Multiplex polymerase chain reaction analysis of Glu-1 high-molecular-mass glutenin genes from wheat by capillary electrophoresis with laser-induced fluorescence detection.
The unique bread-making properties of wheat are closely correlated with composition and quantity of high-molecular-mass (HMW) glutenin subunits encoded by the Glu-1 genes. We report the development of a multiplex polymerase chain reaction (PCR) method to identify bread wheat genotypes carrying HMW glutenin allele composition of Glu-1 complex loci (Glu-A1, Glu-B1 and Glu-D1) by capillary electrophoresis(CE) with laser-induced fluorescence (LIF) detection. Two triplex primer sets of HMW glutenin subunit genes were examined. An automated and rapid CE-LIF technique is helpful in the multiplex PCR optimization process. Two fluorescent intercalating dyes (EnhanCE, and YO-PRO-1) are compared for detection of DNA fragments. Amplified DNA fragments of HMW glutenin Glu-1 genes were well separated both by agarose slab-gel electrophoresis and CE, and revealed minor differences between the sequences of 1Ax2*, 1Axnull, 1Bx6, 1Bx7, 1Bx17 and 1Dx5 genes. Moreover, CE technique requires samples of smaller volumes in comparison to slab-gel electrophoresis, and data can be obtained in less than 20 min. There was a very high concordance in the assessment of the molecular size of PCR-generated DNA markers. Fast and accurate identification of molecular markers of Glu-1 genes by CE-LIF can be an efficient alternative to standard procedure separation for early selection of useful wheat genotypes with good bread-making quality.
Salmanowicz
Boleslaw P
BP
Institute of Plant Genetics, Polish Academy of Sciences, ul. Strzeszynska 34, PL 60-479 Poznan, Poland. bsal@igr.poznan.pl
Moczulski
Marcin
M
D001483:Base Sequence; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D019075:Electrophoresis, Capillary / Q000379:methods*; D004996:Ethidium; D005983:Glutens / Q000031:analogs & derivatives* / Q000235:genetics*; D007834:Lasers; D008970:Molecular Weight; D016133:Polymerase Chain Reaction / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014908:Triticum / Q000235:genetics*
D017931:DNA Primers; D005983:Glutens; D004996:Ethidium; C037302:glutenin