Map-based Cloning of Plant Genes: Principles of Map-based or Positional Cloning

Principles of Map-Based or Positional Cloning

Principles of Map-based or Positional Cloning

The first step of map-based or positional cloning is to identify a molecular marker that lies close to you gene of interest. This procedure typically is done my first finding a marker in the vicinity of the gene (several cM away). For the initial screening smaller population sizes are used (60-150 individuals) The next step is to saturate the region around that original molecular marker with other markers. At this point you are looking for a one that rarely shows recombination with your gene. At this stage, the population size could increase to 300-600 individuals.

The next step is to screen a large insert genomic library (BAC or YAC) with your marker to isolate clones that hybridize to your molecular marker. Once you identify the initial markers that map are near (or better yet) flank your gene and fournd a a clone to which the markers hybridize, you are on your way to determining where that gene resides. The steps that follow are termed chromosomal walking.

This procedure involves creating new markers (usually sequences at the end of the clone) and screening your segregating population with these new markers. Often this population is large (1000-3000 individuals). The goal is to find a set of markers that co-segegate (no recombination) with your gene of interest. Co-segregation means that whenever one allele of your gene is expressed, the markers associated with that allele are also present. In other words, recombination is not seen between your gene and the markers. If these markers do not cosegregate, you select new large insert clones and repeat the process until you have a clone whose markers co-segregates with your gene. To speed the cloning process, it is best to begin with a marker that is tightly linked to the gene with which your are working. Therefore you will not have to do a lot of additional screening.

Because you have your gene flanked on a single clone between two markers, you now know that the gene must be between those two markers. DNA fragments between the flanking markers are cloned and introduced into a genotype mutant for your gene by a genetic engineering technique called plant transformation. If transgenic plant expresses the wild type phenotype, you then know the gene of interest is on that fragment. At this point you must sequence the fragment to find a potential open reading frame (ORF), sequences that most likely will encode a gene product. In the best situation, only a single ORF is found, but this often is not the case. Usually several possible ORFs are found and new transgenic plants are created by transforming with a single ORF. Once this ORF is shown to rescue the mutant phenotype, you then perform an in-depth molecular and biochemical analysis of newly cloned gene.

These steps can be summarized as follows:

Identify a marker tightly linked to your gene in a "large" mapping population
Find a YAC or BAC clone to which the marker probe hybridizes
Create new markers from the large-insert clone and determine if they co-segregate with your gene
If necessary, re-screen the large-insert genomic library for other clones and search for co-segregating markers
Identify a candidate gene from large-inset clone whose markers co-segregate with the gene
Perform genetic complementation (transformation) to rescue the wild-type phenotype
Sequence the gene and determine if the function is known

If you do not have a transformation system for your species of interest, the confirmation is much more difficult. The most powerful approach in this case is recombinational or mutant analysis. Lets say you have two mutants in your gene. You could cross these two mutants and search a large segregating population for an individual with a restored normal phenotype. Next you would clone from each of these three individuals (the two mutants and the restored line) homologouse sequences that you feel contains the correct gene. If you can show that the restored line contains the same sequence as your predicted gene, and that the two mutants have unique changes in the gene sequence not found in the normal gene, you have obtained compelling evidence that the putative sequence is indeed your gene of interest.