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

Molecules

Sun-Hyung Lim¹  Su-Bin Lee²  Jong-Yeol Lee²  Yu-Jeong Yang²  Yong Q. Gu³ 

[1]Division of Horticultural Biotechnology, Hankyong National University, Anseong 17579, Korea

[2]National Institute of Agricultural Science, RDA, Jeonju 54874, Korea

[3]USDA-ARS, Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710, USA

关键词: HMW-GS;    RP-UPLC;    allelic analysis;    large wheat germplasm;   

DOI  :  10.3390/molecules26206174

来源: DOAJ

【 摘 要 】

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 (1Bx7/1Bx7^OE and 1Bx17) and three subunits in 1By (1By8*, 1By9 and 1By15) had overlapping RTs; these were differentiated by SDS-PAGE. To distinguish 1Bx7 and 1Bx7^OE, 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.

【 授权许可】

Unknown