8* most likely to the 1S genome. The divergence between the 6 HMW‐GS genes from the two Aegilops species and those from Triticum species occurred 4.77–23.54 MYA. The authenticity of these isolated endogenous HMW‐GS genes was confirmed through heterologous expression in Escherichia coli and Western blotting.     

Identification of low-molecular weight glutenin subunits of wheat associated with bread-making quality

Although it is known that the compositions of low‐molecular weight glutenin subunits (LMW‐GSs) are important factors for bread‐making quality of wheat, it is still not clear which LMW‐GSs confer improved bread‐making quality and how those LMW‐GSs interact with high‐molecular weight (HMW) GSs. Using a hard red winter wheat line with good bread‐making quality and a Japanese wheat cultivar with poor quality as well as their progeny we identified LMW‐GSs associated with the bread‐making quality. One such LMW‐GS, KS2, which had a molecular weight of 42 kDa and was allelic to HS1, was associated with bread‐making quality. Furthermore, by using four recombinant inbred lines with different HMW‐GS and LMW‐GS combinations, KS2 and HMW‐GS 5+10 showed interaction effects on the bread‐making quality. Two‐dimensional polyacrylamide gel electrophoresis (Page) analysis showed that KS2 consists of two protein components and that HS1 is composed of three components. The N‐terminal amino acid sequences of the five components were identical to the most frequently analysed sequence of LMW glutenin components.     

一年生簇毛麦α-醇溶蛋白基因的分离、原核表达与功能鉴定

醇溶蛋白是面筋的主要成分之一,对小麦品质具有重要影响。根据数据库中全长α-醇溶蛋白基因设计了1对通用引物,从5份一年生簇毛麦(Dasypyrum villosum)品系中共得到52条序列,长度在816~873 bp之间(GenBank登录号为KJ004676~KJ004727)。核酸序列分析表明,其中有8条假基因,有1条(KJ004680)缺失终止密码子。推导氨基酸序列显示,KJ004677、KJ004686、KJ004714和KJ004696含有1个额外的Cys,其中,前3条序列由于Tyr→Cys所致,而KJ004696则由于Ser→Cys突变。序列间的差异主要出现在N-端重复区和多聚谷氨酰胺I区,根据N端重复区多肽序列的差异将一年生簇毛麦α-醇溶蛋白分为5种类型。为了分析具有额外Cys的α-醇溶蛋白所具有的品质效应,选取KJ004708(具有典型的6个Cys)和KJ004714(具有1个额外的Cys)分别构建表达载体,IPTG诱导后均得到分子量约30 kD的蛋白,与理论值相符;目的条带经切胶串联质谱鉴定证明,这2个α-醇溶蛋白基因在大肠杆菌中正确表达。对表达的蛋白亚基进行纯化、复性和低温冷冻干燥,经4 g粉质仪分析表明,KJ004708和KJ004714均能改善面团的加工品质,其中具有1个额外Cys的KJ004714亚基对面粉品质的改善更为显著。     

2··, a new high molecular weight glutenin subunit coded by Glu-A1: its predicted structure and its impact on bread-making quality

The suitability of wheat varieties for bread‐making depends on their glutenin subunits. The amino acid composition of these gluten building‐blocks have a strong influence on the rheology of the dough and, thus, on the suitability of the variety for bread‐making. This study reports a new x‐type high molecular weight glutenin subunit coded by the locus Glu‐A1 and named 2··. To investigate the impact of this allele on 10 quality parameters, a doubled haploid (DH) population of Triticum aestivum, segregating for Glu‐A1, was created. The statistical analysis demonstrates that, at Glu‐A1, the subunit 2·· is as favourable for quality as the subunit 2*. This is in accordance with results showing that the 2·· open reading frame still has the same number of cysteines as 2*. The small differences in the length of the central domain had no detectable effect on the elasticity, tenacity and baking quality, of the dough.     

Detection of HMW glutenin subunit variations among 205 cultivated emmer accessions (Triticum turgidum ssp. dicoccum)

The high molecular weight glutenin subunits (HMW‐GS) encoded by Glu‐1 loci among 205 accessions of cultivated emmer wheat (Triticum turgidum ssp. dicoccum Schrank) collected from different regions of Europe and China were separated and characterized by SDS‐PAGE in combination with two‐dimensional gel electrophoresis (A‐PAGE × SDS‐PAGE) and acidic capillary electrophoresis. High genetic polymorphisms in HMW‐GS compositions were found. A total of 40 alleles (6 for Glu‐A1 and 34 for Glu‐B1) and 62 subunit combinations (genotypes) were detected, some of which were not previously described. At Glu‐A1 locus, two novel alleles, designated Glu‐A1x coding for the subunit 1A × 1.1 and Glu‐A1y coding for the subunit 1A × 2.1′ were found while seven new subunits (1B × 17*, 1B × 6′, 1B × 13′, 1B × 20*, 1By9*, 1By14.1 and 1By8.1) and 20 novel alleles at Glu‐B1 locus were detected. In particular, some additional protein components were detected, which probably were 1Ay subunits encoded by Glu‐A1 locus. The introduction of both Ax and Ay subunits from tetraploid wheats into hexaploid wheats may increase the genetic variability of gluten genes and consequently improve flour technological properties.     

Cloning and characterization of a novel low molecular weight glutenin subunit gene at the Glu-A3 locus from wild emmer wheat (Triticum turgidum L. var. dicoccoides)

Amplification of the coding region, and upstream and downstream sequences of a low-molecular-weight glutenin subunits (LMW-GS) gene from wild emmer wheat (Triticum turgidum L. var. dicoccoides, 2n = 4x = 28, AABB) accession TD22 was carried out using designed allele-specific PCR (AS-PCR) primers. The complete 1,176 bp sequence of a novel LMW-i type subunit gene at the Glu-A3 locus, named LMW-TD22, is described. Analysis of the nucleotide and deduced amino acid sequences showed that this gene possessed striking characteristics although its molecular structure was generally similar to those of previously reported i-type LMW-GS genes that were isolated from common wheat and related species. The deduced amino acid sequence of LMW-TD22 gene contained 390 amino acid residues with the predicted molecular weight being 43,009.3 Da, which appeared to be the longest gene among the cloned LMW-i type genes from bread wheat and related species. The distinct feature of LMW-TD22 was two long polyglutamine stretches of 12 and 17 glutamines occurring in the repetitive and C-terminal domains as well as a cysteine residue present in the seventh amino acid residue of the signal peptide. These polyglutamine repeats are believed to improve the structure of gluten polymer and increase the strength of dough formed from the polymer. In addition, the putative 44 k subunit encoded by LMW-TD22 was verified by N-terminal microsequencing, gel electrophoresis and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis. Certain types of post-translational modifications, such as phosphorylation and glycosylation, may be associated with this LMW-i type subunit. A. Wang and Y. Xiao made equal contribution to the research as the first author.     

Generation and characterization of a high molecular weight glutenin 1Bx14-deficient mutant in common wheat

Significant progress has been made in understanding the structure of high molecular weight (HMW) glutenin subunits and their role in determining the end use quality of wheat grains. However, few reports have dealt with the development and characterization of knock out mutants for HMW glutenin subunit genes. Here, the molecular analysis of MB14, a mutant derived from an elite Chinese wheat variety Xiaoyan 54 through chemical mutagenesis is described. SDS‐PAGE and Western blot experiments revealed that, in the seeds of homozygous MB14 plants, the expression of the 1Bx14 subunit was specifically blocked whereas the remaining four subunits (1Ax1, 1By15, 1Dx2, 1Dy12) accumulated to levels comparable to those in the wild type plants. The 5′‐flanking region and the open reading frame (ORF) of the mutant 1Bx14 allele were amplified and compared to the corresponding regions of wild type 1Bx14. The nucleotide sequences of the 5′‐flanking regions from the mutant and wild type 1Bx14 alleles were identical. However, the ORF of the mutant allele differed from that of the wild type 1Bx14 by three point substitutions, one of which resulted in a premature stop codon in the mutant ORF. Interestingly, the mutant 1Bx14 allele was still transcribed in the developing seeds, but no truncated translation product could be detected by Western blot analysis. Potential application of the 1Bx14 knock out mutant in studying the biological function of 1Bx14 and its contribution to the end use quality control in hexaploid wheat is discussed.     

Identification and isolation of a new x-type HMW glutenin subunit 1Dx1.6t gene from Aegilops tauschii

A new x-type HMW glutenin subunit, designated as 1Dx1.6^t from Aegilops tauschii was identified and characterized by SDS-PAGE and MALDI-TOF-MS. This subunit is located between 1Dx2 and 1Dx1.5^t and possesses a molecular mass ( M _r) of 88565.8 Da. Its complete coding sequence was amplified via allele-specific PCR (AS-PCR), and the amplified product was cloned and sequenced. The authenticity of the cloned 1Dx1.6 ^t gene was confirmed by successful expression of its open reading frame in Escherichia coli. The molecular characterization of 1Dx1.6 ^t gene showed that its coding region consisted of 2541 bp encoding a polypeptide of 845 amino acid residues. Sequence comparison to previously characterized 1Dx1.5^t subunit which is related to good dough quality of bread wheat indicated that the 1Dx1.6^t subunit displayed high homology, but possesses 14 residue substitutions and a nonapeptide insertion. A total of 12 single-nucleotide polymorphisms (1 per 212 bp) was identified in the 1Dx1.6 ^t allele (11 in repetitive domain and 1 in the C-terminal domain), which could facilitate the design of AS-PCR markers.     

Relationship between common wheat (Triticum aestivum L.) gluten proteins and dough rheological properties

This paper reports the correlation between the rheological properties of bread wheat dough and the types and quantities of endosperm proteins in 28 common wheat cultivars. Different methods were used to analyse the allelic composition of these cultivars and the relative quantities of the different proteins contributing to the gluten structure. Neither dough strength (W) nor tenacity/extensibility (P/L) correlated with allelic composition. Different wheats with the same allelic composition (i.e., with respect to glutenins) showed different rheological properties. The glutenins were the most influential components of W and P/L, especially the high molecular weight (HMW) glutenin subunits and in particular the type x form. These proteins seem to increase W and are the main constituents of the gluten network. The gliadins and low molecular weight (LMW) glutenin subunits appear to act as a “solvent”, and thus modify the rheological properties of the dough by either interfering with the polymerisation of the HMW glutenin subunits, or by altering the relative amounts of the different types of glutenin available. Thus, the protein subunits coded for by the alleles Glu-B1x7 and Glu-D1x5 stabilised the gluten network, whereas those coded for by Glu-B1x17 and Glu-D1x2 had the opposite effect. Dough properties therefore appear to depend on the glutenin/gliadins balance, and on the ratio of the type x and type y HMW proteins. The influence of external factors seems to depend on the allelic composition of each cultivar.     

The high and low molecular weight glutenin subunit polymorphism of Algerian durum wheat landraces and old cultivars

The high molecular weight (HMW) and B‐zone low molecular weight (B‐LMW) glutenin subunit composition of 45 Algerian durum wheat (Triticum turgidum L. var. durum) landraces and old cultivars were examined by sodium‐dodecyl‐sulphate polyacrylamide gel electrophoresis (SDS‐PAGE). Nine accessions were heterogeneous and presented two or three genotypes. All together, 33 glutenin patterns were detected, including 12 for HMW and 15 for B‐LMW glutenin subunits. Twenty‐four different alleles were identified for the five glutenin loci studied, Glu‐A1 (3), Glu‐B1 (6), Glu‐A3 (8), Glu‐B3 (5) and Glu‐B2 (2). Five new alleles were found, three at Glu‐A3 and two at Glu‐B3. At the Glu‐1 loci, the Glu‐A1c‐Glu‐B1e allelic composition was predominant (31%). For the B‐LMW glutenins, the most common allelic composition was Glu‐A3a‐Glu‐B3a‐Glu‐B2a (36%). The collection analysed shows a high percentage of glutenin alleles and allele combinations related to high gluten strength, together with some others that have not been tested so far. This information could be useful to select local varieties with improved quality and also as a source of genes to develop new lines when breeding for quality.     

冰草高分子量麦谷蛋白亚基基因的分离及结构特征分析

通过SDS-PAGE分析，在二倍体、四倍体和六倍体冰草[Agropyron cristatum (L.) Gaertn.]中都检测到2个表达的高分子量麦谷蛋白亚基（HMW-GS），但不同倍性的材料之间，以及相同倍性材料的不同种子之间的HMW-GS组成均有所不同。利用PCR技术对冰草的HMW-GS基因进行克隆，结果从二倍体、四倍体和六倍体冰草中分别克隆了3个、1个和3个y型HMW-GS基因的全长编码区序列。序列分析表明，只有来自六倍体冰草中的Bsy2基因具有完整的开放阅读框，编码1个有487氨基酸残基、分子量约为53 kD的HMW-GS，大小相当于SDS-PAGE图谱中的大亚基。而其余6个基因均在中间重复区发生了无义突变。本文对冰草HMW-GS基因的结构特点和进化关系进行了分析。     

Influence of high molecular weight glutenin subunit substitution on rheological behaviour and bread-baking quality of near-isogenic lines developed from Chinese wheats

Three near‐isogenic lines (NILs) of wheat involving Glu‐B1 and Glu‐D1 alleles were used to study the genetic contribution of high molecular weight glutenin subunits (HMW‐GS) to gluten strength. The HMW‐GS composition of each NILs was determined by SDS‐PAGE. No significant differences were found in grain protein contents among the NILs. Gluten strength and dough‐mixing properties were measured by the Farinograph, the Extensograph, and SDS‐sedimentation (SDS‐SE). Results indicated that line 2, containing the Glu‐1B 14 + 15 and Glu‐1D 5 + 10 combination of subunits, had higher values for flour quality, dough rheological parameters, and bread‐baking quality when compared with lines 8 and 13. Line 8, containing Glu‐1B 7 + 9 and Glu‐1D 5 + 10, was better than line 13 with the Glu‐1B 14 + 15 and Glu‐1D 10 combination. Some major parameters appeared significantly different. The presence of Glu‐1B 14 + 15 was associated with higher dough strength based on SDS‐SE volume and several rheological parameters when compared with Glu‐1B 7 + 9. Lines with subunit 10 at Glu‐D1 performed significantly worse than those with 5 + 10 in gluten index, SDS‐SE volume, Farinograph stability time, Extensograph area and bread‐baking quality.     

High molecular weight glutenin subunit composition of Chinese bread wheats

Summary The endosperm storage proteins of 205 Chinese bread wheat cultivars and advanced lines were fractionated by SDS-PAGE to determine their high molecular weight (HMW) glutenin subunit composition. Seventeen alleles were identified: three at Glu-A1, eight at Glu-B1, and six at Glu-D1. The most common alleles were Null, 1, 7+8, 7+9, and 2+12. The results indicate that wheats from different regions differ in their frequencies of HMW glutenin subunits, however, none of the subunits could be related to specific environments. The glutenin quality scores of Chinese wheats ranged from 3 to 10, with an average of 6.7. Increasing quality scores have implications in improving steam-bread making quality for Chinese consumers. On the basis of HMW glutenin subunit composition, Chinese wheats are close to European wheats, especially Italian wheats because several Italian introductions are widely distributed in the pedigrees of Chinese wheat.     

Diversity of five glutenin loci within durum wheat (Triticum turgidum L. ssp. durum (Desf.) Husn.) germplasm grown in Algeria

The HMW and B‐LMW glutenin subunits composition of 120 durum wheat germplasm grown in Algeria was examined using SDS‐PAGE. All together, 39 glutenin patterns were detected, including eight for HMW and 21 for B‐LMW glutenin subunits. Twenty‐six different alleles were identified for the five glutenin loci studied, that is, Glu‐A1 (3), Glu‐B1 (7), Glu‐A3 (5), Glu‐B3 (9) and Glu‐B2 (2). Two new alleles were found at Glu‐B3 locus: Glu‐B3new₁ encodes for five subunits (7 + 8 + 14 + 16 + 18) and Glu‐B3new₂ codes for five subunits (4 + 6* + 12 + 15 + 15*), of which subunit 15* with mobility between bands 15–16 was not described previously. At the Glu‐1 loci, the Glu‐A1c/Glu‐B1e allelic composition was predominant. For the B‐LMW glutenins, the most common allelic composition was Glu‐A3a/Glu‐B3a/Glu‐B2a. The collection analysed shows glutenin alleles and allele combinations related to high gluten strength. This information could be useful to select varieties with improved quality and also as a source of genes to develop new lines when breeding for quality.     

Survey and new insights in the application of PCR‐based molecular markers for identification of HMW‐GS at the Glu‐B1 locus in durum and bread wheat

Wheat, among all cereal grains, possesses unique characteristics conferred by gluten; in particular, high molecular weight glutenin subunits (HMW‐GS) are of considerable interest as they strictly relate to bread‐making quality and contribute to strengthening and stabilizing dough. Thus, the identification of allelic composition, in particular at the Glu‐B1 locus, is very important to wheat quality improvement. Several PCR‐based molecular markers to tag‐specific HMW glutenin genes encoding Bx and By subunits have been developed in recent years. This study provides a survey of the molecular markers developed for the HMW‐GS at the Glu‐B1 locus. In addition, a selection of molecular markers was tested on 31 durum and bread wheat cultivars containing the By8, By16, By9, Bx17, Bx6, Bx14 and Bx17 Glu‐B1 alleles, and a new assignation was defined for the ZSBy9_aF1/R3 molecular marker that was specific for the By20 allele. We believe the results constitute a practical guide for results that might be achieved by these molecular markers on populations and cultivars with high variability at the Glu‐B1 locus.     

Allelic variation at high-molecular-weight glutenin subunit Loci, Glu-A1, Glu-B1 and Glu-D1, in Japanese and Chinese hexaploid wheats

Variation in the electrophoretic banding patterns of high-molecular-weight (HMW) glutenin subunits of 274hexaploid wheat (Triticum aestivum) varieties from China was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 27 different major HMW glutenin subunits were identified. Each variety contained three to five subunits and 29different glutenin subunit patterns were observed in274 Chinese hexaploid wheats. Seventeen alleles were identified based on the comparison of subunits mobility with that previously identified in a set of standard hexaploid wheats. The Chinese hexaploid wheats exhibited allelic variation in HMW glutenin subunit composition and the variation differed from that of Japanese and hexaploid wheats of other countries. This revised version was published online in July 2006 with corrections to the Cover Date.     

Changes in expression of HMW glutenins of wheat (Triticum aestivum L.) induced by nitrosoethylurea

The application of a chemical mutagen, N-nitroso-N-ethylurea, to the grains of wheat (Triticum aestivum L.) cv. ‘Viginta’, provided a mutant line,‘NT-10′, with an altered composition of high molecular weight (HMW) glutenin subunits. The qualitative mutation was detected in the Glu-B1 locus by electrophoretic analyses of glutenins. Instead of the HMW glutenin subunits 7 + 9 present in the original genotype, a separate HMW subunit 6 was expressed in the mutant line. The other glutenin and gliadin proteins of the mutant line remained unchanged. The mutant line is also characterized by several changes in morphological and physiological characters—stronger stem, wider leaf, bigger spike and higher grain hardness. This is the first communication of the possibility of changing the composition of high molecular weight subunits of wheat glutenin by means of mutagenesis.     

Variation of high-molecular-weight glutenin subunits amongst cultivars of Triticum turgidum L. from Portugal

Summary Variation in high-molecular-weight (HMW) glutenin subunit composition amongst 63 varieties of Triticum turgidum L. from Portugal was investigated using SDS-PAGE. A total of thirteen Glu-A1 and Glu-B1 alleles were identified, and three of them were found to be different from those previously described in the literature. A number of the tetraploid wheats examined contained subunits known to have a beneficial effect of the bread-making properties of T.aestivum. Camara, a tetraploid wheat cultivar carrying a 1D/1B chromosomal substitution, is proposed as a bridge for the transfer of Glu-D1 alleles and of other protein fractions controlled by the 1D chromosomes (Gli-D1 locus) from hexaploid to tetraploid wheat.