Department of Plant Bioliogy, Carnegie Institution of Washington
In a review article on “Positional Cloning” published in the July 2000 issue of Plant Physiology (123(3):795-805), Dr.W. R. Scheible and his associates at the Department of Plant Biology, Carnegie Institution of Washington, Stanford, begin by suggesting that molecular mapping can be vastly enhanced by systematically exploiting the available sequence information. As a consequence, it now takes just a few rather basic molecular biology routines and as little as a few months to isolate (almost) any mutation that can be mapped. With a good deal of confidence, the authors hope that this article will prompt researchers with little or no experience in positional cloning to use this novel technique to identify and determine the functional properties of gene(s) under investigation.
The potitional cloning approach is usually a three-step process: (1) localizing a gene to a chromosomal subregion, generally by using traditional linkage analysis; (2) searching databases for an attractive candidate gene within that subregion; and (3) testing the candidate gene for its functional property.
This approach was adopted in Arabidopsis for search of genes in subregions with a physical distance of about 250 kb representing on average a genetic distance of 1% recombination. A population of 1,000 plants yields sufficient data to obtain recombinants in the range of 10-40 kb. Typically a DNA fragment of a physical distance of about 250 kb contains between two and ten genes. It is relatively easy to determine the DNA sequence of such alleles aided by PCR for amplification of the gene of interest. .
The most commonly used combination for mapping purposes is Landsberg erecta X Columbia (Ler X Col). These two accessions have been estimated to differ in 4-11 positions every 1,000 base pairs.
The authors have compared the three kinds of molecular markers used in positional cloning: single sequence length polymorphisms (SSLP), cleaved amplified polymorphic sequence (CAPS) and the latter’s modified version (dCAPS). Another powerful marker, not mentioned in the article, is single nucleotide polymorphisms (SNP), in which DNA sequence analysis reveals one base pair difference between the normal and mutant allele,
To take full advantage of recombinant chromosomes for fine mapping, it is necessary to create new molecular markers closer and closer to the mutation. This can be done by comparing the DNA sequence of the accessions involved. Single- di-, or trinucleotide repeats of 20 or more bp in length are on average found every 10 or 20 kb and provide good targets for new (SSLP).
In many instances, a trait is conditioned by more than one locus as encountered in case of mildew resistance. At least three genes are found involved in an additive manner to confer resistance to this disease. The complication arises in analyzing functional properties of such a locus and assigning it to its correct site in the map because of the presence of modifiers affecting the same trait. Complication in the expression of such loci may be also encountered on account of occurrence of epigenetic mutations which affect the phenotype of an organism without changes in their DNA sequence. These alleles are heritable but unstable and revert to normal phenotype at a low frequency.
Position cloning of some of the genes e.g., GURKE gene in Arabidopsis has been extremely difficult because of its proximity to the centromere where recombination hardly occurs. However, the centromere contains very few expressed genes and consists mainly of repetitive DNA.
Another region where gene mapping in Arabidopsis is very difficult is a short segment on chromosome II where a genetic distance of 1% recombination appears to correspond to 1,000 kb or more. The above chromosome shares several stretches of duplicated DNA with chromosome IV as well as it contains a piece of mitochondrial genome transferred into it in the course of evolution. All the above facts suggest that the Arabidopsis genome is undergoing continual changes involving not only point mutations but also chromosomal rearrangements in which DNA segments are reshuffled. Rearrangements such as chromosomal inversions make it almost impossible to map mutations within the inverted sequence. Irradiation and T-DNA insertions are known to cause DNA rearrangements creating a similar situation for correct gene mapping.
The authors conclude that Arabidopsis researchers are now in a position to clone mutant alleles, induced or natural and that with the vast improvement of techniques over the past few years, positional cloning can be used effectively by interested Arabidopsis researchers to identify and clone the genes generated by natural or induced mutations and their functional properties.