A question of major importance in genetics is why so much genetic poly

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1 ISOZYMES OF A POLYPLOID SERIES OF WHEATP CHARLES F. SING AND GEORGE J. BREWER2 University of Michigan, Ann Arbor, Michigan Received September 9, 1968 A question of major importance in genetics is why so much genetic poly morphism? Use of the electrophoretic technique on random samples of enzymes has resulted in the first efforts to obtain unbiased estimates of the proportion of genetic loci showing variation in outbreeding species (SHAW 1965; HARRIS 1966; and LEWONTIN and HUBBY 1966). Such estimates predict that at least 30 to 40% of genetic loci in*drosophila, Peromyscus, and man are polymorphic. These predictions have led to a flurry of papers presenting mathematical models which show that, contrary to earlier dogma (discussed by KIMURA and CROW 1964; VAN VALEN 1963), populations could theoretically sustain this degree of variation through balanced polymorphism (KING 1967; MILKMAN 1967; SVED, REED, and BODMER 1967). If heterozygote superiority is the responsible factor for maintaining a high degree of genetic polymorphism, one presumes that the increase in number of proteins associated with heterozygosity offers, in general, the selective advantage. In contrast to the attention which polymorphism has received as a mechanism for maintaining an increased number of proteins in heterozygotes, a second type of protein multiplicity, while widely recognized, has received much less attention in terms of its evolutionary significance. It is illustrated by isozyme patterns showing multiplicity of enzymes shared by every individual in the mating population (the lactic dehydrogenase isozymes of the human are an example). Preliminary estimates indicate that this type of protein diversity, probably arising primarily from gene duplication, is very common. For example, we have recently reported (BREWER and SING 1968a) that ten of sixteen randomly selected human red cell enzymes show isozyme multiplicity common to all individuals. We suggest that this type of protein diversity may make a significant contribution to the total protein diversity available to the individual. In a cross fertilizing species such as man, this could be an alternative strategy to allelic variation (FINCHAM 1966), but it may be of prime importance in a species characterized by obligatory inbreeding, as for example, in the self-pollinator, wheat. This paper presents initial studies of the correlation between protein multiplicity, as measured by the zymogram technique, and certain biological characteristics of hexaploid wheat and its tetraploid and diploid progenitor species. The isozyme data reported here were collected to obtain information on three This investigation was supported by contract AT(11 1) 1152 Atomic Energy Commission, USPHS grant AM 09381, and USPHS Career Development Award 1 K3 AM 7959 (GJB) 2 Departments of Human Genetics and Medicine (Simpson Memorial Institute) Unwerslty of Micliigan Melcal School. Geneucs 61: February 1969

2 392 CHARLES F. SING AND GEORGE J. BREWER fundamental questions. First, in view of the almost complete self-fertilizing nature of wheat, does it have a relatively greater proportion, than outbreeding organisms, OP isozymic diversity of the second type, that is, of multiple molecular. forms of enzymes, unrelated to allelic variation? Second, since cultivated wheat, Triticum aestiuum, is a member of a polyploid series (RILEY 1965; RILEY and CHAPMAN 1966; and SEARS 1966), can the contributions of various diploid genomes be recognized in amphidiploid enzyme electrophoretic patterns, as reported for wheat seed proteins by JOHNSON, BARNHART, and HALL (1967) and JOHNSON and HALL (1965). Third, in view of the considerable variation in habitats among species represented by each level of polyploidy (ZOHARY 1965), is there a relationship b2tween the amount of isozymic diversity and ecogeographical range and agricultural utilization of the particular species? Answers to these questions will contribute to the understanding of the influence of protein multiplicity on the evolutionary biology of this polyploid series. MATERIALS AND METHODS Representatives of Triticum aestiuum, designfated AABBDD, and its tetraploid, AABB? and three diploid progenitors; AA, BB, and DD were studied. The species representing each of the members of the polyploid series (designated species group) are given below. More than one species was sampled to represent AA and AABB species groups because of insufficient seed supplies. Intragroup species differences considered in this study proved to be nonsignificant. Species Group AA BB DD AABB AABBDD Triticum monococcum and T. boeoticum Aegilops speltoides Aegilops squarrosa Triticum dicoccoides and T. dicoccurn Triticum aesiiuum Laboratory procedures: Vertical starch gel electrophoresis was employed using a 30 slot gel at 4 C. Electrophoresis at 10 volts per cm was carried out for lengths of time ranging from 4 to 18 hrs. The electrophoretic isozyme systems employed were as follows: Phosphoglucomutase (PGM), method of SPENCER, HOPKINSON and HARRIS (1964), hexokinase (Hexo), method of BREWER and KNUTSEN (1968); m'alic dehydrogenase (MDH), method of BREWER and SING (1969); esterase (Est), method of TASHIAN 1965); acid phosphotase (APh), method of HOP- KINSON, SPENCER and HARRIS (1964) except that the gel buffer was M histidine, ph 6.0, and electrophoresis was carried out for only 4 hrs. For glucose-6-phosphatc dehydrogenase (G-6-PD) the gel buffer was M Tris, M boric acid, and M EDTA, ph 8.3. The bridge buffer was M Tris, 0.75 M boric acid, and M EDTA at ph 8.5. Electrophoresis was carried out for hrs at 4 C. To stain for G-6-PD activity, the gel was incubated in a staining solution containing 4 mg of TPN, 98 mg Mg/Cl,, 10 mg nitro blue tetrazolium, 2 mg of phenozine methosulfate, and 14 mg of glucose-6-phosphate per 100 ml of 0.04 M Tris, ph 7.1. For 6-phosphogluconate dehydrogen'ase (6-PGD) the method was the same as for G-6-PD except that 14 nig of 6-phosphogluconate was substituted for glucose-6-phosphate in the staining solution. For alkaline phosphatase (Alk Ph) the bridge and gel buffers were M histidine and 0.41 M sodium citrate, ph 7.0. Electrophoresis was carried out for 4 hrs. To stain for alkaline phosphatase activity, the gel was incubated in a staining solution containing 10.2 mg of Mg Cl,, 50 mg of sodium alpha-naphthyl phosphate, 500 mg of polyvinyl-pyrolidine, 50 mg of dast blue RR salt, and 2.0 gram NaCl in 100 ml of M Tris buffer, ph 8.5. Two types of tissue, germinated seed and green leaves from seedlings, were employed. Seeds for the study were soaked for 24 hrs in the gel buffer to be utilized in the particular electro-

3 ISOZYMES OF WHEAT 393 phoretic run. Seeds for analysis were selected at a stage when the pericarp was broken but there was no evidence of emergence of the primary root from the coleorhiza. Each seed was then crushed in a test tube with a glass stirring rod in one or two drops of buffer. After settling had taken place, the supernatant was used. With certain exceptions, only extract from an individual seed was placed in each gel slot. Those exceptions involved placement of extract made from four seeds in one slot for each species group to detect any effect of seed size and weight. There were no qualitative changes in the electrophoretic patterns observed which could be attributed to the increase in the quantity of seed material placed in a slot. Plants for leaf tissue were all started on the same day in the greenhouse and treated as uniformly as pmible. Each plant was randomly assigned to a position on the greenhouse bench. To minimize the effects of varymg developmental stage on the comparisons among species groups the samples of gresn leaf for an electrophoretic run were removed from the same relative position on all plants. An extract of a leaf from each pbant was prepared by grinding 400 mg of fresh tissue in a small amount of gel buffer with a ground glass hand mixer. Only leaf extracts from individual plants were placed in each gel slot. Sampling design and statistical procedures: For each tissue, six plants were selected to represent each of the five species groups. The AA species group was represented by three T. monococcum and three T. boeoticum plants for both tissues. For the AABB group, seed was sampled from T. dicoccoides, while three T. dicoccoides and three T. dicoccum plants were used to obtain leaf samples. The number of isozyme bands for each slot was recorded by two independent scorers who had no knowledge of the randomized arrangement of the samples on the gel. The average number of bands recorded by a scorer for a system on the six slots representing a species group was used as the variable for analysis Failure to detect significant differences between the two AA species averages (for the eight systems) and between the two AABB species averages (also for the eight systems) justified pgoling the data within these species groups. A preliminary analysis indicated a sigmficant interaction between species group averages and tissue averages. Therefore, the standard factorial analysis of variance (PENG 1967) with scorer, system, and species group as main effects is given here on seed and on tigsue separately. T'he denominators for the I? statistics involved were determined on the basis of expected mean squares for a model which assumes species group (Sp) as a fixed effect and system (Sy) and storer (Sc) as random effects. Interaction effects involving Sc were assumed to be zero. RESULTS The variable of primary interest in this study is the average number of bands recorded by a scorer on the six slots of each system rzpresenting a species group. Schematic interpretations of the actual isozyme patterns will be shown only to illustrate specific points. A summary of the average of 12 observations by two independent scorers of the six slots is given in Table 1 for each species group for each of eight systems which was evaluated for both seed and leaves. The mean number of bands for each species group (averaged over the eight systems) was more than three bands per individual. With only a few exceptions each enzyme showed more than two bands per individual for each species group. Fractional averages reflect the effects of several sources of random variation. They include differences among slots due to variation among plants within a species group, failure of gel conditions to be identical from slot to slot, and discrepancies between scorers on the same slot. For each system, each diploid species group was characterized by,a distinct pattern oi bands. (A pattern is defined by the number of bands and their pmir tions on the gel.) Comparison of banding patterns of the diploids with the tetra-

4 394 CHARLES F. SING AND GEORGE J. BREWER TABLE 1 A summary of the average number of isozyme bands per slot Species group AA BB DD AABB AABBDD Mean AA BB DD AABB AABBDD Mean Enzyme system PGM IIexo APh Est MDH Alk Ph GRPD IjPGD Mean Seed * Leaves * Insufficient seed available. Missing data estimated using formula given by PENC (1967) ploid and hexaploid representatives showed that in only one (phosphoglucomutase) of the eight enzyme systems studied was the polyploid pattern a simple combination of the contributing diploids (Figure 1). The results with phosphoglucomutase suggest that the AABBDD patterns are the sumation of the AA with the BB or DD pattern. With the other 7 systems no such relationship obtained, as illustrated by alkaline phosphatase (Figure 1 ). Furthermore, the average number of bands was not proportional to the ploidy level for any of the systems studied. A pooled analysis of variance suggested a significant interaction between species groups and type of tissue. For example, the mean of the AA group is higher in the seed than the corresponding BB and DD means but lower in the leaves. Separate analyses (Tables 2 and 3) imply significant differences among species groups in seed but not leaf tissue. With seeds the Triticum species have a significantly greater average number of isozyme bands than the two Aegilops species groups (5.53 us. 3.51). This difference accounts for 92% of the variance among species group averages. DISCUSSION In discussing these results we will make the assumption that a fairly high proportion of the protein diversity seen on starch gel zymograms has biological relevance, i.e. most of the protein bands result from structural differences among the proteins. Some of the banding after electrophoresis may be due to artifact or conformational heterogeneity of protein molecules. An accurate estimate of the proportion of isozyme bands which are due to structural variation is not available, and will probably have to await structural studies of the isozymes of a random selection of enzyme systems. Because of the technical problems involved, these studies will not be available for a considerable time. At present, however, based

5 11 ISOZYMES OF WHEAT ABD A0 D a ;1 0 PGM il. D A AB[ A0 % + I ALK- PH FIGURE 1.-Schematic interpretation of typical starch gel zymograms of an enzyme of wheat leaves (Arlk Ph) and an enzyme of wheat seed (PGM). (A is AA, B is BB, D is DD, AB is AABB, and ABD is AABBDD). The symbol "0' represents the origin. TABLE 2 An analysis of variance of the average number of bands tallied by a scorer for six slots, seed only Source of variation Among species groups (Sp) Triticum us. Aegilops (AA,AABB,AABBDD, us. BB,DD) AABB us. AABBDD Residual Among systems (Sy) Among scorers (Sc) SP x SY Error Mean Probability df square F >F < < > >.IO < > < ~. on the limited number of structural studies which have been done on multiple fnrmc nf nrntninc ciirli ac lartir Jnhvrlrnwnnacn hmnncrlnhin anrl hantnrrlnhin it seems safe to assume that a fairly high proportion of isozymic diversity will be the result of structural variation.

6 396 CHARLES F. SING AND GEORGE J. BREWER TABLE 3 An analysis of uariance of the average number of bands tallied by a scorer for six slots, leaues only Source of variation RIean Probability df square F >F Among species groups (Sp) >.IO Among systems (Syj <,005 Among scxers (Scj >.05 SP x SY <,005 Error The proportion of isozyme system; of the inbreeder, wheat, which show multiplicity in this sample is 100%. So far comparable data have not been gathered on a natural population of a spccies of plant which breeds prinrarily by cross fertilization. The only contrast which can be made at this time is with data gathered on man and Drosophila, which show that 62% and SO%, respectively, of enzyme systems show multiplicity common to all individuals of the population (BREWER and SING 1969). Assuming multiplicity of protein form; has a selective advantage, these data are at least compatible with the suggestion that wheat has used this type of strategy, rather than intrapopulation allelic variation, to acquire protein diversity and associated increased individual fitness. This stratcgy incorporates an increased biochemical flexibility into all individuals of the population, not just heterozygotes. It follows that biochemical flexibility attributable to nonsegregating multiple forms of proteins may be the basis for the examples (cited by BRADSHAW 1965) of stability of phenotypic expression in diverse environments which seems to be determined by gene complexes unrelated to heterozygosity. Therefore, it is reasonable to suggest from the degree of multiplicity reported here that a population of inbreeders may depend primarily upon a process of gene duplication (assuming it to be the major source of nonsegregating isozymes), rather than allelic variation, to attain individuals with a high level of protein diversity. Furthermore, the polyploid nature of wheat suggests one possible mechanism for the origin of duplicate genes in these species. Of the eight enzyme systems studied, only one demonstrated a simple additive relationship between ploidy and isozyme patterns of the type described by JOHNSON, BARNHART and HALL for seed proteins. A simple linear increase in the number of bands of the type suggested by seed proteins was the exception rather than the rule. It is quite probable (see RILEY 1965) that because of mutation and independent evolution of the species involved, the A, B, and D genomes in the diploid species are not nearly as genetically homeologous with their corresponding entities found in the tetraploid and hexaploid as is suggested by chromosome pairing affinities. If this were the case, the identification of specific diploid contributions in the amphidiploid patterns would be difficult, if not impossible. However, one would still expect the number of proteins in polyploids to be an approximate sum of the contributing diploids. One of the more important aspects of the work reported here may be the demonstration of the extent to which mechanisms

7 ISOZYMES OF WHEAT 397 have evolved which regulate gene expression in the polyploids. It is possible that structural genes for storage proteins, because of the large amounts of protein synthesized, in contrast to enzymes, are often constitutive in nature, leading to additivity of patterns as reported by JOHNSON et al. (1967). On the other hand, mechanisms which bring the expression of enzymes in polyploids to resemble, at least in numbers of molecular types, the diploid progenitors seem to be the rule. One mechanism, for example, would be selection of modifiers which reduce the polyploid pattern to one resembling the diploids. The diversification of the wheat species which has been attributed to polyploidy ( ZOHARY 1965 ; RILEY 1965) has been accompanied by some significant interspecific differences in the average number of isozymes. The question of a possible relationship between the amount of isozymic diversity and ecogeographical range occupied by the various wheat species is not easily answered from the data. The seed data are suggestive of such a relationship. The more widely adapted diploid group, AA, has an enzyme multiplicity comparable to the polyploids which have a wide ecogeographical amplitude, while the diploids with a more restricted habitat, BB and DD, show significantly fewer isozymes per system. This would support the suggestion of ZOHARY (1965) that the most successful diploid is preadapted and contributes the pivotal genome or main evolutionary theme for the polyploid cluster. Such a relationship is not, however, apparent in the leaf data. It would appear that in leaves, the AA genome has contributed a level of multiplicity which is affected little in quantity and a yet undetermined degree in quality when combined with the BB and DD genomes. That the amount of multiplicity varies between tissues suggests that intraindividual, as well as ecogeographical selection forces, play a role in determining gene expression. We thank DR. E. N. LARTER, University of Manitoba, and DR. J. C. CRADDOCK, U. S. Department of Agriculture, for supplying seed stock. The space provided by the Botanical Garden, University of Michigan, to increase seed supplies was greatly appreciated. We are grateful to DAVID R. BOWBEER and SASHAINSWORTH for technical assistance. SUMMARY The self-pollinator, wheat, has an extremely high incidence (100% in this study) of enzyme systems which show multiplicity of molecular forms. This type of multiplicity may be a vital evolutionary strategy in self-pollinating plants.- In contrast to reports with crude seed protein extracts, the complement of enzyme proteins in the polyploid is not usually a simple additive function of the contributions of the diploid genomes. The evolution of regulation of gene expression is an intriguing facet of the study of this polyploid series.-data from seed, but not leaves, support the hypothesis that the broadly adapted Triticum species tend to have significantly greater protein diversity than their more narrowly adapted Aegilops relatives. LITERATURE CITED BRADSHAW, A. D., 1965 Evolutionary significance of phenotypic plasticity in plants. Advan. Genet. 13:

8 398 BREWER, G. J., and C. A. KNUTSEN, : CHARLES F. SING AND GEORGE J. BREWER Hexokinase isozymes in human erythrocytes. Science BREWER, G. J., and C. F. SING, 1963 The study evolution through isozymes. J. Clin. Invest. 46: lla (Abstract) 1969 Survey of isozymes of human erythrocytes. In: Biochemical Methods in Red Cell Genetics. Edited by J. YUNIS, Academic Press, N.Y. FINCHAM, J. R. S., 1966 HARRIS, H., 1966 Enzyme polymorphisms in man. Proc. Roy. Soc. London B 164: HOPKINSON, D., N. SPENCER, and H. HARRIS, 1964 Genetical studies on human red cell phosphatase. Am. J. Human Genet. 16: JOHNSON, B. L., and 0. HALL, 1965 Analysis of phylogenetic affinities in the Triticinae by protein electrophoresis. Am. J. Botany 52: JOHNSON, B. L., D. BARNHART, and 0. HALL, 1967 Analysis of genome and species relationships in the polyploid wheats by protein electrophoresis. Am. J. Botany 54: KIMURA, M., and J. F. CROW, 1964 The number of alleles that can be maintained in a finite population. Genetics 49: KING, J. L., 1967 Genetic Complementation. Benjamin. Continuously distributed factors affecting fitness. Genetics 55: LEWONTIN, R. C., and J. L. HUBBY, 1966 A molecular approach to the study of genic heterozygosity in natural populations. 11. Amount of variation and degree of heterozygosity in natural papulations of Drosophila pseudosbscura. Genetics 54: MILKMAN, R. D., 1967 Heterosis as a major cause of heterozygosity in nature. Genetics 55: PENG, K. C., 1967 The Design and Analysis of Scientific Experiments. Addison-Wesley. RILEY, R., 1965 Cytogenetics and the evolution of wheat. In: Crop Plant Euolution. Edited by SIR J. HUTCHINSON, Cambridge University Press, London. RILEY, R., and V. CHAPMAN, 1966 Estimates of the homoeology of wheat chromosomes by measurements of differential affinity at meiosis. In: Chromosome Manipulation and Plant Genetics. Edited by R. RILEY and K. R. LEWIS, Oliver and Boyd, Edinburgh. SEARS, E. R., 1966 Nullisomic-tetrasomic combinations in hexaploid wheat. In: Chromosome Manipulation and Plant Genetics. Edited by R. RILEY and K. R. LEWIS, Oliver and Boyd, Edinburgh. SHAW, C. R., 1965 Electrophoretic variation in enzymes. Science 149: SPENCER, N., D. HOPKINSON, and H. HARRIS, 1964 Nature 204: 74S-745. Phosphoglucomutase polymorphism in man. SVED, J. A., T. E. REED, and W. F. BODMER, 1967 The number of balanced polymorphisms that can be maintained in a natural population. Genetics 55 : TASHIAN, R. E., 1965 Genetic variation and evolution of the carboxylic esterases and carbonic anhydrases of primate erythrocytes. Am. J. Human Genet. 17: VAN VALEN, L., Haldane s dilemma, evolutionary rates, and heterosis. Am. Naturalist 97: ZOHARY, D., 1965 Colonizer species in the wheat group. In: The Genetics of Colonizing Species. Edited by H. G. BAKER and G. L. STEBBINS. Academic Press, N.Y.