**5. Advantages and disadvantages of isozyme analyses**

"The utility of isozymes as genetic markers is generally attributed to their frequent polymorphism, codominance, single gene-Mendelian inheritance, rapid, simple and relatively inexpensive assay and their ubiquity in plant tissues and organs (even in embrios and pollen). Although the selective neutrality of isozymes has been debated, it seems highly probable that they are adaptive under certain circumstances." (Bretting and Widrlechner 1995a).

Other advantages of isozyme analysis are the rapid analyses of samples, a small amount of plant material is sufficient. Young plants can be tested and selected based on their genotypes for features, which morphologically appear later. These can mean significant temporal and financial savings in the case of the breeding of annual plants. Now the best cost-efficient markers are the isozymes (Bretting and Widrlechner, 1995b).

Disadvantages of isozyme analysis as against the DNA markers, that they are organ-, tissueand developmental stage-specific (Fig 4.). They often go through post-transcriptional modifications, which limit their usage (Staub et al., 1996).

Gel Electrophoresis of Grapevine (*Vitis vinifera* L.) Isozymes - A Review 73

Enzyme banding patterns for over 60 varieties of wine and table grapes were determined by gel electrophoresis by Wolfe (1976). Enzymes were extracted from ripe berries of each variety and separated by electrophoresis in a starch gel. Enzyme bands were detected by developing the gels in a buffered solution that produced an insoluble dye at the site of enzyme activity. The varieties were assayed for leucine aminopeptidase, indophenol oxidase, acid phosphatase, catechol oxidase, alcohol dehydrogenase, esterase, and peroxidase. The first four enzymes listed were found the most useful for distinguishing

Enzyme-banding patterns of catechol oxidase, acid phosphatase, esterase, alcohol dehydrogenase, indophenol oxidase, and leucine aminopeptidase obtained by enzyme staining of starch gel electropherograms allow the distinction of berries of the grape cultivars Perlette, Thompson Seedless, Superior Seedless and an early ripening sport of

Twenty-seven cultivars and feral accessions from four Vitis species were examined by SUBDEN et al. (1987) for 12 isozyme systems exhibiting polymorphism. Using extracts from woody tissue and a protocol to avoid isozyme inactivation by polyphenolics and other materials, 27 of 29 strains exhibited unique isozyme banding patterns for glucose-6 phosphate isomerase, peptidase, and acid phosphatase. Implications for genetic homogeneity screening of nursery stock or identifying unknown samples are discussed.

German researchers analysed the isozymes of peroxidase by isoelectric focusing. Purified internodal phloem extracts from dormant wood were used. In the 6-11 pH range 8 bands were found, 71 Vitis species and varieties were identified (Bachmann and Blaich, 1988).

Genetic analysis of 11 allozyme polymorphisms was performed by Weeden (1988) on the progeny of 'Cayuga White' x 'Aurore', two complex interspecific grape (*Vitis*) hybrids. Segregation for most of the polymorphisms closely approximated monogenic Mendelian ratios, and eight new isozyme loci were defined for grape. Joint segregation analysis among the isozyme loci revealed three multilocus linkage groups (ACP-1—PGM-c; ACP-2—AAT-c; GPI-c—LAP-1). These results demonstrate that sufficient allozyme polymorphism exists in grape to establish many multilocus linkage groups and that this genetic analysis can be accomplished using extant progeny or progeny readily produced from highly heterozygous

The pattern of the systems PER and ACP from 8 vines of *Vitis vinifera* L. has been studied in 1988 by Royo et al. (1989). A method to differentiate and characterize 6 clones of *Vitis vinifera* L. has been established by gaining the variability of the PER pattern from the band pattern constantly present in any vine, and the total band pattern from another vine (not only amongst the vines but also along the vegetative cycle). In the vines investigated no

Three enzymes in 5 cultivars of *Vitis vinifera* L. are analyzed by PAGE in young leaves. With acid phosphatase, arylesterase and glutamat-oxalat transaminase more or less different isoenzyme patterns of the different cultivars were obtained. There were no interclonal differences. The most polymorph enzyme was the arylesterase. The best results were

obtained with young leaves from sprouting buds (Eiras-Dias et al., 1989).

**6. Isozyme analysis in grape** 

Superior Seedless (Schwennesen et al., 1982).

difference has been found for the ACP system.

varieties.

clones.

Fig. 4. Schematic representation of the differential expression of GPI (a), LDH (b) and MDH (c) isozymes in adult tissues (Mu, white muscle; Li, liver; Ey, eye; He, heart) and larvae (La) of L. cephalus. The egg pattern is identical to the larval one. Differences in line thickness refer to different staining intensities. (Manaresi et al. 1998)
