Development of tools and strategies towards marker assisted selection and gene cloning / Bart Brugmans

A marker saturated linkage map of potato was used to genetically map a locus involved in resistance against wart disease Synchitrium endobioticum race 1. The locus mapped on the long arm of chromosome 4 and is named Sen1-4 in contrast to a Sen1 locus on chromosome 11. The AFLP markers from the Sen1-4 interval enabled the isolation of BAC clones from an 11 genome equivalent BAC library. This was achieved via fingerprinting of BAC pools with the AFLP primer pairs that resemble to the genetic marker loci. With non-selective AFLP primers fingerprints were generated of individual BAC clones to analyse the overlap between BAC clones using FPC. This resulted in a complete contig and a minimal tiling path of 14 BAC clones enclosing the Sen1-4 locus. The BAC contig has a genetic length of ~6 cM and a physical length of ~1 Mb. Our results demonstrate that map based cloning of Sen1-4 can be pursued on the basis of a strategy of marker saturation alone. Genetic resolution achieved by screening large numbers of offspring for recombination events may not be required. Together with the construction of the BAC contig, a physical map with the position of the markers is accomplished in one step. This provides proof of concept for the utility of the marker saturation that is offered by the ultra dense AFLP map of potato for gene cloning. Introduction Map-based cloning has proven to be a successful method for the isolation of e.g. resistance genes in several plant species such as tomato (Martin et al., 1993; Dixon et al., 1996), Arabidopsis (Bent et al., 1994; Mindrinos et al., 1994; Grant et al., 1995), sugar beet (Cai et al., 1997) and barley (Büschges et al., 1997). Most map-based cloning projects start with a Bulked Segregant Analysis (BSA) (Michelmore et al., 1991) to find bulk specific markers. Subsequently, these markers are mapped in a segregating population to determine the order and distance relative to the trait of interest. Using a graphical display of the marker scores in the offspring genotypes, new pools can be composed using plants with recombination events in the proximity of the target locus for a second cycle of BSA. This second BSA will narrow the window to be saturated, and thus improve the chance to identify of markers that are physically sufficient close to the gene of interest to allow chromosome landing (Tanksley et al., 1995) in order to find the BAC-clone containing the gene of interest. A level of marker saturation which is equivalent to a physical spacing of markers that is less than the average insert size of the genomic library (e.g. BAC or PAC library) that is going to be used, offers an alternative to the screening of