Low Molecular Weight Glutenin Genes Research Articles

Discriminative Features of Some High and Low Molecular Weight Glutenin Genes in Different Wheat Species

Discriminative Features of Some High and Low Molecular Weight Glutenin Genes in Different Wheat Species

Quantitative trait loci influencing end-use quality traits of hard red spring wheat breeding lines

Wheat bread-making quality is influenced by a complex group of traits including dough visco-elastic characteristics. In this study, quantitative trait locus/loci (QTL) mapping and analysis were conducted for endosperm polymeric proteins together with dough mixing strength and bread-making properties in a population of 139 (MN98550 × MN99394) recombinant inbred lines that was evaluated at three environments in 2006. Eleven chromosome regions were associated with endosperm polymeric proteins, explaining 4.2–31.8% of the phenotypic variation. Most of these polymeric proteins QTL coincided with several QTL for dough-mixing strength and bread-making properties. Major QTL clusters were associated with the low-molecular weight glutenin gene Glu-A3, the two high-molecular weight glutenin genes Glu-B1 and Glu-D1, and two regions on chromosome 6D. Alleles at these QTL clusters have previously been proven useful for wheat quality except one of the 6D QTL clusters.

Genome-wide identification of QTL conferring high-temperature adult-plant (HTAP) resistance to stripe rust (Puccinia striiformis f. sp. tritici) in wheat

High-temperature adult-plant (HTAP) resistance to stripe rust (caused by Puccinia striiformis f. sp. tritici) is a durable type of resistance in wheat (Triticum aestivum L.). This study identified quantitative trait loci (QTL) conferring HTAP resistance to stripe rust in a population consisting of 169 F8:10 recombinant inbred lines (RILs) derived from a cross between a susceptible cultivar Rio Blanco and a resistant germplasm IDO444. HTAP resistance was evaluated for both disease severity and infection type under natural infection over two years at two locations. The genetic linkage maps had an average density of 6.7 cM per marker across the genome and were constructed using 484 markers including 96 wheat microsatellite (SSR), 632 Diversity Arrays Technology (DArT) polymorphisms, two sequence-tagged-site (STS) from semi-dwarf genes Rht1 and Rht2, and two markers for low molecular-weight glutenin gene subunits. QTL analysis detected a total of eight QTL significantly associated with HTAP resistance to stripe rust with two on chromosome 2B, two on 3B and one on each of 1A, 4A, 4B and 5B. QTL on chromosomes 2B and 4A were the major loci derived from IDO444 and explained up to 47 and 42% of the phenotypic variation for disease severity and infection type, respectively. The remaining five QTL accounted for 7–10% of the trait variation. Of these minor QTL, the resistant alleles at the two QTL QYrrb.ui-3B.1 and QYrrb.ui-4B derived from Rio Blanco and reduced infection type only, while the resistant alleles at the other three QTL, QYrid.ui-1A, QYrid.ui-3B.2 and QYrid.ui-5B, all derived from IDO444 and reduced either infection type or disease severity. Markers linked to 2B and 4A QTL should be useful for selection of HTAP resistance to stripe rust.

Quantitative trait loci influencing endosperm texture, dough-mixing strength, and bread-making properties of the hard red spring wheat breeding lines

Wheat end product quality is determined by a complex group of traits including dough viscoelastic characteristics and bread-making properties. Quantitative trait loci (QTL) mapping and analysis were conducted for endosperm texture, dough-mixing strength, and bread-making properties in a population of 139 (MN99394 × MN98550) recombinant inbred lines that were evaluated at three environments in 2006. Based on the genetic map of 534 loci, six QTL were identified for endosperm texture, with the main QTL on chromosomes 1A (R(2) = 6.6%-17.3%), 5A (R(2) = 6.1%-17.1%), and 5D (R(2) = 15.8%-22%). Thirty-four QTL were identified for eight dough-mixing strength and bread-making properties. Major QTL clusters were associated with the low-molecular weight glutenin gene Glu-A3, the two high-molecular weight glutenin genes Glu-B1 and Glu-D1, and two regions on chromosome 6D. Alleles at these QTL clusters have previously been proven useful for wheat quality, except one of the QTL clusters on chromosome 6D. A QTL cluster on chromosome 6D is one of the novel chromosome regions influencing dough-mixing strength and bread-making properties. The QTL for endosperm texture on chromosomes 1A, 5A, and 5B also influenced flour ash content (12.4%-23.3%), flour protein content (10.5%-12.5%), and flour colour (7.7%-13.5%), respectively.

Characterization of Low Molecular Weight Glutenin Subunit Gene Representing Glu-B3 Locus of Indian Wheat Variety NP4

Low molecular weight (LMW) glutenin subunits represent major part (30%) of storage proteins in wheat endosperm and determine the quality of dough. Despite their importance few LMW glutenin genes have been characterized so far and none from Indian wheat variety. In the present investigation PCR technique was employed to characterize LMW-GS gene representing Glu-B3 locus from Indian bread wheat cultivar NP4. The deduced protein sequence coded by Glu-B3 locus of LMW-GS gene from NP4 showed the presence of regular structure of the repetitive domain with varying numbers of glutamine (Q) residues and the presence of 1st cysteine residue within the repetitive domain at 40th position in mature polypeptide. Such structure might increase and stabilize the gluten polymer through intermolecular interactions of the large numbers of glutamine side chains and cysteine residues for intermolecular disulphide bond formation leading to stronger dough quality of NP4. Moreover, Glu-B3 specific primers could also be used for identifying 1BL/1RS translocation in addition to amplifying LMW glutenin genes. There was no amplification in 1B/1R translocation lines as short arm of wheat was replaced by short arm of rye chromosome in these lines. Such information can be useful in wheat improvement for dough properties for better chapati and bread quality.

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

AbstractThe low molecular weight (LMW) glutenin subunits 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 glutenin genes (designated LMW‐5, LMW‐7, LMW‐42, LMW‐58, and LMW‐34) were isolated from the Chinese elite wheat cultivar “Xiaoyan 54” by PCR amplification of genomic DNA using a pair of degenerate primers designed from the conserved sequences of the N‐ and C‐terminal regions of published LMW glutenin 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 Escherichia coli. Protein sodium dodecyl sulfate‐polyacrylamide gel electro‐phoresis analysis showed that all proteins expressed in E. coli by the four genes could be related to B‐group LMW glutenin subunits of wheat.(Managing editor: Li‐Hui Zhao)

Rapid genome divergence at orthologous low molecular weight glutenin loci of the A and Am genomes of wheat.

To study genome evolution in wheat, we have sequenced and compared two large physical contigs of 285 and 142 kb covering orthologous low molecular weight (LMW) glutenin loci on chromosome 1AS of a diploid wheat species (Triticum monococcum subsp monococcum) and a tetraploid wheat species (Triticum turgidum subsp durum). Sequence conservation between the two species was restricted to small regions containing the orthologous LMW glutenin genes, whereas >90% of the compared sequences were not conserved. Dramatic sequence rearrangements occurred in the regions rich in repetitive elements. Dating of long terminal repeat retrotransposon insertions revealed different insertion events occurring during the last 5.5 million years in both species. These insertions are partially responsible for the lack of homology between the intergenic regions. In addition, the gene space was conserved only partially, because different predicted genes were identified on both contigs. Duplications and deletions of large fragments that might be attributable to illegitimate recombination also have contributed to the differentiation of this region in both species. The striking differences in the intergenic landscape between the A and A(m) genomes that diverged 1 to 3 million years ago provide evidence for a dynamic and rapid genome evolution in wheat species.

Genetic polymorphism in low-molecular-weight glutenin genes from Triticum aestivum, variety Chinese Spring

Low-molecular-weight (LMW) glutenin subunits consist mainly of two domains, one at the N- terminus which contains repeats of short amino-acid motifs, and a non-repetitive one rich in cysteine, at the C- terminal region. In previous reports, polyacrylamide-gel electrophoresis has been used to show that large size variation exists among LMW and HMW glutenin subunits, and it has been suggested that deletions and insertions within the repetitive region are responsible for these variations in length. In this study, PCR-amplification of genomic DNA (Triticum aestivum variety Chinese Spring) was used to isolate three full-length LMW glutenin genes: LMWG-MB1, LMWG-MB2 and LMWG-MB3. The deduced amino-acid sequences show a high similarity between these ORFs, and with those of other LMW glutenin genes. Comparisons indicate that LMWG-MB1 has probably lost a 12-bp fragment through deletion and that LMWG-MB1 and LMWG-MB2 have an insertion of 81 bp within the repetitive domain. The current study has shown direct evidence that insertions and/or deletions provide a mechanistic explanation for the allelic variation, and the resultant evolution, of prolamin genes. Single-base substitutions at identical sites generate stop codons in both LMWG-MB2 and LMWG-MB3 indicating that these clones are pseudogenes.

PCR‐based assay for detecting IB‐genes for low molecular weight glutenin subunits related to gluten quality properties in durum wheat

A PCR assay has been developed for selecting low molecular weight (LMW) glutenin genes, located on chromosome 1B, and related to durum wheat quality characteristics. Most durum wheat lines possessing good technological properties can be identified on the basis of the presence of specific 1B LMW glutenin components that have been designated LMW‐2 glutenin subunits. On the basis of nucleotide sequences corresponding to LMW glutenin genes, a series of specific oligonucleotide primers were prepared and used in PCR analysis. A pair of these primers gave a single amplification product which can distinguish between durum wheat lines possessing LMW‐2 glutenin subunits and lines possessing other 1B‐LMW allelic variants. Because the proposed PCR analysis can be carried out using part of the endosperm of a single seed, it represents a helpful approach for speeding up the selection of genotypes possessing LMW‐2 glutenin subunits. This part of primers could also be very useful in genome mapping analysis and for testing the purity of wheat flour stock.

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.

Single-seed PCR of LMW glutenin genes to distinguish between durum wheat cultivars with good and poor technological properties.

Polymerase chain reaction (PCR) was used to amplify low-molecular-weight (LMW) glutenin sequences from genomic DNA extracted from a single germinating seed of several durum wheat genotypes. Electrophoretic analysis of PCR reactions showed the presence of amplified products characteristic of durum wheat cultivars with good and poor technological properties. This PCR-based approach is proposed as a very efficient and safe alternative to standard procedures for selecting durum wheat genotypes with good qualitative characteristics.

In vivo footprinting of a low molecular weight glutenin gene (LMWG-1D1) in wheat endosperm.

The quality of the wheat grain is determined by the quantity and composition of storage proteins (prolamins) which are synthesized exclusively in endosperm tissue. We are investigating the mechanisms underlying the regulation of expression of a prolamin gene, the low molecular weight glutenin gene LMWG-1D1. The LMWG-1D1 promoter contains the endosperm box, a sequence motif highly conserved in the promoter region of a large number of storage protein genes, which is thought to confer endosperm-specific expression of prolamin genes. Here we show by in vivo DMS footprinting of wheat endosperm tissue that the endosperm box becomes occupied by putative trans-acting factors during grain ripening. During early stages of development the endosperm motif within the 5' half of the endosperm box becomes occupied first, followed by binding of a second activity to a GCN4/jun-like motif in the 3' half just prior to the stage of maximum gene expression. Occupancy of the endosperm box is highly tissue-specific: no protection was observed in husk and leaf tissues. Several binding activities were identified in vitro from nuclear protein extracts of wheat endosperm which bind specifically to the endosperm and GCN4/jun motifs identified by in vivo footprinting.

Molecular analysis of gliadin and glutenin genes in T. durum cv. Lira. A model system to analyse the molecular bases of quality differences in durum wheat cultivars

As a model system to study the molecular bases of differences between high- and lowquality durum wheat cultivars, designated as biotypes γ-45 and γ-42, respectively, the cultivar Lira, which contains both biotypes, was examined. Polymerase chain reaction analysis of biotypes 42 and 45 was carried out by using oligonucleotide primers from flanking nucleotide sequences coding for α-, β- and γ-gliadin and for high molecular weight (HMW) and low molecular weight (LMW) glutenins. A different amplified band for γ-gliadin sequences was observed between the two biotypes. Southern blot analysis of the same biotypes showed different hybridization patterns for both γ-gliadin and LMW glutenin genes. The hybridization patterns of LMW glutenin sequences revealed the presence of additional bands in biotype 45, suggesting that the quantitative differences in LMW glutenins between biotypes 42 and 45, considered responsible for their quality differences, are most probably due to a higher number of LMW genes in biotype 45. This conclusion seems also to be supported by the results obtained in other durum wheat cultivars belonging to types 42 and 45.

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

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.