Development and validation of a PCR-based marker assay for negative selection of the HMW glutenin allele Glu-B1-1d (Bx-6) in wheat.
Polymorphisms between the coding sequences of high-molecular-weight (HMW) glutenin x-type genes at the Glu-1 locus were used to amplify Glu-1B x-type-specific PCR fragments. PCR analysis in a wheat cultivar subset carrying different Glu-1B x-type alleles resulted in PCR fragments that differed in size for Glu-B1-1d (B-x6) and non -Glu-B1-1d (B-x6) genotypes. Subsequent sequencing analysis revealed a 15-bp in-frame insertion in the coding regions of all Glu-B1-1d (B-x6) genotypes which allowed the development of a B-x6-specific PCR assay for high-throughput allele sizing by ion-pair reversed-phase high-performance liquid chromatography. The assay was validated in a set of 86 German wheat cultivars, and genotyping data unequivocally verified the presence of HMW glutenin subunits GLU-B1-1D (Bx-6) + GLU-B1-2A (By-8) by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis. These results demonstrate that the PCR assay can be applied for the detection and negative selection of the 'poor breadmaking quality' Glu-B1-1d (B-x6) alleles in wheat breeding programs.
Schwarz
G
G
EpiGene GmbH, Biotechnology in Plant Protection, Hohenbachernstrasse 19-21, 85354 Freising, Germany. gschwarz@wzw.tum.de
Felsenstein
F G
FG
Wenzel
G
G
D000483:Alleles; D001483:Base Sequence; D001947:Breeding / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D002874:Chromosome Mapping; D004591:Electrophoresis, Polyacrylamide Gel; D005819:Genetic Markers / Q000235:genetics; D005983:Glutens / Q000031:analogs & derivatives* / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D012641:Selection, Genetic; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014908:Triticum / Q000235:genetics*
D005819:Genetic Markers; D005983:Glutens; C037302:glutenin