Comparison of low molecular weight glutenin subunits identified by SDS-PAGE, 2-DE, MALDI-TOF-MS and PCR in common wheat.
BACKGROUND
Low-molecular-weight glutenin subunits (LMW-GS) play a crucial role in determining end-use quality of common wheat by influencing the viscoelastic properties of dough. Four different methods - sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE, IEF x SDS-PAGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and polymerase chain reaction (PCR), were used to characterize the LMW-GS composition in 103 cultivars from 12 countries.


RESULTS
At the Glu-A3 locus, all seven alleles could be reliably identified by 2-DE and PCR. However, the alleles Glu-A3e and Glu-A3d could not be routinely distinguished from Glu-A3f and Glu-A3g, respectively, based on SDS-PAGE, and the allele Glu-A3a could not be differentiated from Glu-A3c by MALDI-TOF-MS. At the Glu-B3 locus, alleles Glu-B3a, Glu-B3b, Glu-B3c, Glu-B3g, Glu-B3h and Glu-B3j could be clearly identified by all four methods, whereas Glu-B3ab, Glu-B3ac, Glu-B3ad could only be identified by the 2-DE method. At the Glu-D3 locus, allelic identification was problematic for the electrophoresis based methods and PCR. MALDI-TOF-MS has the potential to reliably identify the Glu-D3 alleles.


CONCLUSIONS
PCR is the simplest, most accurate, lowest cost, and therefore recommended method for identification of Glu-A3 and Glu-B3 alleles in breeding programs. A combination of methods was required to identify certain alleles, and would be especially useful when characterizing new alleles. A standard set of 30 cultivars for use in future studies was chosen to represent all LMW-GS allelic variants in the collection. Among them, Chinese Spring, Opata 85, Seri 82 and Pavon 76 were recommended as a core set for use in SDS-PAGE gels. Glu-D3c and Glu-D3e are the same allele. Two new alleles, namely, Glu-D3m in cultivar Darius, and Glu-D3n in Fengmai 27, were identified by 2-DE. Utilization of the suggested standard cultivar set, seed of which is available from the CIMMYT and INRA Clermont-Ferrand germplasm collections, should also promote information sharing in the identification of individual LMW-GS and thus provide useful information for quality improvement in common wheat.
Liu
Li
L
Institute of Crop Science, National Wheat Improvement Center/The National Key Facility for Crop Genetic Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences (CAAS), 12 Zhongguancun South Street, Beijing 100081, China.
Ikeda
Tatsuya M
TM
Branlard
Gerard
G
Peña
Roberto J
RJ
Rogers
William J
WJ
Lerner
Silvia E
SE
Kolman
María A
MA
Xia
Xianchun
X
Wang
Linhai
L
Ma
Wujun
W
Appels
Rudi
R
Yoshida
Hisashi
H
Wang
Aili
A
Yan
Yueming
Y
He
Zhonghu
Z
D000483:Alleles; D018744:DNA, Plant / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005983:Glutens / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014908:Triticum / Q000737:chemistry* / Q000235:genetics
D018744:DNA, Plant; D005983:Glutens; C037302:glutenin