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

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

The characterization and comparative analysis of high-molecular-weight glutenin genes from genomes A and B of a hexaploid bread wheat

Summary Two high-molecular-weight subunit (HMWS) glutenin genes from the A and B genomes of the hexaploid bread wheat Triticum aestivum L. cv Cheyenne have been isolated and sequenced. Both of these genes are of the high M_r class (x-type) of HMW glutenins, and have not been previously reported. The entire set of six HMW genes from cultivar Cheyenne have now been isolated and characterized. An analysis of the Ax and Bx sequences shows that the Ax sequence is similar to the homoeologous gene from the D genome, while the Bx repeat structure is significantly different. The repetitive region of these proteins can be modelled as a series of interspersed copies of repeat modifs of 6, 9, and 15 amino acid residues. The evolution of these genes includes single-base substitutions over the entire coding region, plus insertion/deletions of single or blocks of repeats in the central repetitive domain.     

小麦高分子量谷蛋白亚基及其基因的研究进展

主要介绍了小麦高分子量谷蛋白亚基（HMW－GS）及其基因的研究进展情况,目前,转基因小麦的技术已经逐渐成熟,由于分子生物学领域分子标记技术的迅速发展,尤其是PCR技术的广泛应用,为实现外源优良储藏蛋白基因导入改良品种提供了可能,利用已知小麦品种的基因序列设计引物,从众多的未知小麦品种中扩增出新基因加以研究并做外源优质HMW－GS基因的转入已成为一种趋势。     

Molecular characterization of Syrian date palm cultivars using plasmid-like DNA markers

Date palm (Phoenix dactylifera L.) is one of the most important domesticated fruit trees in the Near East and North African countries. This tree has been, for several decades, in serious threat of being completely destroyed by the “Bayoud” disease caused by Fusarium oxysporum f. sp. albedinis. In this study, 18 Syrian date palm cultivars and four male trees were analyzed according to the identity of mitochondrial plasmid-like DNAs. A PCR strategy that employs plasmid-like DNAs-specific primer pair was used. These primers amplify a product of either 373-bp or 265-bp that corresponds to the S- (Bayoud-susceptible) or the R-plasmid (Bayoud-resistant), respectively. Generated data revealed that only six cultivars (“Medjool”, “Ashrasi”, “Gish Rabi”, “Khineze”, and yellow- and red-“Kabkab”) have the S-plasmid, suggesting their susceptibility to the fusariosis, while the remaining 12 cultivars and the four male trees contain the R-plasmid, suggesting their resistance to the fusariosis. The PCR process applied here has been proved efficient for the rapid screening for the presence of the S and R DNAs in Syrian date palm. PCR markers developed in this study could be useful for the screening of date palm lines growing in the field. The availability of such diagnostic tool for plasmid characterization in date palm would also be of great importance in establishing propagation and breeding programs of date palm in Syria.     

Large-scale expression and purification of high-molecular-weight glutenin subunits

The high-molecular-weight glutenin subunits (HMW-GSs) are considered to be one of the most important components of wheat gluten, contributing to the unique viscoelastic properties of wheat dough. The HMW-GSs are highly homologous in sequence and structure and a mixture of subunits is usually present in wheat flours. Consequently, it is difficult to purify these proteins separately in appreciable amounts. Expression in heterologous systems provides a clear opportunity to produce large amounts of single HMW-GS proteins, amounts (up to 100 mg) which are required for in vitro analysis of these proteins. However, since the first expression studies of HMW-GSs, over 10 years ago, this technology has not been widely utilized. Previous studies have been analytical or small scale (5-100 ml) and in most cases only partial purity was obtained. In the present paper, we describe in detail the expression of the HMW-GSs Glu1-Dx2, Dx5, Dy10, and Dy12 for the first time on a large scale, producing up to 100 mg of target protein from a 2-liter bacterial culture, using a Biostat fermenter. Our results include optimization of expression conditions to increase yield and stability of proteins. Results also include localization, differences between x- and y-type expression and small-scale versus large-scale expression. We also developed a large-scale purification procedure. The bacterially expressed proteins have the same molecular weight on SDS-PAGE and the same retention times on RP-HPLC as their native counterparts extracted from flour. Functionality tests, on the bacterially produced proteins, have shown a clear correlation with the equivalent native proteins from flour. These results provide a clear opportunity to produce protein in amounts necessary for more detailed studies of the structure and function of the HMW-GSs and glutenin polymers on dough development and quality.     

Molecular characterization of Syrian date palm cultivars using plasmid-like DNA markers

Date palm (Phoenix dactylifera L.) is one of the most important domesticated fruit trees in the Near East and North African countries. This tree has been, for several decades, in serious threat of being completely destroyed by the "Bayoud" disease caused by Fusarium oxysporum f. sp. albedinis. In this study, 18 Syrian date palm cultivars and four male trees were analyzed according to the identity of mitochondrial plasmid-like DNAs. A PCR strategy that employs plasmid-like DNAs-specific primer pair was used. These primers amplify a product of either 373-bp or 265-bp that corresponds to the S-(Bayoud-susceptible) or the R-plasmid (Bayoud-resistant), respectively. Generated data revealed that only six cultivars ('Medjool', 'Ashrasi', 'Gish Rabi', 'Khineze', and yellow- and red-'Kabkab') have the S-plasmid, suggesting their susceptibility to the fusariosis, while the remaining 12 cultivars and the four male trees contain the R-plasmid, suggesting their resistance to the fusariosis. The PCR process applied here has been proved efficient for the rapid screening for the presence of the S and R DNAs in Syrian date palm. PCR markers developed in this study could be useful for the screening of date palm lines growing in the field. The availability of such diagnostic tool for plasmid characterization in date palm would also be of great importance in establishing propagation and breeding programs of date palm in Syria.     

Multiplex PCR identification of wheat HMW glutenin subunit genes by allele-specific markers

Wheat bread-making quality is closely correlated with composition and quantity of gluten proteins, in particular with high-molecular weight (HMW) glutenin subunits encoded by the Glu-1 genes. A multiplex polymerase chain reaction (PCR) method was developed to identify the allele composition of HMW glutenin complex Glu-1 loci (Glu-A1, Glu-B1 and Glu-D1) in common wheat genotypes. The study of multiplex PCR to obtain a well-balanced set of amplicons involved examination of various combinations of selected primer sets and/or thermal cycling conditions. One to three simultaneously amplified DNA fragments of HMW glutenin Glu-1 genes were separated by agarose slab-gel electrophoresis and differences between Ax1, Ax2* and Axnull genes of Glu-A1 loci, Bx6, Bx7 and Bx17 of Glu-B1, and Dx2, Dx5 and Dy10 genes of Glu-D1 loci were revealed. A complete agreement was found in identification of HMW glutenin subunits by both multiplex PCR analysis and SDS-PAGE for seventy-six Polish cultivars/strains of both spring and winter common wheat. Rapid identification of molecular markers of Glu-1 alleles by multiplex PCR can be an efficient alternative to the standard separation procedure for early selection of useful wheat genotypes with good bread-making quality.     

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     

Identification of Prunus armeniaca cultivars by RAPD and SCAR markers

Nineteen cultivars of apricot (Prunus armeniaca) were distinguished using random amplified polymorphic DNA (RAPD) markers. One decamer out of 44 used was useful to differentiate cultivars of the Campania Region from those of Northern Italy, North America and Greece. A sequence characterized amplified region (SCAR) marker was obtained. The results provide a protocol to fingerprint DNA of apricots as an efficient way to quality control and fraud prevention.     

Identification and molecular characterization of a large insertion within the repetitive domain of a high-molecular-weight glutenin subunit gene from hexaploid wheat

High-molecular-weight (HMW) glutenin subunits are a particular class of wheat endosperm proteins containing a large repetitive domain flanked by two short N- and C-terminal non-repetitive regions. Deletions and insertions within the central repetitive domain has been suggested to be mainly responsible for the length variations observed for this class of proteins. Nucleotide sequence comparison of a number of HMW glutenin genes allowed the identification of small insertions or deletions within the repetitive domain. However, only indirect evidence has been produced which suggests the occurrence of substantial insertions or deletions within this region when a large variation in molecular size is present between different HMW glutenin subunits. This paper represents the first report on the molecular characterization of an unusually large insertion within the repetitive domain of a functional HMW glutenin gene. This gene is located at the Glu-D1 locus of a hexaploid wheat genotype and contains an insertion of 561 base pairs that codes for 187 amino acids corresponding to the repetitive domain of a HMW glutenin subunit encoded at the same locus. The precise location of the insertion has been identified and the molecular processes underlying such mutational events are discussed.     

Analysis of diversity of Russian and Ukrainian bread wheat (Triticum aestivum L.) cultivars for high-molecular-weight glutenin subunits

The allelic diversity of high-moleculat-weght glutenin subunits (H WIGS) in Russian and Ukrainian bread wheat cultivars was analyzed. The diversity of spring wheat cultivars for alleles of the Glu-1 loci is characterized by medium values of the polymorphism index (polymorphism information content, PlC), and in winter wheats it varies from high at the Glu-A1 locus to low at the Glu-D1 locus. The spring and winter cultivars differ significantly in the frequencies of alleles of the glutenin loci. The combination of the Glu-A1b, Glu-B1c, and Glu-D1a alleles prevails among the spring cultivars, and the combination of the Glu-A1a, Glu-B1c, and Glu-D1d alleles prevails among the winter cultivars. The distribution of the Glu-1 alleles significantly depends on the moisture and heat supply in the region of origin of the cultivars. Drought resistance is associated with the Glu-D1a allele in the spring wheat and with the Glu-B1b allele in the winter wheat. The sources of the Glu-1 alleles were identified in the spring and wheat cultivars. The analysis of independence of the distribution of the spring and winter cultivars by the market classes and by the alleles of the HMWGS loci showed a highly significant association of the alleles of three Glu-1 loci with the market classes in foreign cultivars and independence or a weak association in the Russian and Ukrainian cultivars. This seems to be due to the absence of a statistically substantiated system of classification of the domestic cultivars on the basis of their quality.     

Molecular characterization of novel low-molecular-weight glutenin genes inAegilops longissima

Extensive genetic variations of low-molecular-weight glutenin subunits (LMW-GS) and their coding genes were found in the wild diploid A- and D-genome donors of common wheat. In this study, we reported the isolation and characterization of 8 novel LMW-GS genes fromAe.longissima Schweinf. & Muschl., a species of the sectionSitopsis of the genusAegilops, which is closely related to the B genome of common wheat. Based on the N-terminal domain sequences, the 8 genes were divided into 3 groups. A consensus alignment of the extremely conserved domains with known gene groups and the subsequent cluster analysis showed that 2 out of the 3 groups of LMW-GS genes were closely related to those from the B genome, and the remaining was related to those from A and D genomes of wheat andAe. tauschii. Using 3 sets of gene-group-specific primers, PCRs in diploid, tetraploid and hexaploid wheats andAe. tauschii failed to obtain the expected products, indicating that the 3 groups of LMW-GS genes obtained in this study were new members of LMW-GS multi-gene families. These results suggested that theSitopsis species of the genusAegilops with novel gene variations could be used as valuable gene resources of LMW-GS. The 3 sets of group-specific primers could be utilized as molecular markers to investigate the introgression of novel alien LMW-GS genes fromAe. longissima into wheat.     

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.     

Identification of apple cultivars using RAPD markers

Summary Eleven apple cultivars were differentiated using randomly amplified polymorphic DNA (RAPD) markers obtained by the polymerase chain reaction (PCR). The variability of the technique and of the origin of the DNA extract was analyzed. A set of bands consistent in their presence or absence was chosen to create a differentiating band pattern. A key is proposed by which one can differentiate apple cultivars using commercially available prime.     

PCR analysis of genes encoding allelic variants of high-molecular-weight glutenin subunits at the Glu-D1 locus

Genes encoding high-molecular-weight (HMW) glutenin subunits, present in bread-wheat lines and cultivars, were studied by RFLP (restriction fragment length polymorphism) and PCR (polymerase chain reaction) analyses. In particular, allelic subunits of the x-or y-type, encoded at the Glu-D1 locus present on the long arm of chromosome 1D, were investigated. The variation in size, observed in different allelic subunits, is mainly due to variation in the length of the central repetitive domain, typical of these proteins. Deletions or duplications, probably caused by unequal crossingover, have given rise to the size heterogeneity currently observed. The possibility of using the PCR technique for a detailed analysis of HMW glutenin genes in order to obtain a more accurate estimation of the molecular weight of their encoded subunits, and the detection of unexpressed genes, is also described.     

Identification of in vivo essential genes from Pseudomonas aeruginosa by PCR-based signature-tagged mutagenesis

We adapted PCR-based signature-tagged mutagenesis (STM) to Pseudomonas aeruginosa. A collection of 1056 mutants was screened in a chronic lung infection rat model. Thirteen mutants were confirmed to be attenuated. Analysis revealed that these STM mutants represented transposon insertions into eight genes previously described in databases, three genes encoding proteins sharing identity with hypothetical proteins and two genes that shared no significant identity with sequences in databases. Five strains mutated in genes involved in protein degradation, stress tolerance, cation transport, ABC transporter, and an unknown protein were shown to be highly attenuated when tested individually in the rat chronic lung infection model.     

New DNA markers for high molecular weight glutenin subunits in wheat

End-use quality is one of the priorities of modern wheat (Triticum aestivum L.) breeding. Even though quality is a complex trait, high molecular weight (HMW) glutenins play a major role in determining the bread making quality of wheat. DNA markers developed from the sequences of HMW glutenin genes were reported in several previous studies to facilitate marker-assisted selection (MAS). However, most of the previously available markers are dominant and amplify large DNA fragments, and thus are not ideal for high throughput genotyping using modern equipment. The objective of this study was to develop and validate co-dominant markers suitable for high throughput MAS for HMW glutenin subunits encoded at the Glu-A1 and Glu-D1 loci. Indels were identified by sequence alignment of allelic HMW glutenin genes, and were targeted to develop locus-specific co-dominant markers. Marker UMN19 was developed by targeting an 18-bp deletion in the coding sequence of subunit Ax2* of Glu-A1. A single DNA fragment was amplified by marker UMN19, and was placed onto chromosome 1AL. Sixteen wheat cultivars with known HMW glutenin subunits were used to validate marker UMN19. The cultivars with subunit Ax2* amplified the 362-bp fragment as expected, and a 344-bp fragment was observed for cultivars with subunit Ax1 or the Ax-null allele. Two co-dominant markers, UMN25 and UMN26, were developed for Glu-D1 by targeting the fragment size polymorphic sites between subunits Dx2 and Dx5, and between Dy10 and Dy12, respectively. The 16 wheat cultivars with known HMW glutenin subunit composition were genotyped with markers UMN25 and UMN26, and the genotypes perfectly matched their subunit types. Using an Applied Biosystems 3130xl Genetic Analyzer, four F₂ populations segregating for the Glu-A1 or Glu-D1 locus were successfully genotyped with primers UMN19, UMN25 and UMN26 labeled with fluorescent dyes.