Estudio molecular de gluteninas de alto y bajo peso molecular en Triticum aestivum ssp. vulgare L. y su relación con la calidad panadera

El trigo blando (Triticum aestivum ssp vulgare L., AABBDD, 2n=6x=42) presenta propiedades viscoélasticas únicas debidas a la presencia en la harina de las prolaminas: gluteninas y gliadinas. Ambos tipos de proteínas forman parte de la red de gluten. Basándose en la movilidad en SDS-PAGE, las gluteninas se clasifican en dos grupos: gluteninas de alto peso molecular (HMW-GS) y gluteninas de bajo peso molecular (LMW-GS). Los genes que codifican para las HMW-GS se encuentran en tres loci del grupo 1 de cromosomas: Glu-A1, Glu-B1 y Glu-D1. Cada locus codifica para uno o dos polipéptidos o subunidades. La variación alélica de las HMW-GS es el principal determinante de de la calidad harino-panadera y ha sido ampliamente estudiado tanto a nivel de proteína como de ADN. El conocimiento de estas proteínas ha contribuido sustancialmente al progreso de los programas de mejora para la calidad del trigo. Comparadas con las HMW-GS, las LMW-GS forman una familia proteica mucho más compleja. La mayoría de los genes LMW se localizan en el grupo 1 de cromosomas en tres loci: Glu-A3, Glu-B3 y Glu-D3 que se encuentran estrechamente ligados a los loci que codifican para gliadinas. El número de copias de estos genes ha sido estimado entre 10-40 en trigo hexaploide, pero el número exacto aún se desconoce debido a la ausencia de un método eficiente para diferenciar los miembros de esta familia multigénica. La nomenclatura de los alelos LMW-GS por electroforesis convencional es complicada, y diferentes autores asignan distintos alelos a la misma variedad lo que dificulta aún más el estudio de esta compleja familia. El uso de marcadores moleculares para la discriminación de genes LMW, aunque es una tarea dificil, puede ser muy útil para los programas de mejora. El objetivo de este trabajo ha sido profundizar en la relación entre las gluteninas y la calidad panadera y desarrollar marcadores moleculares que permitan ayudar en la correcta clasificación de HMW-GS y LMW-GS. Se han obtenido dos poblaciones de líneas avanzadas F4:6 a partir de los cruzamientos entre las variedades ‘Tigre’ x ‘Gazul’ y ‘Fiel’ x ‘Taber’, seleccionándose para los análisis de calidad las líneas homogéneas para HMW-GS, LMW-GS y gliadinas. La determinación alélica de HMW-GS se llevó a cabo por SDS-PAGE, y se complementó con análisis moleculares, desarrollándose un nuevo marcador de PCR para diferenciar entre las subunidades Bx7 y Bx7*del locus Glu-B1. Resumen 2 La determinación alélica para LMW-GS se llevó a cabo mediante SDS-PAGE siguiendo distintas nomenclaturas y utilizando variedades testigo para cada alelo. El resultado no fue concluyente para el locus Glu-B3, así que se recurrió a marcadores moleculares. El ADN de los parentales y de los testigos se amplificó usando cebadores diseñados en regiones conservadas de los genes LMW y fue posteriormente analizado mediante electroforesis capilar. Los patrones de amplificación obtenidos fueron comparados entre las distintas muestras y permitieron establecer una relación con los alelos de LMW-GS. Con este método se pudo aclarar la determinación alélica de este locus para los cuatro parentales La calidad de la harina fue testada mediante porcentaje de contenido en proteína, prueba de sedimentación (SDSS) y alveógrafo de Chopin (parámetros P, L, P/L y W). Los valores fueron analizados en relación a la composición en gluteninas. Las líneas del cruzamiento ‘Fiel’ x ‘Taber’ mostraron una clara influencia del locus Glu-A3 en la variación de los valores de SDSS. Las líneas que llevaban el nuevo alelo Glu-A3b’ presentaron valores significativamente mayores que los de las líneas con el alelo Glu-A3f. En las líneas procedentes del cruzamiento ‘Tigre ’x ‘Gazul’, los loci Glu-B1 y Glu-B3 loci mostraron ambos influencia en los parámetros de calidad. Los resultados indicaron que: para los valores de SDSS y P, las líneas con las HMW-GS Bx7OE+By8 fueron significativamente mejores que las líneas con Bx17+By18; y las líneas que llevaban el alelo Glu-B3ac presentaban valores de P significativamente superiores que las líneas con el alelo Glu-B3ad y significativamente menores para los valores de L . El análisis de los valores de calidad en relación a los fragmentos LMW amplificados, reveló un efecto significativo entre dos fragmentos (2-616 y 2-636) con los valores de P. La presencia del fragmento 2-636 estaba asociada a valores de P mayores. Estos fragmentos fueron clonados y secuenciados, confirmándose que correspondían a genes del locus Glu-B3. El estudio de la secuencia reveló que la diferencia entre ambos se hallaba en algunos SNPs y en una deleción de 21 nucleótidos que en la proteína correspondería a un InDel de un heptapéptido en la región repetida de la proteína. En este trabajo, la utilización de líneas que difieren en el locus Glu-B3 ha permitido el análisis de la influencia de este locus (el peor caracterizado hasta la fecha) en la calidad panadera. Además, se ha validado el uso de marcadores moleculares en la determinación alélica de las LMW-GS y su relación con la calidad panadera. Summary 3 Bread wheat (Triticum aestivum ssp vulgare L., AABBDD, 2n=6x=42) flour has unique dough viscoelastic properties conferred by prolamins: glutenins and gliadins. Both types of proteins are cross-linked to form gluten polymers. On the basis of their mobility in SDS-PAGE, glutenins can be classified in two groups: high molecular weight glutenins (HMW-GS) and low molecular weight glutenins (LMW-GS). Genes encoding HMW-GS are located on group 1 chromosomes in three loci: Glu-A1, Glu-B1 and Glu-D1, each one encoding two polypeptides, named subunits. Allelic variation of HMW-GS is the most important determinant for bread making quality, and has been exhaustively studied at protein and DNA level. The knowledge of these proteins has substantially contributed to genetic improvement of bread quality in breeding programs. Compared to HMW-GS, LMW-GS are a much more complex family. Most genes encoded LMW-GS are located on group 1 chromosomes. Glu-A3, Glu-B3 and Glu-D3 loci are closely linked to the gliadin loci. The total gene copy number has been estimated to vary from 10–40 in hexaploid wheat. However, the exact copy number of LMW-GS genes is still unknown, mostly due to lack of efficient methods to distinguish members of this multigene family. Nomenclature of LMW-GS alleles is also unclear, and different authors can assign different alleles to the same variety increasing confusion in the study of this complex family. The use of molecular markers for the discrimination of LMW-GS genes might be very useful in breeding programs, but their wide application is not easy. The objective of this work is to gain insight into the relationship between glutenins and bread quality, and the developing of molecular markers that help in the allele classification of HMW-GS and LMW-GS. Two populations of advanced lines F4:6 were obtained from the cross ‘Tigre’ x ‘Gazul’ and ‘Fiel’ x ‘Taber’. Lines homogeneous for HMW-GS, LMW-GS and gliadins pattern were selected for quality analysis. The allele classification of HMW-GS was performed by SDS-PAGE, and then complemented by PCR analysis. A new PCR marker was developed to undoubtedly differentiate between two similar subunits from Glu-B1 locus, Bx7 and Bx7*. The allele classification of LMW-GS was initially performed by SDS-PAGE following different established nomenclatures and using standard varieties. The results were not completely concluding for Glu-B3 locus, so a molecular marker system was applied. DNA from parental lines and standard varieties was amplified using primers designed in conserved domains of LMW genes and analyzed by capillary electrophoresis. The pattern of amplification products obtained was compared among samples and related to the protein allele classification. It was possible to establish a correspondence between specific amplification products and almost all LMW alleles analyzed. With this method, the allele classification of the four parental lines was clarified. Flour quality of F4:6 advanced lines were tested by protein content, sedimentation test (SDSS) and alveograph (P, L, P/L and W). The values were analyzed in relation to the lines prolamin composition. In the ‘Fiel’ x ‘Taber’ population, Glu-A3 locus showed an influence in SDSS values. Lines carrying new allele Glu-A3b’, presented a significantly higher SDSS value than lines with Glu-A3f allele. In the ‘Tigre ’x ‘Gazul’ population, the Glu-B1 and Glu-B3 loci also showed an effect in quality parameters, in SDSS, and P and L values. Results indicated that: for SDSS and P, lines with Bx7OE+By8 were significantly better than lines with Bx17+By18; lines carrying Glu-B3ac allele had a significantly higher P values than Glu-B3ad allele values. lines with and lower L The analysis of quality parameters and amplified LMW fragments revealed a significant influence of two peaks (2-616 y 2-636) in P values. The presence of 2-636 peak gave higher P values than 2-616. These fragments had been cloned and sequenced and identified as Glu-B3 genes. The sequence analysis revealed that the molecular difference between them was some SNPs and a small deletion of 21 nucleotides that in the protein would produce an InDel of a heptapeptide in the repetitive region. In this work, the analysis of two crosses with differences in Glu-3 composition has made possible to study the influence of LMG-GS in quality parameters. Specifically, the influence of Glu-B3, the most interesting and less studied loci has been possible. The results have shown that Glu-B3 allele composition influences the alveograph parameter P (tenacity). The existence of different molecular variants of Glu-B3 alleles have been assessed by using a molecular marker method. This work supports the use of molecular approaches in the study of the very complex LMW-GS family, and validates their application in the analysis of advanced recombinant lines for quality studies.

Analysis of duplication in the Spanish durum wheat collection maintained in the CRF-INIA on the basis of agro-morphological traits and gliadin proteins

A total of 106 potential duplicate cases involved 277 accessions were detected on the basis of passport data in the durum wheat collection maintained in the CRF-INIA. Similarity between accessions was measured by agro-morphological traits. The 90% of the agro-morphological duplication were verified with gliadin proteins, allowing identification of similar material with greater refinement than agro-morphological data. However, the results indicated not to decide for rationalisation only on the basis of molecular data.

Analysis of duplication in the Spanish durum wheat collection maintained in the CRF-INIA on the basis of agro-morphological traits and gliadin proteins

A total of 106 potential duplicate cases involved 277 accessions were detected on the basis of passport data in the durum wheat collection maintained in the CRF-INIA. Similarity between accessions was measured by agro-morphological traits. The 90% of the agro-morphological duplication were verified with gliadin proteins, allowing identification of similar material with greater refinement than agro-morphological data. However, the results indicated not to decide for rationalisation only on the basis of molecular data

Combined use of gliadins and SSRs to analyse the genetic variability of the Spanish collection of cultivated diploid wheat (Triticum monococcum L. ssp. monococcum)

This work studied the combined use of gliadins and SSRs to analyse inter- and intra-accession variability of the Spanish collection of cultivated einkorn (Triticum monococcum L. ssp. monococcum) maintained at the CRF-INIA. In general, gliadin loci presented higher discrimination power than SSRs, reflecting the high variability of the gliadins. The loci on chromosome 6A were the most polymorphic with similar PIC values for both marker systems, showing that these markers are very useful for genetic variability studies in wheat. The gliadin results indicated that the Spanish einkorn collection possessed high genetic diversity, being the differentiation large between varieties and small within them. Some associations between gliadin alleles and geographical and agro-morphological data were found. Agro-morphological relations were also observed in the clusters of the SSRs dendrogram. A high concordance was found between gliadins and SSRs for genotype identification. In addition, both systems provide complementary information to resolve the different cases of intra-accession variability not detected at the agro-morphological level, and to identify separately all the genotypes analysed. The combined use of both genetic markers is an excellent tool for genetic resource evaluation in addition to agro-morphological evaluation.

Effects of N fertilisation, year and prolamin alleles on guten quality in durum wheat (Triticum turgidum L. ssp. turgidum) landraces from Spain

A subset of durum wheat Spanish landraces, previously evaluated for yield at low and high nitrogen (N) levels, was analysed for quality, protein content (P) and sodium dodecyl sulphate sedimentation (SDSS) test. The evaluation was carried out at the two N rates and in two years. The influence of prolamin alleles at the Glu-1, Glu-3, Glu-B2 and Gli-1 loci on quality parameters was also studied. The non significant Variety-by-Year or Variety-by-N interactions suggested that year and N affected all the varieties in a similar manner. Year and N effects were larger than variety effect for P, which increased with N. In contrast, variety genotype exhibited a stronger influence on SDSS test, which was not affected by year and fertilizer. Variety effects on P did not reflect the variety differences for SDSS test. A high positive influence of some prolamin alleles on quality parameters was detected, mainly for SDSS values. No correlation between yield and P was detected in the landraces adapted to low N. Based on the results of yield and quality evaluations, four landraces with high yield and high gluten strength were pre-selected for low N production.

Genetic redundancy among durum wheat accessions as assessed with SSRs and endosperm proteins

Reducing duplication in ex-situ collections is complicated and requires good quality genetic markers. This study was conducted to assess the value of endosperm proteins and SSRs for validation of potential duplicates and monitoring intra-accession variability. Fifty durum wheat (Triticum turgidum ssp. durum) accessions grouped in 23 potential duplicates, and previously characterised for 30 agro-morphological traits, were analysed for gliadin and high molecular weight glutenin (HMWG) subunit alleles, total protein, and 24 SSRs, covering a wide genome area. Similarity and dissimilarity matrices were generated based on protein and SSRs alleles. For heterogeneous accessions at gliadins the percent pattern homology (PH) between gliadin patterns and the Nei’s coefficient of genetic identity (I) were computed. Eighteen duplicates identical for proteins showed none or less than 3 unshared SSRs alleles. For heterogeneous accessions PH and I values lower than 80 identified clearly off-types with more than 3 SSRs unshared. Only those biotypes differing in no more than one protein-coding locus were confirmed with SSRs. A good concordance among proteins, morphological traits, and SSR were detected. However, the discrepancy in similarity detected in some cases showed that it is advisable to evaluate redundancy through distinct approaches. The analysis in proteins together with SSRs data are very useful to identify duplicates, biotypes, close related genotypes, and contaminations

Analysis of genetic variability in a sample of the durum wheat (Triticum durum Desf.) Spanish collection based on gliadin markers

In this work gliadin proteins were used to analyse the genetic variability in a sample of the durum wheat Spanish collection conserved at the CRF-INIA. In total 38 different alleles were identified at the loci Gli-A1, Gli-A3, Gli-B5, Gli-B1, Gli-A2 and Gli-B2. All the gliadin loci were polymorphic, possessed large genetic diversity and small and large differentiation within and between varieties, respectively. The Gli-A2 and Gli-B2 loci were the most polymorphic, the most fixed within varieties and the most useful to distinguish among varieties. Alternatively, Gli-B1 locus presented the least genetic variability out of the four main loci Gli-A1, Gli-B1, Gli-A2 and Gli-B2. The Gli-B1 alleles coding for the gliadin γ-45, associated with good quality, had an accumulated frequency of 69.7%, showing that the Spanish germplasm could be a good source for breeding quality. The Spanish landraces studied showed new gliadin alleles not catalogued so far. These new alleles might be associated with specific Spanish environment factors. The large number of new alleles identified also indicates that durum wheat Spanish germplasm is rather unique.

Comparison of several models for calculating ozone stomatal fluxes on a Mediterranean wheat cultivar (Triticum durum Desf. cv. Camacho)

Ozone stomatal fluxes were modeled for a 3-year period following different approaches for a commercial variety of durum wheat (Triticum durum Desf. cv. Camacho) at the phenological stage of anthesis. All models performed in the same range, although not all of them afforded equally significant results. Nevertheless, all of them suggest that stomatal conductance would account for the main percentage of ozone deposition fluxes. A new modeling approach was tested, based on a 3-D architectural model of the wheat canopy, and fairly accurate results were obtained. Plant species-specific measurements, as well as measurements of stomatal conductance and environmental parameters, were required. The method proposed for calculating ozone stomatal fluxes (FO(3_3-D)) from experimental gs data and modeling them as a function of certain environmental parameters in conjunction with the use of the YPLANT model seems to be adequate, providing realistic estimates of the canopy FO(3_3-D), integrating and not neglecting the contribution of the lower leaves with respect to the flag leaf, although a further development of this model is needed.

Soil water availability modulation over estimated relative yield losses in wheat (Triticum aestivum L.) due to ozone exposure

The approach developed by Fuhrer in 1995 to estimate wheat yield losses induced by ozone and modulated by the soil water content (SWC) was applied to the data on Catalonian wheat yields. The aim of our work was to apply this approach and adjust it to Mediterranean environmental conditions by means of the necessary corrections. The main objective pursued was to prove the importance of soil water availability in the estimation of relative wheat yield losses as a factor that modifies the effects of tropospheric ozone on wheat, and to develop the algorithms required for the estimation of relative yield losses, adapted to the Mediterranean environmental conditions. The results show that this is an easy way to estimate relative yield losses just using meteorological data, without using ozone fluxes, which are much more difficult to calculate. Soil water availability is very important as a modulating factor of the effects of ozone on wheat; when soil water availability decreases, almost twice the amount of accumulated exposure to ozone is required to induce the same percentage of yield loss as in years when soil water availability is high.

Residual effect of natural and synthetic zinc chelates on zinc in a soil solution of a waterlogged acidic soil. Evolution of the pH and redox potential.

Zinc chelates have been widely used to correct deficiencies in this micronutrient in different soil types and under different moisture conditions. The aging of the metal in soil could cause a change in its availability. Over time the most labile forms of Zn could decrease in activity and extractability and change to more stable forms. Various soil parameters, such as redox conditions, time, soil type and moisture conditions, affect the aging process and modify the solubility of the metal. In general, redox conditions influence pH and also the chemical forms dissolved in the soil solution. Soil pH also affects Zn solubility; at high pH values, most of the Zn is present in forms that are not bioavailable to plants. The objective of this study was to determine the changes in Zn over time in a soil solution in a waterlogged acidic soil to which synthetic and natural chelates were applied

Residual effect of synthetic Zn chelates on the availability of this metal in different soils under waterlogged conditions.

Adding Zn improves crop growth, increases seed yield and also positively affects nutritional quality. After Zn fertilization, there is normally a period of several years in which residual effects provide an adequate supply of Zn to successive crops. Immediately after the application of Zn sources water-soluble Zn slowly but continually decreases. Various factors, including time and moisture conditions, affect the aging process and modify the solubility of the metal in soil and therefore its availability. In previous experiments, we studied the residual effect of synthetic chelates, obtained that the amounts of potentially available Zn decreased in the second cropping year due to aging processes. The present study was undertaken to verify variations in the residual effects of applying four different synthetic Zn sources

Down-Regulating γ-gliadins in bread wheat leads to non-specific increases in other gluten proteins and has no major effect on dough gluten strength.

Background Gliadins are a major component of gluten proteins but their role in the mixing of dough is not well understood because their contribution to wheat flour functional properties are not as clear as for the glutenin fraction. Methodology/Principal Findings Transgenic lines of bread wheat with γ-gliadins suppressed by RNAi are reported. The effects on the gluten protein composition and on technological properties of flour were analyzed by RP-HPLC, by sodium dodecyl sulfate sedimentation (SDSS) test and by Mixograph analysis. The silencing of γ-gliadins by RNAi in wheat lines results in an increase in content of all other gluten proteins. Despite the gluten proteins compensation, in silico analysis of amino acid content showed no difference in the γ-gliadins silenced lines. The SDSS test and Mixograph parameters were slightly affected by the suppression of γ-gliadins. Conclusions/Significance Therefore, it is concluded that γ-gliadins do not have an essential functional contribution to the bread-making quality of wheat dough, and their role can be replaced by other gluten proteins

Effects of the 4N(v) chromosome from Aegilops ventricosa on agronomic and quality traits in bread wheat

Advanced wheat lines carrying the Hessian fly resistance gene H27 were obtained by backcrossing the wheat/Aegilops ventricosa introgression line, H-93-33, to commercial wheat cultivars as recurrent parents. The Acph-N v 1 marker linked to the gene H27 on the 4Nv chromosome of this line was used for marker assisted selection. Advanced lines were evaluated for Hessian fly resistance in field and growth chamber tests, and for other agronomic traits during several crop seasons at different localities of Spain. The hessian fly resistance levels of lines carrying the 4Nv chromosome introgression (4D/4Nv substitution and recombination lines that previously were classified by in situ hybridisation) were high, but always lower than that of their Ae. ventricosa progenitor. Introgression lines had higher grain yields in infested field trials than those without the 4Nv chromosome and their susceptible parents, but lower grain yields under high yield potential conditions. The 4Nv introgression was also associated with later heading, and lower tiller and grain numbers/m2 . In addition, it was associated with longer and more lax spikes, and higher values of grain weight and grain protein content. However, the glutenin and gliadin expression, as well as the bread-making performance, were similar to those of their recurrent parents

Effect of wheat inclusion and xylanase supplementation of the diet on intestinal enzyme activity, nutrient retention and performance in laying hen from 25 to 47 wks of age

A trial was conducted to examine the effects of increasing levels of wheat in the diet and xylanase (ES) supplementation on nitrogen and ether extract retention, pH of the GIT, productive performance from 25 to 47 wks of age, and enzyme activity at the small intestine level. The basal diets (from 25 to 33 wks and from 33 to 47 wks) consisted of soybean meal and corn, and the wheat was introduced in the experimental diets at expenses of corn, primarily.

Multipath Reflectivity Estimation in Urban Environments for Synthetic Aperture Radar Images

Synthetic Aperture Radar (SAR) images a target region reflectivity function in the multi-dimensional spatial domain of range and cross-range. SAR synthesizes a large aperture radar in order to achieve a finer azimuth resolution than the one provided by any on-board real antenna. Conventional SAR techniques assume a single reflection of transmitted waveforms from targets. Nevertheless, today¿s new scenes force SAR systems to work in urban environments. Consequently, multiple-bounce returns are added to directscatter echoes. We refer to these as ghost images, since they obscure true target image and lead to poor resolution. By analyzing the quadratic phase error (QPE), this paper demonstrates that Earth¿s curvature influences the defocusing degree of multipath returns. In addition to the QPE, other parameters such as integrated sidelobe ratio (ISLR), peak sidelobe ratio (PSLR), contrast (C) and entropy (E) provide us with the tools to identify direct-scatter echoes in images containing undesired returns coming from multipath.