The low-molecular-weight glutenin subunit proteins of primitive wheats. II. The genes from A-genome species

 Three accessions of T. boeoticum were selected for the cloning and sequencing of novel low-molecular-weight glutenin subunit (LMW-GS) genes, based on the results of SDS-PAGE and PCR analyses of the LMW-GS diversity in A-genome wheat (Lee et al. 1998 a). A comparison of the nucleotide and deduced amino-acid sequences of three cloned genes, LMWG-E2, LMWG-E4 and LMWG-AQ1, both to each other and to other known LMW-GS genes was carried out. The N-terminal domains showed one variable position; GAG (coding for a glutamic acid) for the E-type, and GAT (coding for an aspartic acid) for the Q-type. The comparisons of the LMW-GSs in the literature and this paper define three different types of N-terminal sequences; METSCIPGLERPW and MDTSCIPGLERPW from the durum and A-genome wheats, and METRCIPGLERPW from the hexaploid and D-genome wheats. The repetitive domains were AC-rich at the nucleotide level and coded for a large number of glutamine residues; this region showed 16 variable positions changing 12 amino-acid residues, three triple nucleotide deletions/additions, a large deletion of 18 nucleotides in LMWG-E4 and a deletion of 12 nucleotides in LMWG-E2. In the C-terminal domains 26 variable positions were found and 12 of these mutations changed amino-acid residues; no deletions/ additions were present in this region. It was shown that the LMWG-E2 and LMWG-E4 genes could be expressed in bacteria and this allowed the respective protein products to be related back to the proteins defined as LMW-GSs in vivo. Received: 24 November 1997 / Accepted: 18 August 1998     

Sequence similarity between allelic Glu-B3 genes related to quality properties of durum wheat

 Low-molecular-weight glutenin subunits (LMW-GSs) are wheat endosperm proteins mostly encoded by genes located at the Glu-3 loci. These proteins are of particular interest in durum wheat because a correlation between LMW-GSs encoded by genes at the Glu-B3 locus and the pasta-making quality of durum wheat semolina has been shown. We isolated and characterized two allelic lmw-gs genes located at the Glu-B3 locus and present in durum wheat lines displaying different qualitative properties. The clones pLMW1CL and λLMW3.1 were found to contain allelic sequences encoding LMW-GSs belonging to the good and poor quality-related groups named LMW-2 and LMW-1, respectively. The LMW-GSs specified by these genes have very large repetitive domains which are composed of repeats regularly distributed along the domain. The main difference between these two proteins is an insertion of 13 amino acids within the repetitive domain which, by itself, seems insufficient to explain the qualitative differences between LMW-2 and LMW-1. These results further support the hypothesis that the greater amount of LMW-2, rather than sequence peculiarities, accounts for the better quality observed in durum wheat cultivars possessing these subunits. The characterization of the complete primary structure of these alleles, other than providing information for an understanding of the structure-function relationship among LMW-GSs and furnishing basic material for wheat engineering, should also assist in our understanding of the evolutionary relationship between the different lmw-gs genes. Received: 8 May 1998 / Accepted: 5 August 1998     

The low-molecular-weight glutenin subunit proteins of primitive wheats. I. Variation in A-genome species

 A Tris-Tricine gel-electrophoresis system (Schaegger and von Jagow 1987), combined with a gradient gel, has been employed to provide an improved resolution of the B and C low-molecular-weight glutenin subunits (LMW-GSs) found in the endosperm of wheat grain. The gel system was used to document the variation in the gluten subunit proteins present in A-genome diploid wheats. The majority of LMW-GSs found in the A-genome diploid wheats were not present in normal bread wheats; the data suggest that they represent a rich source of new variation for the LMW-GSs which are considered to be very important in modulating wheat flour-processing properties. The analysis of variation in the nature of the LMW-GS genes, using PCR, demonstrated that the subclass of C-subunits assayed by primers from a previously published sequence did not show as much variation as the proteins. However, the data collected suggest that sufficient variation may exist in the LMW-GS genes of A-genome diploid wheats to use them as a source of genes for altering the flour-processing properties of hexaploid wheat. Received: 24 November 1997 / Accepted: 18 August 1998     

The low-molecular-weight glutenin subunit proteins of primitive wheats. III. The genes from D-genome species

 The isolation and characterisation by DNA sequencing of two different low molecular weight glutenin subunit (LMW-GS) genes from a genomic library derived from Triticum tauschii is described. These genes are similar (more than 90% similarity) but not identical to previously published LMW-GS gene sequences from cultivated wheats. A comparison of nucleotide sequence of the coding regions revealed the presence of insertions and deletions preferentially located in the region encoding the domains in the LMW-GS proteins rich in proline and glutamine and the middle part of the C-domain. The signal sequences, the amino-terminus and the remaining parts of the C-domain were conserved between all the LMW-GSs compared. The differences detected between the deduced amino-acid sequences in these three regions are only due to single nucleotide substitutions. The most important characteristic of all compared LMW-GS genes is the conservation of eight cysteine residues that could be involved in potential secondary or tertiary structure and disulphide-bond interactions. Comparisons between the 5′ and 3′ non-coding sequences of one of the isolated clones (LMW-16/10) with those of different prolamin genes from wheat, barley and rye led to the distinction of five different gene families, and confirmed the evolutionary relationships determined previously for these genes mainly on the basis of the coding region. In particular, the LMW-GS sequences are more closely related to the B-hordein sequences than to any other prolamin genes from wheat, barley and rye. Formal proof that the isolated genes coded for LMW-GSs, as defined by gel electrophoresis, was obtained by moving one of these genes (LMW-16/10) into a bacterial expression vector based on bacteriophage T7 RNA polymerase. The resulting plasmid directed the synthesis of large amounts of the mature form of the subunit in Escherichia coli. This protein exhibited solubility characteristics identical to those of the LMW-GSs and cross-reacted with antibodies reactive with these proteins. Received: 24 November 1997 / Accepted: 18 August 1998     

Cloning and Expression of Low Molecular Weight Glutenin Genes from the Chinese Elite Wheat Cultivar ＂Xiaoyan 54＂

The low molecular weight （LMW） glutenln subunlts account for 40% of wheat gluten protein content by mass and these proteins are considered to significantly affect dough quality characteristics. Five new full-length LMW glutenln genes （designated LMW-5, LMW-7, LMW-42, LMW-58, and LMW-34） were isolated from the Chinese elite wheat cultivar ＂Xlaoyan 54＂ by PCR amplification of genomlc DNA using a pair of degenerate primers designed from the conserved sequences of the N- and C-terminal regions of published LMW glutenln genes. Deduced amino acid sequence analysis showed that LMW-5 belongs to the LMW-i type genes and that the other four belong to LMW-m type genes. Sequence comparisons revealed that point mutations occasionally occurred in signal peptide and N-terminus domains and often existed in domain III and domain V. Small insertions and deletions are represented in the repetitive domain. There is a stop codon after amino acid position 110 In the repetitive domain of LMW.34, indicating that It is a pseudogene. The other four genes have complete open reading frames and the putative mature regions of these genes were subcloned Into pET-30a expression vector and successfully expressed in Escherlchla coll. Protein sodium dodecyl sulfate-polyacrylamlde gel electro- phoresls analysis showed that all proteins expressed in E. coil by the four genes could be related to B-group LMW glutenln subunits of wheat.     

Development of a new marker system for identifying the complex members of the low-molecular-weight glutenin subunit gene family in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Low-molecular-weight glutenin subunits (LMW-GSs) play an important role in determining the bread-making quality of bread wheat. However, LMW-GSs display high polymorphic protein complexes encoded by multiple genes, and elucidating the complex LMW-GS gene family in bread wheat remains challenging. In the present study, using conventional polymerase chain reaction (PCR) with conserved primers and high-resolution capillary electrophoresis, we developed a new molecular marker system for identifying LMW-GS gene family members. Based on sequence alignment of 13 LMW-GS genes previously identified in the Chinese bread wheat variety Xiaoyan 54 and other genes available in GenBank, PCR primers were developed and assigned to conserved sequences spanning the length polymorphism regions of LMW-GS genes. After PCR amplification, 17 DNA fragments in Xiaoyan 54 were detected using capillary electrophoresis. In total, 13 fragments were identical to previously identified LMW-GS genes, and the other 4 were derived from unique LMW-GS genes by sequencing. This marker system was also used to identify LMW-GS genes in Chinese Spring and its group 1 nulli–tetrasomic lines. Among the 17 detected DNA fragments, 4 were located on chromosome 1A, 5 on 1B, and 8 on 1D. The results suggest that this marker system is useful for large-scale identification of LMW-GS genes in bread wheat varieties, and for the selection of desirable LMW-GS genes to improve the bread-making quality in wheat molecular breeding programmes.     

Molecular characterization of a low-molecular-weight glutenin cDNA clone from Triticum durum

Summary A full-length, low-molecular-weight (LMW) glutenin cDNA clone, pTdUCD1, has been isolated from a Triticum durum cv Mexicali wheat cDNA library. The complete sequence was determined and compared to the LMW glutenin genes that have been isolated from hexaploid wheat, Triticum aestivum. This cDNA codes for a protein of 295 amino acids (33,414 daltons) including a 20-amino acid signal peptide as deduced from the DNA sequence. Northern analysis showed that this cDNA hybridizes to a family of related sequences ranging in length from 1,200 to 1,000 nucleotides. This gene is similar but not identical to previously published LMW glutenin gene sequences. The most striking characteristic of all cloned LMW glutenin genes is the conservation of eight cysteine residues, which could be involved in potential secondary or tertiary structure, disulfide bond interactions. This paper presents a structural map defining distinct regions of the LMW glutenin gene family.     

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.     

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.     

The low-molecular-weight glutenin subunit proteins of primitive wheats. IV. Functional properties of products from individual genes

 Three genes encoding the low-molecular-weight glutenin subunits (LMW-GSs), LMWG-E2 and LMWG-E4, from A-genome diploid wheat species, and LMW-16/10 from a D-genome diploid wheat, were expressed in bacteria. The respective proteins were produced on a relatively large scale and compared with respect to their effects on flour-processing properties such as dough mixing, extensibility and maximum resistance; these are important features in the end-use of wheat for producing food products. The LMWG-E2 and LMWG-E4 proteins caused significant increases in peak resistance and mixing time, compared to the control, when incorporated into dough preparations. The LMWG-16/10 protein was qualitatively less effective in producing these changes. All three proteins also conferred varying degrees of decrease in dough breakdown. LMWG-E2 and LMWG-E4 caused significant increases in dough extensibility, and decreases in maximum resistance, relative to the control. LMW-16/10 did not show a significant effect on extensibility but showed a significant decrease in maximum resistance. The refinement of relating specific features of the structure of the LMW-GS genes to the functional properties of their respective proteins is discussed. Received: 24 November 1997 / Accepted: 18 August 1998     

Characterisation of two gene subunits on the 1R chromosome of rye as orthologs of each of the Glu-1 genes of hexaploid wheat

The visco-elastic properties of bread flour are firmly associated with the presence or absence of certain HMW subunits coded by the Glu-1 genes. Identifying allelic specific molecular markers (AS-PCR) associated with the presence of Glu-1 genes can serve as a valuable tool for the selection of useful genotypes. This paper reports the use of primers designed from nucleotide sequences of the Glu-D1 gene of wheat (AS-PCR for Glu-D1y10) that recognise and amplify homologous sequences of the Glu-R1 gene subunits of rye. The primers amplify the complete coding regions and provided two products of different size in rye, in wheats carrying the substitution 1R(1D) and in rye-wheat aneuploid lines carrying the long arm of chromosome 1R. The location, the molecular characterisation of these sequences and their expression during grain ripening seem to demonstrate that the amplification products correspond to structural genes encoding the high-molecular-weight (HMW) glutenins of rye. The homology of the rye gene to subunits encoding HMW glutenins in wheat was confirmed by Southern blots and sequencing. The amplification-products were cloned, sequenced and characterised, and the sequences compared with the main glutenin subunits of wheat and related species. Further, an RT-PCR experiment was performed using primers designed from the sequence of both amplified products. This assay demonstrated that both sequences are expressed in endosperm during grain ripening. The results of these analyses suggest that both gene subunits correspond to x- and y-type genes of the Glu-R1 locus of rye. Received: 11 December 2000 / Accepted: 17 April 2001     

Locus-specific primers for LMW glutenin genes on each of the group 1 chromosomes of hexaploid wheat

To reveal the chromosomal location of three known low-molecular-weight (LMW) glutenin genes in wheat, we designed and used three sets of sequence-specific primers in polymerase chain reactions (PCR) on Chinese Spring and its derived group 1 aneuploid nullisomic-tetrasomic stocks. Two sets proved to be chromosome specific and amplified sequences from the Glu-A3 and Glu-D3 loci, respectively. The third set was apparently composed of conserved sequences as it produced PCR products in each of the aneuploids. Two of these products were cloned, and their sequences differed from the known LMW glutenin genes at several positions. Again, primer sets specific for these sequences were designed. One set was directed to the Glu-A3 locus, the second set resulted in two PCR products differing in length, one of which was located on chromosome 1B and the other on 1D. Primer sets constructed for the latter two sequences were specific for the Glu-B3 and Glu-D3 loci, respectively. Hence, primer sets specific for each of the three homoeologous chromosomes of the group 1 (1A, 1B, 1D) are available. In addition, these locus-specific primers were assayed for their ability to distinguish among wheat cultivars. PCR products amplified with one of the Glu-A3-specific primer sets showed length polymorphisms in various wheat varieties. Varieties carrying the 1RS.1BL translocated chromosomes could be recognized by the absence of a PCR product when the Glu-B3 primer set was used. These results suggest that PCR with locus-specific primers can be useful in the molecular genetic analysis of hexaploid wheat.     

小麦新品种“川麦42”低分子量谷蛋白亚基新基因的分子克隆

采用PCR方法从小麦（Triticum aestivum L.）新品种“川麦42”中克隆得到一个低分子量谷蛋白亚基（LMW-GS）新基因,暂命名为LMWCM42-1。该基因编码区全长846 bp,编码281个氨基酸,具有LMW-GS基因的典型结构特征。推导氨基酸序列比较显示,尽管LMWCM42-1与已知LMW-GS高度相似,但在N-末端重复区部分重复单元和C-末端区中仍存在明显差异。聚类分析表明,LMWCM42-1可能是由Glu-D3位点编码的。     

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 characterization and genomic organization of low molecular weight glutenin subunit genes at the Glu-3 loci in hexaploid wheat (Triticum aestivum L.)

In this study, we report on the molecular characterization and genomic organization of the low molecular weight glutenin subunit (LMW-GS) gene family in hexaploid wheat (Triticum aestivum L.). Eighty-two positive BAC clones were identified to contain LMW-GS genes from the hexaploid wheat ‘Glenlea’ BAC library via filter hybridization and PCR validation. Twelve unique LMW glutenin genes and seven pseudogenes were isolated from these positive BAC clones by primer-template mismatch PCR and subsequent primer walking using hemi-nested touchdown PCR. These genes were sequenced and each consisted of a single-open reading frame (ORF) and untranslated 5′ and 3′ flanking regions. All 12 LMW glutenin subunits contained eight cysteine residues. The LMW-m-type subunits are the most abundant in hexaploid wheat. Of the 12 LMW-GS, 1, 2 and 9 are i-type, s-type and m-type, respectively. The phylogenetic analysis suggested that the LMW-i type gene showed greater differences to LMW-s and LMW-m-type genes, which, in turn, were more closely related to one another. On the basis of their N-terminal sequences, they were classified into nine groups. Fingerprinting of the 82 BAC clones indicated 30 BAC clones assembled into eight contigs, while the remaining clones were singletons. BAC end sequencing of the 82 clones revealed that long terminal repeat (LTR) retrotransposons were abundant in the Glu-3 regions. The average physical distance between two adjacent LMW-GS genes was estimated to be 81 kb. Most of LMW-GS genes are located in the d-genome, suggesting that the Glu-D3 locus is much larger than the Glu-B3 locus and Glu-A3 locus. Alignments of sequences indicated that the same type (starting with the same N-terminal sequence) LMW-GS genes were highly conserved in the homologous genomes between hexaploid wheat and its donors such as durum wheat and T. tauschii. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Spelt-specific alleles in HMW glutenin genes from modern and historical European spelt ( Triticum spelta L.)

A partial promoter region of the high-molecular weight (HMW) glutenin genes was studied in two wheat specimens, a 300 year-old spelt (Triticum spelta L.) and an approximately 250 year-old bread wheat (Triticum aestivum L.) from Switzerland. Sequences were compared to a recent Swiss landrace T. spelta ’Oberkulmer.’ The alleles from the historical bread wheat were most similar to those of modern T. aestivum cultivars, whereas in the historical and the recent spelt specific alleles were detected. Pairwise genetic distances up to 0.03 within 200 bp from the HMW Glu-A1-2, Glu-B1-1 and Glu-B1-2 alleles in spelt to the most-similar alleles from bread wheat suggest a polyphyletic origin. The spelt Glu-B1-1 allele, which was unlike the corresponding alleles in bread wheat, was closer related to an allele found in tetraploid wheat cultivars. The results are discussed in context of the origin of European spelt. Received: 22 July 2000 / Accepted: 27 April 2001     

Mapping of FASN and ACACA on two chicken microchromosomes disrupts the human 17q syntenic group well conserved in mammals

Fatty acid synthase and Acetyl-CoA carboxylase are both key enzymes of lipogenesis and may play a crucial role in the weight variability of abdominal adipose tissue in the growing chicken. They are encoded by the FASN and ACACA genes, located on human Chromosome (Chr) 17q25 and on Chr 17q12 or 17q21 respectively, a large region of conserved synteny among mammals. We have localized the homologous chicken genes FASN and ACACA coding for these enzymes, by single-strand conformation polymorphism analysis on different linkage groups of the Compton and East Lansing consensus genetic maps and by FISH on two different chicken microchromosomes. Although synteny is not conserved between these two genes, our results revealed linkage in chicken between FASN and NDPK (nucleoside diphosphate kinase), a homolog to the human NME1 and NME2 genes (non-metastatic cell proteins 1 and 2), both located on human Chr 17q21.3, and also between FASN and H3F3B (H3 histone family 3B), located on human Chr 17q25. The analysis of mapping data from the literature for other chicken and mammalian genes indicates rearrangements have occurred in this region in the mammalian lineage since the mammalian and avian radiation. Received: 8 August 1997 / Accepted: 24 November 1997     

Genome-wide identification of PYL gene family in wheat: Evolution,expression and 3D structure analysis

Here, 38 wheat PYL genes (TaPYLs) belonging to 13 homoeologous groups were identified using the genome-search method, with 26 and 12 PYL genes identified in Triticum dicoccoides and Aegilops tauschii, respectively. Phylogenetic relationship, conserved domain and molecular evolution analysis revealed that PYL genes showed highly conservative between wheat and theprogenitors. Interaction network and miRNA target prediction found that TaPYLs could interact with the important components of ABA signaling pathway and Tae-miR966b-3p might be a hub regulator mediating wheat ABA signal network. Furthermore, the tissue-specific and stress-responsive TaPYLs were detected through RNA-seq analysis. Expressions of 10 TaPYLs were validated by QPCR analysis and the homoeologous genes showed significantly differential expression, suggesting subfunctionalization of them has occurred. Finally, 3D structures of the TaPYL proteins were predicted by homology modeling. This study lays the foundation for further functional study of PYL genes for development and stress tolerance improvement in wheat and beyond.