Recombinational removal of linked Mutator elements from lines carrying Mutator-induced mutants in preparation for molecular analysis --Philip S. Stinard Two problems frequently encountered in the use of segregation analyses to identify Mu element tagged genes are the large number of different types of Mu elements that could conceivably be inserted in the gene, and the presence of Mu elements that may be closely linked to the tagged gene, but which are not themselves inserted in the gene. The first problem can be approached by using a Mu end probe, or successively probing segregation analyses with probes homologous to the unique portions of different Mu elements (providing that the mutant is caused by a known Mu element insert). It is the second problem that I address in this report.

The copy number of Mu elements in the line carrying the tagged gene can be reduced by selecting plants in which transposition of Mu elements has ceased, and outcrossing these plants to non-Mu standard lines for several successive generations. The number of unlinked elements should be approximately halved each generation, and an occasional linked Mu element may be removed by recombination. However, it is possible to greatly enrich for the removal of linked elements by selecting for recombinational events. To do this, one can choose non-Mu-containing genetic stocks that carry identifiable markers (e.g. genetic mutations, RFLP's, isozymes, translocations) flanking either side of the wild type counterpart of the tagged gene. F1's are made between the marker stock and the tagged gene stock, followed by the appropriate backcross and identification of plants carrying crossovers between the marker and the tagged gene. Barring the occurrence of a second crossover distal to the marker, all linked Mu elements distal to the marker will be removed, and the probability of the removal of linked Mu elements from the interval between the tagged gene and the marker will be increased. The crosses can be set up to select crossovers between the tagged gene and flanking markers on each side as single crossover events in successive generations of backcrossing, or as double crossover events in a single generation of backcrossing. The latter strategy has the disadvantage of possible chiasma interference limiting the proximity of the crossover events to the tagged gene.

How useful is this method? If the linked marker is 10 map units from the tagged gene, one can cut by a factor of 10 the number of plants analyzed in a segregation analysis where one is trying to determine with a reasonable degree of certainty whether a specific Mu element is inserted at the locus, or whether it is merely linked. If the marker is 2 (or x, where x < 50) map units from the gene, the savings is a factor of 50 (or 100/x). The closer the markers are to the tagged gene, the better. It is also important to obtain crossovers on each side of the gene since one cannot know in advance on which side of the tagged gene linked Mu elements may be.

A slightly different method can be used to select for the removal of unlinked elements on other chromosomes in order to decrease total copy number. The line carrying the tagged gene and in which transposition has ceased is crossed to a non-Mu genetic stock carrying markers on chromosomes different from the one carrying the tagged gene. Backcrosses are made, and segregants carrying the markers plus the tagged gene are selected. The greater the number of different markers selected, the greater will be the Mu copy number reduction. A vigorous Mangelsdorf tester would work well, unless the phenotype of the tagged mutant is masked by one of the Mangelsdorf tester genes.

Although this report concerns Mu elements, a similar approach may be taken for any other transposable element system for which linked elements and copy number are a problem.


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