The construction of testers useful in the detection of dosage series at the kernel stage

For studying the effects of chromosomal dosage on enzyme levels, the B-A translocations have great potential. Roman (1947) suggested that TB-A's could be useful not only for investigating the inheritance of B chromosomes, but also for gene localizations and dosage studies. These translocations have since been extensively applied for localizing genes (Beckett, J. Hered. 69:27) but only minimally in dosage analyses. This has been the case because the appropriately marked genetic stocks have been lacking. In recent years we have begun developing special testers for several regions of the maize genome which, when combined with an appropriate TB-A, will allow the phenotypic distinction of the various doses (1-2-3) of the respective chromosome segment translocated to the B centromere. Some progress has been made, although considerable work remains. These testers involve either isozyme markers or the anthocyanin genes in combination with R-scm alleles, i.e. those alleles of the R locus which permit anthocyanin pigment in the germ in the absence of the appropriate constellation of scutellum color factors (Sprague, U.S. Dept. Ag. Tech. Bull. 292). These systems involve modifications of testers previously suggested by Beckett and by Robertson (MNL 49:83) for identification of TB-A's. Robertson (J. Hered. 58:152) suggested the use of R-scm2 for this purpose and developed TB-10La with this allele and TB-9Sb with C-I for crosses to R-scm. The stocks described here extend its use to other anthocyanin loci. The complete set is not finished, but those that are may be useful to cooperators for dosage studies, induction of new TB-A's, construction of compound TB-A's, studies of nondisjunction or chromosome breakage at the second microspore division and the facilitation of the maintenance of TB-A stocks. The present status of these lines is due in part to the willing assistance of Michael Freeling and Kathleen Newton. The isozyme marker systems will be described elsewhere.

The testers and chromosomal regions are as follows:

1L--A tester of the constitution A A2 C C2 R-scm2 Bz bz2-m has been constructed. This stock is bronze in phenotype, but expresses full-color in the presence of Bz2. The R-scm allele allows the expression of anthocyanin in the scutellum as well as in the aleurone. Thus when crossed by a TB-1La euploid heterozygote carrying Bz2 in 1L, the following phenotypes will correspond to the respective doses of 1L: bronze scutellum, purple aleurone, 1 dose; purple scutellum and aleurone, 2 doses; purple scutellum and bronze aleurone, 3 doses. It is known from systems using Adh (alcohol dehydrogenase-1) as a marker on TB-1La that hyperploid 1 B1 pollen grains cannot compete with the euploid grains under normal pollination conditions.

3L--The A locus, which is required for anthocyanin production, resides in 3L. Consequently three mutant alleles, a-st, a-m, and a-m-1 were used to construct testers for 3L that are homozygous for R-scm on chromosome ten. The constitutions of these testers are a-st A2 C C2 R-scm; a-m A2 C C2 R-scm; and a-m-1 A2 C C2 R-scm. If A is added to the embryo or aleurone genotype, anthocyanin will be expressed. When these testers are crossed by pollen from TB-3La euploid heterozygotes with A in 3L, the following phenotypes will correspond to the various doses of 3L: colorless scutellum and colored aleurone, 1 dose; colored scutellum and aleurone, 2 doses; and colored scutellum and colorless aleurone, 3 doses. E. Ward (Ph.D. dissertation, Indiana) found that hyperploid pollen for 3L could not successfully compete.

4L--An A A2 C c2 R-scm tester has been synthesized. This stock allows anthocyanin production in the scutellum and aleurone when C2 is added to the genotype. It is useful in distinguishing the various doses of 4L when crossed by compounds TB-7Lb-4L4698, TB-9Sb-4L6504, and TB-1Sb-4L4692 in a manner as described for 3L. In these cases it is necessary to use hyperploid heterozygotes since crossing over in euploid heterozygotes will regenerate the original TB-A used to construct the compound. The degree of crossing over between the A and BA-A chromosomes in these hyperploid heterozygotes and the frequency of fertilizations by hyperploid pollen are unknown to the author.

5S--An A a2 C C2 R-scm tester should be completed after another season. It should prove useful for discerning the various doses of 5S using hyperploid heterozygotes of TB-1L-5S8041 with a2 in the normal chromosome and A2 in 5S. The class of hyperploid gametes which are duplicated for the 1L region distal to the 1L-5S(8041) breakpoint are not successful in competition (Birchler, MNL 52:29). The rare crossover derivatives that regenerate TB-1La can only form viable, competitive pollen grains when they segregate with the a2 marked normal chromosome five and will therefore be found in the colorless class of kernels. The complementary product of this recombination (15 with A2) can form a viable gamete in combination with the 51 chromosome. This class of pollen will produce completely colored kernels with two doses of all regions. Most of the kernels of this phenotype however will result from fertilizations by balanced 51 1B B1-5 gametes in which disjunction of the B centromere occurred. These kernels will also have two doses of all regions. Thus the colored scutellum, colored aleurone class will consist of kernels with two doses even though they may be of different chromosomal constitutions. Considering the genetic proximity of the A2 locus to the translocation breakpoint and the complex pairing in the hyperploid heterozygote, it is unlikely that the A2 allele would change its linkage relationship with the B centromere. This parameter has not been determined however.

5L--Dr. J. Kermicle has provided an A A2 C C2 R-scm122 pr stock. This line expresses anthocyanin in both the scutellum and the aleurone. The distinction in this case involves red (pr) versus purple (Pr) anthocyanin. In combination with a TB-5La euploid heterozygote with Pr in 5L, the various doses of 5L can be determined. The frequency of successful fertilizations by hyperploid pollen is not known.

7L--The Dt3 locus has been transferred to TB-7Lb. When this TBA is used in combination with the a-m-1 A2 C C2 R-scm tester described above, the Dotted gene can serve as a marker. At present the usefulness of this marker is limited for dosage studies, because we find that significant numbers of hyperploid pollen grains (7 B7) successfully compete with euploid grains in fertilization when the Esterase-1 isozyme marker is used. Consequently when the a-m-1 tester is crossed by a euploid heterozygote, the resulting kernels with dotted scutellum and aleurone are ambiguous as to whether they are disomic or partially trisomic. Perhaps by using a hyperploid heterozygote, 7 7B B7 B7 with dt3 in the normal 7 and Dt3 on B7, this problem could be circumvented. In this case the occasional transmission of only the chromosome 7 would produce colorless kernels that have two doses of 7L present. A complication in this case might arise from an occasional crossover between the translocation breakpoint and Dt3, which would again introduce ambiguity. We are currently attempting to introduce inversion 7e(SO.89-LO.93) into the a-m-1 R-scm stock to alleviate this problem.

9S--An A A2 c C2 R-scm tester has been synthesized and works in principle like the examples above when used in combination with TB-9Sb marked with C. Robertson (Genetics 55:433) has studied the mechanics of TB-9Sb.

10L--The TB-10L series induced by B-Y. Lin (cited in Beckett, J. of H. 69:27) have an R-scm allele present and are used with r testers.

We are currently attempting to transfer Dt2 onto TB-6Lc and also studying the feasibility of marking TB-1Sb with Mp in combination with the bz2-m tester. Anticipating the construction of a TB-A involving 7S, initial crosses for an in R-scm tester have been made.

For the maintenance of TB-A's, these tester lines have the advantage that they can be used repeatedly with the respective TB-A, obviating the requirement to alternate with other testers involving mutants on the same chromosome arm. That is, the use of hyperploid heterozygotes will generate the same classes of progeny with the same phenotypes generation after generation. Without the R-scm present, the use of an anthocyanin tester for TB-A's will result in kernels with colorless aleurones with hyperploid embryos. If these are used again onto the same tester, the normal chromosome with the recessive allele may be transmitted or an accidental self pollination of the tester would lead to an ambiguity in the classification of the colorless aleurone class, i.e. hyperploid heterozygotes or normal. The use of the R-scm tester series eliminates this ambiguity since the hyperploid heterozygotes will have colored scutella and the normals will be completely colorless. Although it is conceivable that heterofertilization might produce such a phenotype, the frequency of this phenomenon is very low in maize. Moreover, since the majority of successful male gametes are duplicated for the dominantly marked chromosome arm,

the vast majority of heterofertilizations with this phenotype would still be heterozygous hyperploids. Limited supplies of the following anthocyanin testers in combination with R-scm are available upon request: bz2, a-st, a-m, a-m-1 c2, pr, c, and TB-7Lb with Dt3.

James A. Birchler

Present address: Biology Division, Oak Ridge National Lab., Oak Ridge, TN 37830


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