Mapping
From Wikionchus
Mapping or "positional cloning" is our favorite hobby. Pristionchus pacificus does not have a large collection of morphological mutants, like C. elegans has, that could be used for classical 2-point mapping. We rather use batch segregant analysis based on polymorphic strains to positionally map and clone mutants. In principle the Pristionchus dumpies, or pdl for Pristionchus dumpy like as these mutants are called, could be used to map mutants to a chromosome. In practice however it is much faster to run the so called Cocktail Markers on a small number of F2-segregants.
All mutants that are actively worked on have been generated in the reference strain, California PS312 (See also Pristionchus strain list). A genetic linkage map was constructed that is based on the highly polymorphic Washington 1843 strain of Pristionchus pacificus. In short 42 F2 segregants were assayed for their genotype at initial set of ~100 markers. A genetic linkage map was constructed from the resulting data. By now more than 550 Markers falling into 6 linkage groups have been mapped onto the genetic map.
To map recessive mutants hermaphrodites of this mutant strain are crossed with males from the Washington strain. Resulting F1 heterozygotes are controlled for their phenotype (that is wildtype) and singled and allowed to self. The F1 animals are segregating both the maternal (i.e. California) and paternal (i.e. Washington) genotypes. Therefore a randomly picked F2 animal (random in the sense that no phenotypes are scored) shows no preferential genotype at any locus. The chance of being homozygote maternal/paternal or heterozygote at any locus in the genome is simply following Mendelian rules (that is one quarter for either homozygote state and one half for the heterozygote state). If however a mutant animal is picked it will show occurence of maternal loci that are linked to the mutation causing the mutant phenotype. This is because mutants are generated in the maternal genotype background.
To map a mutant to a chromosome is very easy. 21 mutant F2 animals (21 are used because that is exactly the number of slots on one SSCP gel, and for no other reason) are genotyped for 24 markers. These Cocktail Markers are evenly spread over the six chromosome of Pristionchus pacificus and are used to look for markers that show a higher proportion of the maternal genotype. The chromosome on which the mutation is located will show markers that are more often maternal than expected. To give you an example: Among 20 randomly picked animals one would expect 5 to show a maternal pattern, 5 to show a paternal pattern and 10 should be heterozygous at any given locus. If one does the experiment for mutant F2 animals five out of the six linkage groups (i.e. chromosomes) behave like expected under Mendelian segregation. One chromosome however, or to be more precise the cocktail markers located on that chromosome, will show great deviation from the 5-5-10 expectation. This chromosome is the one that carries the phenotype-causing mutation.
Once the chromsome has been identified more animals (sometimes many more) animals have to be tested on markers located along that chromosome. Here the rationale is identical to the inter-chromosome mapping, although for somewhat different reasons:
Different chromsomes are by definition non-linked (that is why the genetic term "linkage group" can be almost intrerchangeably used for the cytological term "chromosome"). This is because their assortment is random. During each Meiosis II chromosomes are assorted independently. Therefore non-linkage is based on random events when comparing markers across chromosomes.
For markers on the same chromosome, that is during intra-chromsome mapping or fine-mapping, linkage is a function of physical distance to the mutated locus. All markers on a chromsome are physically linked. But the extent to which they are genetically linked depends on recombination frequencies. Recombination frequencies are given as centiMorgan (cM) on the linkage map. The map-units in centiMorgan are a a measurement for how likely a recombination between two markers occurs during a meiotic cross over. The closer a marker is to the mutated locus the more likely it will have the maternal genotype. What one does in practice is to exclude markers and to in a step-wise manner narrow down the genomic region where the mutation resides. Or put into other words: One looks for the marker that is maternal in most animals. Once the resolution of the existing markers has been fully used up, which is when a mutation has been mapped between two markers or when in many tested animals one marker is always maternal and never recombines, one has to build a phyical contig around the locus. New markers are then generated at strategic positions in this phyical contig and tested for recombination.
Physical contigs around a given locus are usually made by radioactive hybridizations of PCR products onto the BAC-library filters. Two end-sequenced BAC libraries are available for Pristionchus pacificus. One generated with HindIII and one with EcoRI. The theoretical coverage of the two BAC libraries is 10X the genome. That means that with one hybridazation one would identify around 10 overlapping clones that cover any given locus. In reality between four and 20 are usual. Matrix PCR is then carried out to find out the relative orientation of these BACS.
Once a physical contig has been found with recombination events at its end one can start to either sequence the BACs in this contig to find potential candidate genes or to continue fine mapping.
Links
Back to Pristionchus pacificus Protocols page.
