Specialized Mapping Topics

Specialized Mapping
Topics

Specialized Mapping Topics

Bulk Segregant Analysis

Often a geneticist is not interested in developing a molecular map, but would rather find a few markers that are closely linked to a specific trait. The identification of these markers is often achieved by a procedure called bulk segregant analysis. The essence of this procedure is the creation of a bulk sample of DNA for analysis by pooling DNA from individuals with similar phenotypes. For example, you may be interested in finding a molecular locus linked to a disease resistance locus. You would create two bulk DNA samples, one containing DNA from plants or lines that are resistant to the disease and a second bulk containing DNA from plants or lines that are susceptible to the disease. Each of these bulk DNA samples will contain a random sample of all the loci in the genome, except for those that are in the region of the gene upon which the bulking occurred. Therefore, any difference in RFLP or RAPD pattern between these two bulks should be linked to the locus upon which the bulk was developed. This is a powerful technique that has gained wide acceptance in the few years since it was first described.

Sequence Tagged Sites

For many research labs, RFLPs are not an attractive molecular marker system because of the labor involved and the requirement of radioisotopes. Sequence tagged sites (or STS) may become a popular alternative to RFLP markers because they are PCR-based and do not require radioactive probing. STSs are developed by first sequencing the ends of a RAPD product or a clone used as the RFLP probe. From the sequence information, oligonucleotide primers 18-20 nucleotides long are synthesized that are complementary to each end of the RAPD product or the clone. These new primers are then used to amplify DNA by PCR. Two results could occur. First the size of amplification products among different DNAs (for example, two parents differing for a disease resistance locus, ) could be polymorphic. Alternatively, the amplification products could be monomorphic (of the same size). If this is the case, then it will be necessary to cut the products with various restriction enzymes to identify polymorphisms.

Because the primers are longer than those used for RAPD mapping, the PCR reaction can be run at a higher annealing temperature. This simple change in reaction temperature results in specific and simple amplification pattern that is very reproducible from laboratory to laboratory. (This is often not the case with RAPD technology.) Because they are portable from lab to lab, it is then possible to develop STS markers that can allow the quick location of any gene on a molecular map. First, it is necessary to define from two to three evenly dispersed STS sites for each chromosome of the species with which you are working. Then whenever a new gene of interested is identified, the linkage relationship between that gene and each of the STS loci can be established. The new gene should map within 25 cM of one of the STS loci. Once the new gene is located in relationship to an STS, then you can go to a RFLP or RAPD map and select probes or primers that will allow you to identify markers more closely linked to the new gene.