The F1 hyperploid offspring are crossed as male parents onto a tester stock, su1 in this case, to verify their A-B interchange status and onto a non-Ts5 tester strain for linkage analysis. The 4(Ts5)/4BB4(+)B4(+) F1 hyperploid plant is expected to produce both 4(Ts5)B4(+) and 4BB4(+) microspores. Pollen grains whose tube nuclei are 4BB4(+), having a balanced nuclear genotype, under pollen competition are expected to function to the exclusion of pollen with the unbalanced 4(Ts5)B4(+) genotype and, except for crossovers derived from exchanges between chromosomes 4 and B4 proximal to Ts5, should transmit only the wild-type allele (Ts5+) carried in B4. Testcross progeny should include balanced A-B interchange heterozygotes, 4(+)/4BB4(+), and both products of microspore nondisjunction, the 4(+)/4B hypoploid and the 4(+)/4BB4(+)B4(+) hyperploid. All hypoploid offspring should exhibit normal tassels (Ts5+) since they do not have a B4 chromosome that could, by crossing over, carry Ts5. Balanced and hyperploid offspring are expected to be mainly Ts5+, with occasional crossover Ts5 plants whose frequency is a function of the recombination distance between Ts5 and the B4 breakpoint, and of the effective pairing frequency between chromosomes 4 and B4 in that region.
Needless to say, if the initial cross were to involve the Ts5 strain and a nonchromosome-4 A-B translocation stock, e.g., TB-7Lb, the F1 hyperploid would carry two normal chromosomes 4 and have the genotype Ts5/+; its cross onto the Ts5+ Ts5+ tester strain would be expected to produce a 1:1 ratio of Ts5:+ among the TB-7Lb balanced, hyperploid and hypoploid progeny.
Since the A-B hyperploid testcross procedure described above is based on exclusion of the A chromosome (chromosome 4 in our example) from pollen transmission, it can identify the chromosome, but not the specific arm, that carries the dominant gene in question. For example, it is expected that TB-4S or TB-4L strains would indicate that Ts5 is in chromosome 4 but that neither would necessarily clearly place it in 4S.
The F1 4(Ts5)4B hypoploid from the initial cross produces an unequivocal basis for linkage assignment. Since the 4B chromosome is lethal to both male and female gametophytes, self-pollination of this hypoploid will yield only tassel seed (Ts5/Ts5) offspring, and crosses with nontasselseed testers, involving the F1 hypoploid as both male and female parent, are expected to produce only tassel seed (Ts5/Ts5+) progeny. Again, if the initial cross were to involve the Ts5 strain and a nonchromosome-4 A-B translocation stock, F1 hypoploids would carry two normal chromosomes 4 and have the genotype Ts5/Ts5+; self-pollination of this hypoploid should produce a progeny ratio of 3 Ts5:1 +, and both kinds of testcrosses are expected to produce 1:1 ratios for Ts5:+ among the progeny.
For locating dominant genes to chromosome, compound A-B translocations have a use similar to that of simple A-B translocations. They differ in that a simple A-B translocation is derived from a single chromosome of the "A" complement, whereas a compound A-B translocation includes chromosome segments from two chromosomes of the "A" complement.
The origin of the compound TB-1Sb-2L4464 may serve as an example (Rakha, F. A. and D. S. Robertson, Genetics 65:223, 1970). As a preliminary to extraction of the compound, a female parent carrying TB-1Sb(S.05) may be crossed by a male parent homozygous for T1-2(4464) (1S.53; 2L.28) to produce the F1 combination 1BB12/1221; this combination is completely balanced and homologous pairing can occur in all regions of the "A" chromosomes. Part of this pairing involves the region between the interchange point in TB-1Sb(S.05) and the 1S interchange point in T1-2(4464)(S.53). A crossover in this region leads to formation of a compound interchange chromosome, namely B1,2, which by nondisjunction can lead simultaneously to hyperploidy in progeny for the region between 1S.05 and 1S.53 and for the terminal 2L segment distal to 2L.28, or alternatively may result in hypoploidy for the same segments. Together, a 1B chromosome and a B1,2 chromosome contain the same "A" complement chromatin as the original 12 chromosome. Since the combination 1221 is balanced, the combination 1BB1,221 is also balanced.
Hyperploid stocks of TB-1Sb-2L4464 may be perpetuated by crossing female parents from standard chromosome stocks with hyperploid male plants and again selecting and testing for hyperploid plants in the progeny: 12/211BB1,2B1,2. The nondisjunctional capability of the male parent may be confirmed by the occurrence of virescent progeny plants from crosses onto homozygous (or heterozygous) v4 female testers. If testcrosses are made onto homozygous v4 plants and yield some virescent plants, nonvirescent progeny plants have a good chance of being hyperploid. Hyperploid compound A-B stocks free of the recessive v4 allele may also be perpetuated by using the v4 stocks only to test chromosomal constitution and using nonmutant standard female parent stocks for perpetuation. In the latter instance, the frequency of hyperploid plants from kernels selected for planting may be enhanced by choosing smaller size kernels that have an increased probability of having hypoploid endosperms and hyperploid embryos.
TB-1Sb-2L4464 may be used to test for the chromosome location of the dominant allele Ch (chocolate pericarp; 2L). In the initial cross, a homozygous Ch female parent, when pollinated by hyperploid TB-1Sb-2L4464 plants, is expected to produce hyperploid (1 2(Ch)/211BB1,2B1,2), hypoploid (1 2(Ch)/211B) and balanced (1 2(Ch)/211BB1,2) progeny plants. From hyperploid plants, those microspore tube nuclei which carry a balanced chromosome complement will virtually always be represented by the chromosome combination 211BB1,2; combinations carrying a standard chromosome 1, a standard chromosome 2, or both, and accompanied by a B1,2 chromosome are unbalanced, and presumably noncompetitive. If the Ch allele is carried on either the standard chromosome 1 or the standard chromosome 2, it is expected to be transmitted to progeny only if it is transferred by crossing over to that one of the three interchange chromosome (21, 1B or B1,2) which carries the locus. The frequency of that transfer will be a function of the recombination distance between Ch and the adjacent interchange point.
The F1 hyperploid offspring produced from the initial cross onto the Ch female stock are then crossed onto the tester stock, v4, to verify the A-B nondisjunctional capability and onto a nonchocolate tester strain for linkage analysis. As explained in the previous paragraph, if the Ch allele has been introduced into the F1 hyperploid on a standard chromosome 1 or a standard chromosome 2, linkage analysis of the testcross progeny is expected to reveal transmission of the Ch allele to progeny to be significantly less than 50%.
From an initial cross of a Ch female stock by TB-1Sb-2L4464, hypoploid progeny would be of the constitution 1 2(Ch)/211B. From such hypoploids the only chromosome combination transmissible through either female or male gametophytes is 1 2. If the locus of Ch is represented on a B1,2 chromosome, inasmuch as the hypoploid plant lacks this chromosome, there is no possibility of the transfer of a Ch+ allele to a standard chromosome 1 or a standard chromosome 2 by crossing over. As a result, self-pollinating an hypoploid plant will yield all Ch/Ch progeny and testcrosses to nonchocolate in either direction will yield all Ch/Ch+ progeny. If the Ch+ allele is present in the hypoploid plant on either the 21 or the 1B chromosome, it may by crossing over be transferred to a standard 1 or standard 2 chromosome and be transmitted to progeny with less than 50% frequency.
In brief, when a compound A-B translocation is used to test for location of a dominant gene, the immediate positive evidence of linkage does not distinguish which of the two possible chromosome assignments is correct. However, the question may be resolved in other ways by further evidence from A-B translocation crosses. If there is linkage of Ch to TB-1Sb-2L4464, but not to TB-1Sb itself, then Ch is assigned to chromosome 2. Alternatively, if Ch is linked to TB-1Sb-2L4464 and to a second compound involving chromosome 2 and a chromosome other than chromosome 1, then Ch is assigned to chromosome 2. Finally, Ch is assigned to chromosome 2 if a simple A-B translocation involving chromosome 2 shows linkage.
TB-1La-4L4692 may be used to test for the chromosome location of the dominant allele Tu (tunicate; 4L). In the initial cross, a standard chromosome female parent carrying the Tu allele is pollinated by hyperploid TB-1La-4L4692 to produce hyperploid progeny plants carrying the Tu allele: 1 4/411BB1,4B1,4. The nondisjunctional capability of hyperploids may be demonstrated when crosses onto female c2 R-scm2 tester yield some progeny kernels simultaneously displaying colorless aleurone and colored scutellum. The same tested hyperploid F1 plants may be crossed onto a nontunicate tester strain for linkage analysis. Linkage and transmission characteristics follow the same pattern as detailed for TB-1Sb-2L4464, in that F1 tunicate hyperploid plants produce balanced microspore nuclei whose chromosome complement (411BB1,4) consists entirely of interchange chromosomes. In testcrosses of tunicate hyperploid plants onto nontunicate female testers, linkage of the Tu locus to the compound TB-1La-4L4692 is shown by transmission of the Tu allele to be significantly less than 50% of progeny plants.
We have preliminary data involving the hyperploid method that confirm, or are consistent with, the location of three dominant genes; Ch (chocolate; chromosome 2L), Tu (tunicate; chromosome 4L) and Ts5 (tassel seed-5; chromosome 4S). The respective A-B translocation stocks used were TB-1Sb-2L4464, TB-1La-4L4692 and TB-4Sa. In each case small kernels were taken from ears of the initial cross to select for hyperploid plants that were in turn crossed as male parents onto vigorous nonmutant tester plants.
From the testcross of a single F1 Ch hyperploid plant a total of 43 plants were scored; four had chocolate pericarp and 39 were normal. Included were five hypoploids, all normal.
There were also 43 plants in the testcross progeny of a single F1 Tu hyperploid. Six were tunicate and 37 were normal. Included were six hypoploids, all with normal phenotype.
There were 56 plants in a testcross progeny of a single F1 Ts5 hyperploid. Eight were tassel seed and 48 were normal. There were only two hypoploid plants, both normal.
In all three cases the significant departure from a 1:1 ratio for mutant:normal is consistent with the established location of Ch in chromosome 2 and of Tu and Ts5 in chromosome 4.
We are currently testing the F1 hypoploid
procedure for location of dominant genes. Both this and the F1 hyperploid
method described above are being used to locate Rf genes that restore
male fertility in cms-S and cms-C strains.
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