Map-based analysis of the tenacious glume gene Tg-B1 of wild emmer and its role in wheat domestication

The domestication of wheat was instrumental in spawning the civilization of humankind, and it occurred through genetic mutations that gave rise to types with non-fragile rachises, soft glumes, and free-threshing seed. Wild emmer (Triticum turgidum ssp. dicoccoides), the tetraploid AB-genome progenitor of domesticated wheat has genes that confer tenacious glumes (Tg) that underwent genetic mutations to give rise to free-threshing wheat. Here, we evaluated disomic substitution lines involving chromosomes 2A and 2B of wild emmer accessions substituted for homologous chromosomes in tetraploid and hexaploid backgrounds. The results suggested that both chromosomes 2A and 2B of wild emmer possess genes that inhibit threshability. A population of recombinant inbred lines derived from the tetraploid durum wheat variety Langdon crossed with a Langdon — T. turgidum ssp. dicoccoides accession PI 481521 chromosome 2B disomic substitution line was used to develop a genetic linkage map of 2B, evaluate the genetics of threshability, and map the gene derived from PI 481521 that inhibited threshability. A 2BS linkage map comprised of 58 markers was developed, and markers delineated the gene to a 2.3 cM interval. Comparative analysis with maps containing the tenacious glume gene Tg-D1 on chromosome arm 2DS from Aegilops tauschii, the D genome progenitor of hexaploid wheat, revealed that the gene inhibiting threshability in wild emmer was homoeologous to Tg-D1 and therefore designated Tg-B1. Comparative analysis with rice and Brachypodium distachyon indicated a high level of divergence and poorly conserved colinearity, particularly near the Tg-B1 locus. These results provide a foundation for further studies involving Tg-B1, which, together with Tg-D1, had profound influences on wheat domestication.     

Marker-assisted selection of high molecular weight glutenin alleles related to bread-making quality in Iranian common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Bread-making quality in hexaploid wheats is a complex trait. It has been shown that the amount and composition of protein can influence dough rheological properties. The high-molecular-weight (HMW) glutenins are encoded by a complex locus, Glu-1, on the long arm of group-1 homoeologus chromosome of the A, B and D genomes. In this work we used PCR-based DNA markers as a substitution tool to distinguish wheat bread-making quality. We detected PCR-based DNA markers for coding sequence of Glu-A1x, Glu-B1x and Glu-D1x to be 2300 bp, 2400 bp and 2500 bp respectively. DNA markers related to coding sequence of Glu-A1y, Glu-B1y and Glu-D1y were; 1800 bp, 2100 bp and 1950 bp, however, the repetitive region of their coding sequence were shown to be about 1300 bp, 1500 bp and 1600 bp. The results demonstrate that the size variation was due to different lengths of the central repetitive domain. Good or poor bread-making quality in wheat is associated with two allelic pairs of Glu-D1, designated 1Dx5-1Dy10 and 1Dx2-1Dy12. The 1Bx7 allele has moderate-to-good quality score. The specific DNA markers, of 450 bp, 576 bp, 612 bp and 2400 bp respectively were characterized for 1Dx5, 1Dy10, 1Dy12 and 1Bx7 alleles. These markers are very important in screening of wheat for bread-making quality.     

Genetic diversity for morphological traits and seed storage proteins in Spanish rivet wheat

The objective of the current work was to analyse the variability of high and low molecular mass (HMM and LMM) glutenin subunits, along with some morphological characteristics in sixty Spanish accessions of rivet wheat (Triticum turgidum L. ssp. turgidum). The lines were grouped in sixteen botanical varieties and five additional types, according the morphological criteria. Up to 13 allelic variants (four alleles for the Glu-A1 locus and nine alleles for the Glu-B1 locus) and 34 B-LMMGs patterns were found in the evaluated lines. The current data indicated a clear reduction of morphological variability, along with an asymmetric distribution of the alleles and patterns for seed storage proteins. This polymorphism could be useful for enlarging the genetic background of modern durum wheat.     

Cloning and molecular characterization of a novel x-type glutenin gene Glu-D(t)1 from Aegilops tauschii

A novel gene encoding an x-type high molecular weight glutenin subunit (HMW-GS), designated 1Dx1.1 ^t , was isolated from Aegilops tauschii. It is the largest HMW-GS gene reported so far in this species and its product has a slower mobility than that of subunit 1Ax1 in SDS-PAGE. The open reading frame (ORF) of the gene was 2,628 bp, encoding a protein of 874 amino acid residues. Comparisons of amino acid sequences showed that subunit 1Dx1.1^t had high similarity with other 1Dx subunits but also had two unique characteristics. Firstly, a tripeptide of consensus LQE present in the N-terminal domains of other 1Dx subunits was absent from subunit Dx1.1^t. Secondly, three copies of tandem duplications of the tripeptide motif GQQ and a novel tripeptide sequence (GQL) were present in its central repetitive domain. Phylogenetic analysis showed that subunit 1Dx1.1^t clustered with other known 1Dx subunits.     

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.     

Chromosomal structural changes and microsatellite variations in newly synthesized hexaploid wheat mediated by unreduced gametes

Allohexaploid wheat was derived from interspecific hybridization, followed by spontaneous chromosome doubling. Newly synthesized hexaploid wheat by crossing Triticum turgidum and Aegilops tauschii provides a classical model to understand the mechanisms of allohexaploidization in wheat. However, immediate chromosome level variation and microsatellite level variation of newly synthesized hexaploid wheat have been rarely reported. Here, unreduced gametes were applied to develop synthesized hexaploid wheat, NA0928, population by crossing T. turgidum ssp. dicoccum MY3478 and Ae. tauschii SY41, and further S0–S3 generations of NA0928 were assayed by sequential cytological and microsatellite techniques. We demonstrated that plentiful chromosomal structural changes and microsatellite variations emerged in the early generations of newly synthesized hexaploid wheat population NA0928, including aneuploidy with whole-chromosome loss or gain, aneuploidy with telosome formation, chromosome-specific repeated sequence elimination (indicated by fluorescence in situ hybridization) and microsatellite sequence elimination (indicated by sequencing), and many kinds of variations have not been previously reported. Additionally, we reported a new germplasm, T. turgidum accession MY3478 with excellent unreduced gametes trait, and then succeeded to transfer powdery mildew resistance from Ae. tauschii SY41 to synthesized allohexaploid wheat population NA0928, which would be valuable resistance resources for wheat improvement.     

Molecular Characterization of a HMW Glutenin Subunit Allele Providing Evidence for Silencing of x-type Gene on Glu-B1

Understanding the molecular structure of high-molecular-weight glutenin subunit (HMW-GS) may provide useful evidence for the study on the improvement of quality of cultivated wheat and the evolution of Glu-1 alleles. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) shows that the subunits encoded by Glu-B1 were null, named 1Bxm, in a Triticum turgidum var. dicoccoides line PI94640. Primers based on the conserved regions in wheat HMW-GS gene promoter and coding sequences were used to amplify the genomic DNA of line PI94640. The PCR products were sequenced, and the total nucleotide sequence of 3 442 bp including upstream sequence of 1 070 bp was obtained. Compared with the reported gene sequences of Glu-1Bx alleles, the promoter region of the Glu-1Bxm showed close resemblance to 1Bx7. The Glu-1Bxm coding region differs from the other Glu-1Bx alleles for a deduced mature protein with only 212 residues, and a stop codon (TAA) at 637 bp downstream from the start codon was present, which was probably responsible for the silencing of x-type subunit genes at the Glu-B1 locus. Phylogenetic tree based on the nucleotide sequence alignment of HMW glutenin subunit genes showed that 1Bxm was the most ancient type of Glu-B1 alleles, suggesting that the evolution rates are different among Glu-1Bx genes. Further study on the contribution of the unique silenced Glu-B1 alleles to quality improvement was also discussed.     

Comparative analysis of the D genome-encoded high-molecular weight subunits of glutenin

Four genes encoding novel 1Dx-type high-molecular weight (HMW) subunits were amplified by polymerase chain reaction, two each from Aegilops tauschii and bread wheat Triticum aestivum. The two subunits from Ae. tauschii (1Dx2.1^t and 1Dx2^t) were both very similar in sequence to subunit 1Dx2 from bread wheat. In contrast, the two novel bread wheat subunits (1Dx2.2 and 1Dx2.2*) differed from subunit 1Dx2 in having different internally duplicated regions (of 132 and 186 amino acid, respectively) within their repetitive domains. These duplicated sequences were located adjacent to the regions from which they had been duplicated and had complete intact repeat motifs at each end. The implications of these results for HMW subunit evolution and wheat quality improvement are discussed.     

Characterisation of high- and low-molecular weight glutenin subunits associated to the D genome of Aegilops tauschii in a collection of synthetic hexaploid wheats

Synthetic hexaploid wheats (2n=6x=42, AABBDD) involving genomes from Triticum turgidum (2n= 4x=28, AABB) and Aegilops tauschii (2n=2x=14, DD) have been produced as a means for introducing desirable characteristics into bread wheat. In the present work we describe the genetic variability present at the Glu-D ^t 1 and Glu-D ^t 3 loci, encoding high- (HMW) and low-molecular-weight (LMW) glutenin subunits respectively, derived from Ae. tauschii, using electrophoretic and chromatographic methods, in a collection of synthetic hexaploid wheats. A wide variation both in mobility and surface hydrophobicity of HMW glutenin subunits was observed between different accessions of Ae. tauschii used in the production of the synthetic hexaploids. A combination of electrophoretic and chromatographic methods improves the identification of HMW glutenin subunits; in fact subunits with identical apparent mobility were revealed to have a different surface hydrophobicity by reversed-phase high performance liquid chromatography. None of the Dx5^t subunits present in Ae. tauschii showed the presence of the extra cysteine residue found in the HMW glutenin subunit Dx5 of Triticum aestivum, as revealed by selective amplification with polymerase chain reaction (PCR). The wide variability and the high number of subunits encoded by the Glu-D ^t 3 locus suggests that Ae. tauschii may be a rich source for enhancing the genetic variability of glutenin subunits in bread wheat and improving bread-making properties. Received: 3 March 2001 / Accepted: 23 March 2001     

Molecular characterization of two y-type high molecular weight glutenin subunit alleles 1Ay12 and 1Ay8 from cultivated einkorn wheat (Triticum monococcum ssp. monococcum)

Two y-type high molecular weight glutenin subunits (HMW-GSs) 1Ay12^? and 1Ay8^? from the two accessions PI560720 and PI345186 of cultivated einkorn wheat (Triticum monococcum ssp. monococcum, AA, 2n = 2x = 14), were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The mobility of 1Ay12^? and 1Ay8^? was similar to that of 1Dy12 and 1By8 from common wheat Chinese Spring, respectively. Their ORFs respectively consisted of 1812 bp and 1935 bp, encoding 602 and 643 amino acid residues with the four typical structural domains of HMW-GS including signal peptide, conserved N-, and C-terminal and central repetitive domains. Compared with the most similar active 1Ay alleles previous published, there were a total of 15 SNPs and 2 InDels in them. Their encoding functions were confirmed by successful heterogeneous expression. The two novel 1Ay alleles were named as 1Ay12^? and 1Ay8^? with the accession No. JQ318694 and JQ318695 in GenBank, respectively. The two alleles were classed into the two distinct groups, Phe-type and Cys-type, which might be relevant to the differentiation of Glu-A1-2 alleles. Of which, 1Ay8^? belonged to Cys-type group, and its protein possessed an additional conserved cysteine residue in central repetitive region besides the six common ones in N- and C-terminal regions of Phe-type group, and was the second longest in all the known active 1Ay alleles. These results suggested that the subunit 1Ay8^? of cultivated einkorn wheat accession PI345186 might have a potential ability to strengthen the gluten polymer interactions and be a valuable genetic resource for wheat quality improvement.     

Homoeologous recombination within bread wheat to develop novel combinations of HMW-GS genes: transfer of the <Emphasis Type="Italic">Glu-A1</Emphasis> locus to chromosome 1D

In an attempt to improve the bread-making quality within hexaploid wheat by elaborating novel high-molecular weight glutenin subunits (HMW-GS) combinations useful in wheat-breeding programmes, a 1A chromosome fragment carrying the Glu-A1 locus encoding the subunit Ax2*, was translocated to the long arm of chromosome 1D. The partially isohomoeoallelic line, designated RR239, had a meiotic behaviour as regular as cv. Courtot. It was characterised using genomic in situ hybridization and microsatellite markers as well as biochemical and proteomic approaches. The translocated 1D chromosome had an interstitial 1AL segment representing in average 30% of the recombinant arm length that was confirmed by molecular analysis. The genetic length of the removed segment in chromosome 1DL was estimated to be at least 51 cM, and that of the interstitial 1AL translocation to be at least 33 cM. Proteome analysis performed on total endosperm proteins revealed variation in amounts, 8 spots and 1 spot being up- and downregulated, respectively. Quantitative variations in HMW-GS were observed for the Glu-A1 (Ax2*) and Glu-B1 (Bx7 + By8) loci in response to duplication of the Glu-A1 locus.     

Photoperiod and Vernalization Responses in Triticum turgidumxT. tauschii Synthetic Hexaploid Wheats

Studies of synthetic hexaploid wheat developed from Triticumturgidum(AABB genomes) and T. tauschii(DD genome) can provideinformation on potentially useful characters in T. tauschiiand/or T. turgidum for genetic improvement of hexaploid wheat(T. aestivum). Synthetic hexaploid wheats and the T. turgidumand T. tauschii parents were assessed for their developmentalresponses to photoperiod and vernalization for days to ear emergence,final leaf number and the number of spikelets per spike. Theresponses to photoperiod and vernalization of the synthetichexaploids were generally intermediate between those of theparents but in some instances the levels of expression exhibitedby the T. tauschii or T. turgidum parents were epistatic inthe synthetic hexaploids. The relatively strong photoperiodresponse of the T. tauschii accessions was not expressed inthe synthetic hexaploids, but rather the synthetic hexaploidsreflected the photoperiod response of the respective T. turgidumparents. The synthetic hexaploids had vernalization responsesstronger than those of the T. turgidum and bread wheats usedin the study. The expression of ear emergence in response tovernalization of these synthetic hexaploids appeared to be modifiedby the T. turgidum parent. Copyright 2001 Annals of Botany Company Photoperiod, synthetic hexaploids, Triticum aestivum, Triticum tauschii, Triticum turgidum, vernalization     

High Molecular Weight Glutenin Subunit Composition of Indian Wheats and Their Relationships with Dough Strength

One hundred and seventy two wheat varieties including twenty-five durum wheat cultivars were evaluated for high molecular weight glutenin subunit (HMW-GS) composition using SDS-PAGE. The relationship between HMW-GS and sedimentation tests for dough strength was studied. Three alleles were present at the Glu-A1 locus, eight at Glu-B1 and two at Glu-D1 in bread wheat. The data indicated the prevalence of the Glu-A1b allele (63.5%) at the Glu-A1 and Glu-D1a (71.4%) at Glu-D1 loci. Three alleles, namely Glu-B1b (30.61%), Glu-B1c (25.85%) and Glu-B1i (34.00%) represented about 90% of the alleles at Glu-B1 locus. The combination of Glu-A1b, Glu-B1i and Glu-D1d alleles exhibited highest dough strength as measured by sedimentation value in comparison to other combinations (p<0.001). However, this combination was present only in 7% of the samples evaluated. In durum wheat, the null allele (Glu-A1c) was observed more frequently (76%) than the Glu-A1b allele (24%). Glu-B1f and Glu-B1e alleles represented equally (32% each). Protein subunits 13+16 and 6+8 were found correlated positively (p<0.05) with improved dough strength as compared to subunit 20 in durum wheat. This information can be a valuable reference for designing breeding programme for the improvement of bread and pasta making quality of bread and durum wheats, respectively in India.     

Identification and molecular characterization of a novel y-type Glu-Dt 1 glutenin gene of Aegilops tauschii

A novel y-type high-molecular-weight glutenin subunit possessing a slightly faster mobility than that of subunit 1Dy12 in SDS-PAGE, designated 1Dy12.1^t in Aegilops tauschi, was identified by one- and two-dimensional gel and capillary electrophoresis. Its coding gene at the Glu-D ^t 1 locus was amplified with allele-specific-PCR primers, and the amplified products were cloned and sequenced. The complete nucleotide sequence of 2,807 bp containing an open reading frame of 1,950 bp and 857 bp of upstream sequence was obtained. A perfectly conserved enhancer sequence and the –300 element were present at positions of 209–246 bp and 424–447 bp upstream of the ATG start codon, respectively. The deduced mature protein of 1 Dy12.1^t subunit comprised 648 amino acid residues and had a Mr of 67,518 Da, which is slightly smaller than the 1Dy12 (68,695 Da) but larger than the 1Dy10 (67,495 Da) subunits of bread wheat, respectively, and corresponds well with their relative mobilities when separated by acid-PAGE. The deduced amino acid sequence indicated that the 1Dy12.1^t subunit displayed a greater similarity to the 1Dy10 subunit, with only seven amino acid substitutions, suggesting that this novel gene could have positive effect on bread-making quality. A phenetic tree produced by nucleotide sequences showed that the x- and y-type subunit genes were respectively clustered together and that the Glu-D ^t 1y12.1 gene of Ae. tauschii is closely related to other y-type subunit genes from the B and D genomes of hexaploid bread wheat.Communicated by H.F. Linskens     

47份外引小麦种质资源Waxy和HMW-GS分子检测与品质性状分析

为有效利用外引小麦种质资源,本研究对收集的47份外引小麦种质材料进行Waxy和HMW-GS等位基因的分子检测,并分析了其直链淀粉、支链淀粉、湿面筋等品质参数.结果 表明,在Wx-A1位点存在3种类型:Wx-A1a、Wx-A1g和Wx-A1b,39份材料(82.98％)为Wx-A1a类型;Wx-B1位点3种类型:Wx-B...     

Stably expressed <Emphasis Type="SmallCaps">d</Emphasis>-genome-derived HMW glutenin subunit genes transformed into different durum wheat genotypes change dough mixing properties

Durum wheat (Triticum turgidum L. var. durum) is traditionally used for the production of numerous types of pasta, and significant amounts are also used for bread-making, particularly in southern Italy. The research reported here centres on the glutenin subunits 1Dx5 and 1Dy10 encoded by chromosome 1D, and whose presence in hexaploid wheats is positively correlated with higher dough strength. In order to study the effects of stable expression of the 1Dx5 and 1Dy10 glutenin subunits in different durum wheat genotypes, four cultivars commonly grown in the Mediterranean area (‘Svevo’, ‘Creso’, ‘Varano’ and ‘Latino’) were co-transformed, via particle bombardment of cultured immature embryos, with the two wheat genes Glu-D1-1d and Glu-D1-2b encoding the glutenin subunits, and a third plasmid containing the bar gene as a selectable marker. Protein gel analyses of T₁ generation seed extracts showed expression of one or both glutenin genes in four different transformed durum wheat plants. One of these transgenic lines, DC2-65, showed co-suppression of all HMW-GS, including the endogenous ones. Transgene stability in the transgenic lines has been studied over four generations (T₁–T₄). Fluorescence in situ hybridization (FISH) analysis of metaphase chromosomes from T₄ plants showed that the integration of transgenes occurred in both telomeric and centromeric regions. The three plasmids were found inserted at a single locus in two lines and in two loci on the same chromosome arm in one line. The fourth line had two transgenic loci on different chromosomes: one with both glutenin plasmids and a different one containing only the construct with the gene encoding the 1Dy10 glutenin subunit. Segregation of these two loci in subsequent generations allowed establishment of two sublines, one containing both 1Dx5 and 1Dy10 and the other containing only 1Dy10. Small-scale quality tests showed that accumulation of Dx5, Dy10 or both in transgenic durum wheat seeds resulted in doughs with stronger mixing characteristics. A. Gadaleta and A. E. Blechl have contributed equally to this work.     

Variation in the HMW and LMW glutenin subunits from Spanish accessions of emmer wheat (Triticum turgidum ssp. dicoccum Schrank)

Emmer wheat (Triticum turgidum ssp. dicoccum Schrank) is hulled wheat that survives in marginal areas of the Mediterranean Region. The HMW and LMW glutenin subunit composition of 97 accessions of emmer wheat from Spain have been analysed by SDS-PAGE. For the HMW glutenin subunits, four allelic variants were detected for the Glu-A1 locus; one of them has not been previously described. For the Glu-B1 locus, three of the nine alleles detected have not been found before. A high degree of variation was evident for the LMW glutenin subunits, and up to 23 different patterns were detected for the B-LMW glutenin subunits. Considering both types of proteins (HMW and LMW), 30 combinations were found between all the evaluated lines. This wide polymorphism can be used to transfer new quality genes to wheat, and to widen its genetic basis. Received: 13 June 2000 / Accepted: 3 July 2000     

Dissemination of the highly expressed Bx7 glutenin subunit (<Emphasis Type="Italic">Glu-B1al</Emphasis> allele) in wheat as revealed by novel PCR markers and RP-HPLC

Increased expression of the high molecular weight glutenin subunit (HMW-GS) Bx7 is associated with improved dough strength of wheat (Triticum aestivum L.) flour. Several cultivars and landraces of widely different genetic backgrounds from around the world have now been found to contain this so-called over-expressing allelic form of the Bx7 subunit encoded by Glu-B1al. Using three methods of identification, SDS-PAGE, RP-HPLC and PCR marker analysis, as well as pedigree information, we have traced the distribution and source of this allele from a Uruguayan landrace, Americano 44D, in the mid-nineteenth century. Results are supported by knowledge of the movement of wheat lines with migrants. All cultivars possessing the Glu-B1al allele can be identified by the following attributes: (1) the elution of the By sub-unit peak before the Dx sub-unit peak by RP-HPLC, (2) high expression levels of Bx7 (>39% Mol% Bx), (3) a 43 bp insertion in the matrix-attachment region (MAR) upstream of the gene promoter relative to Bx7 and an 18 bp nucleotide duplication in the coding region of the gene. Evidence is presented indicating that these 18 and 43 bp sequence insertions are not causal for the high expression levels of Bx7 as they were also found to be present in a small number of hexaploid species, including Chinese Spring, and species expressing Glu-B1ak and Glu-B1a alleles. In addition, these sequence inserts were found in different isolates of the tetraploid wheat, T. turgidum, indicating that these insertion/deletion events occurred prior to hexaploidization.     

Allelic variation of high molecular weight glutenin subunits of bread wheat in Hebei province of China

In common wheat (Triticum aestivum L.), allelic variations of Glu-1 loci have important influences on grain end-use quality. The allelic variations in high molecular weight glutenin subunits (HMW-GSs) were identified in 151 hexaploid wheat varieties representing a historical trend in the cultivars introduced or released in Hebei province of China from the years 1970s to 2010s. Thirteen distinct alleles were detected for Glu-1. At Glu-A1, Glu-B1 and Glu-D1, we found that the most frequent alleles were the 1 (43.0%), 7+8 (64.9%), 2+12 (74.8%) alleles, respectively, in wheat varieties. Twenty two different HMW-GS compositions were observed in wheat. Twenty-five (16.6%) genotypes possessed the combination of subunits 1, 7+8, 2+12, 25 (16.6%) genotypes had subunit composition of 2*, 7+8, 2+12; 20 (13.2%) genotypes had subunit composition of null, 7+8, 2+12. The frequency of other subunit composition was less than 10%. The Glu-1 quality score greater than or equal to 9 accounted for 20.6% of the wheat varieties. The percentage of superior subunits (1 or 2* subunit at Glu-A1 locus; 7+8, 14+15 or 17+18 at Glu-B1 locus; 5+10 or 5+12 at Glu-D1 locus) was an upward trend over the last 40 years. The more different superior alleles correlated with good bread-making quality should be introduced for their usage in wheat improvement efforts.     

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.