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Using a gel‐free PCR‐ELISA for the molecular identification of wheat genotypes carrying wheat–rye translocations
Author(s) -
Zuñiga J.,
Soto B.,
Campos H.
Publication year - 2008
Publication title -
plant breeding
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.583
H-Index - 71
eISSN - 1439-0523
pISSN - 0179-9541
DOI - 10.1111/j.1439-0523.2007.01377.x
Subject(s) - biology , amplicon , digoxigenin , gel electrophoresis , genotype , chromosome , genetics , polymerase chain reaction , chromosomal translocation , microbiology and biotechnology , gene , messenger rna , in situ hybridization
Current techniques to identify wheat lines possessing the 1RS chromosome are generally unsuitable in relation to the speed and cost needs in modern wheat breeding programmes. A gel‐free, direct amplicon capture PCR‐ELISA assay was developed and evaluated, aiming at speeding the identification of wheat genotypes possessing the 1RS chromosome arm in breeding programmes. The chosen target sequence was the repetitive, interspersed rye‐specific element RIS‐1. Primers were end‐labelled with digoxigenin and biotin, and amplicons captured on to straptavidine‐coated microplates. Subsequent immunodetection of the digoxigenin moiety readily distinguished 1RS from non‐1RS control genotypes tested. When a nursery consisting of 120 winter and spring wheat lines was screened by PCR‐gel electrophoresis and the PCR‐ELISA, a perfect agreement between both techniques was observed. Test robustness, as measured by the tolerance to variations in DNA input, was better for PCR‐ELISA than PCR‐gel electrophoresis. In conclusion, a simple, robust, fast and scalable technique for the detection of 1RS chromosome carriers in wheat breeding programmes is now available.