Development of STS markers and establishment of multiplex PCR for Glu-A3 alleles in common wheat (Triticum aestivum L.)

Low-molecular-weight glutenin subunits (LMW-GS) play a key role in determining the processing quality of the end-use products of common wheat. The objectives of this study were to identify genes at Glu-A3 locus, develop the STS markers, and establish multiplex PCR with the STS markers for Glu-A3 alleles. Gene-specific PCR primers were designed to amplify six near-isogenic lines (NILs) and Glenlea with different Glu-A3 alleles (a, b, c, d, e, f and g) defined by the protein electrophoretic mobility. Three Glu-A3 genes with complete coding sequence were cloned, designated as GluA3-1, GluA3-2 and GluA3-3, respectively. Seven dominant allele-specific STS (sequence tagged sites) markers were designed based on the SNPs (single nucleotide polymorphisms) among different allelic variants for the discrimination of the Glu-A3 protein alleles a, b, c, d, e, f and g. Four multiplex PCRs were established including Glu-A3b + Glu-A3f, Glu-A3d + Glu-A3f, Glu-A3d + Glu-A3g, and Glu-A3b + Glu-A3e. These markers and multiplex-PCR systems were validated on 141 CIMMYT wheat varieties and advanced lines with different Glu-A3 alleles, confirming that they can be efficiently used in marker-assisted breeding.     

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.     

Relationships between dough strength,polymeric protein quantity and composition for diverse durum wheat genotypes

Five different Glu-B1 HMW-GS patterns were identified among a collection of diverse durum wheat genotypes grown in 2001 in two locations in western Canada. The durum wheat lines exhibited a wide range of dough and gluten strength characteristics as measured by alveograph and 2 g mixograph parameters, gluten index (GI), and protein composition as measured by unextractable polymeric protein (UPP) content and the ratio of high-molecular weight (HMW) glutenin subunits (GS) to low-molecular weight (LMW) GS. HMW-GS subunits patterns represented within the genotypes were 6+8, 7+8, 7+16, 14+15 and 20. Two of the genotypes expressed Glu-A1 HMW-GS 2* in combination with other HMW-GS. Approximately 95% of the durum genotypes were γ-gliadin 45 types. Analysis of variance indicated that genotype was a greater source of variation in all measurements than was growing location, with the exception of protein content which showed less variation contributed by genotype and more contributed by location than for other quality parameters. UPP was strongly associated with all strength measurements. All of the γ-gliadin 42 types were low in UPP and weak. Among the γ-gliadin 45 types, those possessing HMW-GS 20 were typically in the lower half of the UPP and strength range. There was no clear evidence of an association between any of the other HMW-GS patterns and gluten strength. The majority exhibited HMW to LMW-GS ratios that were within the relatively narrow range of 0.15–0.25, yet there were wide variations in dough strength among genotypes within that range. Increasing proportions of HMW-GS resulting in ratios of greater than 0.30 were generally associated with weak dough and gluten and low UPP content.     

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.     

Effects of Fusarium culmorum and water stress on durum wheat in Tunisia

The effects of water stress on Fusarium foot and root rot in durum wheat were investigated in growth chamber, greenhouse and field tests in Tunisia. In the seedling stage, emergence of six durum wheat cultivars in the growth chamber was significantly reduced by inoculation with Fusarium culmorum and water stress (P<0.0001), with more disease under drier conditions. Additionally, the tiller number per mature plant, the 1000 grain weight and disease severity in mature stage were reduced by inoculation in greenhouse studies. In a field test, inoculation with F. culmorum significantly reduced the yield (P<0.001), by more than 17% for Om Rabiaa and 38% for Karim, the two cultivars tested. Yield was also significantly affected by precipitation and irrigation levels. The severity of the disease, estimated by the percentage of white heads, was separately affected by the cultivar (P<0.001) and inoculation (P = 0.0004). Percentage of white heads was 1.5 and 2 × higher in inoculated plants than non-inoculated for Om Rabiaa and Karim cultivars, respectively. Disease severity was highest in treatments with the greatest water stress. This is the first detailed study of water stress and F. culmorum on durum wheat in Tunisia, and indicates that cultivar resistance and irrigation management may be important in the management of Fusarium foot rot.     

Partial purification and characterization of polyphenoloxidase from durum wheat (Triticum durum L.)

A bright yellow color of pasta is an important qualitative trait for the durum wheat industry. Final color is the result of the balance between yellow and brown components in semolina. Polyphenoloxidase (PPO) is implicated as playing a significant role in darkening. This study aimed to characterize PPO activity of durum wheats. PPO was extracted and partially purified by ion-exchange chromatography on a column packed with diethyaminoethyl cellulose (DEAE). This procedure led to 26.33-fold purification with 24.7% recovery. The optimum temperature and pH of PPO were found to be 40 °C and 6.5, respectively. Heat stability of durum wheat PPO decreased as the temperatures increased from 30 to 80 °C. The z-value was calculated as 23.4 °C. It increased to 26.3 and 48.4 °C in the presence of 40% sucrose and 1 M NaCl, respectively. Durum wheat PPO was shown to use several phenolic compounds as substrate. Among the substrates used, the greatest substrate specificity was observed with catechol. Durum wheat PPO was sensitive to inhibitors such as ascorbic acid, cysteine, oxalic acid and citric acid. Ascorbic acid was the most effective inhibitor.     

A transgenic durum wheat line that is free of marker genes and expresses 1Dy10

As currently practiced, genetic engineering of monocots requires the use of selective agents, such as herbicides and antibiotics, and marker genes for resistance to favor the multiplication of the initially transformed cells. In the present paper we have used “minimal gene cassettes” and positive selection to generate transgenic durum wheat lines free of herbicide and antibiotic resistance marker genes. Two biolistic transformation experiments were carried out using three “minimal gene cassettes” consisting of linear DNA fragments each excised from the source plasmids. The targeted trait genes were two bread wheat sequences encoding the Dx5 and Dy10 high-molecular-weight (HMW) glutenin subunits, which have been associated with superior bread-making quality and which are absent from durum wheats. The positive selectable marker was the Escherichia coli phosphomannose isomerase (pmi) gene, whose product catalyzes the reversible interconversion of mannose-6-phosphate and fructose-6-phosphate, allowing plant cells to utilize mannose as a carbon source. PCR assays of genomic DNA from regenerated plants identified 15 T₀ plants that contained the pmi marker gene for an overall transformation efficiency of 1.5%, which is similar to biolistic transformation efficiencies of durum wheat with intact circular plasmids. Line TC-52, which initially contained pmi, non-expressed 1Dx5, and expressed 1Dy10 HMW glutenin subunit transgenes, was further investigated. PCR was used to follow inheritance of the pmi marker gene and 1Dx5 from the T₁ to T₃ generations. Transgene expression was monitored by the chlorophenol-red assay for pmi and SDS-PAGE of seed proteins for 1Dy10. From these analyses, we observed that the 1Dy10, 1Dx5 and pmi transgenes were not linked, allowing us in the T₃ generation to identify 1Dy10 transgenic segregants that contained no marker or silent 1Dx5 transgenes. Homozygotes containing and expressing only the 1Dy10 transgene were identified in the T₄ generation. These experiments show that it is possible to combine biolistic transformation by minimal gene cassettes with genetic segregation to make marker-free transgenic wheat plants with new traits.     

Comparative proteomic analysis of kernel proteins of two high amylose transgenic durum wheat lines obtained by biolistic and Agrobacterium-mediated transformations

In this work, we compared the proteome of mature and immature kernels of transgenic and untransformed durum wheat lines in which genes of the starch branching enzymes of class II (SBEIIa) were silenced by RNA interference using two different methods of genetic transformation. The comparative analysis of granule bound and metabolic protein fractions of Svevo and Ofanto and their derived transgenic lines highlighted twenty four and thirty three differentially accumulated spots, respectively, in the line MJ16-112 (obtained by biolistic transformation of Svevo) and in the line A431_4p1a (obtained by Agrobacterium-mediated transformation of Ofanto).     

Diversity of sequences encoded by the Gsp-1 genes in wheat and other grass species

The Gsp-1 genes of wheat encode two components, the “grain softness protein” (whose role in determining texture has not been substantiated) and a 15 residue arabinogalactan peptide (AGP) sequence which is O-glycosylated and is also of unknown function. We have determined genomic Gsp-1 sequences from 29 species within the Triticeae tribe and an additional 12 species from the major subfamilies of the Poaceae (Anomochlooideae, Bambusoideae, Ehrhartoideae, Chloridoideae and Panicoideae). Twelve new AGP sequence types were identified with forms present in Agropyron mongolicum, Secale cereale, Oryza sativa subsp. japonica and Sorghum bicolor containing an extra ten amino acids within the AGP sequence. Phylogenetic analysis showed distinct groupings of AGP/GSP sequence types which had no apparent relationship to the species or even the genus. However, individual forms of AGP forms were associated with specific groups of GSP sequences, providing no evidence that the AGP and GSP-1 parts of the protein have diverged at different rates or in different ways.     

Effect of nitrogen application rate on content of glutenin macropolymer and high molecular weight glutenin subunits in grains of two winter wheat cultivars

Two winter wheat (Triticum aestivum L.) cultivars differing in grain protein content were selected to study the effect of N application rate on changes in contents of glutenin macropolymer (GMP) and high molecular weight glutenin subunits (HMW-GS) during grain filling. Contents of GMP and HMW-GS were much higher in the high GPC cultivar, Xuzhou 26, than those in low GPC cultivar, Ningmai 9. N increased contents of GMP and HMW-GS in Xuzhou 26 with N rate between 0 and 300 kg ha⁻¹, while at the very high N rate of 300 kg ha⁻¹ the contents of GMP and HMW-GS in Ningmai 9 decreased. The high contents of GMP and HMW-GS at maturity were closely related to the rapid increase in contents of GMP and HMW-GS during the initial period of their synthesis. HMW-GS and GMP content were closely correlated. The total HMW-GS content was important in determining GMP content than the content of any HMW-GS pair or any individual HMW-GS present in the selected cultivars. The pattern of response of GMP content to N application rate was closely related to the regulatory effect of N on HMW-GS synthesis.     

Characterisation of polyclonal and monoclonal anti-peptide antibodies specific for some lowMrsubunits of wheat glutenin and their use in the detection of allelic variants atGlu-3loci

Polyclonal and monoclonal antibodies (Mabs) were produced against the major type ofN-terminal amino acid sequence of lowM_rglutenin subunits. The reactivities of these antibodies were determined using glutenin extracts of several bread wheat cultivars of known allelic composition. Analyses were performed by immunoblotting after one or two-dimensional electrophoresis. One Mab (Mab 6x1) was found to react with lowM_rglutenin subunits encoded by chromosomes 1B and 1D but not with subunits controlled by chromosome 1A. Only some of the subunits encoded at theGlu-D3locus were recognised. In contrast, this Mab reacted with all the subunits controlled by theGlu-B3locus. After single dimension SDS–PAGE, we observed significant differences between immunoblot patterns of cultivars expressing different lowM_rglutenin subunits from chromosome 1B. Mab6 x1 is a useful reagent for analysing the allelic composition at theGlu-B3locus.     

Segregation analysis indicates that Puroindoline b-2 variants 2 and 3 are allelic in Triticum aestivum and that a revision to Puroindoline b-2 gene symbolization is indicated

Genetic and kernel texture relationships between Puroindoline b-2 variants 2 and 3 have not been fully established in wheat (Triticum aestivum L.). Here, 480 F₂ plants, derived from three hard spring wheat populations were used to test the segregation of Puroindoline b-2 (Pinb-2) variants 2 and 3. Chi-square analysis indicated that Pinb-2 variants 2 and 3 in all three F₂ populations segregated as a single bi-allelic locus, with segregation ratios fitting a 1:2:1 ratio. Using 448 of the 480 plants derived from these three F₂ populations, the average SKCS hardness index of plants homozygous for Pinb-2 variant 2 vs. those homozygous for variant 3 was not significantly different (67.5 vs. 67.9). Results indicated that plants with Pina-D1b/Pinb-D1a were on average 10.0 Single Kernel Characterization System (SKCS) hardness index units harder than those carrying the Pina-D1a/Pinb-D1b haplotype. In conclusion, Pinb-2 variants 2 and 3 are allelic and exert little effect on kernel texture in hard-kernel T. aestivum germplasm. Further, the designation of Pinb-2v2 and Pinb-2v3 should be changed to Pinb-B2a and Pinb-B2b, respectively. We propose that Pinb-2 variants 1 and 4 of Chinese Spring be designated Pinb-D2a and Pinb-A2a, respectively.     

Association of Puroindoline b-B2 variants with grain traits,yield components and flag leaf size in bread wheat (Triticum aestivum L.) varieties of the Yellow and Huai Valleys of China

A total of 169 wheat (Triticum aestivum L.) varieties (landraces and cultivars) were used to asses the relationship between Puroindoline D1 alleles and Puroindoline b-B2 variants and grain hardness, other grain traits, yield components, and flag leaf size. Results indicated that the average SKCS hardness of Pinb-B2v3 varieties was significantly greater than that of Pinb-B2v2 varieties within the soft Puroindoline D1 haplotype sub-group. Conversely, no statistically significant difference was obtained for SKCS hardness between varieties with the Pinb-B2v3 vs. Pinb-B2v2 alleles within the two hard Puroindoline D1 haplotypes (Pinb-D1b and Pinb-D1p sub-groups). Therefore, the Puroindoline b-B2 gene may have a bigger impact on soft wheat varieties than hard. Across all varieties, thousand-kernel weight, grain weight per spike, grain diameter, grain number per spike, flag leaf width and area of Pinb-B2v3 varieties were significantly greater than those possessing Pinb-B2v2. These results indicated that the Pinb-B2v3 allele was associated with preferable grain yield traits compared to the Pinb-B2v2 allele in bread wheat. This study provides evocative information for better understanding the molecular and genetic basis of wheat grain yield.     

Contribution of common wheat protein fractions to dough properties and quality of northern-style Chinese steamed bread

Thirty-three cultivars and advanced lines originated from China, Mexico, and Australia were sown in four environments in Chinese spring wheat regions to investigate the association between gluten protein fractions determined by reversed-phase high-performance liquid chromatography (RP-HPLC), and dough properties and northern-style Chinese steamed bread (CSB) quality. The genotypes were divided into two groups of 10 and 23 entries with and without the 1B/1R translocation, respectively. 1B/1R translocation lines had significantly high amounts of ω  -gliadins, and low levels of glutenin and low molecular weight glutenin subunits (LMW-GS), but no significant difference in dough properties and CSB quality from non-translocation lines. The association between protein fractions and dough properties, and CSB quality largely depended upon the presence of 1B/1R translocation. Gliadin contributed more in quantity to flour protein content (FPC) than glutenin, while glutenin and its fractions contributed more to dough strength and CSB quality. Among non-translocation lines, moderate to high correlation coefficients between quantified glutenin and its fractions, and farinograph development time (DT, r=0.85$r=0.85$–0.92) and stability (ST, r=0.81$r=0.81$–0.93), extensograph maximum resistance (R_max, r=0.90$r=0.90$–0.93), CSB stress relaxation (SR, r=0.55$r=0.55$–0.61) and CSB score (r=0.56$r=0.56$–0.62), were observed. Gliadin:glutenin ratios showed significant and negative associations with dough properties and CSB quality. Correlation coefficients between gliadin:glutenin, gliadin:HMW-GS, gliadin:LMW-GS ratios, and CSB score were −0.79, −0.73, and −0.79 among non-translocation lines, respectively. HMW-GS and LMW-GS, x-type HMW-GS and y-type HMW-GS contributed similarly to dough properties and CSB quality for non-translocation lines. Weak correlations between protein fractions and dough properties, and CSB quality were observed among translocation lines. This information should be useful for improvement of dough properties and CSB quality.     

Kernel vitreousness and protein content: Relationship,interaction and synergistic effects on durum wheat quality

Four sets of durum samples were used in this study to further understand the interrelationships among hard vitreous kernels (HVK), protein content, and pigment concentration, with a focus on the interaction and synergistic effects of protein content and vitreousness on durum quality. HVK level increases with higher protein content in the range of 9.5–12.5%, but this relationship is less evident in durum samples with high protein content (12.5–14.5%). Both protein content and kernel vitreousness can significantly affect durum milling quality. White starchy kernels (WSK) in low protein durum have a very detrimental impact on milling and pasta processing quality, but high protein content can mitigate the adverse impact of WSK on durum quality. Although protein content plays a dominant role, higher HVK might contribute positively to pasta firmness. There was no significant difference in yellow pigment content between HVK and WSK. However, pigment loss from semolina to dough was higher for WSK than HVK. Despite the difference in protein content, HVK and WSK have little difference in gluten strength. The monomeric protein was preferentially accumulated in HVK. The glutenin proteins of HVK and WSK were similar in the ratios of 1Bx/1By and HMW/LMW-GS.     

Effect of the 1BL.1RS translocation and of the Glu-B3 variation on fifteen quality tests in a doubled haploid population of wheat (Triticum aestivum L.)

To investigate the impact of 1BL.1RS translocation on protein content, starch quality, dough rheology, RMT volumes and other quality traits, a doubled haploid population was created and sown in a two-year field experiment. Translocated genotypes accumulated more proteins in the endosperm than non-translocated genotypes. Decrease in the gelatinization of starch was associated with the 1BL.1RS translocation. As for rheological parameters, adapted to bread types not requiring high mixing energy, the 1BL.1RS translocation significantly reduced the elasticity, tenacity and strength of the dough compared to allele c of Glu-B3. Tolerance to over-mixing was also significantly lower in translocated DH lines. In contrast to previously published work, the presence of allele Glu-D3 c resulted in significantly higher tenacity, and thus strength, compared with the allele Glu-D3 b in the present DH population. The final baking test performed on the DH lines of the population, combining favourable alleles for dough rheology and high protein content, demonstrated that in some cases lower tenacity induced by the 1BL.1RS translocation or by Glu-B3 b increases the volume of the loaves.     

Comparison of predictions of baking volume using large deformation rheological properties

Three large deformation rheological tests, the Kieffer dough extensibility system, the D/R dough inflation system and the 2 g mixograph test, were carried out on doughs made from a large number of winter wheat lines and cultivars grown in Poland. These lines and cultivars represented a broad spread in baking performance in order to assess their suitability as predictors of baking volume. The parameters most closely associated with baking volume were strain hardening index, bubble failure strain, and mixograph bandwidth at 10 min. Simple correlations with baking volume indicate that bubble failure strain and strain hardening index give the highest correlations, whilst the use of best subsets regression, which selects the best combination of parameters, gave increased correlations with R²=0.865 for dough inflation parameters, R²=0.842 for Kieffer parameters and R²=0.760 for mixograph parameters.     

Differential mixing action effects on functional properties and polymeric protein size distribution of wheat dough

A micro Z-arm mixer and a 2g-Mixograph were used to compare the effect of pin and Z-arm-type mixing actions on mixing properties of wheat flour dough. Although the two mixing curves obtained with pin- and Z-arm-type mixing action showed a very similar mixing trace, no significant correlation was found between the two mixers other than the number of revolutions required for optimal dough consistency (peak resistance). Mixing requirement was described by a rate-independent parameter, the number of revolutions to peak dough development and was found to be greater in a Z-arm mixer than in a pin mixer. Mixing requirement showed significant correlation with stability, which is therefore a dough strength parameter. The change in the polymeric structure of gluten proteins of dough as indicated by %UPP (unextractable polymeric protein percentage) was monitored and showed a smaller decrease with Z-arm mixing than with pin mixing. Therefore, pin-mixing action is more energetic than Z-arm mixing. At peak resistance, Z-arm mixing gives a larger quantity of polymeric protein content in the dough relative to pin mixing. The degree of dough development at maximum resistance in the different mixers was shown to be different. A new parameter, delta-_UPPMZ${\mathrm{UPP}}_{\mathrm{MZ}}$ (the difference between %UPP of dough obtained with pin vs Z-arm mixing actions) was identified and proposed to have some relationship to the stability of the polymeric proteins in the dough.