Map-based Cloning of Plant Genes: An Example of Plant Map-based Cloning

Principles of Map-Based or Positional Cloning

An Example

An Example of Plant Map-based Cloning

Recently map-based cloning was employed to clone the first plant resistance gene that follows the gene-for-gene interaction. The tomato gene that was cloned was Pto and it provides resistance against bacterial speck disease of tomato caused by Pseudomonas syringae pv. tomato. This pathogen expresses the avirulence gene avrPto that interacts with Pto to provide resistance in tomato. This gene was cloned by Martin et al. [Science (1993) 262:1432]. The following is the list of the various steps Martin et al. used to clone the Pto gene.

A genetic population of 251 F2 plants was screened with DNA probes, and the locus TG538 cosegregated with Pto.
A YAC library was screened with the TG538 probe, and the clone PTY538-1 was identified.
Primers that marked the end of the 400 kb clone were created, and the population was screened. The left end of the PTY538-1 (PCR marker PTY538-1L was 1.8 cM from Pto, and the right end of the clone (PTY538-1R) cosegregated with Pto. The right end of the clone maybe to left or right of Pto, so it is important to determine if the YAC clone spans the Pto gene.
1300 plants from F2, F3 and cultivars were screened, and one plant that contains the Pto allele at the TG538 and the PTY538-1R pto allele was found. For this result to occur, Pto must be located on the YAC clone PTY538-1.
To find a candidate clone on the YAC clone, DNA from the PTY538-1 clone was hybridized to a leaf cDNA library. 200 clones were found and 30 analyzed. A population of 50 plants with recombination in the Pto region was screened. cDNA clone CD127 cosegregated with Pto.
Because Southern hybridization with CD127 detected many polymorphic fragments, the clone could be detecting exons or a multigene family. 14 other cross hybridizing clones were found that all represented six different classes of related genes. Analyses of PTY538-1 detected all (but one) of the genomic fragments that were detected with Southern hybridization. It was concluded that PTY538-1 contained a multigene family.
To determine if the cDNAs contained the Pto gene, transformation vectors were created with CD127 (1.2 kb insert; vector pPTC5) and CD186 (2.4 kb insert; vector pPTC8). Two pPTC8 resistant plants were found. No pPTC5 resistant plants were found.
One resistant plant (genotype=Pto/Pto) was crossed to a susceptible cultivar (genotype= pto/pto). Of 22 plants 9 contained the CD186 sequence and were resistant to the bacterial speck pathogen, and the 13 without CD186 were susceptible to bacterial speck.
The clone CD186 was sequenced, and a 321 amino acid opening reading frame was found. The sequence of the open reading frame was similar to a serine-threonine protein kinase.

The steps outlined for this experiment demonstrate the general approach needed to clone a gene using map-based cloning. The specific steps may vary between this and other map-cloning experiments, but the same general steps must be met. For example, finding a cosegregating clone may require a high density map developed with RAPD or AFLP markers. The availability of techniques to screen a high volume of potential markers has changed one aspect of map-based cloning. It was originally thought that it would be necessary to perform a chromosome walk into the gene. The major difficulty with this technique in plants is the abundance of repetitive sequence. If the walk went through such a region, the walk could be diverted to a different chromosome, and the gene would not be found. This new technique of using high volume marker screening to move close to the gene has been called chromosome landing.