Glu-D1 Alleles Research Articles

ASSESSMENT OF HIGH MOLECULAR WEIGHT PROTEINS IN SELECTED INDIGENOUS BREAD WHEAT VARIETIES OF PAKISTAN

Higher molecular weight gluten subunits (HMW-GS) articulate variation in allelic frequency, reflecting a strong correlation with bread quality. In order to determine this quality trait, 23 different Pakistani indigenous wheat varieties were screened that may be used as selection criteria in a breeding program. For detecting variations in HMW-GS, sodium dodecyl sulfate acrylamide gel electrophoresis was used. In all, 77 Glu-1 were identified including 25 at Glu-A1, 26 at Glu-B1 and 26 at Glu-D1. Alleles at Glu-1 were 2* with a frequency of 53.3% and N with 60% frequency at Glu-A1, 6+8 with 60%, 17+18 with 45 % at Glu-B1 and 5+10 with 60% and 72% at Glu-D1. The highest frequency of subunits 20 and 5+10 were found at Glu-B1-e and Glu-D1 (a), followed by a frequency of 2* and Null at Glu-A1 (b, c), which is then followed by 2+12 at GluD-1 (a). The lowest frequency of 13+16, 17+18 and 6+8 were observed at Glu-B1 (f, i, d), respectively. Glu-A1, Glu-B1, and Glu-D1 alleles showed genetic index (i.e. 0.587, 0.54), (0.587. 0.065 and 0.48, 0.39, respectively) with an average of 0.55 and 0.37. The glu-D1 locus has a lower genetic index comparatively. The composition N, 20, 5+10 was found in maximum number (4) in Fakhr-e-Sarhad, Pasban, Pirsabak-2004 and Tatara genotypes, followed by the composition of 2*, 20, 2+12 in one check (Marvi-2000) and two varieties i.e. Chakwal-97 and Bakhtawar-92. The results describing the allelic frequency and composition of bread wheat would benefit further breeding plans for wheat to select good quality parameters.

Identification of Glu-D1 Alleles and Novel Marker-Trait Associations for Flour Quality and Grain Yield Traits under Heat-Stress Environments in Wheat Lines Derived from Diverse Accessions of Aegilops tauschii.

Heat stress during grain filling is considered one of the major abiotic factors influencing wheat grain yield and quality in arid and semi-arid regions. We studied the effect of heat stress on flour quality and grain yield at moderate and continuous heat stress under natural field conditions using 147 lines of wheat multiple synthetic derivatives (MSD) containing Aegilops tauschii introgressions. The study aimed to identify the marker–trait associations (MTAs) for the quality traits and grain yield under heat-stress conditions and identify stress-resilient germplasm-combining traits for good flour quality and grain yield. The MSD lines showed considerable genetic variation for quality traits and grain yield under heat-stress conditions; some lines performed better than the recurrent parent, Norin 61. We identified two MSD lines that consistently maintained relative performance (RP) values above 100% for grain yield and dough strength. We found the presence of three high-molecular-weight glutenin subunits (HMW-GSs) at the Glu-D1 locus derived from Ae. tauschii, which were associated with stable dough strength across the four environments used in this study. These HMW-GSs could be potentially useful in applications for future improvements of end-use quality traits targeting wheat under severe heat stress. A total of 19,155 high-quality SNP markers were used for the genome-wide association analysis and 251 MTAs were identified, most of them on the D genome, confirming the power of the MSD panel as a platform for mining and exploring the genes of Ae. tauschii. We identified the MTAs for dough strength under heat stress, which simultaneously control grain yield and relative performance for dough strength under heat-stress/optimum conditions. This study proved that Ae. tauschii is an inexhaustible resource for genetic mining, and the identified lines and pleiotropic MTAs reported in this study are considered a good resource for the development of resilient wheat cultivars that combine both good flour quality and grain yield under stress conditions using marker-assisted selection.

High molecular weight glutenin gene diversity in Aegilops tauschii demonstrates unique origin of superior wheat quality

Central to the diversity of wheat products was the origin of hexaploid bread wheat, which added the D-genome of Aegilops tauschii to tetraploid wheat giving rise to superior dough properties in leavened breads. The polyploidization, however, imposed a genetic bottleneck, with only limited diversity introduced in the wheat D-subgenome. To understand genetic variants for quality, we sequenced 273 accessions spanning the known diversity of Ae. tauschii. We discovered 45 haplotypes in Glu-D1, a major determinant of quality, relative to the two predominant haplotypes in wheat. The wheat allele 2 + 12 was found in Ae. tauschii Lineage 2, the donor of the wheat D-subgenome. Conversely, the superior quality wheat allele 5 + 10 allele originated in Lineage 3, a recently characterized lineage of Ae. tauschii, showing a unique origin of this important allele. These two wheat alleles were also quite similar relative to the total observed molecular diversity in Ae. tauschii at Glu-D1. Ae. tauschii is thus a reservoir for unique Glu-D1 alleles and provides the genomic resource to begin utilizing new alleles for end-use quality improvement in wheat breeding programs.

Characterization of high- and low-molecular-weight glutenin subunits from Chinese Xinjiang wheat landraces and historical varieties.

Landraces and historical varieties are necessary germplasms for genetic improvement of modern cereals. Allelic variations at the Glu-1 and Glu-3 loci in 300 common wheat landraces and 43 historical varieties from Xinjiang, China, were evaluated by Sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and allele-specific molecular markers. Among the materials investigated, three, nine, and seven alleles were identified from the Glu-A1, Glu-B1, and Glu-D1 loci, respectively, and a total of 26 high-molecular-weight glutenin subunit (HMW-GS) combinations were found, of which 18 combinations were identified in landraces and historical varieties. Allelic frequency of HMW-GS combinations null, 7 + 8, 2 + 12 was found to be the highest in both the landraces (63.3%) and historical varieties (39.5%). Besides, some distinctive HMW-GS alleles, such as the novel Glu-B1 allele 6.1* + 8.1* and Glu-D1 alleles 2.6 + 12, 2.1 + 10.1, and 5** + 10 were observed in Xinjiang wheat landraces. Among the Glu-A3 and Glu-B3 loci of landraces and historical varieties, a total of eight and nine alleles were found, respectively. At each locus, two novel alleles were identified. A total of 33 low-molecular-weight glutenin subunit (LMW-GS) combinations of Glu-A3 and Glu-B3 were identified, with 31 and 14 combinations occurring in landraces and historical varieties, respectively, but only 10 combinations shared by both of them. As Glu-D1, Glu-A3, and Glu-B3 have highest contribution to the end-use quality and processing properties as compared to Glu-A1, Glu-B1, and Glu-D3 locus, the novel or distinctive HMW-GS and LMW-GS alleles in these loci could potentially be utilized for the improvement in the quality of modern wheat.

Development of 1AS.1AL-1DL durum wheat chromosome carrying Glu-D1a locus encoding high molecular weight glutenin subunits 2 + 12

High molecular weight glutenin subunits (HMW-GS) are well-known to directly influence the bread wheat quality. In a previous paper, our group reported the replacement of the locus Glu-A1 by the locus Glu-D1—allele Glu-D1a encoding the HMW-GS (2–12)—in a partial isohomoeoallelic lines of the French bread wheat variety cv. Courtot. We plan to exploit the novel translocated 1AS.1AL-1DL chromosome in durum wheat in order to develop durum lines presenting novel properties. The translocated chromosome was first introduced into durum wheat. However, as the distal translocated 1DL segment represents 25% (i.e., 84 Mb) of the recombinant 1AL length, it appeared worthwhile to shorten it through homoeologous recombination induced by the ph1c mutation. Two rounds of recombination were made. The shortest interstitial 1DL segment detected through SNP genotyping measured 9.19 Mb and could not be visualized by genomic in situ hybridization. Such reduced transfer not previously achieved hitherto provides regularity of the meiotic pairing and will allow the use of those subunits absent in durum wheat species for direct exploitability in breeding.

Identification of Glu-A1 and Glu-D1 High Molecular Weight Glutenin Subunits of Common Wheat (Triticum aestivum L.) Using Genetic Markers

Abstract High molecular weight glutenin subunits (HMW-GS) of wheat are important factors in the determination of bread-making quality. They are responsible for elasticity and polymer formation of wheat dough. In the present study, 43 winter and 40 spring common wheat (Triticum aestivum L.) cultivars originated from Estonia, Belarus, Finland, Denmark, France, Germany, the Great Britain, Latvia, Lithuania, the Netherlands, Norway, Poland, Russia, Sweden, and New Zealand were characterised for Glu-A1 and Glu-D1 allelic composition using PCR method. Analyses were conducted with one DNA marker for identification of Glu-D1 allele encoding subunit Dx5, three DNA markers for Glu-A1 Ax1, Ax2* and AxNull subunits. It was determined that 32 (74.4%) winter and 35 (83.3%) spring cultivars had allele Glu-D1d, and 23 (53.5%) winter and 33 (78.6%) spring — Glu-A1a or Glu-A1b alleles, which have positive effect on dough properties. Polymorphism at Glu-A1 locus was detected in 15 cultivars, and 9 cultivars were polymorphic for locus Glu-D1. The obtained results were compared with published SDS-PAGE data. Complete or partial agreements were found for 78.1% of Glu-A1 and 70.6% of Glu-D1 alleles. Rapid and accurate identification of wheat Glu-1 alleles by molecular markers can be used for selection of wheat genotypes with good bread-making potential.

Allelic variation of LMW-GS composition in Chinese wheat landraces of the Yangtze-River region detected by MALDI-TOF-MS.

Low molecular weight glutenin subunits are important components of wheat storage proteins, which play an important role in determining end-use quality of common wheat. A newly established matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) procedure was used to analyze 478 landraces of bread wheat collected from the Yangtze-River region in China. Results indicated that 17 alleles at three loci: Glu-A3, Glu-B3 and Glu-D3 were identified, resulting in 87 different allele combinations. Of the 17 alleles detected at all the Glu-3 loci, five belonged to Glu-A3, seven to Glu-B3 and five to Glu-D3 locus. MALDI-TOF-MS indicated Glu-A3a/c was present in 72.8%, Glu-A3b in 8.4%, Glu-A3d in 8.4%, Glu-A3f in 5.2% and Glu-A3e in 3.6% lines. Seven types of alleles were identified at the Glu-B3 locus: Glu-B3d/i (25.5%), Glu-B3b (21.3%), Glu-B3c (16.9%), Glu-B3h (13.8%), Glu-B3f (8.4%), Glu-B3a (8.2%), and Glu-B3g (5.2%). Five types of Glu-D3 alleles were detected: Glu-D3a (58.4%), Glu-D3c (22.6%), Glu-D3d (15.5%), Glu-D3b (3.3%) and Glu-D3f (0.2%). Four new alleles that showed abnormal MALDI-TOF spectrum patterns were identified at the Glu-A3 and Glu-B3 loci. A detailed study is needed to further characterize these alleles and their potential usage for wheat improvement.

The Interactive Effects of Transgenically Overexpressed 1Ax1 with Various HMW-GS Combinations on Dough Quality by Introgression of Exogenous Subunits into an Elite Chinese Wheat Variety

Seed storage proteins in wheat endosperm, particularly high-molecular-weight glutenin subunits (HMW-GS), are primary determinants of dough properties, and affect both end-use quality and grain utilization of wheat (Triticum aestivum L). In order to investigate the interactive effects between the transgenically overexpressed 1Ax1 subunit with different HMW-GS on dough quality traits, we developed a set of 8 introgression lines (ILs) overexpressing the transgenic HMW-glutenin subunit 1Ax1 by introgression of this transgene from transgenic line B102-1-2/1 into an elite Chinese wheat variety Chuanmai107 (C107), using conventional crossing and backcrossing breeding technique. The donor C107 strain lacks 1Ax1 but contains the HMW-GS pairs 1Dx2+1Dy12 and 1Bx7+1By9. The resultant ILs showed robust and stable expression of 1Ax1 even after five generations of self-pollination, and crossing/backcrossing three times. In addition, overexpression of 1Ax1 was compensated by the endogenous gluten proteins. All ILs exhibited superior agronomic performance when compared to the transgenic parent line, B102-1-2/1. Mixograph results demonstrated that overexpressed 1Ax1 significantly improved dough strength, resistance to extension and over-mixing tolerance, in the targeted wheat cultivar C107. Further, comparisons among the ILs showed the interactive effects of endogenous subunits on dough properties when 1Ax1 was overexpressed: subunit pair 17+18 contributed to increased over-mixing tolerance of the dough; expression of the Glu-D1 allele maintained an appropriate balance between x-type and y-type subunits and thereby improved dough quality. It is consistent with ILs C4 (HMW-GS are 1, 17+18, 2+12) had the highest gluten index and Zeleny sedimentation value. This study demonstrates that wheat quality could be improved by using transgenic wheat overexpressing HMW-GS and the feasibility of using such transgenic lines in wheat quality breeding programs.

Composition and functional analysis of low-molecular-weight glutenin alleles with Aroona near-isogenic lines of bread wheat

BackgroundLow-molecular-weight glutenin subunits (LMW-GS) strongly influence the bread-making quality of bread wheat. These proteins are encoded by a multi-gene family located at the Glu-A3, Glu-B3 and Glu-D3 loci on the short arms of homoeologous group 1 chromosomes, and show high allelic variation. To characterize the genetic and protein compositions of LMW-GS alleles, we investigated 16 Aroona near-isogenic lines (NILs) using SDS-PAGE, 2D-PAGE and the LMW-GS gene marker system. Moreover, the composition of glutenin macro-polymers, dough properties and pan bread quality parameters were determined for functional analysis of LMW-GS alleles in the NILs.ResultsUsing the LMW-GS gene marker system, 14–20 LMW-GS genes were identified in individual NILs. At the Glu-A3 locus, two m-type and 2–4 i-type genes were identified and their allelic variants showed high polymorphisms in length and nucleotide sequences. The Glu-A3d allele possessed three active genes, the highest number among Glu-A3 alleles. At the Glu-B3 locus, 2–3 m-type and 1–3 s-type genes were identified from individual NILs. Based on the different compositions of s-type genes, Glu-B3 alleles were divided into two groups, one containing Glu-B3a, B3b, B3f and B3g, and the other comprising Glu-B3c, B3d, B3h and B3i. Eight conserved genes were identified among Glu-D3 alleles, except for Glu-D3f. The protein products of the unique active genes in each NIL were detected using protein electrophoresis. Among Glu-3 alleles, the Glu-A3e genotype without i-type LMW-GS performed worst in almost all quality properties. Glu-B3b, B3g and B3i showed better quality parameters than the other Glu-B3 alleles, whereas the Glu-B3c allele containing s-type genes with low expression levels had an inferior effect on bread-making quality. Due to the conserved genes at Glu-D3 locus, Glu-D3 alleles showed no significant differences in effects on all quality parameters.ConclusionsThis work provided new insights into the composition and function of 18 LMW-GS alleles in bread wheat. The variation of i-type genes mainly contributed to the high diversity of Glu-A3 alleles, and the differences among Glu-B3 alleles were mainly derived from the high polymorphism of s-type genes. Among LMW-GS alleles, Glu-A3e and Glu-B3c represented inferior alleles for bread-making quality, whereas Glu-A3d, Glu-B3b, Glu-B3g and Glu-B3i were correlated with superior bread-making quality. Glu-D3 alleles played minor roles in determining quality variation in bread wheat. Thus, LMW-GS alleles not only affect dough extensibility but greatly contribute to the dough resistance, glutenin macro-polymers and bread quality.

Comparative analyses of LMW glutenin alleles in bread wheat using allele-specific PCR and SDS-PAGE

One hundred and eighty-two bread wheat cultivars developed in India were characterized for low molecular weight (LMW) glutenins using SDS-PAGE and allele-specific polymerase chain reaction (PCR) to assess allelic diversity encoded by Glu-3 loci, as well as their utility for correctly identifying different alleles. SDS-PAGE indicated Glu-A3c is present in 64.6% of the cultivars, Glu-A3b in 13.8%, Glu-A3d in 12.7% and Glu-A3e/f in 8.8%. Seven types of alleles were present at the Glu-B3 locus: Glu-B3b (29.3%), Glu-B3g (27.0%), Glu-B3h (13.8%), Glu-B3i (16.1%), Glu-B3j (12.1%), Glu-B3c (0.6%) and Glu-B3d (1.1%). SDS-PAGE found three types of Glu-D3 alleles: Glu-D3a (30.2%), Glu-D3b (67.1%) and Glu-D3c (2.7%). However, PCR found two different alleles in cultivars classified as carrying Glu-D3a and three alleles in those identified as carrying Glu-D3b cultivars, indicating a more complex nature of the Glu-D3 locus. In conclusion, the data found greater consistency between the SDS-PAGE and PCR amplification patterns of alleles such as Glu-A3c, Glu-A3d, Glu-B3g, Glu-B3h and Glu-B3i, and less consistency between those same patterns in the Glu-A3b, Glu-A3e/f and Glu-B3b alleles. More studies are needed in order to achieve unambiguous identification of the Glu-3 alleles and thereby allow their greater utility in germplasm evaluation and breeding.

Comparison of low molecular weight glutenin subunits identified by SDS-PAGE, 2-DE, MALDI-TOF-MS and PCR in common wheat

BackgroundLow-molecular-weight glutenin subunits (LMW-GS) play a crucial role in determining end-use quality of common wheat by influencing the viscoelastic properties of dough. Four different methods - sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE, IEF × SDS-PAGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and polymerase chain reaction (PCR), were used to characterize the LMW-GS composition in 103 cultivars from 12 countries.ResultsAt the Glu-A3 locus, all seven alleles could be reliably identified by 2-DE and PCR. However, the alleles Glu-A3e and Glu-A3d could not be routinely distinguished from Glu-A3f and Glu-A3g, respectively, based on SDS-PAGE, and the allele Glu-A3a could not be differentiated from Glu-A3c by MALDI-TOF-MS. At the Glu-B3 locus, alleles Glu-B3a, Glu-B3b, Glu-B3c, Glu-B3g, Glu-B3h and Glu-B3j could be clearly identified by all four methods, whereas Glu-B3ab, Glu-B3ac, Glu-B3ad could only be identified by the 2-DE method. At the Glu-D3 locus, allelic identification was problematic for the electrophoresis based methods and PCR. MALDI-TOF-MS has the potential to reliably identify the Glu-D3 alleles.ConclusionsPCR is the simplest, most accurate, lowest cost, and therefore recommended method for identification of Glu-A3 and Glu-B3 alleles in breeding programs. A combination of methods was required to identify certain alleles, and would be especially useful when characterizing new alleles. A standard set of 30 cultivars for use in future studies was chosen to represent all LMW-GS allelic variants in the collection. Among them, Chinese Spring, Opata 85, Seri 82 and Pavon 76 were recommended as a core set for use in SDS-PAGE gels. Glu-D3c and Glu-D3e are the same allele. Two new alleles, namely, Glu-D3m in cultivar Darius, and Glu-D3n in Fengmai 27, were identified by 2-DE. Utilization of the suggested standard cultivar set, seed of which is available from the CIMMYT and INRA Clermont-Ferrand germplasm collections, should also promote information sharing in the identification of individual LMW-GS and thus provide useful information for quality improvement in common wheat.

Evaluation and characterization of high-molecular weight 1D glutenin subunits from Aegilops tauschii in synthetic hexaploid wheats

The high-molecular weight (HMW) glutenin subunits of bread wheat are major determinants of end-use quality. The objective of this study was to determine the 1Dx and 1Dy subunits present in 43 synthetic hexaploid wheat (SHW) lines derived by crossing durum ‘Langdon’ to 43 Aegilops tauschii accessions. Protein samples were initially electrophoresed multiple times on SDS-PAGE gels to arrange subunits into similar groups and then were electrophoresed on urea/SDS-PAGE gels. Initial results with SDS-PAGE gels indicated that there were six 1Dx and six 1Dy subunits in these SHW lines. However, results of the urea/SDS-PAGE indicated that some of the subunit groups could be further differentiated into additional subunits. A total of eleven 1Dx and eight 1Dy subunits including the newly designated subunits 1Dx2 t-1, 1Dx2 t-2, 1Dx2 t-3, 1Dx1.5 t-1, 1Dx2.1 t-1, 1Dy10 t-1, and 1Dy12 t-1 were identified, and they composed 17 1Dx and 1Dy combinations in the SHW lines. Eight of the combinations included at least one novel subunit and hence they were novel Glu-D1 alleles. Our results indicated that urea/SDS-PAGE can be very useful in identifying new HMW glutenin subunits. Quality testing of the SHW lines will determine if any of the alleles are useful in improving wheat-baking quality.

Effects of the replacement of Glu-A1 by Glu-D1 locus on agronomic performance and bread-making quality of the hexaploid wheat cv. Courtot

The aim of this study was to evaluate the cumulative and interactive effects on wheat ( Triticum aestivum L.) gluten strength and mixing properties of dough associated with the duplication of the Glu-D1 locus. A partially isohomoeoallelic line RR240, in which a segment of the wheat chromosome 1D containing the Glu-D1 locus encoding the Dx2 + Dy12 subunits and translocated to the long arm of the chromosome 1A through homoeologous recombination, was assessed. Agronomic traits and yield components were studied in the translocated line RR240 and compared with the control line cv. Courtot. Both lines were evaluated under field conditions in two experimental years. Technological effects resulting from the duplication of HMW glutenin subunits Dx2 and Dy12 were evaluated using the Alveograph test, the Mixograph test and the baking test. The RR240 line was shown to have a lower agronomic performance for 1000-kernel weight and grain yield. However the duplication of the Glu-D1 allele was associated with a significant effect on dough strength and mixing resistance, and on the Zeleny sedimentation volume. Baking parameters were not significantly modified between both lines although the score values of the CNERNA test were observed to be slightly higher in RR240 than in Courtot.

Allelic variation at theGlu-D3locus in Chinese bread wheat and effects on dough properties, pan bread and noodle qualities

Glutenin subunit alleles at the Glu-D3 locus and their effects on dough properties, pan bread, and dry white Chinese noodle (DWCN) qualities were investigated using 106 winter and facultative wheat cultivars and advanced lines. Allele Glu-D3c (42.5%) was the most frequent glutenin subunit, followed by Glu-D3b (25.5%) and Glu-D3a (23.6%). Glu-D3d and Glu-D3f occurred in only three and six cultivars, respectively. The effect of Glu-D3 was significant forDWCNquality, accounting for up to 16% of the variation, but there were no significant differences between individual Glu-D3 alleles on dough properties and qualities of DWCNand pan bread. Interaction effects Glu-A1 × Glu-D3 and Glu-B1 × Glu-D3 were significant for DWCN quality and loaf volume. More work is needed to understand the effects of Glu-D3 variation on the determination of dough properties and end-use quality.

Novel allelic variants encoded at the Glu-D3 locus in bread wheat

Low-molecular weight glutenin subunits (LWM-GS) are important components of wheat ( Triticum aestivum L.) gluten, with important effects on end-use quality. The LMW-GS are encoded at Glu-3 loci ( Glu-A3, Glu-B3 and Glu-D3, on the short arms of chromosomes 1A, 1B and 1D), each of which exhibits extensive allelic variation. Each locus encodes numerous LMW-GS, some of which have similar electrophoretic mobilities, making it difficult to distinguish among Glu-3 loci. Alleles of the Glu-D3 locus of bread wheat are considered the most problematic to assign. To date, six Glu-D3 alleles, designated a, b, c, d, e and f, have been reported. We report five previously undescribed alleles ( g, h, i, j and k), and describe a method for characterizing them using a combination of SDS-PAGE and multiplexed PCR-based DNA markers. This method could be used for accurate identification of Glu-D3 alleles, permitting the estimation of the effects of these alleles on end-use quality and the selection of desirable alleles and allelic combinations in wheat breeding.

Wheat breeding for the improved bread-making quality using PCR based markers of glutenins

The relation between high-molecular-weight (HMW) glutenin subunits and bread-making quality could enable selection for improved bread-making quality in early stages of breeding process. The composition of HMW glutenin subunits was investigated in F<sub>2</sub> and F<sub>7</sub> progenies derived from the cross between winter wheat varieties Sulamit and Clever. The presence of Glu-A1 (AxNull and Ax1), Glu-B1 (Bx6+By8 and Bx 17+By18) and Glu-D1 alleles (x 5+Dy10 and Dx 2+Dy12) was monitored using a PCR based assay. Segregation of alleles corresponded with the theoretically assumed 1:2:1 Mendelian ratio in F<sub>2</sub> generation, however, the values of χ<sup>2</sup>-test in F<sub>7</sub> generation indicated a strong affection of allelic frequencies by the breeding process. Significant variation was also observed in Glu-1 score frequency between F<sub>2</sub> and F<sub>7</sub> generation. These changes were probably caused by deliberate phenotypic selection for important agronomical traits. SDS and Zeleny sedimentation tests, mixographic parameter breakdown and HMW glutenin composition were analyzed in F<sub>7</sub> to reveal the effects of different combinations of HMW glutenin alleles on the bread-making quality characters. The results showed statistically significant differences in the contribution of HMW glutenin alleles. In general, the alleles Ax1, Bx17+By18 and Dx5+Dy 10 can be considered as markers of good baking quality. The data presented in this paper suggest that heterozygous constitution may also have a positive effect on bread-making quality.

Identification of SNPs and development of functional markers for LMW-GS genes at Glu-D3 and Glu-B3 loci in bread wheat (Triticum aestivum 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.

Effects of High-Molecular-Weight Glutenin Subunits on the Texture of Yellow Alkaline Noodles Using Near-Isogenic Lines

The effects of high-molecular-weight glutenin (HMWG) subunits on the physical properties of yellow alkaline noodles (YAN) were investigated by using 10 near-isogenic lines (NILs) that only differed in HMWG composition and the recurrent parent Harunoakebono (HA). Although there were some differences among NILs, including HA, regarding protein content, other characteristics of the flours, such as the ash content and the starch traits, were not notably different. Differences in HMWG composition had a significant effect on the micro SDS-sedimentation volume (MSDSS), breaking force (BF, an index of hardness) and breaking deformation (BD, an index of extensibility) of raw noodles and the BF and BF/BD (an index of elasticity) of boiled noodles. In particular, the BF of raw and boiled noodles were significantly and positively correlated with MSDSS, which is an index of flour protein strength (the quality of flour protein). There were no significant differences in the physical properties of YAN between HMWG subunits 1 and 2 at the Glu-A1 allele. In a comparison of all subunits at the Glu-B1 allele, subunits 17+18 and 20 had significantly higher and lower BF than subunits 7+9 (HA) in raw noodles, respectively, and the BD of subunits 6+8 and 20 was also significantly lower than that of other subunits. No large differences were observed in the physical properties of subunits at the Glu-B1 allele in boiled noodles, with a few exceptions. At the Glu-D1 allele, subunits 2+12, 4+12, and 2.2+12 basically had smaller BF and BF/BD in raw and boiled noodles than subunits 5+10 (HA). The physical properties of NILs with subunits 17+18 and 2.2+12 were similar to those of HA with subunits 7+9 and 5+10. Conversely, those of NIL with subunits 20 and 2.2+12 were extremely small compared to those of HA. The results obtained in this study indicate that the dough strength (the quality of flour protein), as evaluated by MSDSS, has a significant effect on the physical properties of YAN, and that a desirable texture of boiled YAN with a large BF and BF/BD, especially BF, was obtained from flour with HMWG subunits with positive effects on dough strength. Regarding HMWG composition, subunits 2+12, 4+12, and 2.2+12 had the most negative effect on texture, particularly BF, of YAN, followed by subunits 20 and 7+8. In contrast, subunits 5+10 and 17+18, especially the former, had positive effects. NILs with both subunits 5+10 and 17+18 had the best YAN texture (the largest BF and moderate BF/BD values for boiled noodles) among all NILs.

Variation for glutenin and waxy alleles in the US hard winter wheat germplasm

We conducted a survey of high- and low-molecular-weight glutenin subunits (HMW-GS and LMW-GS, respectively) and waxy ( Wx) allele composition in 111 winter wheat cultivars and advanced lines developed in the US Hard Winter Wheat Region since 1991. At the HMW-GS Glu-A1 locus, 76.1% of entries had allele b (encoding subunit 2*), 21.2% carried allele a (subunit 1), and 2.7% had allele c (null). Glu-B1 alleles were c (subunits 7+9; 47.7% of entries), b (subunits 7+8; 33.3%), e (subunits 20x+20y; 7.7%), i (subunits 17+18; 6.3%), and w (subunits 6*+8*; 5%). At Glu-D1, 80.6% of the entries carried allele d (subunits 5+10), 11.3% had allele a (subunits 2+12), 7.2% carried allele b (subunits 3+12), and 0.9% had allele e (subunits 2+10). Glu-B1 and Glu-D1 allele frequency differed significantly ( P<0.01) from a previous study of the US Hard Winter Wheat germplasm developed from 1970 to 1990. At LMW-GS loci, five Glu-A3 alleles, nine Glu-B3 alleles, and five Glu-D3 alleles were identified. Ten entries with the null Wx-B1b allele and seven with the null Wx-A1b allele were detected. This survey allows comparisons of HMW-GS and waxy allele compositions with the US Hard Winter Wheat germplasm from previous eras and provides baseline data on LMW-GS composition.

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.