Evidence of intralocus recombination at the <Emphasis Type="Italic">Glu-3</Emphasis> loci in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Key message

Recombination at the Glu-3 loci was identified, and strong genetic linkage was observed only between the amplicons representing i-type and s-type genes located, respectively, at the Glu-A3 and Glu-B3 loci.

Abstract

The low-molecular weight glutenin subunits (LMW-GSs) are one of the major components of wheat seed storage proteins and play a critical role in the determination of wheat end-use quality. The genes encoding this class of proteins are located at the orthologous Glu-3 loci (Glu-A3, Glu-B3, and Glu-D3). Due to the complexity of these chromosomal regions and the high sequence similarity between different LMW-GS genes, their organization and recombination characteristics are still incompletely understood. This study examined intralocus recombination at the Glu-3 loci in two recombinant inbred line (RIL) and one doubled haploid (DH) population, all segregating for the Glu-A3, Glu-B3, and Glu-D3 loci. The analysis was conducted using a gene marker system that consists of the amplification of the complete set of the LMW-GS genes and their visualization by capillary electrophoresis. Recombinant marker haplotypes were detected in all three populations with different recombination rates depending on the locus and the population. No recombination was observed between the amplicons representing i-type and s-type LMW-GS genes located, respectively, at the Glu-A3 and Glu-B3 loci, indicating tight linkage between these genes. Results of this study contribute to better understanding the genetic linkage and recombination between different LMW-GS genes, the structure of the Glu-3 loci, and the development of more specific molecular markers that better represent the genetic diversity of these loci. In this way, a more precise analysis of the contribution of various LMW-GSs to end-use quality of wheat may be achieved.

     

Flow karyotyping and chromosome sorting in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Previously, we reported on the development of procedures for chromosome analysis and sorting using flow cytometry (flow cytogenetics) in bread wheat. That study indicated the possibility of sorting large quantities of intact chromosomes, and their suitability for analysis at the molecular level. However, due to the lack of sufficient differences in size between individual chromosomes, only chromosome 3B could be sorted into a high-purity fraction. The present study aimed to identify wheat stocks that could be used to sort other chromosomes. An analysis of 58 varieties and landraces demonstrated a remarkable reproducibility and sensitivity of flow cytometry for the detection of numerical and structural chromosome changes. Changes in flow karyotype, diagnostic for the presence of the 1BL·1RS translocation, have been found and lines from which translocation chromosomes 5BL·7BL and 4AL·4AS-5BL could be sorted have been identified. Furthermore, wheat lines have been identified which can be used for sorting chromosomes 4B, 4D, 5D and 6D. The ability to sort any single arm of the hexaploid wheat karyotype, either in the form of a ditelosome or a isochromosome, has also been demonstrated. Thus, although originally considered recalcitrant, wheat seems to be suitable for the development of flow cytogenetics and the technology can be applied to the physical mapping of DNA sequences, the targeted isolation of molecular makers and the construction of chromosome- and arm-specific DNA libraries. These approaches should facilitate the analysis of the complex genome of hexaploid bread wheat.     

Genetic architecture of seed longevity in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The deterioration in the quality of ex situ conserved seed over time reflects a combination of both physical and chemical changes. Intraspecific variation for longevity is, at least in part, under genetic control. Here, the grain of 183 bread wheat accessions maintained under low-temperature storage at the IPK-Gatersleben genebank over some decades have been tested for their viability, along with that of fresh grain subjected to two standard artificial ageing procedures. A phenotype–genotype association analysis, conducted to reveal the genetic basis of the observed variation between accessions, implicated many regions of the genome, underling the genetic complexity of the trait. Some, but not all, of these regions were associated with variation for both natural and experimental ageing, implying some non-congruency obtains between these two forms of testing for longevity. The genes underlying longevity appear to be independent of known genes determining dormancy and pre-harvest sprouting.     

Structure and expression of the <Emphasis Type="Italic">TaGW7</Emphasis> in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

OsGW7 (also known as OsGL7) is homologous to the Arabidopsis thaliana gene that encodes LONGIFOLIA protein, which regulates cell elongation, and is involved in regulating grain length in rice. However, our knowledge on its ortholog in wheat, TaGW7, is limited. In this study, we identified and mapped TaGW7 in wheat, characterized its nucleotide and protein structures, predicted the cis-elements of its promoter, and analysed its expression patterns. The GW7 orthologs in barley (HvGW7), rice (OsGW7), and Brachypodium distachyon (BdGW7) were also identified for comparative analyses. TaGW7 mapped onto the short arms of group 2 chromosomes (2AS, 2BS, and 2DS). Multiple alignments indicated GW7 possesses five exons and four introns in all but two of the species analysed. An exon–intron junction composed of introns 3–4 and exons 4–5 was highly conserved. GW7 has a conserved domain (DUF 4378) and two neighbouring low complexity regions. GW7 was mainly expressed in wheat spikes and stems, in barley seedling crowns, and in rice anthers and embryo-sacs during early development. Drought and heat significantly increased and decreased GW7 expression in wheat, respectively. In barley, GW7 was significantly down-regulated in paleae and awns but up-regulated in seeds under drought treatment and down-regulated under Fusarium and stem rust inoculation. In rice, OsGW7 expression differed significantly under drought treatments. Collectively, these results provide insights into GW7 structure and expression in wheat, barley and rice. The GW7 sequence structure and expression data are the foundation for manipulating GW7 and uncovering its roles in plants.     

A high-density microsatellite consensus map for bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

A microsatellite consensus map was constructed by joining four independent genetic maps of bread wheat. Three of the maps were F₁-derived, doubled-haploid line populations and the fourth population was Synthetic × Opata, an F₆-derived, recombinant-inbred line population. Microsatellite markers from different research groups including the Wheat Microsatellite Consortium, GWM, GDM, CFA, CFD, and BARC were used in the mapping. A sufficient number of common loci between genetic maps, ranging from 52 to 232 loci, were mapped on different populations to facilitate joining the maps. Four genetic maps were developed using MapMaker V3.0 and JoinMap V3.0. The software CMap, a comparative map viewer, was used to align the four maps and identify potential errors based on consensus. JoinMap V3.0 was used to calculate marker order and recombination distances based on the consensus of the four maps. A total of 1,235 microsatellite loci were mapped, covering 2,569 cM, giving an average interval distance of 2.2 cM. This consensus map represents the highest-density public microsatellite map of wheat and is accompanied by an allele database showing the parent allele sizes for every marker mapped. This enables users to predict allele sizes in new breeding populations and develop molecular breeding and genomics strategies.Electronic Supplementary Material Supplementary material is available for this article at     

Interactions between <Emphasis Type="Italic">Glu-1</Emphasis> and <Emphasis Type="Italic">Glu-3</Emphasis> loci and associations of selected molecular markers with quality traits in winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) DH lines

The quality of wheat depends on a large complex of genes and environmental factors. The objective of this study was to identify quantitative trait loci controlling technological quality traits and their stability across environments, and to assess the impact of interaction between alleles at loci Glu-1 and Glu-3 on grain quality. DH lines were evaluated in field experiments over a period of 4 years, and genotyped using simple sequence repeat markers. Lines were analysed for grain yield (GY), thousand grain weight (TGW), protein content (PC), starch content (SC), wet gluten content (WG), Zeleny sedimentation value (ZS), alveograph parameter W (APW), hectolitre weight (HW), and grain hardness (GH). A number of QTLs for these traits were identified in all chromosome groups. The Glu-D1 locus influenced TGW, PC, SC, WG, ZS, APW, GH, while locus Glu-B1 affected only PC, ZS, and WG. Most important marker-trait associations were found on chromosomes 1D and 5D. Significant effects of interaction between Glu-1 and Glu-3 loci on technological properties were recorded, and in all types of this interaction positive effects of Glu-D1 locus on grain quality were observed, whereas effects of Glu-B1 locus depended on alleles at Glu-3 loci. Effects of Glu-A3 and Glu-D3 loci per se were not significant, while their interaction with alleles present at other loci encoding HMW and LMW were important. These results indicate that selection of wheat genotypes with predicted good bread-making properties should be based on the allelic composition both in Glu-1 and Glu-3 loci, and confirm the predominant effect of Glu-D1d allele on technological properties of wheat grains.     

The endophytic fungi from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

In order to study the species composition of endophytes from wheat healthy plants in Buenos Aires Province (Argentina) and to determine their infection frequencies from leaves, stems, glumes and grains, wheat plants were collected from five cultivars at five growth stages from crop emergence to harvest. A total of 1,750 plant segments (leaves, stems, glumes and grains) were processed from the five wheat cultivars at five growth stages, and 722 isolates of endophytic fungi recovered were identified as 30 fungal genera. Alternaria alternata, Cladosporium herbarum, Epicoccum nigrum, Cryptococcus sp., Rhodotorula rubra, Penicillium sp. and Fusarium graminearum were the fungi that showed the highest colonization frequency (CF%) in all the tissues and organs analysed. The number of taxa isolated was greater in the leaves than those in the other organs analysed.     

Formation of phenolic compounds in various cultivars of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The formation of soluble phenolic compounds, including flavonols, was studied in winter (Erythrospermum , Lutescens 230, and R 47-28) and spring cultivars (Lada) of wheat (Triticum aestivum L.). The contents of soluble phenolic compounds and flavonols were 1.8–2.6 and 0.5–1.3 mg/g fresh weight, respectively. These results illustrate the similarity of phenol metabolism in leaves of winter and spring wheat cultivars. The exception was the cultivar R 47-28 that accumulated the maximum amount of phenolic compounds (e.g., flavonols). In this cultivar the ratio of flavonols reached 50% of total soluble phenol content. In other cultivars, this parameter did not exceed 25–35%. The data indicate that the cultivar R 47-28 differs from other wheat cultivars in metabolism of phenolic compounds. The observed differences are probably related to genetic modifications of the cultivar R 47-28 during selection.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 113–116.Original Russian Text Copyright © 2005 by Zagoskina, Olenichenko, Chzhou Yunvei, Zhivukhina.     

QTL mapping for some grain traits in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Grain traits are important agronomic attributes with the market value as well as milling yield of bread wheat. In the present study, quantitative trait loci (QTL) regulating grain traits in wheat were identified. Data for grain area size (GAS), grain width (GWid), factor form density (FFD), grain length-width ratio (GLWR), thousand grain weight (TGW), grain perimeter length (GPL) and grain length (GL) were recorded on a recombinant inbred line derived from the cross of NW1014?×?HUW468 at Meerut and Varanasi locations. A linkage map of 55 simple sequence repeat markers for 8 wheat chromosomes was used for QTL analysis by Composite interval mapping. Eighteen QTLs distributed on 8 chromosomes were identified for seven grain traits. Of these, five QTLs for GLWR were found on chromosomes 1A, 6A, 2B, and 7B, three QTLs for GPL were located on chromosomes 4A, 5A and 7B and three QTLs for GAS were mapped on 5D and 7D. Two QTLs were identified on chromosomes 4A and 5A for GL and two QTLs for GWid were identified on chromosomes 7D and 6A. Similarly, two QTLs for FFD were found on chromosomes 1A and 5D. A solitary QTL for TGW was identified on chromosome 2B. For several traits, QTLs were also co-localized on chromosomes 2B, 4A, 5A, 6A, 5D, 7B and 7D. The QTLs detected in the present study may be validated for specific crosses and then used for marker-assisted selection to improve grain quality in bread wheat.     

Drought-induced changes in photosynthetic membranes of two wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) cultivars

Two wheat (Triticum aestivum L.) cultivars contrasting in architectonics and differing in drought resistance, Azamatli-95 (short stature, vertically oriented small leaves, drought-tolerant) and Giymatli-2/17 (short stature, broad and drooping leaves, drought-sensitive), were studied. It was found that the content of CP I (115 kDa) and 63-kDa apoprotein P700 and also LHC II polypeptides increases slightly in the drought-resistant cv. Azamatli-95 under extreme water supply limitation, while their content decreases in drought-sensitive cv. Giymatli-2/17. The intensity of synthesis of α- and β-subunits of CF₁ (55 and 53.5 kDa) and 33–30.5 kDa proteins also decreases in the sensitive genotype. The intensity of short wavelength peaks at 687 and 695 nm sharply increases in the fluorescence spectra (77K) of chloroplasts from Giymatli-2/17 under water deficiency, and there is a stimulation of the ratio of fluorescence band intensity F687/F740. After exposure to drought, cv. Giymatli-2/17 shows a larger reduction in the actual PS II photochemical efficiency of chloroplasts than cv. Azamatli-95. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 8, pp. 1109–1116.     

QTL mapping for quantities of protein fractions in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

One of the key targets of breeding programs in bread wheat is to improve the end-use quality. The relationships between quantities of protein fractions and dough rheological characters have been well established, but there is little information on the genetic control of quantities of protein fractions. Two hundred and forty F₆ recombinant inbred lines derived from a cross between two Chinese wheat cultivars, PH82-2 and Neixiang 188, were sown at Jiaozuo in Henan province in the 2005–2006 and 2006–2007 cropping seasons, and inclusive composite interval mapping was used to dissect main effect quantitative trait loci (M-QTLs) and digenic epistatic QTLs (E-QTLs) for quantities of protein fractions. A total of 55 M-QTLs and 77 pairs of E-QTLs affecting the quantities of protein fractions including GLU-A1 (QGA1), GLU-B1 (QGB1), GLU-D1 (QGD1), HMW-GS (QHMW), GLU-A3 (QGA3), GLU-B3 (QGB3), LMW-GS (QLMW), glutenin (QGLU) and the ratio of the quantity of glutenin to those of gliadin were identified, with M-QTLs contributing 39.3–95.6% of the phenotypic variance explained (PVE), and E-QTLs accounting for 1.4–33.5% of the PVE. Among the M-QTLs, 33 were consistent in two seasons and in the mean value of two seasons with similar effects in both magnitude and direction, including major genes on HMW and LMW glutenin loci linked to Sec1 and Glu-B1c, Glu-D1d, Glu-A3a, and grain hardness locus Ha, indicating that these genes were the most important determinants of gluten strength, and they might have significant effects on dough properties not only through effects on allelic composition, but also by influencing quantities of protein fractions. The effects of E-QTLs were more influenced by environments, compared with those of M-QTLs, with only two pairs of E-QTLs consistent in two seasons and in the mean value of two seasons. The M-QTLs were detected in 12 marker intervals, all of which involved E-QTLs on quantities of protein fractions, whereas only 40 of 77 pairs of E-QTLs involved intervals in which M-QTLs were detected. The results indicated that besides main effects, epistatic effects were also important factors in determining quantities of protein fractions in wheat.     

Molecular mapping of genes for Coleoptile growth in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Successful plant establishment is critical to the development of high-yielding crops. Short coleoptiles can reduce seedling emergence particularly when seed is sown deep as occurs when moisture necessary for germination is deep in the subsoil. Detailed molecular maps for a range of wheat doubled-haploid populations (Cranbrook/Halberd, Sunco/Tasman, CD87/Katepwa and Kukri/Janz) were used to identify genomic regions affecting coleoptile characteristics length, cross-sectional area and degree of spiralling across contrasting soil temperatures. Genotypic variation was large and distributions of genotype means were approximately normal with evidence for transgressive segregation. Narrow-sense heritabilities were high for coleoptile length and cross-sectional area indicating a strong genetic basis for differences among progeny. In contrast, heritabilities for coleoptile spiralling were small. Molecular marker analyses identified a number of significant quantitative trait loci (QTL) for coleoptile growth. Many of the coleoptile growth QTL mapped directly to the Rht-B1 or Rht-D1 dwarfing gene loci conferring reduced cell size through insensitivity to endogenous gibberellins. Other QTL for coleoptile growth were identified throughout the genome. Epistatic interactions were small or non-existent, and there was little evidence for any QTL × temperature interaction. Gene effects at significant QTL were approximately one-half to one-quarter the size of effects at the Rht-B1 and Rht-D1 regions. However, selection at these QTL could together alter coleoptile length by up to 50 mm. In addition to Rht-B1b and Rht-D1b, genomic regions on chromosomes 2B, 2D, 4A, 5D and 6B were repeatable across two or more populations suggesting their potential value for use in breeding and marker-aided selection for greater coleoptile length and improved establishment.     

Functional gene markers for polyphenol oxidase locus in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Higher polyphenol oxidase (PPO) activity in wheat kernels and flour has been implicated in the time dependent darkening of various end-products. Previous study conducted on a bread wheat (Triticum aestivum L.) doubled haploid (DH) mapping population derived from Chara (medium-high PPO) and WW2449 (low PPO) identified a major QTL for PPO activity located on the long arm of chromosome 2A. Physical mapping of SSR markers accounting for up to 84% of phenotypic variation for PPO activities suggests that the candidate PPO locus is localised in the deletion bin delimited by 2AL 0.77–0.85. In order to develop functional gene markers, nine wheat ESTs mapped to this deletion bin and partial PPO reference genes were explored for their sequence identities and linkage with PPO locus in a mapping population. In the present study, two markers: one SNP and one CAPS based upon BQ161439 sequence variation between the parents were identified which exhibited a tight linkage (0–0.6 cM) with the PPO loci designated as XTc1 and XPPO- _LDOPA. We also mapped the reference PPO gene (GenBank AY526268) characterised from developing kernels of wheat, on the long arm of chromosome 2A which exhibited a complete linkage with XPPO- _L DOPA locus. Results suggest that PPO variation displayed in the DH population from Chara/WW2449 is due to the same reference PPO gene. Allelic homoplasy of tightly linked markers, indicated that these markers are ‘diagnostic’ for the selection of low PPO gene in a range of germplasm being used in different Australian breeding programs. Identification and validation of ‘functional gene markers’ would facilitate in enhancing the selection efficiency for low PPO activity in wheat breeding programs.     

New data on the distribution of hybrid necrosis genes in winter bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) cultivars

Hybrid necrosis genotypes have been identified in 125 Russian cultivars of winter bread wheat. More than half of them (56%) carry the Ne2 gene (genotype ne1ne1Ne2Ne2); others are free of necrosis genes (genotype ne1ne1ne2ne2). The possible causes of the increase in the Ne2 allele frequency and the loss of the Ne1Ne1ne2ne2 genotype in modern Russian cultivars of winter wheat are discussed. The principal component method has been used to compare the structures of the genetic diversity of cultivars differing in the hybrid necrosis genotype. It has been found that the Ne2 allele in winter wheat cultivars from northern Russia has originated from the cultivar Mironovskaya 808, whereas the cultivar Bezostaya 1 is not a source of this gene. In cultivars from southern Russia, the presence of the Ne2 allele is also mainly accounted for by the use of Mironovskaya 808 wheat in their breeding. The recessive genotype is explained by the presence of descendants of the cultivar Odesskaya 16 in the pedigrees of southern Russian winter wheats. The genetic relationship of cultivars with identical and different necrosis genotypes has been analyzed in nine regions of the Russian Federation.     

Analysis of diversity of russian and Ukrainian bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) cultivars for high-molecular-weight glutenin subunits

The allelic diversity of high-moleculat-weght glutenin subunits (HMWGS) 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 polymorphism information content (PIC), 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.     

Overexpression of <Emphasis Type="Italic">AtHDG11</Emphasis> enhanced drought tolerance in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Drought is one of the major abiotic stresses restricting the yield of wheat (Triticum aestivum L.). Breeding wheat varieties with drought tolerance is an effective and durable way to fight against drought. Here we reported introduction of AtHDG11 into wheat via Agrobacterium-mediated transformation and analyzed the morphological and physiological characteristics of T2 generation transgenic lines under drought stress. With drought treatment for 30 days, transgenic plants showed significantly improved drought tolerance. Compared with controls, the transgenic lines displayed lower stomatal density, lower water loss rate, more proline accumulation and increased activities of catalase and superoxide dismutase. Without irrigation after booting stage, the photosynthetic parameters, such as net photosynthesis rate, water use efficiency and efficiency of excitation energy, were increased in transgenic lines, while transpiration rate was decreased. Moreover, the kernel yield of transgenic lines was also improved under drought condition. Taken together, our data demonstrate that AtHDG11 has great potential in genetic improvement of drought tolerance of wheat.     

Assessment of genetic diversity among Syrian durum (<Emphasis Type="Italic">Triticum</Emphasis> ssp. <Emphasis Type="Italic">durum</Emphasis>) and bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) using SSR markers

Genetic diversity among 49 wheat varieties (37 durum and 12 bread wheat) was assayed using 32 microsatellites representing 34 loci covering almost the whole wheat genome. The polymorphic information content (PIC) across the tested loci ranged from 0 to 0.88 with average values of 0.57 and 0.65 for durum and bread wheat respectively. B-genome had the highest mean number of alleles (10.91) followed by A genome (8.3) whereas D genome had the lowest number (4.73). The correlation between PIC and allele number was significant in all genome groups accounting for 0.87, 074 and 0.84 for A, B and D genomes respectively, and over all genomes, the correlation was higher in tetraploid (0.8) than in hexaploid wheat varieties (0.5). The cluster analysis discriminated all varieties and clearly divided the two ploidy levels into two separate clusters that reflect the differences in genetic diversity within each cluster. This study demonstrates that microsatellites markers have unique advantages compared to other molecular and biochemical fingerprinting techniques in revealing the genetic diversity in Syrian wheat varieties that is crucial for wheat improvement.     

Identification of SNPs and development of functional markers for LMW-GS genes at <Emphasis Type="Italic">Glu-D3</Emphasis> and <Emphasis Type="Italic">Glu-B3</Emphasis> loci in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Low-molecular-weight glutenin subunits (LMW-GS) have great effect on wheat processing quality, but were numerous and difficult to dissect by SDS-PAGE. The development of functional markers may be the most effective way for a clear discrimination of different LMW-GS genes. In the present study, three different approaches were used to identify SNPs of different genes at Glu-D3 and Glu-B3 loci in bread wheat for the development of six STS markers (3 for Glu-D3 and 3 for Glu-B3 genes) that were validated with distinguished wheat cultivars. Firstly, seven LMW-GS gene sequences ( AY585350, AY585354, AY585355, AY585356, AY585349, AY585351 and AY585353 ) from Aegilops tauschii, the diploid donor of the D-genome of bread wheat, were chosen to design seven pairs of AS-PCR primers for Glu-D3 genes. By amplifying the corresponding genes from five bread wheat cultivars with different Glu-D3 alleles (a, b, c, d and e) and Ae. tauschii, a primer set, S13F2/S13R1, specific to the gene AY585356, was found to be positive to cultivars with alleles Glu-D3c and d. Nevertheless, the other five pairs of primers designed from AY585350, AY585349, AY585353, AY585354 and AY585355, respectively, did not produce specific PCR products to the cultivars tested. Secondly, all the PCR products from the five primer sets without specific characteristics were sequenced and an SNP from the gene AY585350 was detected in the cultivar Hartog, which resulted in the second STS marker S1F1/S1R3 specific to the allelic variant of AY585350. Thirdly, three Glu-D3 sequences (AB062851, AB062865 and AB062872) and three Glu-B3 sequences (AB062852, AB062853 and AB062860) defined by Ikeda et al. (2002) were chosen to query wheat EST and NR databases, and DNA markers were developed based on the putative SNPs among the sequences. Using this approach, four STS markers were developed and validated with 16-19 bread wheat cultivars. The primer set T1F4/T1R1 was also a Glu-D3 gene-specific marker for AB062872, while T2F2/T2R2, T5F3/T5R1 and T13F4/T13R3 were all Glu-B3 gene specific markers for AB062852, BF293671 and AY831800, respectively. The chromosomal locations of the six markers were verified by amplifying the genomic DNA of Ae. tauschii (DD), T. monococcum (AA) and T. turgidum (AABB) entries, as well as Chinese Spring and its group 1 chromosome nulli-tetrasomic lines. The results are useful to discriminate the corresponding Glu-D3 and Glu-B3 genes in wheat breeding programs.