Glu-B1 Alleles Research Articles

Development of PCR-based markers for identification of wheat HMW glutenin Glu-1Bx and Glu-1By alleles

BackgroundIn common wheat (Triticum aestivum L.), allelic variations in the high-molecular-weight glutenin subunits Glu-B1 locus have important effects on grain end-use quality. The Glu-B1 locus consists of two tightly linked genes encoding x- and y-type subunits that exhibit highly variable frequencies. However, studies on the discriminating markers of the alleles that have been reported are limited. Here, we developed 11 agarose gel-based PCR markers for detecting Glu-1Bx and Glu-1By alleles.ResultsBy integrating the newly developed markers with previously published PCR markers, nine Glu-1Bx locus alleles (Glu-1Bx6, Glu-1Bx7, Glu-1Bx7*, Glu-1Bx7OE, Glu-1Bx13, Glu-1Bx14(−), Glu-1Bx14(+)/Bx20, and Glu-1Bx17) and seven Glu-1By locus alleles (Glu-1By8, Glu-1By8*, Glu-1By9, Glu-1By15/By20, Glu-1By16, and Glu-1By18) were distinguished in 25 wheat cultivars. Glu-1Bx6, Glu-1Bx13, Glu-1Bx14(+)/Bx20, Glu-1By16, and Glu-1By18 were distinguished using the newly developed PCR markers. Additionally, the Glu-1Bx13 and Glu-1Bx14(+)/Bx20 were distinguished by insertions and deletions in their promoter regions. The Glu-1Bx6, Glu-1Bx7, Glu-1By9, Glu-1Bx14(−), and Glu-1By15/By20 alleles were distinguished by using insertions and deletions in the gene-coding region. Glu-1By13, Glu-1By16, and Glu-1By18 were dominantly identified in the gene-coding region. We also developed a marker to distinguish between the two Glu-1Bx14 alleles. However, the Glu-1Bx14(+) + Glu-1By15 and Glu-1Bx20 + Glu-1By20 allele combinations could not be distinguished using PCR markers. The high-molecular-weight glutenin subunits of wheat varieties were analyzed by ultra-performance liquid chromatography and sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the findings were compared with the results of PCR analysis.ConclusionsSeven Glu-1Bx and four Glu-1By allele detection markers were developed to detect nine Glu-1Bx and seven Glu-1By locus alleles, respectively. Integrating previously reported markers and 11 newly developed PCR markers improves allelic identification of the Glu-B1 locus and facilitates more effective analysis of Glu-B1 alleles molecular variations, which may improve the end-use quality of wheat.

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.

Evaluation of Agronomic Traits and Allele Specific DNA Markers Related to Some Disease and Quality Traits in Mutant Karakılçık M4 Individuals

Karakılçık (KK) is a landrace, of which both bread and durum wheat forms exist. This is the first study that reports mutation induction and detection with agronomic traits and allele specific markers on KK durum wheat landrace. In the study, KK landrace was induced with chemical mutation using sodium azide (NaN3) (3 mM) mutagen to improve agronomic traits. In the research, KK genotype and the 13 M4 individuals (KK-1 to KK-13) were used as plant materials. According to the results, the shortest mutant genotype was KK-10 (125.80 cm), with the highest protein ratio (18.50%) and wet gluten ratio (37.10%), while KK-9 genotype had the highest grain yield (4285.6 kg ha-1). The average polymorphism information content (PIC) was calculated as 0.97, while the average allele number was 15.2 per marker. Glu-B1 (Bx7OE primer) allele was determined on KK-11 and KK-13 genotypes. Wx-A1 allele was found on KK-1, KK-2, KK-4, KK-5 and KK-7 genotypes. Sr49 allele (Sun209) was determined on KK-5 genotype, while Yr45 allele (Xwgp118) was detected on KK-10 genotype. In addition, the high protein content allele Gpc-B1 (UHW89 primer) was found on KK-1, KK-2, KK-3 and KK-4 genotypes. Based on the principal component biplot analysis, it was determined that there was a positive relationship between grain yield (GY) with test weight (TW), grain number and weight per spike (GNS and GWS), and negative relationship with the other traits.

Evaluating selection efficacy of high molecular weight glutenin subunits (HMWGs) by relating gluten quality parameters

The allelic combinations of high molecular weight glutenin subunits (HMWGs) encoded by the Glu-1 loci are related to the variability in the viscoelastic properties of dough. Subunit 5 + 10 is associated with strong gluten in Glu-D1. It is also necessary to determine usability in the wheat line selection of the most efficient Glu-B1 and Glu-A1 alleles. In this study, 519 materials were divided into two main groups, 5 + 10 (strong) and 2 + 12 (weak) corresponding to the subunits of Glu-D1. In both main groups, subgroups were formed with the alleles of Glu-A1 (1, 2*, null) and Glu-B1 (7, 7 + 8, 7 + 9, 17 + 18). This study confirmed the efficacy of the combinations of HMWGs on the gluten quality with location-based studies. The gluten quality characteristics were significantly higher in the subgroup with allele 1. Subunit 17 + 18 was found as the most efficient allele, and 7 + 8 was better than 7 + 9 in terms of bread-making quality, however, subunit 7 was associated with weak gluten strength. Generally, the efficacy of the allele 7 + 9 was variable, and increased with the frequency of the allele 2*.

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.

Dough properties and bread-making quality-related characteristics of Yumechikara near-isogenic wheat lines carrying different Glu-B3 alleles.

We investigated the relationships of three allelic variations in Glu-B3 (ab, g, and h) with dough properties and bread-making quality-related characteristics using near-isogenic lines (NILs) of ‘Yumechikara’ that commonly carry Glu-A1a, Glu-B1b, Glu-D1d, Glu-A3f, Glu-B3ab and Glu-D3a. Measurement of peak time (PT) in a 2-g mixograph indicated that Glu-B3g was the most effective for a strong dough property, followed by Glu-B3ab, with Glu-B3h being the least effective. The results of measurement of mixing time during bread-making were similar to those for PTs, i.e., the lines carrying Glu-B3g showed the longest mixing time, followed by those of Glu-B3ab, and those of Glu-B3h showed the shortest mixing time. Since two parameters of bread-making quality, loaf volume (LV) and specific loaf volume (SLV), were affected by flour protein contents in all groups of the Glu-B3 genotype, we compared the effects of the three Glu-B3 alleles on those parameters using analysis of covariance (ANCOVA) to remove the effect of protein content. The results indicated that the Glu-B3h group showed the largest SLV, followed by the Glu-B3ab group, and the Glu-B3g group showed the smallest SLV. These results suggest that the introduction of Glu-B3h into ‘Yumechikara’ makes it possible to breed varieties with good bread-making quality-related characteristics.

Evaluation of genetic variations at glutenin loci in Korean wheat landraces using allele-specific DNA markers

The allelic variations at glutenin loci could significantly affect the bread baking quality, and specific glutenin alleles might be closely associated with greater gluten strength, which, in turn, is related to superior bread baking quality. In this study, allelic variations at Glu-1, Glu-A3 and Glu-B3 loci were evaluated in 222 Korean wheat landraces using gene-specific polymerase chain reaction (PCR) markers. Ten alleles were identified at Glu-1 loci. Glu-A1c, Glu-B1b, and Glu-D1a or Glu-D1f alleles were predominantly found at the respective loci and their frequencies were 86.5, 87.8 and 96.9 %, respectively. Seven Korean wheat landraces carried the Glu-D1d allele, and only one Korean wheat landrace (IT173162) achieved 10 points for the Glu-1 score. Fifteen alleles were identified at Glu-A3 and Glu-B3 loci; Glu-A3c and Glu-B3d or Glu-B3i alleles were commonly found at the respective loci and their frequencies were 77.0, 33.3 and 37.8 %, respectively. Glu-B3 alleles exhibited the highest genetic diversity than other alleles, while Glu-B1 and Glu-A1 alleles exhibited the lowest genetic diversity than other alleles. Twenty Korean wheat landraces had the Glu-A3d and Glu-B3b, Glu-B3d, Glu-B3f, Glu-B3g or Glu-B3i alleles, which were correlated with superior bread baking quality. Among these wheat lines, two (IT59787 and IT236544) carried the Glu-D1d allele.

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.

A second ‘overexpression’ allele at the Glu-B1 high-molecular-weight glutenin locus of wheat: sequence characterisation and functional effects

Bread is one of the major constituents of the human diet and wheat (Triticum aestivum L.) is the most important cereal for bread making. The gluten proteins (glutenins and gliadins) are recognised as important components affecting the processing quality of wheat flour. In this research, we investigated a particular glutenin subunit allele in an Australian cultivar, H45. Based on protein and DNA assays, the Glu-B1 allele of H45 seems to be Glu-B1al, an allele that includes a functional duplication of a gene encoding an x-type high-molecular-weight glutenin subunit, and is thought to increase dough strength through overexpression of that subunit. Yet H45 does not have the dough properties that would be expected if it carries the Glu-B1al allele. After confirming that H45 overexpresses Bx subunits and that it has relatively low un-extractable polymeric protein (an indicator of weak dough), we cloned and sequenced two Bx genes from H45. The sequences of the two genes differ from each other, and they each differ by four single-nucleotide polymorphisms (SNPs) from the sequence that has been reported for the Glu-B1al x-type glutenin genes of the Canadian wheat cultivar Glenlea. One of the SNPs leads to an extra cysteine residue in one of the subunits. The presence of this additional cysteine may explain the dough properties of H45 through effects on cross-linkage within or between glutenin subunits. We propose that the Glu-B1 allele of H45 be designated Glu-B1br, and we present evidence that Glu-B1br is co-inherited with low un-extractable polymeric protein.

Applicability of 'glume color' controlled by Rg-B1 locus as a field-marker to detect Glu-B3 alleles encoding low-molecular-weight glutenin subunits in common wheat

Glu-B3 alleles on chromosome 1BS of common wheat (Triticum aestivum L.) encoding low-molecular-weight glutenin subunit are associated with dough property. Among them, Glu-B3g is known to increase dough strength. To incorporate efficiently Glu-B3g allele into breeders’ lines, DNA marker-assisted selection (DNA-MAS) is considered to be a powerful tool. In the present study, we examined the applicability of ‘glume color’ (GC) controlled by Rg-B1 (Rg1) locus on 1BS as a ‘field-marker’ to detect Glu-B3 alleles. Three populations of F4 breeding lines were used. In each cross, both genotypes of Glu-B3 and Rg-B1 loci of each parent were different. Glu-B3 allele-specific DNA markers were applied to determine the genotype of Glu-B3 locus of each line. The genotype of Rg-B1 locus of each line was visually determined by GC. Significantly tight cosegregations between Glu-B3 and Rg-B1 loci were found in all the populations of breeding lines. It is considered that GC controlled by Rg-B1 locus is a useful ‘field-marker’ to detect Glu-B3 alleles, as it is less time-consuming and more cost effective than DNA-MAS. By using the ‘field-marker’, breeders can select elite lines carrying Glu-B3g allele in breeding fields only by observing the GC of each line/plant without laboratorial procedures for DNA-MAS.

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.

Identification of 1BL/1RS Translocation Based on Mixograph Parameters in Common Wheat

1BL/1RS translocation has been widely used for improving agronomic performance and disease resistance in wheat(Triticum aestivum L.),however,it has strong negative effect on processing quality.To develop a method for 1BL/1RS transloca-tion identification with mixograph parameters,404 advanced lines from 146 crosses in 2005–2006(experiment I) and 175 ad-vanced lines and main cultivars of Shandong province(experiment II) in 2005–2006 and 2006–2007 cropping seasons were used in this study.All materials were sown under irrigation condition in a randomized complete design with 1 replication in Jinan.The genetic effect of 1BL/1RS translocation on mixograph parameters was investigated.The variations of mixograph parameters under different combinations of the high molecular weight glutenin subunits(HMW-GS) and low molecular weight glutenin subunits(LMW-GS) were also analyzed.1BL/1RS translocation lines showed significantly shorter mixing time,less bandwidth of peak and bandwidth after 1 min peak,and higher angle of descent and the bandwidth ratio(the ratio of bandwidth of peak/bandwidth after 1 min peak) in comparison with non-1BL/1RS translocation lines.It indicated that the 1BL/1RS translocation has deleterious effects on mixograph parameters.Mixograph of the 1BL/1RS translocation was characterized with the bandwidth sharply declin-ing and narrowing after 1 min peak,and increasing the bandwidth ratio,whereas the bandwidth of non-1BL/1RS translocations declined gently after 1 min peak or had a longer mixing tolerance,and had a little variation about the bandwidth ratio.Further-more,85.2%(experiment I) and 96.8%(experiment II) accuracies were achieved in grouping the 1BL/1RS translocation and non-1BL/1RS translocation on the basis of the band width ratio,i.e.,1BL/1RS translocation line had a value more than or equal to 1.6,and non-1BL/1RS translocation line had a value smaller than 1.6.Although the Glu-B3 alleles showed better quality parame-ters when HMW-GS 5+10 was presented,it was still the most unfavorable allele on mixograph parameters among all Glu-B3 al-leles.Therefore,mixograph parameters could be used to determine the rheological properties and the presence of 1BL/1RS translocation.

Screening Selected Spring Wheat Cultivars for Productivity, Protein Content and the Glu-B1 Allele Using PCR

Spring wheat cultivars (Sama, Yocra Rojeo, Sids 1, Sakha 8, Sakha 61, Giza 164, Giza 168, Bani Swaf 1, Bani Swaf 3, and Shandawel) were evaluated for their productivity in two field experiments during 2005/2006 and 2006/2007 winter seasons. The results indicated that cultivars (Sids 1 and Giza 164) produced the highest grain yield. While, cultivars (Bani Swaf 3, and Bani Swaf 1) produced the lowest grain yield. Also, the last cultivars gave the lowest harvest index. Wheat cultivars Sama, Giza-164 and Sids 1 were the tallest plants. Moreover, there were significant differences in yield components among wheat cultivars. The highest protein cultivar Sakha 61 had a mean protein percentage of 15.65%. Cultivar Sakha 61 had genetic potential to produce grain with higher protein content than the other cultivars. Bani Swaf 3 cv. had the highest protein content and the lowest grain yield. A set of PCR-based markers for specific HMW glutenin genes encoding By-subunits were used to identify wheat genotypes carrying By genes at the Glu-B1 locus for its bread-making quality. The presence of primer pair ZSBy9aF1/R3 in the reaction mixture, which is an allele-specific marker for the wheat HMW glutenin By9 allele, showed one band of 650 bp in wheat cultivars Sama, Yocra Rojeo, Sids 1, Sakha 8, Sakha 61, and Giza 168. Also, primer set ZSBy9F2/R2, specific for By16 and By-null subunits in wheat cultivars, amplified three different banding patterns for wheat cultivars. Wheat cultivars Sama, Sids 1, and Sakha 61 produced three PCR fragments for the By16 gene, which exists in Glu-B1f (Bx13 + By16). Cultivars Yecora Rojo, Sakha 8, Giza 164, Giza 168, and Bani Swaf 3 produced two PCR fragments. These two PCR fragments were found to be specific for By8, By9, By18 and By15 genes. Cultivars Bani Swaf 1, and Shandawel did not produce any PCR product that was found to be specific for By-null or 20 gene. It was concluded that wheat genotypes Sakha 61 produced the highest grain yield and had the highest protein content By16 gene, which are associated with a superior bread-making quality.

Characterization of low-molecular-weight glutenin subunit Glu-B3 genes and development of STS markers in common wheat (Triticum aestivum L.)

Low-molecular-weight glutenin subunit (LMW-GS) Glu-B3 has a significant influence on the processing quality of the end-use products of common wheat. To characterize the LMW-GS genes at the Glu-B3 locus, gene-specific PCR primers were designed to amplify eight near-isogenic lines and Cheyenne with different Glu-B3 alleles (a, b, c, d, e, f, g, h and i) defined by protein electrophoretic mobility. The complete coding regions of four Glu-B3 genes with complete coding sequence were obtained and designated as GluB3-1, GluB3-2, GluB3-3 and GluB3-4. Ten allele-specific PCR markers designed from the SNPs present in the sequenced variants discriminated the Glu-B3 proteins of electrophoretic mobility alleles a, b, c, d, e, f, g, h and i. These markers were validated on 161 wheat varieties and advanced lines with different Glu-B3 alleles, thus confirming that the markers can be used in marker-assisted breeding for wheat grain processing quality.

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.

Composition of polymeric proteins and bread-making quality of wheat lines with allelic HMW-GS differing in number of cysteines

Three lines of Australian wheat variety Avocet, two biotypes of Australian variety Halberd, and the Italian bread wheat cultivar Fiorello and its derived line were used to study the possible role of the Glu-B1 HMW-GS in polymeric protein composition and flour functionality. These sets of lines contain HMW-GS 7+8, 7+9, 20x+20y or 26+27. Subunit Bx7 has four cysteine residues, while subunit Bx20 has only two. Compared to Avocet A (7+8), line Avocet C (20x+20y) exhibited an increase in polymeric protein, a decrease in the gliadin-to-glutenin ratio, and a marked decrease in unextractable polymeric protein (UPP). Avocet C doughs exhibited greater extensibility and shorter Mixograph dough development times, and baked into smaller loaves compared to those from Avocet A. Similar differences were observed in comparisons between the two Fiorello lines and between the two Halberd biotypes that had differing Glu-B1 alleles. These results show that in closely related genotypes, the presence of the Bx7+By8 or Bx7+By9 HMW-GS instead of Bx20+By20 or Bx26+By27 is associated with higher UPP, higher dough strength and better bread-making performance. It is suggested that this could be due to the greater number of cysteines in the Bx7 subunit that are available for forming higher-molecular-weight glutenin polymer. This could give the possibility to manipulate dough properties and bread-making quality by utilizing HMW-GS alleles with varying numbers of cysteine residues.

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.

Identification of Glu-B1 alleles in bread wheat cultivars using PCR

High dough strength is used as a predictor of good quality bread wheat and it has been attributed largely to the type of allele present at the Glu-D1 locus, where the Glu-D1d allele is most favorable. Recently, it has been observed that cultivars with over expressed subunit Bx7OE at the Glu-B1 have enhanced dough strength. In order to implement recently developed PCR assay for Glu-B1 x-type allele discrimination in marker-assisted selection, a set of wheat cultivars from 11 countries was analyzed. The PCR results matched the known Glu-B1 HMW GS genotypes of the cultivars possessing subunits Bx7 or Bx17. It was shown that this molecular marker also differentiated some other Glu-B1 alleles: Bx6, Bx13, Bx20 and Bx22. These results create the opportunity for an improved method of polymorphism scoring at the Glu-B1 in bread wheat germplasm. .

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.

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.