In order to manipulate the dosage of Adh and investigate the nature of ADH compensation (Birchler, 1977, MGNL 51:13), it was deemed necessary to localize the Adh locus relative to selected translocation breakpoints. It was previously known that the Adh locus was uncovered by TB1La and genetically localized to approximately 1.5 m.u. from lw (D. Schwartz, 1971, Genetics 67:411-425).

Experiments were designed to further localize Adh by producing interstitial chromosomal trisomics by the method outlined by D. M. Gopinath and C. R. Burnham (1956, Genetics 41:382) and by testing for the inclusion of Adh in compound B-A translocations (F. A. Rakha and D. S. Robertson, 1970, Genetics 65:223).

Basically, the experimental design was to cross together two different translocations, such that the breakpoints of both translocations are in the same two chromosome arms. Yet the breakpoint in one chromosome is proximal in one translocation and distal in the other and the breakpoint in the other chromosome must be distal in the first translocation and proximal in the second.

When a plant, heterozygous for interchanges of the types just described, proceeds with meiosis four types of gametophytes are formed, if homologous centromeres segregate during meiosis: 1) balanced translocation #1; 2) balanced translocation #2; 3) deficiencies for the regions between the breakpoints; 4) duplications for the regions between the breakpoints. Type three gametophytes usually abort. Thus if the heterozygote is used as a female, the resulting ear is about 25% sterile, and has 33% of the kernels with interchange #1, 33% of the kernels with interchange #2 and 33% segmentally trisomic for all the regions between the translocation breakpoints. Crossing over between the breakpoints will result in the production of normal chromosomes and insertion of the region between the breakpoints of one chromosome between a duplication for the region between the breakpoints on the other chromosome. Only when the complementary crossover strands segregate together would a viable gametophyte be produced. This gamete type would be duplicate for the regions between the breakpoints. Adjacent-2 segregations would produce severely deficient gametophytes which would be expected to abort.

In order to locate the Adh locus cytologically, heterozygotes of two appropriate interchanges were crossed as females by normal pollen from plants that were homozygous for an Adh-C (or W) electrophoretic variant. Since the C variant is not found in natural populations of maize, there will be a detectable heterozygosity for ADH mobility in the resulting scutella from such a cross. A majority of maize varieties have an Adh-F allele. If the Adh locus is located between the breakpoints of the two translocations, approximately a third of the kernels should show a 4:4:1 zymogram ratio for the FF homodimer, FC heterodimer, CC homodimer isozyme bands. These kernels are segmentally trisomic for the regions of the two chromosome arms between the breakpoints. The remaining two-thirds (euploids) of the kernels will show a zymogram ratio of approximately 1:2:1 FF:FC:CC isozyme bands.

If one of the translocations carries an Adh-F variant and the other carries an Adh-S variant, the resulting segmental trisomic scutella would show an Adh F/S/C constitution. The remaining scutella will be either C/S or C/F in genotype. If the Adh locus lies outside the region spanned by the breakpoints, none of the scutella would give zymograms that indicate trisomy for the Adh locus. That is, all kernels will show a 1FF:2FC:1CC isozyme band ratio if both translocation stocks have the Adh-F variant. If the heterozygote of two interchanges is also heterozygous for Adh-F and S variants, there will be only F/C and S/C scutella. Figure 1 lists the combinations of translocations tested for the inclusion of the Adh locus between the breakpoints. The reported breakpoints are also listed.

Figure 1.

These combinations were crossed by homozygous Adh-C (or W) males, and the resulting genotypes from each combination are given in Table 1. Two of the combinations (#6,8) reported here produced segmental trisomics for Adh. Both of these had an Adh-F linked to one translocation and an Adh-S linked to the other. Consequently, both combinations produced approximately 33% F/C, 33% S/C and 33% F/S/C scutellar genotypes. Both of these combinations involve 1-3 translocations. The regions which are made trisomic, as based on the reported breakpoints only, are for combination #8:1L 0.72-0.90; 3L 0.65-0.73 and for combination #6: 1L 0.66-0.90; 3L 0.65-0.87. These data then place Adh distal to the breakpoint of T1-3(5267) and T1-3(5476) and proximal to the breakpoint of T1-3(5242).

Table 1.

As a further check on this order, a compound TB-1L-3L (5267) translocation was synthesized. This was accomplished by crossing a hyperploid TB-1La (1 B1 B1 1B) female by the homozygous translocation 1-3(5267) and then crossing the F1 plants, which include among their number balanced euploids heterozygous for both TB-1La and T1-3(5267), as males onto an a A2 C C2 R-scm-2 tester. This tester will allow the detection of newly arising compound TB-1L-3L's if they carry the A locus because the scutellum will be colored but not the aleurone if the sperm carrying the nondisjoined A locus fertilizes the egg instead of the polar nuclei. Such kernels were found. They were crossed again as males onto the a A2C C2 R-scm-2 tester to confirm their identity as a compound Tb-1L-3L (5267) translocation as opposed to a heterofertilization event. All such kernels so tested produced progeny indicative of the synthesis of a TB-1L-3L (5267) translocation, i.e., kernels with A scutellum and a endosperm; kernels with a scutellum and A endosperm; kernels with a scutellum and a endosperm; and kernels with A scutellum and A endosperm. The last two classes were less frequent than the first two classes.

When one of these confirmed cases of TB-1L-3L was crossed to an Adh-C tester, there were no scutella out of 72 analyzed which indicated nondisjunction of the Adh locus at the second microspore division. That is, there were no C/- or C/F/F, only C/F scutella.

These observations indicate the following: (1) The translocation T1-3(5267) has a breakpoint proximal to the A locus in 3L. (2) The breakpoint of this translocation is proximal to Adh in 1L. Therefore, in the combination T1-3(5267) and T1-3(5242), the former translocation has a breakpoint proximal to Adh and the latter must be distal to Adh. This conclusion is in agreement with the relative position of the reported breakpoints.

Further studies on the localization of Adh involved a test of whether the locus was included in TB-1La-5S(8041). This compound TB-A involves TB-1La and T1-5(8041)(1L 0.80; 5S 0.10). It was synthesized by Dr. D. S. Robertson and kindly supplied to me by him. The translocation stocks carried Adh-S and Adh-F. When hyperploid plants heterozygous for the translocation were crossed as males onto Adh-C or W silks, the results shown in Table 2 were found. As one can see, there is a low frequency of cases in which only the maternal electrophoretic variant was present for two of the four ears analyzed. Otherwise, there are no ADH genotypes which would be expected if the locus were included in the portion of 1L remaining in the compound. If the Adh locus were included, scutella with only the maternal allele as well as scutella which show ratios typical of trisomics (i.e., C F F or C S S) in the defective endosperm (hypoploid) class of kernels would be found. Isozyme ratios in the hyperploid scutella were typical of diploid heterozygotes. They showed a ratio of CC:CF:FF of approximately 1:2:1 in the mature scutellum. The isozyme ratios of the normal kernels on the ear did not differ from those in the defective kernel class. When seedlings were grown from these two classes of kernels and ADH ratios compared from epicotyl and root tissues there was still no difference in isozyme band ratios. The exceptional scutella which had only the maternal allele are therefore believed to have arisen from self pollination or from an occasional crossing over event in the translocation parent to reconstitute the original TB-1La, which would then uncover the Adh locus. As stated above, the reported 1L breakpoint of T1-5(8041) is 0.80. Thus, the Adh locus is approximately between 0.80 and 0.90 on 1L when the data from overlapping translocations and compound TB-A's are considered together. This determination is, of course, only as accurate as the reported cytological determinations.

Table 2.

James A. Birchler*

*Present address: Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830

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