Genetic markers can efficiently be obtained by using amplified fragment length polymorphism (AFLP) fingerprinting because no prior information on DNA sequence is required. However, the conversion of AFLP markers from complex fingerprints into simple single locus assays is perceived as problematic because DNA sequence information is required for the design of new locus-specific PCR primers. In addition, single locus polymorphism (SNP) information is required to design an allele-specific assay. This paper describes a new and versatile method for the conversion of AFLP markers into simple assays. The protocol presented in this paper offers solutions for frequently occurring pitfalls and describes a procedure for the identification of the SNP responsible for the AFLP. By following this approach, a high success rate for the conversion of AFLP markers into locus-specific markers was obtained.

A marker-saturated linkage map of potato was used to genetically map a locus involved in the resistance against wart disease Synchytrium 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 the genetic marker loci. With non-selective AFLP primers, fingerprints of individual BAC clones were generated 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.

SummaryExpression pro®ling by cDNA-AFLP is commonly used to display the transcriptome of a speci®c tissue, treatment or developmental stage. In this paper, cDNA-AFLP has been used to study transcripts expressed in segregating populations from Arabidopsis thaliana and potato (Solanum tuberosum). The genetic differences between the offspring genotypes are thus visualized as polymorphisms in the transcriptome. We show that polymorphic transcripts can be used as genetic markers and allow the construction of a linkage map. The resulting map shows that, in contrast to genomic markers, the transcriptome-derived markers did not cluster in particular areas of the chromosome, and that cDNA-AFLP markers are targeted speci®cally to transcriptionally active regions. The cDNA-AFLP markers used in mapping are derived from DNA polymorphisms in transcripts, rather than differences in expression regulation. The high potential of transcriptome markers as opposed to (anonymous) genomic markers for applications in genetic analyses, marker-assisted breeding and map-based cloning is discussed.

NBS proWling is a method for the identiWcation of resistance gene analog (RGA) derived fragments. Here we report the use of NBS proWling for the genome wide mapping of RGA loci in potato. NBS proWling analyses on a minimal set of F1 genotypes of the diploid mapping population previously used to generate the ultra dense (UHD) genetic map of potato, allowed us to eYciently map polymorphic RGA fragments relative to 10,000 existing AFLP markers. In total, 34 RGA loci were mapped, of which only 13 contained RGA sequences homologous to RGAs genetically positioned at approximately similar positions in potato or tomato. The remaining RGA loci mapped either at approximate chromosomal regions previously shown to contain RGAs in potato or tomato without sharing homology to these RGAs, or mapped at positions not yet identiWed as RGA-containing regions. In addition to markers representing RGAs with unknown functions, segregating markers were detected that were closely linked to four functional R genes that segregate in the UHD mapping population. To explore the potential of NBS proWling in RGA transcription analyses, RNA isolated from diVerent tissues was used as template for NBS proWling. Of all the fragments ampliWed approximately 15% showed putative intensity or absent/present diVerences between diVerent tissues suggesting putative tissue speciWc RGA or R gene transcription. Putative absent/present diVerences between individuals were also found. In addition to being a powerful tool for generating candidate gene markers linked to R gene loci, NBS proWling, when applied to cDNA, can be instrumental in identifying those members of an R gene cluster that are transcribed, and thus putatively functional.

BackgroundPotato is the world's third most important food crop, yet cultivar improvement and genomic research in general remain difficult because of the heterozygous and tetraploid nature of its genome. The development of physical map resources that can facilitate genomic analyses in potato has so far been very limited. Here we present the methods of construction and the general statistics of the first two genome-wide BAC physical maps of potato, which were made from the heterozygous diploid clone RH89-039-16 (RH).ResultsFirst, a gel electrophoresis-based physical map was made by AFLP fingerprinting of 64478 BAC clones, which were aligned into 4150 contigs with an estimated total length of 1361 Mb. Screening of BAC pools, followed by the KeyMaps in silico anchoring procedure, identified 1725 AFLP markers in the physical map, and 1252 BAC contigs were anchored the ultradense potato genetic map. A second, sequence-tag-based physical map was constructed from 65919 whole genome profiling (WGP) BAC fingerprints and these were aligned into 3601 BAC contigs spanning 1396 Mb. The 39733 BAC clones that overlap between both physical maps provided anchors to 1127 contigs in the WGP physical map, and reduced the number of contigs to around 2800 in each map separately. Both physical maps were 1.64 times longer than the 850 Mb potato genome. Genome heterozygosity and incomplete merging of BAC contigs are two factors that can explain this map inflation. The contig information of both physical maps was united in a single table that describes hybrid potato physical map.ConclusionsThe AFLP physical map has already been used by the Potato Genome Sequencing Consortium for sequencing 10% of the heterozygous genome of clone RH on a BAC-by-BAC basis. By layering a new WGP physical map on top of the AFLP physical map, a genetically anchored genome-wide framework of 322434 sequence tags has been created. This reference framework can be used for anchoring and ordering of genomic sequences of clone RH (and other potato genotypes), and opens the possibility to finish sequencing of the RH genome in a more efficient way via high throughput next generation approaches.