Development of tools and strategies towards marker assisted selection and gene cloning

In this thesis research is described aiming at alleviation of the perceived limitations in the standard protocol which encompasses: mapping a trait, followed by marker saturation, genetic resolution, and finally BAC landing and walking to span the physical distance between the markers. In Chapter 2 proof of concept is presented that the level of marker saturation offered by the current version of the ultra dense map of potato is sufficient for map based cloning efforts. This is demonstrated by the assembly of a contig enclosing the Sen1-4 gene involved in wart disease resistance. Current linkage maps generated with AFLP show the unpleasant feature of centromeric clustering of genomic markers due to centromeric repression of recombination. Methods to target markers in the heterochromatic parts of the genome and to avoid the centromeric euchromating depend on e.g. methylation. AFLP markers based on the methylation sensitive enzyme Pst I show a more uniform coverage of the linkage map. Another method to target the gene rich parts of the genome is the use of cDNA as template for marker development. In the past RFLP probes were derived from Pst I digested libraries or cDNA clones. In the era of PCR based marker techniques it is evident that cDNA-AFLP might be a useful method; not only for transcript profiling but also for marker development. Furthermore this may also help to circumvent the time consuming BAC walking, when gene landing in stead of BAC landing might be an option. To enable gene landing, a marker has to be derived from the gene itself. When cDNA-AFLP patterns are generated from a series of offspring genotypes from a mapping population, then the polymorphisms in these cDNA-AFLP fingerprints should serve as a genetic marker of a specific chromosomal position. In Chapter 3 markers are generated in a mapping population using the cDNA-AFLP technique to evaluate and test the applicability of these markers. AFLP fingerprinting, both with genomic DNA as well as cDNA as the source material for template production, is a random technique. It does not allow specific targeting of gene rich regions in general or certain DNA sequences in particular. However, at this moment novel combinations of selective AFLP primers and specific primers allow us to generate fingerprints with amplification products that represent sequence tags of specific classes of genes. One of these AFLP-derived gene-family-specific fingerprinting techniques is called NBS profiling. This enables the specific amplification of parts of resistance genes or resistance gene analogs. In Chapter 4 NBS profiling is performed on templates prepared from DNA isolated from a mapping population to evaluate the potential to genetically map resistance gene clusters. In addition, cDNA can be used to evaluate the expression of R-genes between tissues in combination with NBS-profiling. AFLP and anchor related markers are rather laborious and expensive because several enzymes and amplification steps are needed before a product is ready for separation. This has to be performed on a polyacrylamide gel based system to create the resolution that is needed for accurate separation of the fragments. This time consuming and expensive protocol make anchor related techniques not suitable for recombinant screenings or marker assisted selection (MAS). Moreover, the required equipment is often unavailable to breeding companies. Therefore, a protocol for the efficient conversion of AFLP markers into simple PCR markers was developed (Chapter 5). Finally in chapter 6 the potential of the different methods to improve the efficiency for gene mapping and gene cloning is discussed.