Interpretation of Isozyme Banding Patterns in Diploids Dikaryons and Polyploids

Interpretation of isozyme banding patterns in diploids and dikaryons is much more complicated, and is aided by knowing whether a particular enzyme is mono- or multimeric. For monomeric enzymes, homozygous diploid individuals should produce a single band on a gel as described for haploids. Diploid individuals that are heterozygous at a locus for a monomeric enzyme will give two bands in a properly stained isozyme gel following electrophoresis. Each band can be interpreted easily as a different allele at a single genetic locus. Banding patterns are more complicated for enzymes composed of two or more subunits. In these cases the subunits must join together to form the active enzyme. Because dimeric enzymes (those composed of two subunits) are the most common, they will be the subject of this discussion. However, the concepts covered here are directly applicable to enzymes composed of three or more subunits. To correctly interpret isozyme banding patterns produced by dimeric enzymes, it is necessary to understand how the subunits combine to form the active enzyme. In heterozygous diploid individuals, the subunits produced by the two alleles combine at random in the cytoplasm of the cell to form the active molecule. For the hypothetical case of a diploid with two alleles, one coding for a subunit with a — 2 charge, the other with a — 3 charge, there are four possible ways the subunits can be joined to form the active dimer (Figure 2A). Three types of dimer can result: two homodimers (when identical subunits combine) and a heterodimer (composed of two different subunits). In Figure 2A, each — 2 subunit can join with another — 2 subunit (to form a — 2/— 2 homodimer) or with a — 3 subunit (for a — 2/— 3 heterodimer). Similarly, half of the — 3 subunits will pair with a — 2 subunit and half with a

— 3 subunit. Notice that for every — 2/— 2 homodimer (total charge = — 4) there are two — 2/— 3 heterodimers (total charge = — 5) and one — 3/— 3 homodimer (total charge = — 6), for a ratio of 1:2:1.

When these molecules are separated according to charge in an electric field, they will migrate to three regions on the gel, which after staining will be visualized as three distinct bands (Figure 2B). Notice that the middle (heterodimer) band is approximately twice as intense as either homodimer band, reflecting the 1:2:1 ratio in the numbers of each molecule. This pattern is unmistakable on isozyme gels (Figure 3).

The pattern is slightly more complicated for trisomic or polyploid individuals possessing three alleles. In this case, the subunits pair at random as before, only now there are nine possibilities (Figure 4A). If the different alleles vary by single-step charge differences, for example with charges of

— 2, — 3, and — 4, then the — 3/— 3 homodimer will have the same — 6 charge as the — 2/—4 heterodimers and will migrate to the same place on the gel during electrophoresis. This will produce a five-banded phenotype in which the intensities of the bands should be in a ratio of approximately 1:2:3:2: 1 (Figure 4B). This pattern is seen in the US-8 genotype of the

Figure 3 Banding patterns of glucose-6-phosphate isomerase in a diploid. Heterodimers are seen as more intensely staining bands between two other bands. The gel shows interspecific hybrids between the oomycetes Phytophthora infestans and its close relative P. mirabilis. The P. infestans parent was heterozygous 86/122and the P. mirabilis parent was homozygous l08/108. Interspecific hybrids are 86/108 (lanes 1,2,4,7, and 8) or 108/122 (lanes 3,5,6, and 9). Lanes 10 (homozygous 108/108) and 11 (86/122) indicate self fertilization of the P. mirabilis and P. infestans parents, respectively.

Figure 3 Banding patterns of glucose-6-phosphate isomerase in a diploid. Heterodimers are seen as more intensely staining bands between two other bands. The gel shows interspecific hybrids between the oomycetes Phytophthora infestans and its close relative P. mirabilis. The P. infestans parent was heterozygous 86/122and the P. mirabilis parent was homozygous l08/108. Interspecific hybrids are 86/108 (lanes 1,2,4,7, and 8) or 108/122 (lanes 3,5,6, and 9). Lanes 10 (homozygous 108/108) and 11 (86/122) indicate self fertilization of the P. mirabilis and P. infestans parents, respectively.

oomycete Phytophthora infestans at the Gpi locus, and was confirmed by thorough genetic analyses (Goodwin et al. 1992).

Other banding patterns can be produced by individuals containing three or more alleles separated by unequal charges. For example, an individual heterozygous for three alleles in uneven steps, e.g., — 2, — 3, and — 5, would yield a six-banded pattern in a 1:2:1:2:2:1 ratio. Band-intensity ratios in polyploids also can be affected by the number of copies of particular alleles at a locus. For example, an individual with three alleles, two of which are identical, e.g., — 2, — 4, and — 4, should give rise to a three-banded pattern in a 1:4:4 ratio. This has been documented in the US-1 genotype of P. infestans which has two copies of the 100 and one copy of the 86 allele at the Gpi locus (Goodwin et al. 1992). Even more complicated banding patterns are produced by tetrameric enzymes and by multiple loci that share alleles. Interpretation of these patterns was covered in detail elsewhere (Micales et al. 1992). Fortunately, most enzymes are dimeric so the principles discussed previously will apply directly. An understanding of these basic principles combined with genetic analyses should allow unambiguous interpretation of virtually any isozyme pattern.

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