Genetic analysis of isoenzyme phenotypes using single tree progenies

Heredity 63 (1989) 135—141 The Genetical Society of Great Britain Received 17 February 1989 Genetic analysis of isoenzyme phenotypes using single tree progenies Elizabeth Gillet and Hans H. Hattemer Abteilung für Forstgenetik und Forstpflanzenzüchtung, Georg-August-Universität Göttingen, Büsgenweg 2, 3400 Göttingen, Federal Republic of Germany A method of genetic analysis is proposed for determination of the mode of inheritance of environmentally and ontogenetically stable isoenzyme phenotypes as expressed in angiospermous forest trees. This method also applies to higher plant and animal species characterized by multiple matings of single female parents. The modes of inheritance considered are codominance in the absence and the presence of a (recessive) null allele. The analyzed material coRsists of zymograms of single maternal trees and their progenies (as seeds or seedlings) from open pollination. Such data is more easily obtained than controlled crosses and can represent the total variation in the population. The genetic analysis requires only the basic assumptions of classical Mendelian analysis, which make use only of the elementary mechanisms of meiosis and fertilization. Additional assumptions on the mating system, such as those required by the mixed mating model, are not needed. The results confirm the need for explicit genetic analysis of zymograms. THE NECESSITY OF GENETIC ANALYSIS OF ENZYME PHENOTYPES Complexities can arise in the interpretation of enzyme phenotypes, some of which are not at all visible in the zymograms alone. The following are of importance: (a) Null alleles may exist which code for an enzyme of reduced or no activity in vivo, in vitro, or both. All types of null alleles are operationally recessive under routine procedures of laboratory analysis. Thus, if the Therefore, it is not clear from the zymogram alone whether or not the presence of double bands can be interpreted as heterozygosity. (c) The differences in electrophoretic mobility of the products of multiple gene loci controlling an enzyme system are not always greater than differences among allozymes (Stuber and Goodman, 1984, for 6-PGDH in maize). Thus the "zones" of a zymogram can overlap, causing problems in assigning the variation in one zone to the genetic variation at one gene locus. This is particularly true if the enzymes are for the null allele will appear to be homozygous for its active allele, and thus its null monomers. (d) Intergenic (or interlocus) heterodimers among multiple gene loci make it difficult to discriminate between zones of a given zymogram and thus between possible modes of transmission involving differing numbers of gene loci. MDH allele will not be detected. Furthermore, homo- in pine seeds (O'Malley et aL, 1979; El- modes of extraction and staining are not sensi- tive to the amount of active enzyme in the zymogram bands, an individual heterozygous zygosity for a null allele can be a lethal condition. Since only viable genotypes can be observed, analysis of the zymogram patterns alone can never reveal the existence of the null allele in such cases. (b) Some alleles of gene loci controlling monomers code for double bands even in haploid tissue, as is known from both acid phosphatase and leucine aminopeptidase in conifer endosperm (Bergmann, 1973, 1974). Kassaby, 1981; Müller-Starck, 1985a) and in spruce seeds (Cheliak et a!., 1985; Pitel et a!., 1987) may serve as an example. If intergenic heterodimers occur together with null alleles, as is the case with MDH in maize (Goodman et a!., 1980) and Douglas-fir (El-Kassaby, 1981) as well as 6-PGDH in maize (Stuber and Goodman, 1984) and beech (Müller-Starck, personal communication), the zymograms may be uninterpretable. 136 These complexities exist in only a few enzyme E. GILLET AND H. H. HATTEMER molecule helps to avoid ambiguities of genetic cite here (cf. Rudin, 1986), deal with the mode of inheritance of enzyme phenotypes in conifers. In contrast, comparatively few studies have been published on the mode of inheritance of interpretation. For instance, appropriate bio- enzyme phenotypes in angiospermous tree species. systems (cf. Shields et al., 1983). In most systems, information on the structure of the enzyme chemical methods consisting of inhibition of enzymes migrating into one of two different zones might be applied to prove that a certain enzyme system is controlled by two gene loci. Nevertheless, such complexities do arise, sometimes coinciding with post-translational modification of the isoenzyme phenotype. If they go unnoticed and thus are not incorporated into the postulated mode of inherit- ance, all further interpretations based on the erroneous mode of inheritance, such as charac- terization of the mating system, population differentiation, genetic distance between populations, or degree of heterozygosity, can be worthless. For this reason, genetic analysis of zymograms is essential. For one, analysis of their tissue usually requires special extraction techniques (Torres, 1983; Arulsekar et a!., 1983). Furthermore, analysis of the triploid endosperm depends upon the detectability of allele dosage differences (Schoen, 1979, 1980). Most existing studies have used progeny from controlled crossings. Among these are the investigations by Feret and Stairs (1971) and Feret (1972) on Ulmus species, Guzina (1978) and Rajora (1986) on Populus species, Kim (1979, 1980), Thiebaut et a!. (1982), and Müller-Starck (1985b) on Fagus sylvatica, Wendel and Parks (1982) on Camellia japonica, Linares-Bensimón (1984) on Alnus glutinosa, and Arulsekar et a!. (1985) on Juglans species. Genetic analysis of enzyme phenotypes in various fruit trees using controlled crossings was reviewed by Torres GENETIC ANALYSIS OF ENZYME PHENOTYPES IN TREE SPECIES In most tree species, classical Mendelian analysis, which requires offspring from controlled crosses as well as parental and offspring tissue of the same type and ontogenetic stage, is problematical. Con- (1983). Several investigators utilized single tree offspring from open pollination but postulated the mode of inheritance on the basis of comparison with other species as well as comparison of total progeny and maternal gene frequencies (Brown et a!. (1975) and Phillips and Brown (1980) on trolled crosses in trees are often technically difficult Eucalyptus species; reviewed in Moran and Bell (1983)) or comparison of the genotypic distribu- to perform, and the numbers of offspring obtainable from controlled crosses ae often too small tions within population samples with HardyWeinberg-proportions (Saidman and Naranjo for statistical testing. Yet even if controlled crosses succeed, the long generation intervals in trees (1982) in the leguminous tree Prosopis ruscifolia, O'Malley eta!. (1988) in Bertholletis exce!sa). Brotschol (1983) also used the former method in her ontogenetic stage can rarely be sampled from both (...truncated)