University of Minnesota

Chromosome hopping: using intragenic recombination to positionally clone maize genes

— Okagaki, RJ

Chromosome walking strategies have been used to clone genes from Drosophila melanogaster, Arabidopsis thaliani, and other organisms with relatively small genomes. In these species, chromosome walking has been a time-consuming and difficult process, although having the complete genome sequence has simplified the process. An alternative to the standard chromosome walking approach may be available in maize; this approach relies on intragenic recombination to “hop” into a gene of interest.

Chromosome hopping requires at least two independent mutant alleles of the gene of interest. A double mutant heterozygote is made and crossed onto a tester to recover revertants. Revertants are produced by recombination within the gene of interest. The rate of intragenic recombination between a pair of alleles is generally high enough that this is a reasonable undertaking. As most recombinants are crossovers, locating the recombination event to a small region is straightforward. Markers distal to the crossover will come from one parent, and proximal markers come from the second parent. The crossover event will produce a non-parental allele. This recombinant allele will usually be detectable by Southern blot analysis, and this is a means to identify the gene of interest.

Finding the crossover itself, and the gene of interest, will depend on the development of the BAC contig map. Much of the maize genome has been anchored on a BAC contig map, and it should soon be possible to attempt chromosome hopping.

The potential for chromosome hopping can be illustrated by a study of intragenic recombination at the maize wx locus (Okagaki and Weil, Genetics 147:815, 1997). First, intragenic recombinants between wx-B1 and wx-I were recovered at respectable frequencies (>1 × 10-4). Second, almost all of the recombinants were crossover events. Third, crossovers between wx-B1 and wx-I produce novel bands in a Southern blot. These properties make chromosome hopping possible. Similar results have been found in other studies, for example, Dooner and Martinez-Ferez (Plant Cell 9:1633, 1997). Obviously, not all genes can be cloned using this approach. Two mutant alleles may not exist, or lesions in the mutant alleles may be too close for recombination to occur between them. However, for some genes, chromosome hopping could prove an efficient way to associate sequences with a phenotype.

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