Bolesław P. Salmanowicz

The relationship between grain hardness, dough mixing parameters and bread-making quality in winter wheat.

The influence of grain hardness, determined by using molecular markers and physical methods (near-infrared (NIR) technique and particle size index—PSI) on dough characteristics, which in turn were determined with the use of a farinograph and reomixer, as well as bread-making properties were studied. The material covered 24 winter wheat genotypes differing in grain hardness. The field experiment was conducted at standard and increased levels of nitrogen fertilization. Results of molecular analyses were in agreement with those obtained by the use of physical methods for soft-grained lines. Some lines classified as hard (by physical methods) appeared to have the wild-type Pina and Pinb alleles, similar to soft lines. Differences in dough and bread-making properties between lines classified as hard and soft on the basis of molecular data appeared to be of less significance than the differences between lines classified as hard and soft on the basis of physical analyses of grain texture. Values of relative grain hardness at the increased nitrogen fertilization level were significantly higher. At both fertilization levels the NIR parameter determining grain hardness was significantly positively correlated with the wet gluten and sedimentation values, with most of the rheological parameters and bread yield. Values of this parameter correlated with quality characteristics in a higher degree than values of particle size index.

Identification and characterization of high-molecular-weight glutenin genes in Polish triticale cultivars by PCR-based DNA markers

Molecular markers were used to identify the allele/gene composition of complex lociGlu-A1 andGlu-B1 of high-molecular-weight (HMW) glutenin subunits in triticale cultivars. Forty-six Polish cultivars of both winter and spring triticale were analysed with 7 PCR-based markers. Amplified DNA fragments of HMW gluteninGlu-1 genes were separated by agarose slab-gel electrophoresis. Differences between all 3 alleles at the locusGlu-A1 [Glu-A1a (encoding Ax1),1b (Ax2*), and1c (AxNull)], 4 alleles atGlu-B1-1 [Glu-B1-1a (Bx7),1b (Bx7*),1d (Bx6),1ac (Bx6.8)], and 5 alleles atGlu-B1-2 [Glu-B1-2a (By8),2b (By9),2o (By8*),2s (By 18*), and2z (By20*)] were revealed. In total, 16 allele combinations were observed. Molecular markers are particularly helpful in distinguishing the wheatGlu-A1a andGlu-A1b alleles from the ryeGlu-R1a andGlu-R1b alleles in triticale genotypes, respectively, as well as subunits Bx7 from Bx7* and By8 from By8*, which could not be distinguished by SDS-PAGE. Novel glutenin subunits By 18* and By20* (unique to triticale) were identified. HMW glutenin subunit combinations of Polish triticale cultivars, earlier identified by SDS-PAGE analyses, were verified by PCR-based DNA markers. Rapid identification of wheatGlu-1 alleles by molecular markers can be an efficient alternative to the standard separation procedure for early selection of useful triticale genotypes with good bread-making quality.

Capillary electrophoresis of seed 2S albumins from Lupinus species

Two modes of capillary electrophoresis (CE)--free-solution capillary zone electrophoresis (CZE) and sodium dodecyl sulfate capillary electrophoresis (SDS-CE) using a non-gel sieving matrix--have been developed for comparative analysis of low-molecular-mass 2S albumin isoforms from lupins. The albumin fraction and 2S albumins were separated in uncoated fused-silica capillary by CZE with 0.02 M phosphate buffer, pH 7.3, containing the sodium salt of phytic acid. The use of phytic acid (0.025 M) as buffer modifier and ion-pairing agent improved migration reproducibility, peak shape and separation efficiency. The reduced 2S albumins were separated by SDS-CE using a high concentration (0.3-0.5 M) mixture of tris(hydroxymethyl)aminomethane and borate buffers in uncoated fused-silica capillary. Of the various polymers used as non-gel sieving matrix, SDS-CE with a 10% dextran solution was found to be suitable for separation of 2S albumin polypeptides with molecular masses of 4,000-7,000 and 8,000-11,000. The addition of glycerol or ethylene glycol to the SDS separating buffer improved the resolution of polypeptides. The examined Lupinus species showed species-specific CZE and SDS-CE migration profiles of the 2S albumins.

Effect of Genotype, Environment and Their Interaction on Quality Parameters of Wheat Breeding Lines of Diverse Grain Hardness

Abstract Understanding the contribution of genotype, environment and genotype-by-environment interaction to wheat grain quality facilitates the selection for quality in breeding programs. Stability of grain quality characteristics is an important requirement in the baking industry. We assessed 24 winter wheat genotypes with different grain hardness in multienvironment trials at four locations and two levels of fertilization in each location. Grain samples were analyzed for hardness, protein and starch content, and wet gluten content, Zeleny sedimentation value, alveograph parameter (W) and hectoliter weight. All parameters were evaluated on whole grains using the near infrared transmittance technique. Differences between hard and soft genotypes appeared to be significant, apart from grain hardness, for protein content, Zeleny test and alveograph parameter. Genotype was found to have a major influence only on grain hardness; for protein content, wet gluten and Zeleny sedimentation value environment prevailed the influence of genotype, and for starch content, alveograph W parameter and hectoliter weight both sources of variation had similar importance. Genotype-by-environment interaction was of smaller size relative to genotype and environment in terms of all the studied quality parameters. Stable genotypes predominate the breeding lines studied. Response of unstable genotypes to environmental conditions was nonlinear in most cases.

Charge-based characterisation of high-molecular-weight glutenin subunits from common wheat by capillary isoelectric focusing

In this study, the capillary isoelectric focusing (CIEF) method for the separation and charge characterisation of the heterogeneity of high molecular-weight-glutenin subunits (HMW-GS) in common wheat (Triticum aestivum L.) using linear polyacrylamide (LPA) and polyvinyl alcohol (PVA) coated capillaries was developed. Particularly good repeatability and well-resolved charge isoform profiles were obtained by introducing a mixture of carrier ampholytes (pH 3-10 and pH 5-8), a high concentration of urea (6M) and SB3-12 as detergent in a sample solution during separation in a PVA-coated capillary. One major and one or two minor isoforms were observed for the individual HMW-GS. These isoforms were satisfactorily separated using a pH gradient into two groups: y-type isoforms and x-type isoforms encoded by the Glu-B1 locus with shorter migration times and remaining x-type isoforms with longer times. The method produced from eight to twelve isoforms of wheat HMW-GS with pI points in the range of 4.72-6.98. Generally, the minor isoforms were more acidic compared with the major isoform. The y-type subunits had an approximately neutral character (pI 6.70-6.98); however, x-types showed a weakly acidic character (pI 4.72-5.23), with the exception of subunits encoded by the Glu-B1 locus. The isoelectric point peak profiles were compared with capillary zone electrophoresis (CZE) electropherograms. Generally, the number of detected isoforms for the particular HMW-GS detected using both methods were similar.

Identification and characterization of high-molecular-weight secalins from triticale seeds by capillary zone electrophoresis.

A rapid and reliable method for separation and characterization of the variability of high‐molecular‐weight secalin subunits (HMW‐SS) in hexaploid triticale (× Triticosecale Wittmack) by CZE has been developed. In this method, a mixture of two poly(ethylene oxide) polymers differing in molecular weight and a high concentration of ACN in isoelectric buffer was applied as the running electrolyte. For dynamic coating of the capillary inner wall, a low‐concentration mixture of poly(vinylpyrrolidone) and hydroxypropylmethylcellulose was employed. Wide allelic variations in rye HMW‐SS composition, including some novel x‐ and y‐type HMW‐SS, were detected by CZE. The CZE electropherograms of HMW‐SS showed two groups of peaks in accordance with y‐ and x‐type subunits, with migration times of 8.0–8.8 and 11.0–13.3 min, respectively. HMW‐SS differed in migration times from the simultaneously resolved HMW glutenin subunits, but frequently had very similar electrophoretic mobilities during separation by SDS‐PAGE. Each of the two rye subunits 2r and 6.5r detected by SDS‐PAGE represents in fact two subunits (5.1r or 5.2r, and 6.4r or 6.5r, respectively). After analyzing 106 European triticale cultivars, 12 HMW‐SS were identified (six x‐type and six y‐type). They form six allelic variants of these subunits. The simultaneous separation and identification of triticale HMW glutenin and secalin subunits by CZE is an efficient alternative to SDS‐PAGE and should facilitate breeding of valuable cultivars.

Variation of High-Molecular-Weight Secalin Subunit Composition in Rye (Secale cereale L.) Inbred Lines

In this study, identification and characterization of the rye HMW secalin subunit (HMW-SS) composition in 68 inbred rye (Secale cereale L.) lines was performed by capillary zone electrophoresis (CZE). The HMW-SS were separated in an uncoated fused-silica capillary using an isoelectric iminodiacetic buffer in combination with poly(ethylene oxide), lauryl sulfobetaine, and acetonitrile as the separation buffer. The separations of the nonalkylated HMW-SS provided very good resolution and high reproducibility. Generally, the x-type rye HMW-SS were more abundant and have longer migration times than the y-type subunits. Both types of rye HMW-SS were separated into the major protein peak and one or two minor peaks. In total, seven x-type HMW-SS, five of which were newly identified subunits, and six y-type subunits, four of which were new, were distinguished on the basis of their CZE migration times. The migration order of the rye HMW-SS using CZE differed considerably from the relative electrophoretic mobilities in the SDS-PAGE gels.

Molecular, physicochemical and rheological characteristics of introgressive Triticale/Triticum monococcum ssp. monococcum lines with wheat 1D/1A chromosome substitution.

Three sets of hexaploid introgressive triticale lines, with Triticum monococcum ssp. monococcum (cultivated einkorn wheat) genes and a bread wheat chromosome 1D substituted for chromosome 1A, and one set of secondary triticale lines were evaluated for grain and flour physicochemical and dough rheological characteristics in two generations (F7 and F8). Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) confirmed the 1D/1A chromosome substitution. The presence or absence of einkorn high-molecular-weight (HMW) glutenin subunits and the wheat Glu-D1d locus encoding the 5 + 10 subunits was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), capillary zone electrophoresis, and allele-specific molecular markers. Significant differences were found among physicochemical properties (with the exception of the Hagberg falling number) of all introgressive Triticale/T. monococcum lines and the secondary triticale lines. The wheat 1D/1A chromosome substitution also affected these properties. The results showed that in all introgressive triticale lines, the protein and gluten content, Zeleny sedimentation value, and water absorption capacity, were increased. The rheological parameters estimated using micro-farinograph, reomixer, and Kieffer dough extensibility systems also showed an appreciable increase in dough-mixing properties, maximum resistance to extension (Rmax), and dough extensibility. Introgressive Triticale/T. monococcum lines with 5 + 10 subunits have particularly favorable rheological parameters. The results obtained in this study suggest that the cultivated einkorn genome Am, in the context of hexaploid secondary triticale lines and with a wheat 1D/1A substitution, has the potential to improve gluten polymer interactions and be a valuable genetic resource for triticale quality improvement.

CE determination of secaloindoline allelic forms in hexaploid triticale (×Triticosecale Wittmack)

Differences in kernel texture are mainly caused by specific secaloindoline (SIN) proteins occurring in friabilin fraction of hexaploid triticale (x Triticosecale Wittmack) grain. SINs were isolated using Triton X-114 partitioning from either kernels/flour or starch of five triticale cultivars with wide range of different hardness. Crude SIN fraction was obtained by size-exclusion HPLC. SINs were separated on an uncoated fused-silica capillary using the iminodiacetic (IDA) buffer in conjunction with lower-concentrated poly(ethylene oxide) and ACN. A low-concentrate mixture of hydrophilic polymers, PVP and hydroxypropylmethylcellulose in IDA buffer was employed for dynamic coating of capillary inner wall. In total, on the basis of CZE profiles, two SIN-a proteins and two SIN-b proteins were identified. Allelic forms SIN-a1 and SIN-b1 have both two soft and one medium hard genotypes, however other allelic forms, designed as SIN-a2 and SIN-b2, were identified in hard and other medium hard cultivars. The CZE profiles showed that the ratio of the peak areas of SIN-b proteins isolated from triticale starch can be preliminarily used to distinguish cultivars with soft and hard grain.

Rheological characterisation of gluten from triticale (x Triticosecale Wittmack)

BACKGROUND Triticale gluten still remains very poorly characterised rheologically. In this study the mechanical spectra of gluten isolated from four triticale cultivars were registered and fitted with Cole-Cole functions yielding the visco-elastic plateau parameters. Master spectra were calculated. A retardation test was performed and used to calculate the composite mechanical spectra and the width of visco-elastic plateau l. Protein fractional composition of triticale flour and gluten was studied using capillary zone electrophoresis. RESULTS Differentiated HMW-GS/SS compositions were identified in the triticale cultivars studied. The rheological parameters reached the following values: JN0 1.05·10-3 to 2.69·10-3 Pa-1 , GN0 372 to 956 Pa, ω0 0.003 to 0.06 rad s-1 , l 169 to 3121, Je0 1.57·10-3 to 5.03·10-3 Pa-1 , Ge0 199 to 637 Pa and η0 1.06·107 to 3.93·107 Pa s. CONCLUSIONS Visco-elastic properties of triticale gluten correspond to the lower end of medium visco-elasticity shown by common wheat gluten. Master spectra and the composite mechanical spectra prove that four triticale glutens exhibit practically an identical type of visco-elastic behaviour of a biopolymeric visco-elastic liquid similar to wheat gluten. The visco-elastic plateau parameters GN0 , JN0 , ω0 and l appeared significantly correlated with the contents of prolamins and secaloglutenins in triticale flours and glutens.