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Abstract: White clover (Trifolium repens L.) is a forage legume widely used in combination with grass in pastures because of its ability to fix nitrogen. We have constructed a bacterial [...]

An ultradense genetic linkage map with > 10,000 AFLP loci was constructed from a heterozygous diploid potato population. To our knowledge, this is the densest meiotic recombination map ever constructed. A fast marker-ordering algorithm was used, based on the minimization of the total number of recombination events within a given marker order in combination with genotyping error-detection software. This resulted in 'skeleton bin maps,' which can be viewed as the most parsimonious marker order. The unit of distance is not expressed in centimorgans but in 'bins.' A bin is a position on the genetic map with a unique segregation pattern that is separated from adjacent bins by a single recombination event. Putative centromeres were identified by a strong clustering of markers, probably due to cold spots for recombination. Conversely, recombination hot spots resulted in large intervals of up to 15 cM without markers. The current level of marker saturation suggests that marker density is proportional to physical distance and independent of recombination frequency. Most chromatids (92%) recombined once or never, suggesting strong chiasma interference. Absolute chiasma interference within a chromosome arm could not be demonstrated. Two examples of contig construction and map-based cloning have demonstrated that the marker spacing was in accordance with the expected physical distance: approximately one marker per BAC length. Currently, the markers are used for genetic anchoring of a physical map of potato to deliver a sequence-ready minimal tiling path of BAC contigs of specific chromosomal regions for the potato genome sequencing consortium (http://www.potatogenome.net).

Segregation data were obtained for 1260 potato linkage group I-specific AFLP loci from a heterozygous diploid potato population. Analytical tools that identified potential typing errors and/or inconsistencies in the data and that assembled cosegregating markers into bins were applied. Bins contain multiple-marker data sets with an identical segregation pattern, which is defined as the bin signature. The bin signatures were used to construct a skeleton bin map that was based solely on observed recombination events. Markers that did not match any of the bin signatures exactly (and that were excluded from the calculation of the skeleton bin map) were placed on the map by maximum likelihood. The resulting maternal and paternal maps consisted of 95 and 101 bins, respectively. Markers derived from EcoRI/MseI, PstI/Msel, and SacI/ MseI primer combinations showed different genetic distributions. Approximately three-fourths of the markers placed into a bin were considered to fit well on the basis of an estimated residual 'error rate' of 0-3%. However, twice as many PstI-based markers fit badly, suggesting that parental PstI-site methylation patterns had changed in the population. Recombination frequencies were highly variable across the map. Inert, presumably centromeric, regions caused extensive marker clustering while recombination hotspots (or regions identical by descent) resulted in empty bins, despite the level of marker saturation.

The type and frequency of simple sequence repeats (SSRs) in plant genomes was investigated using the expanding quantity of DNA sequence data deposited in public databases. In Arabidopsis, 306 genomic DNA sequences longer than 10 kb and 36,199 EST sequences were searched for all possible mono-to pentanucleotide repeats. The average frequency of SSRs was one every 6.04 kb in genomic DNA, decreasing to one every 14 kb in ESTs. SSR frequency and type differed between coding, intronic, and intergenic DNA. Similar frequencies were found in other plant species. On the basis of these findings, an approach is proposed and demonstrated for the targeted isolation of single or multiple, physically clustered SSRs linked to any gene that has been mapped using low-copy DNA-based markers. The approach involves sample sequencing a small number of subclones of selected randomly sheared large insert DNA clones (e.g., BACs). It is shown to be both feasible and practicable, given the probability of fortuitously sequencing through an SSR. The approach is demonstrated in barley where sample sequencing 34 subclones of a single BAC selected by hybridization to the Big1 gene revealed three SSRs. These allowed Big1 to be located at the top of barley linkage group 6HS.