BS-AFLP ANALYSIS ENABLES A RAPID AND PRECISE GENOMIC LOCALISATION OF MUTANT LOCI

Dep of Genetics, UG/VIB, Ledeganckstraat 35, B9000 Gent, Belgium

Map-based cloning strategies depend on accurately defining the position of the mutated locus. AFLP analysis facilitates mapping experiments tremendously. Twelve AFLP primer combinations were sufficient to generate and map around one hundred AFLP markers on the existing RI map. In combination with a bulked segregant analysis, this set of markers allows to localise a mutant locus to any genomic region within an interval of 10 cM. After DNA isolation, this can be achieved within a one-week period. In a practical example, the analysis of 4 DNA bulks of 10 specific F₂ mutant plants each, and DNA samples from 4 individual wild type F₂ plants and the two parental lines, produced 3 markers on chromosome 5, spanning a 7 cM region, including the particular mutation. Analysis of the individual mutant plants for these markers, enabled to define the flanking markers. Further analysis using other AFLP pcs allows to identify new, completely linked markers within the defined region. It thus should be possible to position the mutation onto a region which is spanned by only one or a few overlapping BAC clones in a short period. Analysis of the sequences underlying the identified BAC clones can be used for an in-silico AFLP analysis. Preliminary results show an almost perfect match between predicted and experimental AFLP patterns. This approach thus allows to specifically generate new markers for further narrowing down of the flanking recombination points. Gene predictions on the sequences in between the flanking markers will indicate a limited number of candidate genes.