An attempt to tag rhm with transposable elements
--Ru-Ying Chang and Peter A. Peterson
The rhm gene controls resistance of maize plants to the disease southern leaf blight, which is caused by the fungus Helminthosporium maydis (Bipolaris maydis). The homozygous recessive allele rhm/rhm confers resistance, while the dominant allele Rhm is associated with susceptibility. Lines are susceptible (Rhm/Rhm) unless bred for resistance. This greatly accelerates the transposon tagging process due to ease of constructing element-laden genotypes for mutant screening.
Transposable elements randomly insert into a locus at a frequency of 10-6 to 10-5 (Peterson, MNL 59:3, 1985; Doering, Maydica 34:73-78, 1989). This project is designed to tag the rhm gene with transposable elements based on random insertion of transposable elements into the gene. Because normal lines are susceptible to the fungus, transposable element lines are of the allele composition of Rhm/Rhm El El (El, abbreviation of transposable elements). Element insertion into the Rhm gene will abolish the function of the Rhm allele. Hybridization of genomic Southerns from the mutant with a probe from the element and subsequent cloning will enable us to isolate the gene.
In order to detect an element insertion into the Rhm allele, the genotype Rhm/Rhm El El was testcrossed by an rhm/rhm tester. This cross yields an Rhm/rhm El F1, which is the genotype used for mutant screening. This genotype is susceptible to the fungus, while the mutants (designated rhm*/rhm) with an element insertion are resistant.
As of now, eighteen mutants have been obtained out of approximately four hundred thousand seedlings screened. The results are listed in Table 1 by element categories.
Table 1. Screening results of rhm tagging. The screening material,
El, was the progeny from the crosses of Rhm/Rhm El El x rhm/rhm.
Artificial inoculation was used at 3-5 leaf stage in a greenhouse.
|Pop'n||Element||Total screened||No. of mutants||Designation||Mutation rate|
|2||CyTEL||78,200||6||rhm-m-1 -rhm-m- 7||7.67x10-5|
|3||Cy||263,840||11||rhm-m-8 -rhm-m- 18||4.17x10-5|
|5||T4-6~c2- m1~Rhm/Cy bz-rcy||26,282||many||~5.00x10-2|
The mutation rates are similar for the first three classes, En, Cy and CyTEL. No mutants were found out of 47,391 seedlings screened from the T4-6(033)-16~c2-m1~Rhm line (line with c2-m1 [En in the C2 gene] labeled 6S). The labeling was to increase the frequency of transposon insertion into the Rhm allele by taking advantage of the transposition preference of transposons to closely linked sites. These results with unexpected low frequency of mutation may be explained in the following two ways. i) It was originally suspected that the rhm gene was at the centromere of chromosome 6. Recently it was shown to be located at the end of 6S (Zaitlin et al., Genome 36:555-564, 1993). T4-6(033-16) has a break point of 0.9 on 6S. It is possible that the rhm locus is not linked to c2-m1, but moved to 4L, which has the other break point of the translocation. The c2-m1 allele was moved from 4L to 6S. Thus the enhancement expected with close linkage was not in effect. ii) Based on random insertion, 10-5 to 8 x 10-5 as shown in Table 1, 0.5 to 4 mutants are expected. Because of this expectation, the screened population may not be large enough to ensure a mutant to be found.
Another unexpected result deserves careful examination. When Cy and T4-6(033-16)~c2-m1~Rhm line were used separately in the screening, the mutation rates were 4.17 x 10-5 and zero, respectively. However, when the two lines were crossed together to increase vigor and the F1 was testcrossed by rhm/rhm to develop the screening seed, mutants were found at a high frequency of around 5% from screening the testcross population. This result was unexpected.
To explain these results, it is hypothesized that two closely linked recessive genes are involved in determining resistance in this case. Each of the two parents has a dominant allele of one gene and a recessive allele of the other. This aspect is discussed in some detail in an accompanying report (see Chang and Peterson in this issue).
The mutants obtained (rhm*/rhm) were crossed to an Rhm/Rhm line in order to separate rhm* from rhm. The two genotypes yielded from this cross, Rhm/rhm* and Rhm/rhm in equal ratio, can be distinguished using RFLP analysis provided a suitable probe is chosen. A probe, umc85, which detected polymorphisms between Rhm/Rhm and rhm/rhm lines, was chosen in this case. Because rhm* was derived from Rhm, it should possess the band specific to the Rhm/Rhm line, while rhm should possess a band specific to the rhm/rhm line. Nine of the 18 mutants were analyzed in this way by our collaborators in Dr. A. Gierl, A's lab in Köln, Germany. Five of the nine showed the expected banding pattern: the 1/2 Rhm/rhm* possessed a single band while the 1/2 Rhm/rhm possessed both bands. When genomic Southerns from the mutants as well as from the wild-type lines were probed with an element sequence, no cosegregating band was detected.
Failure to detect a cosegregating band may stem from several reasons. First, the number of mutants tested is not large enough. Secondly, the RFLP analysis used distinguishes between rhm* and rhm. It tells us whether rhm* is derived from the Rhm/Rhm line. It does not show the nature of the mutation. The rhm* allele might have been derived from spontaneous mutations from Rhm to rhm or due to another transposon. This project is currently underway. More mutants are being generated and analysed at this time.
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