Amplified fragment length polymorphism (AFLP) first described by Zabeau & Vos (1993) is a reproducible, highly multiplex assay with the ability to screen a large number of loci for polymorphisms thereby facilitating efficient marker detection and marker saturation. Traditionally, the detection of AFLP fragments is based on using radioactively labelled primers in the final amplification step and separation on polyacrylamide gels. To avoid the use of radioactivity which strongly hampers the application of this technique in practical barley breeding AFLP products can also be identified with an automatic DNA sequencer using fluorescence labelled primers (cf. Schondelmaier et al. 1996). Therefore, attempts were made to optimize the protocol described by Vos et al. (1995) for detection of AFLPs on a LI-COR DNA Analyzer Genereadir4200 (MWG Biotech) in order to establish this technique for marker detection and saturation concerning resistance genes to the barley yellow mosaic virus complex.

DNA restriction and ligation was performed using the AFLP Core Reagent Kit (Gibco Life Technologies). 150 ng genomic DNA (determined by the use of DNA fluorometer TKO 100, Hoefer) were digested with EcoRI an MseI. Ligation was followed by two pre-amplification steps. First non- selective AFLP primers E00 (5'-GAC TGC GTA CCA ATT C-3') and M00 (5'-GAT GAG TCC TGA GTA A-3') were used in order to reduce unspecific background on polyacrylamide gels (+0- pre-amplification) followed by primers E01 (5'-GAC TGC GTA CCA ATT CA-3') and M02 (5'- GAT GAG TCC TGA GTA AC-3') with one additional selective nucleotide (+1-pre-amplification). Both pre-amplifications were performed in a total volume of 50 l containing 5.0 l of a 1:10 dilution (with TE buffer) of the digested and ligated DNA and of the +0-pre-amplification product, respectively, 75 ng of each primer (MWG Biotech), 0.2 mM dNTP mix (MBI Fermentas) and 1 U Taq DNA polymerase (Qiagen) and its corresponding reaction buffer. The cycle profile for each pre- amplification was as follows; after an initial denaturation step (94°C/3 min) 20 cycles of 94°C/30 s, 56°C/1 min, and 72°C/1 min were carried out followed by an additional extension of 5 min at 72°C. The PCR reaction of the +1-pre-amplification was diluted 1:20 with TE buffer and used as template for the selective amplification. Selective amplification was carried out using primers with three additional selective nucleotides. In each case the EcoRI primer was 5'-end labeled with fluorescence dye IRD700 or IRD800 (MWG Biotech). The selective PCR was performed in a total volume of 20 l, comprising 30 ng MseI primer, 5 ng 5'-IRD700 labeled EcoRI primer or 10 ng 5'- IRD800 labeled EcoRI primer, 0.2 mM dNTP mix (MBI Fermentas) and 0.4 U Taq DNA polymerase (Qiagen), and its corresponding reaction buffer. PCR conditions were set according to Vos et al. (1995) with minor modifications. An initial denaturation step of 94°C for 3 min was followed by one cycle of 94°C for 30 s, 65°C for 30 s, 72°C for 60 s, 11 cycles in which the annealing temperature is decreased by 0.7°C per cycle and by 22 cycles of 94°C/30 s, 56°C/30 s, 72°C/1 min and an ultimate extension at 72°C for 5 min. All PCR reactions were carried out in a GeneAMP PCR System 9700 (Perkin Elmer). An equal volume of formamide loading buffer (95% formamide (v/v), 10 mM EDTA pH 8, 0.1% basic fuchsin, 0.01% bromephenol blue) was added and the samples were denatured at 94°C for 1.5 min; 1.0 l of each sample was loaded on to 25 cm, 8% denaturing polyacrylamide gel (Long Ranger, FMC Biozym) which had been pre-heated for 30 min. Electrophoresis was conducted in 1.0 Long Run TBE buffer at 1500 V, 40 W, 40 mA and 48°C. After about 4 h the gel was in general loaded a second time resulting in the separation of 192 probes per gel. To get very clear banding patterns using the LI-COR DNA Sequencer 4200 it turned out that the additional +0-pre-amplification is of great importance.

Using the protocol and technique described above distinct and clear fluorescence detected AFLP-patterns could be generated routinely and first analysis concerning AFLP-marker detection for the resistance locus rym5 which is located on chromosome 3H and confers resistance to BaMMV, BaYMV and BaYMV-2 (Graner et al. 1999) have been carried out. In a first step, 128 primer combinations were screened for polymorphisms using bulked segregant analysis (Michelmore et al. 1991) and out of 34 polymorphic fragments identified linkage of fragments of three primer combinations, i.e. E31/M49, E39/M48 and E45/M61 to rym5 was detected (Fig. 1), using a subpopulation of the original mapping population (Graner et al. 1999) consisting of 81 DH-lines of the cross 'W122/37.1' (rym5) × 'Interbell' (susceptible). Although, linkage to rym5 is not very tight and these markers are only of limited value for marker based selection, they have been developed in a very short period of time after optimizing the fluorescence detected AFLP-technique and demonstrate the power of this method avoiding the use of radioactivity and time consuming detection on X-ray films. Therefore, after having constructed a high resolution mapping population for rym5 based on 1026 F₂-plants additional fluorescence detected AFLPs will be analysed for efficient marker saturation around this locus.

References:

Graner, A., S. Streng, A. Kellermann, A. Schiemann, E. Bauer, R. Waugh, B. Pellio, F. Ordon, 1998. Molecular mapping of the rym5 locus encoding resistance to different strains of the barley yellow mosaic virus complex. Theor. Appl. Genet. (in press).

Schondelmaier; J, G. Koch, C. Jung, 1996. Die Einsatzmöglichkeiten von AFLPs in der Pflanzenzüchtung. Vortr. Pflanzenzchtg. 33, 112-125.

Michelmore, R.W., I. Paran, R.V. Kesseli, 1991. Identification of markers linked to disease-resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating populations. Proc. Natl. Acad. Sci. USA 88, 9828-9832.

Vos, P., R. Hogers, M. Bleeker, M. Reijans, T. van de Le, M. Hornes, A. Frijiters, J. Pot, J. Peleman, M. Kuiper, M. Zabeau, 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23, 4407-4414.

Zabeau, M, P. Vos, 1993. Selective restriction fragment amplification: a general method for DNA fingerprinting.

European patent application number 92402629.7, Publication number 0 534 858 A1.