A reliable assay for the detection of soft wheat adulteration in Italian pasta is based on the use of new DNA molecular markers capable of discriminating between Triticum aestivum and Triticum durum

Italian pasta must be prepared using exclusively durum wheat. According to current Italian rules, only a maximum of 3% Triticum aestivum is allowed to account for cross-contamination that may occur during the agricultural process. Efficient methods for the detection of accidental or intentional contamination of common wheat to durum wheat products are therefore required. This article describes a novel approach for the detection and quantification of soft wheat adulteration in whole grain durum flours and dried pasta. The assay relies on the presence of intron-specific DNA length polymorphisms in the plant β-tubulin gene family, which can be highlighted through the PCR-based TBP (Tubulin-Based Polymorphism) method. In wheat, the TBP method produces species-specific amplification products, which can be either directly used as new DNA molecular markers capable of discriminating between T. aestivum and Triticum durum or analyzed at the sequence level for the design of species-specific probes. The latter approach allowed the development of new sequence-specific targets that can be exploited in RT-PCR assays for a rapid and accurate quantification of soft wheat adulteration in durum wheat pasta.     

Phenolic acids composition,total polyphenols content and antioxidant activity of Triticum monococcum, Triticum turgidum and Triticum aestivum: A two-years evaluation

Wheat is a good source of polyphenols, plant metabolytes with beneficial effects on human health. However, little information is available on phenolic acid composition and concentration in different Triticum species, as well as on possible environmental effects. To shed some light on this issue, thirty-nine wheat accessions cropped for two years and belonging to different Triticum species (Triticum monococcum, Triticum turgidum ssp. dicoccum, turanicum and durum, Triticum aestivum ssp. spelta and aestivum) were assessed for phenolic acids (ferulic, p-coumaric, vanillic, syringic, p-hydroxybenzoic, caffeic acids and syringaldehyde), total polyphenols and antioxidant activity in soluble conjugated and insoluble bound extracts. Ferulic acid was the most abundant compound in both extracts. Insoluble bound phenolic acids represented >90% of total phenolic acids. Einkorn showed the maximum concentration of conjugated phenolic acids (50.5 mg/kg DM), while durum and bread wheats presented the highest content of bound phenolic acids (651.8 and 629.2 mg/kg DM, respectively). Cropping year influenced the concentration of conjugated but not of bound phenolic acids. A survey of phenolic acid distribution in the kernel showed that they are rare in endosperm, but abundant in germ and bran. Total polyphenols and antioxidant activity were highly correlated to phenolic acid content.     

Baking quality of emmer-derived durum wheat breeding lines

Durum wheat (Triticum turgidum L. var. durum) is used predominantly for pasta products, but there is increasing interest in using durum for bread-making. The goal of this study was to assess the bread-making potential of 97Emmer19, an Emmer wheat (Triticum turgidum L. var. dicoccum) and in breeding lines derived from crosses of 97Emmer19 with adapted durum wheat cultivars. 97Emmer19 and its progeny were evaluated in 2005 and 2006 along with five durum wheat cultivars. Three bread wheat (Triticum aestivum L.) cultivars were included as checks to provide a baseline of bread making quality observed in high quality bread wheat cultivars. 97Emmer19 exhibited higher LV than all the durum wheat checks and approached the LV achieved with the bread wheat cultivar ‘AC Superb’. Breeding lines derived from 97Emmer19 had higher LV than those of the durum wheat checks, confirming that this trait was heritable. In general, durum wheat cultivars with elevated gluten strength and/or increased dough extensibility were noted to have higher LV. Dough extensibility appeared to be a more critical factor as gluten strength increased. These results indicate that there is potential to select for genotypes with improved baking quality in durum breeding programs.     

A deletion at the Lpx-B1 locus is associated with low lipoxygenase activity and improved pasta color in durum wheat (Triticum turgidum ssp. durum)

The concentration of yellow carotenoid pigments in durum wheat grain is an important quality criterion and is determined both by their accumulation and by their degradation by lipoxygenase enzymes (Lpx loci). The existence of a duplication at the Lpx-B1 locus and the allelic variation for a deletion of the Lpx-B1.1 copy is reported. This deletion was associated with a 4.5-fold reduction in lipoxygenase activity and improved pasta color (P<0.0001) but not semolina color, suggesting reduced pigment degradation during pasta processing. A molecular marker for the deletion was mapped on chromosome 4B in a population obtained from the cross between durum line UC1113 and variety Kofa. A second lipoxygenase locus, designated Lpx-A3, was mapped on the homoeologous region on chromosome 4A and was associated with semolina and pasta color (P<0.01) but not with lipoxygenase activity in the mature grain. Selection for both the UC1113 allele for Lpx-A3 and the Kofa Lpx-B1.1 deletion resulted in a 10% increase in yellow scores for dry pasta relative to the opposite allele combination. This result indicates that the markers and the new allelic variants reported here will be useful tools to manipulate the wheat Lpx loci and to improve pasta color.     

In vitro pullulanase activity of wheat (Triticum aestivum L.) limit-dextrinase type starch debranching enzyme is modulated by redox conditions

Expression of a limit-dextrinase (LD) type starch debranching enzyme (EC 3.2.1.41) was observed in developing wheat (Triticum aestivum L.) endosperm and germinating grains, indicating a role for the enzyme in both biosynthesis and degradation of starch. A full-length cDNA, TaLD1, encoding LD in wheat developing kernels was isolated and predicted to encode a 98.6 kDa mature protein active in amyloplasts. Isolated cDNA was expressed in Escherichia coli to produce a recombinant His-tagged LD, which mainly accumulated in inclusion bodies as an inactive enzyme. Extraction of His-tagged LD from the inclusion bodies followed by dialysis under thiol/disulphide redox conditions allowed partial refolding of the protein and detection of pullulanase specific activities by zymogram analysis and enzyme assays. Several active conformations were demonstrated by the recombinant TaLD1 and pullulanase activity could be modulated by redox conditions in vitro. The results suggest that cellular redox conditions may regulate the level of LD activity in wheat tissues.     

Identification,mapping and evolutionary course of wheat lipoxygenase-1 genes located on the A genome

Durum wheat (Triticum turgidum) is the cereal of preference for semolina and pasta production. Bright yellow color, which is desirable for pasta making, depends on the amount of carotenoid pigments present in the grain. Lipoxygenases (LPXs) account for most of the carotenoid degradation activity. Although B genome Lpx genes have been extensively described, little information about the A genome has been reported. Here, we demonstrate that the Lpx-A1 locus is represented by a single gene in the diploid Triticum urartu, the tetraploid T. turgidum and the hexaploid Triticum aestivum wheats in contrast to the multiple copies reported in the B genome. The Lpx-A1_like pseudogene previously identified in T. turgidum genome A was also identified in the T. aestivum cv Chinese Spring wheat, whereas T. urartu possesses a complete copy, suggesting that pseudogenization occurred after the formation of the tetraploid and then passes to the hexaploid wheat. The nucleotide sequence of T. urartu Lpx is more closely related to genome B Lpx-1 than to Lpx genes of genome A from T. turgidum and T. aestivum, probably due to the deletions and insertions that occur. Thus, the present paper extends our knowledge of lipoxygenase gene organization and evolution in the wheat A genome.     

A PCR-based Method for the Detection of Hexaploid Bread Wheat Adulteration of Durum Wheat and Pasta

This paper presents a new method for the detection of the adulteration by hexaploid bread wheat of pasta products described as being manufactured solely from durum wheat. This problem has, in the past, been addressed using a variety of methods based mainly upon the analysis of proteins, the detection of which are problematical owing to the wide range of processing conditions employed throughout the industry. The new method is based upon the use of the polymerase chain reaction, whereby the presence of a previously described DNA sequence amplified in the D-genome of wheat can be detected at low levels. The method described is rapid, inexpensive, and can be readily replicated for accuracy. This new method has been applied to a large number of wheat cultivars from around the world to demonstrate that it has general applicability. It is hoped that this method can be further extended to form the basis of a quantitative assay for the adulteration of durum-wheat products.     

Effect of the grain protein content locus Gpc-B1 on bread and pasta quality

Grain protein concentration (GPC) affects wheat nutritional value and several critical parameters for bread and pasta quality. A gene designated Gpc-B1, which is not functional in common and durum wheat cultivars, was recently identified in Triticum turgidum ssp. dicoccoides. The functional allele of Gpc-B1 improves nitrogen remobilization from the straw increasing GPC, but also shortens the grain filling period resulting in reduced grain weight in some genetic backgrounds. We developed isogenic lines for the Gpc-B1 introgression in six hexaploid and two tetraploid wheat genotypes to evaluate its effects on bread-making and pasta quality. In common wheat, the functional Gpc-B1 introgression was associated with significantly higher GPC, water absorption, mixing time and loaf volume, whereas in durum wheat, the introgression resulted in significant increases in GPC, wet gluten, mixing time, and spaghetti firmness, as well as a decrease in cooking loss. On the negative side, the functional Gpc-B1 introgression was associated in some varieties with a significant reduction in grain weight, test weight, and flour yield and significant increases in ash concentration. Significant gene × environment and gene × genotype interactions for most traits stress the need for evaluating the effect of this introgression in particular genotypes and environments.     

Quality Characteristics of Durum Wheat Lines Deriving High Protein From aTriticum dicoccoides(6b) Substitution

One of the objectives in the Canadian durum wheat (Triticum turgidumL.) breeding programs has been the selection of lines having higher protein content. The Langdon-dicoccoides(Triticum dicoccoides6B) substitution, a source of high protein, has been introgressed into two high yielding, but lower protein Canadian lines of durum wheat. The resulting lines with protein content similar to registered cultivars were evaluated for protein quality. The introgression had no detrimental effects upon pasta cooking quality, and thus the dicoccoides 6B chromosome substitution will be a valuable route to increasing protein level for the durum wheat breeding program.     

Discriminant analysis of selected yield components and fatty acid composition of chosen Triticum monococcum, Triticum dicoccum and Triticum spelta accessions

This study analyses the variability of key yield components, the content of protein and crude fat in grain and the fatty acid composition of 50 spring accessions of Triticum monococcum, Triticum dicoccum and Triticum spelta of various origins. The average protein content of the grain of T. monococcum was 20.8%, of T. dicoccum 19.7%, and of spelt 17.0%. The crude fat content of T. monococcum grain (2.7%) was significantly higher compared with T. spelta (2.4%) and T. dicoccum (2.3%). In crude fat, fatty acids C18:2, C18:1 and C16 predominated. T. spelta was characterised by the highest concentrations of C18:2 and C16 (55.89% and 18.77% respectively), while T. monococcum had the highest content of C18:1 (26.35%). The structure of analysed fatty acids proved to be highly desirable in this species. A discriminant analysis performed separately for five variables: protein and fat content and three biometrical characters and separately for fatty acid composition enabled three Triticum species to be distinguished. These species also differed significantly with respect to the C18:1/C16 ratio which was equal to 1.78, 1.06, 1.47 and 0.99 in T. monococcum, T. dicoccum, T. spelta and Triticum aestivum respectively.     

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.     

Development and characterization of a Triticum aestivum-H. villosa T5VS•5DL translocation line with soft grain texture

The Hardness locus on the short arm of chromosome 5D is the main determinant of grain texture in bread wheat. The Pina and Pinb genes are tightly linked at this locus, and the soft kernel texture phenotype results when both genes are present and encode the wild-type puroindoline proteins PINA and PINB. In this study a compensating T5VS•5DL Triticum aestivum-Haynaldia villosa translocation line, NAU415, was characterized by chromosome C-banding, genomic in situ hybridization and molecular markers. Single Kernel Characterization System (SKCS) analysis and scanning electron microscopy indicated that NAU415 had soft endosperm although it lacked the wheat Pina-D1a and Pinb-D1a genes, suggesting the presence of functional Pin gene orthologs on chromosome 5VS. Using a PCR approach, Pina-related (designated Dina) and Pinb-related (Dinb) genes in H. villosa and NAU415 were identified and sequenced. The nucleotide and predicted amino acid sequences showed close similarities to the wild-type puroindolines of T. aestivum cv. Chinese Spring. The tryptophan-rich regions of both Dina and Dinb showed a sequence change from lysine-42 to arginine, a feature that may have an effect on grain texture. The potential of T5VS•5DL translocation line as a source of genes that may be used for modulation of endosperm texture and other valuable traits in wheat breeding is discussed.     

Development of analytical systems based on real-time PCR for Triticum species-specific detection and quantitation of bread wheat contamination in semolina and pasta

The cereal composition of specific foods is always a key factor in the quality and safety of the final product. It is important to introduce new control methods for certain special foods, such as those for coeliac consumers, where the contamination of different cereal species must be accurately determined. Moreover, pasta made from durum wheat is considered superior in several qualitative aspects to that manufactured with bread wheat or a mixture of the two species. This work has been directed toward the development of analytical systems for the qualitative and quantitative detection of specific cereals in food. More specifically, the primary aim of this work has been to develop analytical tools based on end-point and real-time PCR to detect the presence of Triticum species in flour and food. Furthermore, qualitative and quantitative PCR-based methods are proposed to detect hexaploid wheat adulteration in pasta.     

Isolation and characterization of EMS-induced Dy10 and Ax1 high molecular weight glutenin subunit deficient mutant lines of elite hexaploid wheat (Triticum aestivum L.) cv. Summit

The mixing properties of the dough are critical in the production of bread and other food products derived from wheat. The high molecular weight glutenin subunits (HMW-GS) are major determinants of wheat dough processing qualities. The different alleles of the HMW-GS genes in hexaploid wheat vary in their effect on dough quality. To determine the contribution of the individual HMW-GS alleles, lines deficient in HMW-GS proteins were generated by chemical mutagenesis in the elite bread wheat Triticum aestivum cv. Summit. In this report we describe the identification and characterization of Dy10 and Ax1 deficient lines. Examination of the effect of Dy10 and Ax1 deficiency on dough rheological properties by mixography showed shorter mixing time to reach peak resistance, and weaker and less extensible doughs relative to the wild type control. This is the first time that the role of Dy10 in vivo has been examined apart from the Dx5 + Dy10 allelic pair combination.     

Antibodies against wheat xylanase inhibitors as tools for the selective identification of their homologues in other cereals

Polyclonal antibodies (PAbs) were raised against wheat (Triticum aestivum L.) TAXI- and XIP-type xylanase inhibitors by rabbit immunization. A small contaminant in both antigens, not detected by SDS-PAGE and later identified through Western blot as a recently discovered third type of xylanase inhibitor from wheat, i.e. thaumatin-like xylanase inhibitor (TL-XI), led to the coproduction of PAbs against this protein in the rabbits. To obtain inhibitor-specific PAbs, the PAbs against TAXI, XIP and TL-XI were separated by affinity chromatography using immobilised recombinant and native xylanase inhibitors. The purified PAbs allowed the immunoquantification of each type of wheat xylanase inhibitor using Western blot and densitometric analysis against purified inhibitor standards. The method allowed the detection of the purified inhibitors at the 20 ng level. As the PAbs against the wheat xylanase inhibitors cross-reacted with their homologous targets from other cereals, immunoprobing allowed identification of XIP homologues in oats (Avena sativa L.) and TL-XI homologues in durum wheat (Triticum durum Desf.) and rye (Secale cereale L.).     

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.     

The antioxidant potential of milling fractions from breadwheat and durum

The effect of primary processing, namely milling, on the phenolic content and antioxidant capacity of two wheat cultivars, namely Canada Western Amber Durum (Triticum turgidum L. var. durum) and Canada Western Red Spring (Triticum aestivum L.) was studied. The milling products: bran, flour, shorts and feed flour fractions were examined. In addition, semolina was an end-product of durum wheat milling. Antioxidant activity of wheat phenoliocs was evaluated using oxygen radical absorbance capacity, inhibition of photochemiluminescence, the Rancimat method, and inhibition of oxidation of low-density lipoprotein cholesterol and deoxyribonucleic acid. The phenolic composition of wheat extracts was determined using high performance liquid chromatography. Bran showed the highest antioxidant activity whereas the endosperm possessed the lowest in both cultivars examined. The phenolic compounds are concentrated in the outermost layers namely the bran. The consumption of wheat with bran in the form of whole grain may provide beneficial health effects.     

Effects of suni-bug (Eurygaster spp.) damage on semolina properties and spaghetti quality characteristics of durum wheats (Triticum durum L.)

Effects of suni-bug (Eurygaster spp.) damage on semolina properties and spaghetti quality characteristics of durum wheats (Triticum durum L.) were investigated. The semolinas obtained from sound (control), medium damage (around 20%) and high damage (around 40%) samples of five durum wheat cultivars (cvs. Diyarbakir, Firat, Ege, Svevo and Zenith) were processed into spaghetti. As the bug damage level increased, Glutograph stretch values of all cultivars decreased significantly probably due to deteriorative effects of bug damage on gluten quality. Glutograph relaxation values and gluten spread values of the damaged samples were considerably higher compared to those of sound samples in all cultivars, due to proteolytic degradation. The breaking force of the uncooked spaghetti samples decreased significantly with increasing bug damage level indicating that they were susceptible to breakage and not suitable for handling, packaging and shipment. Panel tests indicated significant deterioration in sensory properties (stickiness, firmness and bulkiness) generally at the medium damage level.     

Metabolic profiling and analysis of volatile composition of durum wheat semolina and pasta

Although pasta is generally not considered for its aromatic properties, some evidence proves that cereal flours release volatile compounds and they might have an effect on the aroma of the transformed products. This work reports on the characterization of the volatile components of semolina and pasta obtained from four durum wheat cultivars (Triticum durum Desf., cvs. PR22D89, Creso, Cappelli, Trinakria). Semolina samples were characterized through polar metabolite profiling and fatty acid analysis to identify potential precursors of the volatile components. The results show significant differences among the samples tested with cv. Trinakria characterized by the highest content of sugars and fatty acids. Volatile composition was investigated both in semolina and in cooked pasta using headspace solid-phase micro-extraction (HS-SPME) and identified by GC–MS. Thirty-five volatile compounds including aldehydes, ketones, alcohols, terpenes, esters, hydrocarbons and a furan were identified. Significant differences were observed between semolina and pasta samples in terms of composition and amount of the volatile compounds. During cooking an increase in aldehyde content, the appearance of ketones and a decrease in alcohol content were observed. Correlations between metabolites and volatiles demonstrate that the flavour of cooked pasta may differ significantly depending on the durum wheat cultivar employed.     

Development of simple and co-dominant PCR markers to genotype puroindoline a and b alleles for grain hardness in bread wheat (Triticum aestivum L.)

Grain hardness is one of the most important quality characteristics of cultivated bread wheat (Triticum aestivum L.). A large deletion in the puroindoline a (Pina) gene or single nucleotide polymorphisms (SNPs) in the puroindoline b (Pinb) gene results in hard grain texture. So far, nine Pina alleles (Pina-D1a–Pina-D1b, Pina-D1k–Pina-D1q) and seventeen Pinb alleles (Pinb-D1a–Pinb-D1g, Pinb-D1p–Pinb-D1ab) have been identified in bread wheat. The major Pina and Pinb alleles identified in hard wheat cultivars are Pina-D1b, Pinb-D1b, Pinb-D1c and Pinb-D1d. In this study, a three-primer PCR system was employed to develop nine co-dominant STS markers for genotyping Pina-D1a and Pina-D1b, whereas temperature-switch (TS) PCR was used to develop six co-dominant SNP markers for genotyping the Pinb-D1a, Pinb-D1b, Pinb-D1c and Pinb-D1d alleles. These STS and TS-PCR markers were used to verify the grain hardness genotype of 100 wheat cultivars. The reliability and genotyping accuracy of TS-PCR markers were confirmed through sequencing of PCR products and a comparison with previously published results. Therefore, STS and TS-PCR markers offer a simple, cost-effective and reliable method for high-throughput genotyping Pina and Pinb alleles to select grain hardness in wheat quality breeding programs and for wheat market classification.