Abstract
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.
Highlights
Gluten, composed of polymeric glutenin and monomeric gliadin, is the primary factor that produces visco-elasticity in wheat dough and is important for wheat end-use quality [1,2]
Under Condition 4, all subunits of High-molecular-weight glutenin subunits (HMW-GS) in Chinese Spring (CS) were effectively resolved, and all peaks eluted within about 5 min, substantially reducing the analysis time
We identified the composition for 22 HMW-GS in 16 standard wheat cultivars, all reported in various earlier publications, using our optimized reversed-phase ultra-performance liquid chromatography (RP-UPLC) method along with auxiliary methods (Table 1, Figures 2 and 3, Figures S3 and S4)
Summary
Gluten, composed of polymeric glutenin and monomeric gliadin, is the primary factor that produces visco-elasticity in wheat dough and is important for wheat end-use quality [1,2]. To determine the HMW-GS allelic composition of bread wheat, techniques such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), polymerase chain reaction (PCR) analysis and reversed-phase high-performance liquid chromatography (RPHPLC) are commonly and widely used as alternatives to techniques requiring expensive instruments, such as high-performance capillary electrophoresis (HPCE) or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) [18,19,20,21,22,23,24,25,26] Each of these three methods has drawbacks, . We developed an optimized RP-UPLC method to identify these molecules by adopting a recently developed column to separate high-molecular-weight proteins and using stepwise gradient conditions to markedly shorten the time and increase resolution for bread wheat samples With this optimized method and auxiliary methods, we successfully identified HMW-GS alleles of a large, worldwide collection of 1787 wheat lines, providing valuable resources for integrating wheat end-use quality data into breeding programs in the future
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