PAG-VI: COMPARISON OF DIFFERENT METHODS TO SCORE RADIOACTIVE AFLP GELS

PAG-VI  Plant & Animal Genome VI Conference

Town & Country Hotel, San Diego, CA, January 18-22, 1998.

P78

COMPARISON OF DIFFERENT METHODS TO SCORE RADIOACTIVE AFLP GELS

Isabel Roldán-Ruiz1, Ann Depicker2, Jochen Dendauw1, Erik Van Bockstaele1 3, MARC DE LOOSE1

  1. Department for plant genetics and breeding, Centre for Agricultural Research-Gent (CLO)., Burg. Van Gansberghelaan 109, B-9820 Merelbeke, Belgium.
  2. Laboratory of Genetics, University of Gent., Ledeganckstraat 35, B-9000 Gent, Belgium.
  3. Department of Plant Production, University of Gent, Coupure Links 653, B-9000 Gent, Belgium.

AFLP was originally developed as a radioactive system. In its original description, the developers of the technique (Keygene, NL) used radioactively labelled primers and a phosphorimager for the visualisation of the AFLP products. Keygene also developed a computer program for the automatic scoring of the gels that is not yet commercially available. Other authors have used light-sensitive films to develop their AFLP patterns, usually combined with visual scoring or a flat bed scanner. Our objective in this study was to compare different methods of scoring radioactive AFLP gels in terms of accuracy and efficiency. AFLP gels of ryegrass plants displaying a very high degree of polymorphism were used. Radioactive gels were handled according to one of the following procedures: (i) brought in contact with a light sensitive film during 4 days, developed and scored visually (visual inspection), (ii) brought in contact with a light sensitive film during at least 4 days, developed and scanned in a HP-Scanjet IIcx desktop scanner at high resolution (scanner inspection), (iii) placed in a phosphorimager cassette during 24 hours and read in a phosphorimager from Molecular Dynamics (phosphorimager inspection). Image files produced for the same gel by both the scanner and the phosphorimager were analysed using GelCompar and the software developed by Keygene. This allowed us to compare the performance of the two computer programs. The visual scoring of the gels resulted in accurate and reproducible results, but it was rather time consuming and not feasible for the analysis of either a large number of cultivars or a large number or gels. In this latter case it is advisable to follow an automatic procedure as standardised as possible. The two computer programs compared resulted in different results regarding accurateness of the scoring.

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