Ears from the above cross produced kernels which were all solid (slightly dilute) purple colored. They were examined for single kernel exceptions such as colored-colorless mosaics indicating repeated loss of the B locus which should occur if a Ds with chromosome breaking properties were present somewhere near the B-Peru locus. Two were found and reported (MNL63:61, 1989).

Two other types of kernels were also found. The first was a colorless kernel with tiny colored dots; it was designated b-m1. The second kernel was pale purple with darker colored dots; it was designated as b-md-2. Subsequent tests showed that both kernels were mutable alleles of a color factor on chromosome 2S. Both map to the position of the B locus in 3-point linkage tests. Both b-m1 and b-md-2 were shown genetically to be Ds mutants, since neither activates the Ds at C-I, and both produce purple sectored kernels in the presence of the Ac at P-vv but not in its absence.

The amount of pigment in b-md-2 mutant aleurones depends on the direction of the cross: kernels of the constitution b-md-2/b/b, +Ac show deeply colored sectors against a pale purple background, while in b-md-2/b-md-2/b +Ac kernels the background is so dark that the sectors cannot be distinguished. Homozygous b-md-2 kernels lacking Ac show intense pigmentation that is visually indistinguishable from that of wild-type B-Peru. The color differences may indicate a dosage relationship; however, we have not done the appropriate experiments to determine whether the difference in color intensity among the various classes of kernels is due to dosage effects, or is the result of reduced expression in male versus female transmitted gametes, as was found for R mottling (J. Kermicle, Genetics 66:69-85, 1970).

Molecular analysis: b-md-2: Restriction mapping based on genomic Southern blots indicated that b-md-2 contains a 400-base insertion, which was cloned and sequenced. This was accomplished by cloning a 3.5 kb SpeI fragment of B-Peru containing the insertion into the XbaI site of the lambda vector LongC (Stratagene), and subcloning a HindIII fragment from this clone into pTZ vectors (U.S. Biochemical) for restriction mapping and sequencing. The insertion in b-md-2 proved to be a Ds1 element of 400 bases. This Ds1 contains the 11 bp perfect Ac inverted repeats and is flanked by an 8 bp target site duplication. This Ds1 has 85-95% homology with the sequences of the 11 maize and Tripsacum Ds1 elements that have been published (Sutton et al., Science 223:1265-1268, 1984; Wessler et al., EMBO J. 5:2427-2432, 1986; Schiefelbein et al., Genetics 120:767-777, 1988; Gerlach et al., J. Mol. Evol. 26:329-334, 1987). It shares with the maize Ds101 element a 10 bp deletion not found in the other Ds1 elements sequenced to date.

The b-md-2 insertion is in the opposite orientation with regard to the direction of gene transcription as the Ds1's in Adh-Fm335 (Sutton et al., 1984), wx-m1 (Wessler et al., 1986), and bz-wm (Schiefelbein et al., l988). It is inserted in the 5' leader region of the B-Peru gene (Fig. 1). Preliminary RNaseA protection experiments with wild-type B alleles suggest that this region is in an intron (D. Turks, personal communication). The reduced level of pigmentation observed in the mutant may be due to a B-Peru message that is altered in either structure or stability. Two other Ds1 insertions into the upstream regions of genes have been described; each was associated with a leaky phenotype as is b-md-2. A Ds1 insertion 30 bases upstream of the TATA box in bz1 is associated with a reduced steady state level of bz1 mRNA and protein (Schiefelbein et al., 1988). At Adh, Ds1 has inserted into the 5' leader where it is spliced out as an intron using a 5' donor site within the element and an acceptor site in immediately adjacent flanking DNA. The altered message is apparently reduced in stability (E.S. Dennis et al., Nucl. Acids Res. 16:3815-3828, 1988). Experiments are in progress to determine the nature of the interaction of Ds1 and B-Peru in b-md-2.

Figure 1. Restriction map of the Ds insertions in the B-Peru gene. The 2.2 kb message and direction of transcription are shown below the 4.3 kb genomic DNA. B = BamHI, Bc = BclI, H = HindIII, P = PstI, S = SacI(SstI), Sp = SpeI.

b-m1: Restriction mapping based on genomic Southern blots indicates that b-m1 contains an insertion of approximately 400 bases in the coding region of B-Peru. This insertion is contained in a 1.0 kb SpeI/SacI fragment that contains several exons and introns in the progenitor B-Peru allele (Fig. 1).

It is not possible to identify unambiguously the Ds element in b-m1 using genomic Southern blots because of the large number of Ds elements in the genome. However, because of its size and because b-m1 arose in the same experiments as b-md-2, our working hypothesis is that the Ds in b-m1 is a Ds1. Cloning of the b-m1 allele is underway.

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