scholarly journals A Rapid, Reliable RP-UPLC Method for Large-Scale Analysis of Wheat HMW-GS Alleles

Molecules ◽  
2021 ◽  
Vol 26 (20) ◽  
pp. 6174
Author(s):  
Su-Bin Lee ◽  
Yu-Jeong Yang ◽  
Sun-Hyung Lim ◽  
Yong Q. Gu ◽  
Jong-Yeol Lee
Keyword(s):  
Large Scale ◽  
Storage Proteins ◽  
Ultra Performance Liquid Chromatography ◽  
Reversed Phase ◽  
Wheat Breeding ◽  
Pcr Analysis ◽  
Large Scale Analysis ◽  
End Use ◽  
Acquity Uplc ◽  
Wheat Storage Proteins

High-molecular-weight glutenin subunits (HMW-GS) account for only 10% of total wheat storage proteins, but play an important role in the processing quality of wheat flour. Therefore, identifying HMW-GS alleles associated with good end-use quality provides important information for wheat breeders. To rapidly, accurately and reproducibly identify HMW-GS, we established an optimized reversed-phase ultra-performance liquid chromatography (RP-UPLC) method. Separation parameters were optimized using an ACQUITY UPLC Protein BEH C4 column and stepwise ACN gradient, and the separation patterns and retention times (RTs) of 22 subunits were comparatively analyzed in 16 standard wheat cultivars. All HMW-GS proteins were well separated within about 5.5 min, and all analyses were complete within 12 min. We distinguished the 16 subunits based on RT, although three subunits in 1Bx (1Bx7/1Bx7OE and 1Bx17) and three subunits in 1By (1By8*, 1By9 and 1By15) had overlapping RTs; these were differentiated by SDS-PAGE. To distinguish 1Bx7 and 1Bx7OE, which differ in protein abundance, RP-UPLC was combined with PCR analysis of DNA junction markers. The optimized method was successfully applied to determine HMW-GS alleles in a large collection of bread wheat germplasm (1787 lines). This protocol is an appropriate option for selecting lines harboring favorable HMW-GS alleles in wheat breeding.

scholarly journals Fast identification of wheat 1BL.1RS translocation by reversed-phase ultra-performance liquid chromatography (RP-UPLC)

2013 ◽  
Vol 64 (9) ◽  
pp. 865 ◽  
Author(s):  
Jianwen Zhou ◽  
Caixia Han ◽  
Hui Cao ◽  
Shoumin Zhen ◽  
Zitong Yu ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Large Scale ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ultra Performance Liquid Chromatography ◽  
Reversed Phase ◽  
Wheat Breeding ◽  
Parental Line ◽  
Wheat Cultivars ◽  
Protein Markers ◽  
Fast Identification

The 1BL.1RS chromosomal translocation in wheat is the result of replacement of the short arm of chromosome 1B of wheat by the short arm of chromosome 1R of rye, which had been widely used as a parental line in worldwide wheat breeding, resulting in a high percentage of wheat cultivars containing this translocation. A fast and reliable approach to identify this translocation is highly desirable in modern wheat breeding. This study compared reversed-phase ultra-performance liquid chromatography (RP-UPLC), acidic polyacrylamide gel electrophoresis (A-PAGE), liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), allelic-specific PCR, and reversed-phase high-performance liquid chromatography (RP-HPLC) approaches to identify the 1BL.1RS translocation in 76 bread wheat cultivars. Two gliadin bands in the Gli-B1 region of A-PAGE separation were confirmed by LC-MS/MS to be omega secalins from the 1BL.1RS translocation, and they can be used as reliable protein markers for identifying the translocation. A few specific minor peaks eluted at 12–13 min on the RP-UPLC patterns can readily differentiate the 1BL.1RS translocation. Of the 76 wheat cultivars tested, 40 were identified as carrying the 1BL.1RS translocation by RP-UPLC, which was consistent with the results of A-PAGE, HPLC, and PCR. Compared with other established methods, RP-UPLC showed a clear advantage in fast identification of the 1BL.1RS translocation with higher reliability and lower costs, and it is therefore ideal for large-scale screening of the 1BL.1RS translocation in wheat breeding.

The Industrial Use of EU Wheats

1996 ◽  
Vol 25 (4) ◽  
pp. 243-251
Author(s):  
Jean-Claude Autran
Keyword(s):  
European Union ◽  
Storage Proteins ◽  
Research Programme ◽  
Wheat Breeding ◽  
The European Union ◽  
Processing Industry ◽  
Milling Quality ◽  
Industrial Use ◽  
Sprouting Resistance ◽  
Wheat Storage Proteins

A four-year coordinated wheat research programme was recently conducted with the aim of advancing understanding of wheat processing and quality under the specific conditions of the European Union. The main areas examined included milling quality, starch/gluten separation, the basis of breadmaking quality, the basis of biscuit quality, flour composition, dough development, the genetics of wheat storage proteins and sprouting resistance. The programme produced a range of results which will contribute to developments In the processing industry, wheat breeding and trade.

Involvement of a Novel Peptide in the Heat Shock Response of Australian Wheats

1990 ◽  
Vol 17 (4) ◽  
pp. 441 ◽  
Author(s):  
CS Blumenthal ◽  
IL Batey ◽  
CW Wrigley ◽  
EWR Barlow
Keyword(s):  
Heat Shock ◽  
High Performance ◽  
Storage Proteins ◽  
Seed Proteins ◽  
Reversed Phase ◽  
Terminal Sequence ◽  
Chinese Spring Wheat ◽  
End Use ◽  
Peptide Gene

A low molecular weight peptide, induced by exposure of coleoptiles to heat stress at 41°C, has been detected by reversed-phase high performance liquid chromatography of extracts from coleoptiles of five wheat (Triticum aestivum) cultivars. This component is detected within 1 h of a 41°C heat shock, is not detected 48 h after cessation of the heat shock, and remains present during a continuous 24 h heat treatment. The appearance of this component is also induced by exposure of the coleoptile to 0.1 M sodium arsenite or 10% ethanol. When other species such as barley (Hordeum vulgare), soybean (Glycine max), sorghum (Sorghum bicolor), maize (Zea mays), mungbean (Vigna radiata), or rice (Oryza sativa) were examined for the presence of a component eluting in the same position, it was only detected in maize. The amino acid sequence for the heat-induced peptide from wheat was determined to be: V-L-V-P-V-P-Q-L-Q-P-Q-N-Q-P/Q. The sequence of 12 of these amino acids is the same as the N-terminal sequence of α- and β gliadins (wheat endosperm storage proteins). The production of this heat-induced peptide in aneuploids of Chinese spring wheat indicated that the peptide gene was located on the same chromosome arm as one of the gliadin genes. The presence of this gliadin-like peptide in heat-stressed coleoptiles may be due to the presence of five heat shock elements in the gene sequence of gliadins. The potential heat inducibility of the gliadin gene has important implications for end-use quality of wheat. The results also imply that seed proteins may have a function other than storage of nitrogen.

scholarly journals Structural elucidation of triacylglycerol using online acetone Paternò–Büchi reaction coupled with reversed-phase liquid chromatography mass spectrometry

The Analyst ◽  
2020 ◽  
Vol 145 (20) ◽  
pp. 6532-6540 ◽  
Author(s):  
Elissia T. Franklin ◽  
Yu Xia
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Large Scale ◽  
Reversed Phase ◽  
Structural Elucidation ◽  
Scale Analysis ◽  
Liquid Chromatography Mass Spectrometry ◽  
Reversed Phase Liquid Chromatography ◽  
Large Scale Analysis ◽  
Phase Liquid

The developed online RPLC-PB-MS/MS system allows large scale analysis of isomeric triacylglycerol lipids differing in CC locations.

scholarly journals Analytical Method development and Validation of Lamivudine in Formulation by using Reversed Phase Ultra Performance Liquid Chromatography

2020 ◽  
Vol 13 (2) ◽  
pp. 1-6
Author(s):  
Anshul kumar ◽  
Chandana Majee ◽  
Vivek Namdev
Keyword(s):  
Method Development ◽  
Ultra Performance Liquid Chromatography ◽  
Reversed Phase ◽  
Array Detector ◽  
Solution Stability ◽  
Solution Stability And Robustness ◽  
Acquity Uplc ◽  
Development And Validation ◽  
Tablet Dosage ◽  
Liquid Chromatographic

Aim of the experiment was to develop a simple, specific and accurate reverse phase ultra-performance liquid chromatographic (UPLC) method for the determination of lamivudine in the tablet dosage forms. The chromatographic separation was achieved on Acquity UPLC HSST3 (2.1 x 100mm) 1.8 um particle size and the mobile phase containing 0.1%TFA: MeOH for lamivudine. The run time was 10 min and the retention time of lamivudine was about 4.6. The detection was carried out 215nm using photo diode array detector (PDA) with a flow rate 0.6 ml/min. The linearity of lamivudine with correlation coefficient 0.9998. The recovery was found in the range (100±10%). The developed method was validated as per International Conference on Harmonization guidelines (ICH) with respect to specificity, linearity, accuracy, method precision, system precision, solution stability and robustness

Measuring polyphenol oxidase activity in a wheat breeding program

1999 ◽  
Vol 79 (4) ◽  
pp. 507-514 ◽  
Author(s):  
T. N. Mccaig ◽  
D. Y. K. Fenn ◽  
R. E. Knox ◽  
R. M. Depauw ◽  
J. M. Clarke ◽  
...  
Keyword(s):  
Polyphenol Oxidase ◽  
Large Scale ◽  
Water Extract ◽  
Wheat Breeding ◽  
Screening Tools ◽  
Breeding Programs ◽  
Spring Triticale ◽  
The Mean ◽  
Whole Seed ◽  
End Use

High levels of polyphenol oxidase (PPO) have been associated with discoloration of end-use products of wheat, especially certain noodle types. Two whole-seed methods of measuring PPO, one based on 10 mM tyrosine as substrate and the other on 90 mM catechol, were examined and modified to determine their potential as screening tools in large-scale breeding programs. Thirteen spring wheat and two spring triticale genotypes were used to compare the methods. Both methods could measure PPO on individual seeds. All genotypes displayed large seed-to-seed variation for PPO with both substrates. The mean coefficient of variation for the PPO values of individual seeds within genotypes was 39% with tyrosine and 34% with catechol. Furthermore, the PPO values of individual seeds within genotypes were not normally distributed for most genotypes. Identifying genotypes with incremental improvements in PPO would probably require measurement of 70–100 seeds. Approximately 50% of the catecholase activity was associated with the water extract after soaking seeds for 16 h, while all of the tyrosinase activity was still associated with the seed, suggesting that different enzymes are responsible for oxidizing tyrosine and catechol in wheat. While the 10 mM tyrosine assay was nondestructive and allowed plants to be generated from seeds low in PPO, 90 mM catechol reduced germination to less than 20%. Reducing the catechol to 30 mM improved germination to 85%, did not substrate-limit the reaction, and reduced the health risk associated with the assay. Spectral and kinetic differences between the assays were also considered. Key words: Triticum sp., wheat, polyphenol oxidase, catechol, tyrosine

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