ALCOHOL DEHYDROGENASE POLYMORPHISM IN MAIZE- SIMPLE AND COMPOUND LOCI' DREW SCHWARTZ AND TORU END02 Department of Botany, Indiana University, Bloomington Received November 22, 1965 OMPOUND loci have previously been described in maize ( LAUGHNAN1949; STADLERand EMMERLING1956). The evidence for the duplicate nature of the locus usually comes from recombination experiments in which the two com- ponents of the complex are separated by rare crossovers. It is ordinarily difficult if not impossible to recognize the compound nature of a locus strictly on the basis of phenotype. If the action of the two genes in the complex are different enough to control different phenotypes, they are usually considered to be separate loci, even though they may have arisen by duplication of a single gene. The distinction between the single or compound form of a locus is easier in those cases where genetic differences in protein structure are analyzed, since single amino acid re- placements can be detected electrophoretically if the replacement alters the net charge of the protein (PAULING,ITANO, SINGER and WELLS1949). A single cistron will specify a single protein form, whereas the compound locus composed of two cistrons will specify two distinguishable forms of the protein, isozymes, if the cistrons differ in having codons which code for amino acids of unequal charge. Such a condition has been found to exist for the alcohol dehydrogenase (ADH) gene in maize. The gene which specifies this enzyme occurs singly or in duplicate. The cistrons which make up the compound locus specify different alcohol dehy- drogenase isozymes (EC 1.1.1.1). The two allelic forms found in the complex also occur singly. MATERIALS AND METHODS Alcohol dehydrogenase in maize occurs in the developing kernel, scutellum of the mature kernel, and plumule and root of the very young seedling. In most of the work reported in this paper the scutellum was used as the enzyme source, since enzyme concentration is highest in that tissue. A simple procedure was adopted for the electrophoretic analysis of the scutellar enzyme. Kernels were soaked in water at 30°C for 24 hours. Each scutellum was excised from the kernels with a scalpel and squashed on a 6 x 6 mm square of Whatman #3 mm filter paper. The filter paper squares were inserted in the starch gels for electrophoretic separation of the scutellar proteins. The electrophoretic procedure was similar to that described previously (SCHWARTZ1960) for our esterase studies. Ethanol was used as the enzyme substrate. The ADH bands were developed by a modified staining technique for lactate dehydrogenase (DEWEYand CONKLIN1960). The gels were immersed in a solution composed of the following: 15 ml 1.0~ Tris-HC1 buffer pH 8.0, 1.5 ml 0.5 M KCN solution, 1.5 ml 0 01 M nicotinamide adenine dinucleo- ' This work was supported by National Science Foundatlon Grant GB 2833 ' OR leave from the National Institute of Genetm, Mmma, Japan. Genetics 53: 709-715 Apnl 1966 71 0 D. SCHWARTZ AND T. END0 tide solution, 1.5 ml 0.01 M nitro-blue tetrazolium solution, 0.9 ml ethanol (95%), 1.5 ml 0.01 M phenazine methosulfate solution, and 128.0 ml distilled water. The zymograms were developed at room temperature in the dark. The ADH bands started to appear within G-hr and development was allowed to progress until the bands had reached the desired intensity. RESULTS AND DISCUSSION Three electrophoretically distinguishable allelic forms of an alcohol dehydro- genase have been found in maize. These forms specify isozymes with different migration rates. The isozymes migrate to the anode in starch gel electrophoresis at pH 8.5. They are designated AdW, AdhF,and Adhc, with the isozyme specified by the S allele showing the slowest migration rate, and the isozyme specified by the C allele the most rapid (Figure 1a, b, c) . The enzyme behaves as a dimer. In plants homozygous for any one of these alleles, only a single ADH band is observed in one of three positions, depending on the allele. In heterozygotes an allodimer or hybrid enzyme with an inter- mediate migration rate is observed in addition to the two autodimers (Figure lc, e, f). This is analogous to the situation observed for the E, (SCHWARTZ1960) and E, (SCHWARTZ1964) esterases in maize as well as for a number of other enzyme systems. The analogy with the E, esterase system extends beyond hybrid FICUFIE1.-Zymograms of scutellar alcohol dehydrogenase isozymes from homozygous and heterozygous genotypes. (a) and (d) = AdhF/AdhF, (b) = AdP/AdhS, (c) = AdhC(')/AdhF, (e) = AdhC(tJ/AdhF,(f) = AdhF/Adhs. 0 denates the origin. MAIZE ALCOHOL DEHYDROGENASE 71 1 enzyme formation. The three North American alleles of the pH 7.5 esterase, ElF,E,’. and ElS form a series in that the FS allodimer composed of monomers specified by the two extreme alleles, ElFand ElShas a migration rate identical to the NN autodimer formed by the intermediate allele, E,”. This also holds for the other four E, alleles in the second series (SCHWARTZ,FUCHSMAN and MCGRATH 1965). In the case of the Adh alleles, the SC allodimer isozyme formed in AdN/ AdhCheterozygotes migrates at the same rate as the FF isozyme found in AdhF homozygotes (Figure IC,d). This suggests that the magnitude of the charge differ- ence between S and F is the same as that between F and C. The genes which specify the three ADH isozymes are considered to be allelic, since in heterozygotes the two genes always segregate at meiosis, with the one exception to be noted later in the paper. For example, if a plant carrying AdhS and AdhCis crossed with a plant containing only the AdhFgenes, two classes of progeny are found: AdhF/AdPheterozygotes which form the FF, FS, and SS isozyme bands, and AdhC/AdhFheterozygotes which form the CC, CS, and SS bands, 92 and 88 respectively in a typical cross. This is the case regardless of the gene combination in the heterozygotes. If the cistrons specifying the three iso- zymes were closely linked but not allelic, each chromosome would have to carry a null allele at two of the three loci. All plants examined showed at least one of the three isozymes. The exception to segregation of allelic types mentioned above came from a strain of maize from Colombia, South America. This strain carries a compound locus which contains two alleles, AdhFand AdhC (AdhFC).The duplicate nature of the gene was deduced from the observation that the two alleles are inherited as a unit and do not segregate from each other. In crosses of AdhFC/AdhSby Adhs/ Adhs, all the progeny which form isozymes containing the C monomer also form isozymes with the F monomer. Segregation could readily be detected from these crosses, since it would result in progeny of the constitution AdhC/AdhSwhich would form only the CC, CS, and SS isozymes. No recombinations separating the two alleles in the complex have been observed. The backcrosses yielded 255 ker- nels which carried all three alleles (AdhS/AdhE)and 228 kernels that formed only the SS isozyme (AdhS/AdhS).Self-pollination of the Adhs/Adhz plants gave rise to three classes of progeny, 95 AdhS/AdhS,146 AdhS/Adh”, and 74 AdNy/AdhE. Zymograms from kernels heterozygous for the AdhE duplication and an AdN allele should show three isozyme bands, FF, FC, and CC. Only two distinct bands are seen with a very faint third band appearing if zymograms are allowed to develop for a prolonged period (Figure 2a). Kernels heterozygous for Adhz and AdhS should form six isozymes, SS, SF, FF, CS, CF, and CC. Only four distinct hands are observed with a very faint fifth band appearing with prolonged devel- opment of zymogram (Figure 2b). Actually only five isozyme bands are ex- pected, since the FF and SC isozymes have identical migration rates. From a com- parison with AdhF and Ad@ (Figure 3). The intensity of the CmCmautodimer those formed in AdhF/AdhSand AdhF/AdhCheterozygotes, it is clear that the very faint band in each case represents the CC isozyme. The strain of maize from 712 D. SCHWARTZ AND T. ElVDO I I.- 0. ab FIGURE2.-Zymograms of scutellar tissue FIGURE3.4omparison of banding patterns cawing the AdhE complex (a) = of heterozygotes for the Adhc alleles of maize AdhF/Atfh=, (b) = Adhz/Adhs. and teosinte origin. (a) = AdhC(m)/Adhs, (b) = Adhc(t)/Adhs. which the complex AdhE locus was recovered was also segregating for an AdhC allele in simplex form not associated with AdttF. In these AdhC homozygotes, a weak band at the CC position is seen. The AdhC/AdhFor AdhC/AdhS hetero- zygotes both show only a faint band at the CC position and two strong bands at the FC and FF, or SC and SS positions, respectively (Figure 3a). The allele which forms the weak band has been designated Adhc(") to indicate its maize origin and to distinguish it from the other allele designated Adhc(t),which was recovered from teosinte. The CC autodimer specified by the Adhcct) allele migrates at the same rate as the autodimer formed by the Adhc(") allele. Furthermore, the allo- dimers formed in heterozygotes between Adhcct) and Adhc(") wi"& the other alleles have identical migration rates. The alleles differ as far as we can deter- mine only in that Adhc(t)produces isozyme bands of normal intensity in com- parison with AdhF and Ad@ (Figure 3). The intensity of the C"C" autodimer is low in all plant parts where the dehydrogenase is found, as in endosperm, scutellum, and plumules and roots of young seedlings. Even when the C"C" auto- dimer is too faint to be seen in the zymogram, the presence of the Adhc('")allele can be detected in AdhS/AdhCcm)or AdhF/AdhCfm)heterozygotes because of the appearance of allodimers involving a Cmmonomer as SC" or FC" bands.