Characterization of low-molecular-weight glutenin subunit Glu-B3 genes and development of STS markers in common wheat (Triticum aestivum L.)

Low-molecular-weight glutenin subunit (LMW-GS) Glu-B3 has a significant influence on the processing quality of the end-use products of common wheat. To characterize the LMW-GS genes at the Glu-B3 locus, gene-specific PCR primers were designed to amplify eight near-isogenic lines and Cheyenne with different Glu-B3 alleles (a, b, c, d, e, f, g, h and i) defined by protein electrophoretic mobility. The complete coding regions of four Glu-B3 genes with complete coding sequence were obtained and designated as GluB3-1, GluB3-2, GluB3-3 and GluB3-4. Ten allele-specific PCR markers designed from the SNPs present in the sequenced variants discriminated the Glu-B3 proteins of electrophoretic mobility alleles a, b, c, d, e, f, g, h and i. These markers were validated on 161 wheat varieties and advanced lines with different Glu-B3 alleles, thus confirming that the markers can be used in marker-assisted breeding for wheat grain processing quality. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. L. H. Wang and X. L. Zhao contributed equally to this study.     

Characterization of low-molecular-weight glutenin subunit genes and their protein products in common wheats

To characterize the low-molecular-weight glutenin subunit (LMW-GS), we developed specific PCR primer sets to distinguish 12 groups of LMW-GS genes of Norin 61 and to decide their loci with nullisomic–tetrasomic lines of Chinese Spring. Three, two, and ten groups were assigned to Glu-A3, Glu-B3, and Glu-D3 loci, respectively. To identify the proteins containing the corresponding amino acid sequences, we determined the N-terminal amino acid sequence of 12 spots of LMW-GSs of Norin 61 separated by two-dimensional gel electrophoresis (2DE). The N-terminal sequences of the LMW-GS spots showed that 10 of 12 groups of LMW-GSs were expressed as protein products, which included LMW-i, LMW-m, and LMW-s types. Four spots were encoded by Glu-A3 (LMW-i). Three spots were encoded by Glu-B3 (LMW-m and LMW-s). Five spots were encoded by Glu-D3 (LMW-m and LMW-s). A minor spot of LMW-m seemed to be encoded by the same Glu-B3 gene as a major spot of LMW-s, but processed at a different site. Comparing among various cultivars, there were polymorphic and non-polymorphic LMW-GSs. Glu-A3 was highly polymorphic, i.e., the a, b, and c alleles showed one spot, the d allele showed four spots, and the e allele had no spot. Insignia used as one of the Glu-A3 null standard cultivars had a LMW-GS encoded by Glu-A3. We also found that Cheyenne had a new Glu-D3 allele. Classification of LMW-GS by a combination of PCR and 2DE will be useful to identify individual LMW-GSs and to study their contribution to flour quality.     

小麦近缘种低分子量麦谷蛋白亚基基因Glu-B3克隆及系统发育分析

发掘小麦近缘种低分子量麦谷蛋白基因, 可为小麦品质改良提供更多的基因资源。文章利用Glu-B3位点特异性标记LB1F/LB1R、LB2F/LB2R、LB3F/LB3R和 LB4F/LB4R, 对普通小麦B染色体组的7个可能供体近缘种, 即硬粒小麦(T. durum)、栽培二粒小麦(T. dicoccum)、野生二粒小麦(T. dicoccoides)、拟斯卑尔脱山羊草(Ae. speltoides)、高大山羊草(Ae. longissima)、西尔斯山羊草(Ae. searsii)和双角山羊草(Ae. bicornis)共20份材料进行PCR扩增, 克隆小麦近缘种中GluB3-1、GluB3-2、GluB3-3和GluB3-4基因的等位变异, 并对Glu-B3位点基因进行系统发育分析。共获得16个新等位变异, 其中GluB3-1基因的新等位变异1个, 命名为GluB3-16, 其推导氨基酸分子量为39.2 kDa; GluB3-3的新等位变异有3个, 分别命名为GluB3-35、GluB3-36和GluB3-37, 其推导氨基酸分子量为44.5 kDa(GluB3-36)或44.6 kDa(GluB3-35和GluB3-37); GluB3-4的新等位变异12个, 分别命名为GluB3-46、GluB3-47、GluB3-48、GluB3-49、GluB3-410、GluB3-411、GluB3-412、GluB3-413、GluB3-414、GluB3-415、GluB3-416和GluB3-417, 其推导氨基酸分子量变化在38.6(GluB3-414)~ 42.5 kDa(GluB3-413)之间; 16个新等位变异都包含单一的完整开放阅读框, 具有低分子量麦谷蛋白亚基的典型结构。文章进一步拓展了低分子量麦谷蛋白基因资源, 揭示不同Glu-B3基因的进化过程不完全相同, 为有效地利用小麦近缘种材料和转基因育种提供了新的基因资源。     

Identification of new low-molecular-weight glutenin subunit genes in wheat

To clarify the composition of low-molecular-weight glutenin subunits (LMW-GSs) in a soft wheat cultivar, we cloned and characterized LMW-GS genes from a cDNA library and genomic DNA in Norin 61. Based on alignment of the conserved N- and C- terminal domains of the deduced amino-acid sequences, these genes are classified into 12 groups. One of these groups (group 5), the corresponding gene of which has not been reported previously, contains two additional hydrophobic amino-acid clusters interrupting the N-terminal repetitive domain. Other groups (groups 11 and 12), which were not identified in other cultivars as a protein product, showed all eight cysteines in the C-terminal conserved domain. With specific primer sets for these groups it was revealed that Glu-D3 and Glu-A3 encoded the former and the latter, respectively. Both groups of genes were expressed in immature seeds. The presence of these groups of LMW-GSs may affect the dough strength of soft wheat. Received: 26 March 2001 / Accepted: 16 July 2001     

Novel DNA variations to characterize low molecular weight glutenin Glu-D3 genes and develop STS markers in common wheat

Low-molecular-weight glutenin subunits (LMW-GS) play an important role in bread and noodle processing quality by influencing the viscoelasticity and extensibility of dough. The objectives of this study were to characterize Glu-D3 subunit coding genes and to develop molecular markers for identifying Glu-D3 gene haplotypes. Gene specific primer sets were designed to amplify eight wheat cultivars containing Glu-D3a, b, c, d and e alleles, defined traditionally by protein electrophoretic mobility. Three novel Glu-D3 DNA sequences, designated as GluD3-4, GluD3-5 and GluD3-6, were amplified from the eight wheat cultivars. GluD3-4 showed three allelic variants or haplotypes at the DNA level in the eight cultivars, which were designated as GluD3-41, GluD3-42 and GluD3-43. Compared with GluD3-42, a single nucleotide polymorphism (SNP) was detected for GluD3-43 in the coding region, resulting in a pseudo-gene with a nonsense mutation at the 119th position of deduced peptide, and a 3-bp insertion was found in the coding region of GluD3-41, leading to a glutamine insertion at the 249th position of its deduced protein. The coding regions for GluD3-5 and GluD3-6 showed no allelic variation in the eight cultivars tested, indicating that they were relatively conservative in common wheat. Based on the 12 allelic variants of three Glu-D3 genes identified in this study and three detected previously, seven STS markers were established to amplify the corresponding gene sequences in wheat cultivars containing five Glu-D3 alleles (a, b, c, d and e). The seven primer sets M2F12/M2R12, M2F2/M2R2, M2F3/M2R3, M3F1/M3R1, M3F2/M3R2, M4F1/M4R1 and M4F3/M4R3 were specific to the allelic variants GluD3-21/22, GluD3-22, GluD3-23, GluD3-31, GluD3-32, GluD3-41 and GluD3-43, respectively, which were validated by amplifying 20 Chinese wheat cultivars containing alleles a, b, c and f based on protein electrophoretic mobility. These markers will be useful to identify the Glu-D3 gene haplotypes in wheat breeding programs. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Characterization of low-molecular-weight glutenin subunit genes from Hordeum brevisubulatum ssp. turkestanicum

Three novel low-molecular-weight glutenin subunit (LMW-GS) genes (designated as Ht1, Ht2, and Ht3) were isolated from the genomic DNA of Hordeum brevisubulatum ssp. turkestanicum by PCR amplification (accession no. Y0695). The coding regions of Ht1, Ht2, and Ht3 were 924, 924, and 903 bp, respectively. The deduced amino acid sequences were 306, 306, and 299 amino acid residues each with a signal peptide, a central repetitive region rich in proline and glutamine, and N-and C-terminal non-repetitive domains. A comparison was carried out of these genes with other known B hordein genes from cultivated barley and LMW glutenin genes from wheat. The results indicated that Ht1, Ht2, and Ht3 had a more similar structure and a higher level of homology with the LMW-GS genes than the B hordein genes. In order to investigate the evolutionary relationship of the novel genes with the prolamin genes from barley and wheat, the phylogenetic tree was constructed and the subfamilies of these prolamin genes were identified. The results suggested that the three novel genes were glutenin-like proteins designated as LMW-m type genes. The text was submitted by the authors in English.     

Isolation of low-molecular-weight glutenin subunit genes from wild emmer wheat (Triticum dicoccoides)

Three low-molecular-weight glutenin subunit (LMW-GS) genes, designated LMW-Td1, LMW-Td2 and LMW-Td3, were isolated from wild emmer wheat (Triticum dicoccoides), which is the tetraploid progenitor of common wheat (T. aestivum). The complete nucleotide sequence lengths of LMW-Td1, LMW-Td2 and LMW-Td3 are 858, 900 and 1062 bp, respectively. LMW-Td1 and LMW-Td3 can encode proteins with 284 and 352 amino acid residues, respectively, whereas LMW-Td2 is a putative pseudogene due to the presence of 3 inframe stop codons in its C-terminal domain. The deduced protein sequences of the 3 genes share the same typical polypeptide structures with known LMW-GS genes containing 8 cysteines in the mature protein domains. LMW-Td1 was clearly distinguished from all known LMW-GS genes, and considered as a novel LMW-GS gene. Two hydrophobic motifs (i.e. PIIIL and PVIIL) were observed in the repetitive domain of LMW-Td3. Sequence comparison indicates that sequences of the 3 LMW-GS genes from this study are strongly similar to known LMW-GS genes. Our phylogenetic analysis suggests that LMW-Td1 and LMW-Td2 are homologous with genes on chromosome 1A, and LMW-Td3 is closely related to genes on chromosome 1B.     

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     

Chromosomal location of genes for Rubisco small subunit and Rubisco-binding protein in common wheat

Summary The genes coding for the Rubisco small subunit (SSU) and for the -subunit of the Rubisco-binding protein were located to chromosome arms of common wheat. HindIII-digested total DNA from the hexaploid cultivar Chinese Spring and from ditelosomic and nullisomic-tetrasomic lines was probed with these two genes, whose chromosomal location was deduced from the disappearance of or from changes in the relative intensity of the relevant band(s). The Rubisco SSU pattern consisted of 14 bands, containing at least 21 different types of DNA fragments, which were allocated to two homoeologous groups: 15 to the short arm of group 2 chromosomes (4 to 2AS, 7 to 2BS, and 4 to 2DS) and 6 to the long arm of group 5 chromosomes (2 on each of arms 5AL, 5BL, and 5DL). The pattern of the Rubisco-binding protein consisted of three bands, each containing one type of fragment. These fragments were located to be on the short arm of group 2 chromosomes. The restriction fragment length polymorphism (RFLP) patterns of several hexaploid and tetraploid lines were highly conserved, whereas the patterns of several of their diploid progenitors were more variable. The variations found in the polyploid species were mainly confined to the B genome. The patterns of the diploids T. monococcum var. urartu and Ae. squarrosa were similar to those of the A and D genome, respectively, in polyploid wheats. The pattern of T. monococcum var. boeoticum was different from the patterns of the A genome, and the patterns of the diploids Ae. speltoides, Ae. longissima, and Ae. Searsii differed from that of the B genome.     

Analysis and validation of genome-specific DNA variations in 5′ flanking conserved sequences of wheat low-molecular-weight glutenin subunit genes

The thirty-three 5′ flanking conserved sequences of the known low-molecular-weight subunit (LMW-GS) genes have been divided into eight clusters, which was in agreement with the classification based on the deduced N-terminal protein sequences. The DNA polymorphism between the eight clusters was obtained by sequence alignment, and a total of 34 polymorphic positions were observed in the approximately 200 bp regions, among which 18 polymorphic positions were candidate SNPs. Seven cluster-specific primer sets were designed for seven out of eight clusters containing cluster-specific bases, with which the genomic DNA of the ditelosomic lines of group 1 chromosomes of a wheat variety ‘Chinese Spring’ was employed to carry out chromosome assignment. The subsequent cloning and DNA sequencing of PCR fragments validated the sequences specificity of the 5′ flanking conserved sequences between LMW-GS gene groups in different genomes. These results suggested that the coding and 5′ flanking regions of LMW-GS genes are likely to have evolved in a concerted fashion. The seven primer sets developed in this study could be used to isolate the complete ORFs of seven groups of LMW-GS genes, respectively, and therefore possess great value for further research in the contributions of a single LMW-GS gene to wheat quality in the complex genetic background and the efficient selections of quality-related components in breeding programs.     

Analysis and validation of genome-specific DNA variations in 5' flanking conserved sequences of wheat low-molecular-weight glutenin subunit genes

The thirty-three 5' flanking conserved sequences of the known low-molecular-weight subunit (LMW-GS) genes have been divided into eight clusters, which was in agreement with the classification based on the deduced N-terminal protein sequences. The DNA polymorphism between the eight clusters was obtained by sequence alignment, and a total of 34 polymorphic positions were observed in the approximately 200 bp regions, among which 18 polymorphic positions were candidate SNPs. Seven cluster-specific primer sets were designed for seven out of eight clusters containing cluster-specific bases, with which the genomic DNA of the ditelosomic lines of group 1 chromosomes of a wheat variety 'Chinese Spring' was employed to carry out chromosome assignment. The subsequent cloning and DNA sequencing of PCR fragments validated the sequences specificity of the 5' flanking conserved sequences between LMW-GS gene groups in different genomes. These results suggested that the coding and 5' flanking regions of LMW-GS genes are likely to have evolved in a concerted fashion. The seven primer sets developed in this study could be used to isolate the complete ORFs of seven groups of LMW-GS genes, respectively, and therefore possess great value for further research in the contributions of a single LMW-GS gene to wheat quality in the complex genetic background and the efficient selections of quality-related components in breeding programs.     

Length variations of i-type low-molecular-weight glutenin subunit genes in diploid wheats

Allelic variation of the low-molecular-weight glutenin subunit (LMW-GS) is associated with the significant differences of dough quality in bread and durum wheat, and has been widely evaluated at protein level in wheat and its relatives. In this study, a PCR primer set, targeting the high variable repetitive domains, was employed to assay the length variation of i-type LMW-GS genes in the A-genomes of diploid wheats, the diploid progenitors of tetraploid and hexaploid wheat. A total of 71 accessions of diploid wheats, belonging to two wild and one cultivated species, were investigated. The higher variations of repetitive length in i-type LMW-GS genes were found in diploid wheats with Nei's genetic variation index (H) of 0.834. The two wild species, T. boeoticum and T. urartu, were found to possess the similar degree of variability, with the Nei's genetic variation index of 0.806 and 0.783, respectively. Less variations were detected in T. monococcum (H = 0.680), a cultivated species domesticated from T. boeoticum. The sufficient variations found in this study could be used as valuable sources for the enrichment of the genetic variations and the alteration of flour-processing properties of the cultivated wheat. To our knowledge, it was the first time that an analysis of length variation targeting a particular group of genes of LMW-GS complex multigene families was conducted.     

Length variation of i-type low-molecular-weight glutenin subunit genes in diploid wheats

Allelic variation of the low-molecular-weight glutenin subunit (LMW-GS) is associated with the significant differences of dough quality in bread and durum wheat, and has been widely evaluated at protein level in wheat and its relatives. In this study, a PCR primer set, targeting the high variable repetitive domains, was employed to assay the length variation of i-type LMW-GS genes in the A-genomes of diploid wheats, the diploid progenitors of tetraploid and hexaploid wheat. A total of 71 accessions of diploid wheats, belonging to two wild and one cultivated species, were investigated. The higher variations of repetitive length in i-type LMW-GS genes were found in diploid wheats with Nei’s genetic variation index (H) of 0.834. The two wild species, T. boeoticum and T. urartu, were found to possess the similar degree of variability, with the Nei’s genetic variation index of 0.806 and 0.783, respectively. Less variation was detected in T. monococcum (H = 0.680), a cultivated species domesticated from T. boeoticum. The sufficient variation found in this study could be used as valuable source for the enrichment of genetic variations and the alteration of flour-processing properties of the cultivated wheat. To our knowledge, it was the first time that an analysis of length variation targeting a particular group of genes of LMW-GS complex multigene families was conducted. This article was submitted by the authors in English.     

Variation and classification of B low-molecular-weight glutenin subunit alleles in durum wheat

 The B low-molecular-weight (LMW) glutenin subunit composition of a collection of 88 durum wheat cultivars was analyzed. Extensive variation has been found and 18 different patterns were detected. Each cultivar exhibited 4–8 subunits, and altogether 20 subunits of different mobility were identified. The genetic control of all these subunits was determined through the analysis of nine F₂ populations and one backcross. Five subunits were controlled at the Glu-A3 locus, 14 at Glu-B3 and 1 at Glu-B2. At the Glu-A3 locus each cultivar possessed from zero to three bands and eight alleles were identified. At the Glu-B3 locus each cultivar showed four or five bands and nine alleles were detected. Only one band was encoded by the Glu-B2 locus. A nomenclature for these alleles is proposed and the relationship between them and the commonly used LMW-model nomenclature is discussed. Received: 10 February 1997 / Accepted: 25 April 1997     

Characterization of three homoeologous cDNAs encoding chloroplast-targeted aminolevulinic acid dehydratase in common wheat

In the tetrapyrrole biosynthetic pathway of higher plants, 5-aminolevulinic acid (ALA) is metabolized by ALA dehydratase (ALAD). Here, we isolated ALAD1 cDNA from common wheat (Triticum aestivum L.) and its diploid progenitors, and produced transgenic tobacco plants expressing the wheat ALAD1 gene. The ALAD1 genes were highly conserved among wheat relatives, and three homoeologous loci of wheat ALAD1 (TaALAD1) were equally transcribed in common wheat. A transient expression assay of a TaALAD1-GFP (green fluorescent protein) fusion protein suggested that TaALAD1 is localized in chloroplasts. Overexpression of TaALAD1 in transgenic tobacco resulted in a significant increase in ALAD activity in leaves. Moreover, the transgenic tobacco showed vigorous growth and increased survival rate on medium containing ALA at herbicidal concentrations. These results indicate that wheat ALAD1 has catalytic activity in metabolizing ALA in plastids, and that ectopic expression of TaALAD1 in transgenic plants increases their tolerance to ALA application at high concentrations.     

Identification and chromosomal location of four subfamilies of the rubisco small subunit genes in common wheat

Summary Three different 3 noncoding sequences of wheat rubisco small subunit (SSU) genes (RbcS) were used as probes to identify the gene members of different RbcS subfamilies in the common wheat cultivar Chinese Spring (CS). All genes of the wheat RbcS multigene family were previously assigned to the long arm of homoeologous group 5 and to the short arm of homoeologous group 2 chromosomes of cv CS. Extracted DNA from various aneuploids of these homoeologous groups was digested with four restriction enzymes and hybridized with three different 3 noncoding sequences of wheat SSU clones. All RbcS genes located on the long arm of homoeologous group 5 chromosomes were found to comprise a single subfamily, while those located on the short arm of group 2 comprised three subfamilies. Each of the ancestral diploid genomes A, B, and D has at least one representative gene in each subfamily, suggesting that the divergence into subfamilies preceded the differentiation into species. This divergence of the RbcS genes, which is presumably accompanied by a similar divergence in the 5 region, may lead to differential expression of various subfamilies in different tissues and in different developmental stages, in response to different environmental conditions. Moreover, members of one subfamily that belong to different genomes may have diverged also in the coding sequence and, consequently, code for distinguishable SSU. It is assumed that such utilization of the RbcS multigene family increases the adaptability and phenotypic plasticity of common wheat over its diploid progenitors.     

Characterization of low-molecular-weight glutenin subunit genes of Aegilops section Sitopsis and comparative analysis with those of wheat (Triticum aestivum L.) and some Aegilops species

Journal of Genetics -