--Ru-Ying Chang and Peter A. Peterson
Transposable elements insert into random sites in the genome. The frequency that a transposable element inserts into a particular locus usually ranges between 10-5 to 10-6. In order to tag a disease resistance gene such as Rp, a functioning element is introduced into a homozygous dominant line for the particular gene, e.g., Rp-Rp (in this case, the Rp-D allele). This is then made homozygous by selfing and selecting for both the dominant trait and element activity. Once the homozygous dominant line with an element is obtained, it can be crossed by a recessive tester as shown below. The progeny seedlings (Rp rp) are then tested for resistance and screened for a mutant type which is expected to be susceptible.
In this experiment, the Uq (along with others) element was introduced into the Rp/Rp line. Following the procedure described in the preceding paragraph, the resulting Rp/Rp Uq/Uq line was then crossed by rp-o/rp-o to produce seeds for screening.
The resulting offspring are resistant. However, if the element has inserted into the Rp gene (designated rp-m), a susceptible individual will be produced. This seedling test uncovers any susceptible individuals which will be candidates for insertional mutations. The screening results are shown in Table 1.
Table 1. Screening results for Rp tagging
| 1) Total seedlings screened | 600,826 |
| 2) Seedlings from contaminated suscept. | 142,738 |
| lines (discarded) | |
| 1)-2) | 458,088 |
| Mutants found | 25 |
| Mutation rate | 5.5 x 10-5 |
The susceptible lines (2) are probably due to contamination of pollen or to misclassification in making the Rp/Rp UqUq line. These seedlings are subtracted from the total because they do not contribute to the population. The mutants found are designated rp-m-1 through rp-m-25.
After the mutants are obtained they are verified by a co-segregation test. Two types of mutants are expected as to the status of Uq. One is co-segregating with Uq, i.e., Uq inserted into the Rp gene; the other is with a receptor element controlled by an independent Uq, i.e., Uq is outside the Rp locus. In the Uq insert case, a strategy is used to test co-segregation. Figure 1 shows the strategy and the expected results for the co-segregation test.
The two cases can be distinguished readily. In the former, rows from spotted kernels are susceptible and rows from non-spotted kernels are resistant. In the latter, all rows will be mixtures of susceptible and resistant seedlings. In the latter element relation can not be determined. The results of co-segregation tests are shown in Table 2.
Of the 25 mutants identified in the screening test, 2 died, and 12 have been tested for co-segregation. Four out of the 12 tested had no element activity at all. The other 8 had element activity. But element activity was independent of the mutant phenotype. The remaining mutants are currently being tested.
There are a few possible reasons for the mutants with independent Uq. First, the mutants might be due to spontaneous mutation of Rp to rp. Secondly, the element in Rp is a defective derivative of the Uq element.
The identity of the alleles in the mutants is being verified by RFLP analysis in Köln,, i.e rp-m vs rp-o-line 2 in Figure 1. This analysis is to distinguish the rp-m vs. rp-o types following outcrosses to Rp-Rp.
Figure 1. Tests of co-segregation.
Table 2. Results of co-segregation test for Uq*
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* RpM-15 to RpM-25 are still being tested.
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