**6. Isozyme analysis in grape**

72 Gel Electrophoresis – Advanced Techniques

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)

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 varieties.

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 Superior Seedless (Schwennesen et al., 1982).

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 clones.

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 difference has been found for the ACP system.

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).

Gel Electrophoresis of Grapevine (*Vitis vinifera* L.) Isozymes - A Review 75

Shiraishi et al. (1994) used GPI and PGM isozyme banding patterns for the detection of hybrid origin of seedlings during their triploide grape breeding. First they analysed 99 diploide cultivars, 20 diploide plants from 8 wild *Vitis* species and populations from the crosses between them . In the GPI-2 locus 13 in the GPM locus 11 allels was found. Data showd high genetical differences between Vitis species . After that, the GPI-2 and PGM-2 genotype of 6 diploide and 4 tetraploide cultivars (used for the crosses) were determined. 15 diploide x tertraploide crosses were made. Trisomy gene expression was detected in 92 out

Seed proteins and enzymes (AcP, ADH, EST, G-6-PDH, MDH, PGM, POD) from several cultivars and wild ecotypes of *Vitis vinifera* L. have been used to evaluate taxonomic differences between *V. vinifera* sspp. sativa and sylvestris (Scienza et al., 1994). Only total proteins in the pH range of 4.0-5.5 and AcP, EST and G-6-PDH were useful for genotype differentiation. The cluster analysis (UPGMA), based on Jaccard genetic distance and determined on the presence/absence of electrophoretic profiles, reveals 2 distinct groups, supporting the hypothesis of the authors that *V. sativa* and *V. silvestris* should be regarded as

Studies on the induction characteristics and the fine structure of grapevine cells cultured in vitro were undertaken with cultivar Monastrell berry samples of different developmental stages between fruit set and veraison (Zapata et al., 1996). Medium composition, electron microscopy application and protoplast isolation procedures are explained. It could be shown that the intensity of cell development and callus induction percentage depended on the berry growth stage; the de-differentiation process is mainly located in meso-carp tissues. Cultured cells showed to be highly vacuolated with their cytoplasm reduced to a very thin

Ros Barceló et al. (1996) studied the gene expression of isozymes of providase in downy mildew resistant (*Vitis vinifera* x *Vitis rupestris*) x *Vitis riparia* hybrids and in the susceptible *Vitis vinifera* parent. The peroxidase isoenzyme type B3 (PI=8,9) expressed in the phloem

To test whether the basic peroxidase isoenzyme B3 may be considered as a molecular marker of disease resistance in *Vitis* species, suspension cell cultures derived from the downy mildew susceptible *V. vinifera* parent species were treated with an elicitor (cellulase Onoztika R-10) from the soil fungus *Trichoderma viride*, a specific and wellknown elicitor of disease resistance reactions in grapevines. The results showed that treatment with the elicitor induces, simultaneously with the activation of the disease resistance mechanism, the appearance of B3 in the cell cultures. These results suggest that the basic peroxidase isoenzyme B3 may be considered as a marker of disease resistance in

Isoenzymes from grapevine woody stems and shoots were evaluated for their use in identification of varieties and clones by Royo et al. (1997). Plant extracts were separated by polyacrylamide gel electrophoresis. Isoenzyme analysis was carried out for esterases, peroxidases, catechol oxidase, glutamate oxalacetate transaminase and acid phosphatase. The plant material was grown and sampled at two localities in Spain, with different climatic conditions. Sampling was carried out bimonthly for two consecutive years in order to find out the influence of the environment and time of the year. Each isozyme system had a

and leaves of resistant hybrids was completely absent in the susceptible parent.

of 98 seedlings, as 6 showed diploide patterns.

peripheral layer (containing golgi sacks).

2 separate taxa.

Vitis species.

Starch-gel electrophoresis was used by Walters et al (1989) for the analysis of *Vitis vinifera* L. cultivars, interspecific *Vitis* hybrids and wild individuals of *Vitis riparia* Michx. They suggest a simple and inexpensive procedure for the extraction of active enzymes from grape, which is rapid and efficient. Starch-gel electrophoresis with different optimized gel-electrode buffer systems is used for 40 different isoenzymes, 14 of which were consistently resolvable and showed variation among different cultivars.

Isozyme analysis is one of the means suitable to characterize clonally propagated cultivars. Isoelectric focusing was used to reveal differences in isozyme patterns between tissuecultured plants and mother plants, for the cultivars Barbera, Queen of the Vineyards, Dolcetto and Delight. In cultivar Barbera both 2n and 4n plants were considered. Leaf samples were collected from shoots grown on cuttings under controlled environmental conditions and from plants obtained by tissue culture. The buds used for tissue culture were taken from the same shooted cuttings. Leaf extracts were analyzed by isoelectric focusing considering the following isozymes: AcPH (acid phosphatase), GPI (glucose phosphate isomerase) and PGM (phosphoglucomutase). The banding patterns of GPI and PGM showed differences among the cultivars, while for AcPH there seemed to be no differences among them in the pH range considered. There were no differences between isozyme patterns of the Barbera 2n and Barbera 4n. The main difference between in vitro plants and mother plants was the amount of isozyme evaluated by densitometric measurements. In all the cultivars, the amount of isozymes for AcPH was higher in mother plants than in in vitro ones, while for PGM and GPI it was the opposite. This can be due to the different environmental conditions affecting cellular metabolism (Botta et al., 1990).

The idea of using woody stems during the resting period instead of leaves for the isozyme analysis arose in 1990. Kozma et al. (1990) analysed the esterase isozymes of varieties from different convarietas and interspecific hybrid families by poliacrilamide gel electrophoresis and isoelectric focusing. Based on their results they established, that the phloem extracts from woody stems collected in the resting period or shoots colleting in spring give good reproducible patterns, but the leaf extracts give irreproducible patterns.

Tests were carried out on different types of calli and somatic embryos of V. rupestris using 2-D electrophoresis. The investigation carried out by Martinelli et al. (1993) is focused on the isozyme patterns of AcP (acid phosphatase), ADH (alcohol dehydrogenase), EST (esterase), G6PDH (gluconate-6-phosphate dehydrogenase) and PGM (phosphoglucomutase). A typical variation of isozyme pattern could be observed during the different steps of somatic embryogenesis. De-differentiated callus showed other types of isoenzyme pattern compared to those obtained during the development of somatic embryos.

Similarly to Kozma et al. Bachmann (1994) used extracts from phloem of dormant canes for the isozyme analysis. This comprehensive study has analysed the peroxidase isozyme banding patterns of 313 different cultivars and species of *Vitis* using isoelectric focusing on polyacrylamid gels. The author reports that acidic peroxidases were characteristic for *Vitis vinifera* L. cultivars with only a 5 % frequency of occurrence in other *Vitis* species. Variation in neutral to basic peroxidases could be used to group together similar cultivars independent of berry colour, e.g. Pinot noir, Pinot gris, Pinot blanc and Pinot meunier. However, other examples of colour variants, e.g. Merlot noir and blanc were clearly different using peroxidase banding.

Starch-gel electrophoresis was used by Walters et al (1989) for the analysis of *Vitis vinifera* L. cultivars, interspecific *Vitis* hybrids and wild individuals of *Vitis riparia* Michx. They suggest a simple and inexpensive procedure for the extraction of active enzymes from grape, which is rapid and efficient. Starch-gel electrophoresis with different optimized gel-electrode buffer systems is used for 40 different isoenzymes, 14 of which were consistently resolvable

Isozyme analysis is one of the means suitable to characterize clonally propagated cultivars. Isoelectric focusing was used to reveal differences in isozyme patterns between tissuecultured plants and mother plants, for the cultivars Barbera, Queen of the Vineyards, Dolcetto and Delight. In cultivar Barbera both 2n and 4n plants were considered. Leaf samples were collected from shoots grown on cuttings under controlled environmental conditions and from plants obtained by tissue culture. The buds used for tissue culture were taken from the same shooted cuttings. Leaf extracts were analyzed by isoelectric focusing considering the following isozymes: AcPH (acid phosphatase), GPI (glucose phosphate isomerase) and PGM (phosphoglucomutase). The banding patterns of GPI and PGM showed differences among the cultivars, while for AcPH there seemed to be no differences among them in the pH range considered. There were no differences between isozyme patterns of the Barbera 2n and Barbera 4n. The main difference between in vitro plants and mother plants was the amount of isozyme evaluated by densitometric measurements. In all the cultivars, the amount of isozymes for AcPH was higher in mother plants than in in vitro ones, while for PGM and GPI it was the opposite. This can be due to the different

The idea of using woody stems during the resting period instead of leaves for the isozyme analysis arose in 1990. Kozma et al. (1990) analysed the esterase isozymes of varieties from different convarietas and interspecific hybrid families by poliacrilamide gel electrophoresis and isoelectric focusing. Based on their results they established, that the phloem extracts from woody stems collected in the resting period or shoots colleting in spring give good

Tests were carried out on different types of calli and somatic embryos of V. rupestris using 2-D electrophoresis. The investigation carried out by Martinelli et al. (1993) is focused on the isozyme patterns of AcP (acid phosphatase), ADH (alcohol dehydrogenase), EST (esterase), G6PDH (gluconate-6-phosphate dehydrogenase) and PGM (phosphoglucomutase). A typical variation of isozyme pattern could be observed during the different steps of somatic embryogenesis. De-differentiated callus showed other types of isoenzyme pattern compared

Similarly to Kozma et al. Bachmann (1994) used extracts from phloem of dormant canes for the isozyme analysis. This comprehensive study has analysed the peroxidase isozyme banding patterns of 313 different cultivars and species of *Vitis* using isoelectric focusing on polyacrylamid gels. The author reports that acidic peroxidases were characteristic for *Vitis vinifera* L. cultivars with only a 5 % frequency of occurrence in other *Vitis* species. Variation in neutral to basic peroxidases could be used to group together similar cultivars independent of berry colour, e.g. Pinot noir, Pinot gris, Pinot blanc and Pinot meunier. However, other examples of colour variants, e.g. Merlot noir and blanc were clearly

environmental conditions affecting cellular metabolism (Botta et al., 1990).

reproducible patterns, but the leaf extracts give irreproducible patterns.

to those obtained during the development of somatic embryos.

different using peroxidase banding.

and showed variation among different cultivars.

Shiraishi et al. (1994) used GPI and PGM isozyme banding patterns for the detection of hybrid origin of seedlings during their triploide grape breeding. First they analysed 99 diploide cultivars, 20 diploide plants from 8 wild *Vitis* species and populations from the crosses between them . In the GPI-2 locus 13 in the GPM locus 11 allels was found. Data showd high genetical differences between Vitis species . After that, the GPI-2 and PGM-2 genotype of 6 diploide and 4 tetraploide cultivars (used for the crosses) were determined. 15 diploide x tertraploide crosses were made. Trisomy gene expression was detected in 92 out of 98 seedlings, as 6 showed diploide patterns.

Seed proteins and enzymes (AcP, ADH, EST, G-6-PDH, MDH, PGM, POD) from several cultivars and wild ecotypes of *Vitis vinifera* L. have been used to evaluate taxonomic differences between *V. vinifera* sspp. sativa and sylvestris (Scienza et al., 1994). Only total proteins in the pH range of 4.0-5.5 and AcP, EST and G-6-PDH were useful for genotype differentiation. The cluster analysis (UPGMA), based on Jaccard genetic distance and determined on the presence/absence of electrophoretic profiles, reveals 2 distinct groups, supporting the hypothesis of the authors that *V. sativa* and *V. silvestris* should be regarded as 2 separate taxa.

Studies on the induction characteristics and the fine structure of grapevine cells cultured in vitro were undertaken with cultivar Monastrell berry samples of different developmental stages between fruit set and veraison (Zapata et al., 1996). Medium composition, electron microscopy application and protoplast isolation procedures are explained. It could be shown that the intensity of cell development and callus induction percentage depended on the berry growth stage; the de-differentiation process is mainly located in meso-carp tissues. Cultured cells showed to be highly vacuolated with their cytoplasm reduced to a very thin peripheral layer (containing golgi sacks).

Ros Barceló et al. (1996) studied the gene expression of isozymes of providase in downy mildew resistant (*Vitis vinifera* x *Vitis rupestris*) x *Vitis riparia* hybrids and in the susceptible *Vitis vinifera* parent. The peroxidase isoenzyme type B3 (PI=8,9) expressed in the phloem and leaves of resistant hybrids was completely absent in the susceptible parent.

To test whether the basic peroxidase isoenzyme B3 may be considered as a molecular marker of disease resistance in *Vitis* species, suspension cell cultures derived from the downy mildew susceptible *V. vinifera* parent species were treated with an elicitor (cellulase Onoztika R-10) from the soil fungus *Trichoderma viride*, a specific and wellknown elicitor of disease resistance reactions in grapevines. The results showed that treatment with the elicitor induces, simultaneously with the activation of the disease resistance mechanism, the appearance of B3 in the cell cultures. These results suggest that the basic peroxidase isoenzyme B3 may be considered as a marker of disease resistance in Vitis species.

Isoenzymes from grapevine woody stems and shoots were evaluated for their use in identification of varieties and clones by Royo et al. (1997). Plant extracts were separated by polyacrylamide gel electrophoresis. Isoenzyme analysis was carried out for esterases, peroxidases, catechol oxidase, glutamate oxalacetate transaminase and acid phosphatase. The plant material was grown and sampled at two localities in Spain, with different climatic conditions. Sampling was carried out bimonthly for two consecutive years in order to find out the influence of the environment and time of the year. Each isozyme system had a

Gel Electrophoresis of Grapevine (*Vitis vinifera* L.) Isozymes - A Review 77

Isozyme and SSR analysis were carried out for the differentiation of the grapevine cultivars Kéknyelű and Picolit. The name of the grapevine cultivar 'Kéknyelű' has become inseparable from the name of the Badacsony vine region (Hungary), whose fame is also well known beyond the Hungarian frontier. In the Vitis International Variety Catalogue (http://www.genres.de/idb/vitis/) 'Kéknyelű' is reported, as the synonym of the Italian grapevine cultivar 'Picolit'. Vertical poliacrylamide-gel electrophoresis was used for the investigation isoenzymes of catechol-oxidase (CO) and acid phosphatase (AcP). Microsatellite analyses were carried out at 6 loci (VVS2, VVS16, VrZag79, VVMD7, VMC4A1, VMC4G6). The results of the isoenzymatic and microsatellite analyses confirmed,

Jahnke et al. (2009) investigated the genetic diversity of Hungarian grapevine cultivars with biochemical and molecular markers (isoenzyme and SSR). The isoenzyme patterns of 4 enzyme systems (catechol-oxidase, glutamate-oxalacetate-transaminase, acid phosphatase and peroxidase) and the microsatellite profile in 6 loci (VVS2, VVS16, VVMD7, VMC4A1,

The results with CO, GOT, AcP and PER enzymes were reproducible and the zymograms obtained from the woody stems were independent from the time of sampling during the

Based on the isoenzyme patterns of these 4 enzymes most of the investigated varieties (40/48) were identified. A correlation was found between the isoenzyme patterns and the

Fig. 5. Characteristic interpretative zymograms observed for CO, GOT, AcP and PER enzymes. The letters mark the different types of isoenzyme patterns, while numbers refer to

It was established, that while the varieties of the convarietas pontica differed from those of the convarietas orientalis and occidentalis, the two latter groups could have not been differentiated from each other. Based on the SSR (simple sequence repeat) analyses 46 of the

the number of different isoenzyme bands (Jahnke et al. 2009.)

that this two cultivars are different (Jahnke et al., 2007).

VMC4G6, VrZag79) of 48 grapevine varieties were analysed.

dormant period of the grape (Fig 5.).

classification to convarietas of the varieties.

pattern defined by 'fixed' bands that were always present at both localities and during the resting period of the plant (autumn —winter).

An evaluation of the genetic diversity of 'Albariño' (*Vitis vinifera* L.) was carried out by Vidal et al. (1998). The 73 isozyme and 308 RAPD markers were common in the samples tested. The results show the existence of a genetic homogeneity within 'Albariño'cultivated in Galicia. Minor ampelographic differences among samples could be due to external factors rather than to genetic differences.

DNA and isoenzyme analyses were used to characterize 20 table grape cultivars including Moscato d'Amburgo, Italia, Sultanina, Bicane and some recently released new cultivars (Crespan et al., 1999). GPI and PGM isoenzyme systems were able to separate the cultivars into 9 groups whereas the 8 microsatellite loci that were analysed revealed a higher discriminating power. Parentage analysis confirmed that the cultivar Italia was obtained from the crossing Bicane x Moscato d'Amburgo.

Hungarian researchers used isoelectric focusing for the peroxidase and esterase isozymes of some grapevine cultivars. Samples were gathered at different times of the year. The leaf samples after blooming were found the best for the identification of varieties, but they found the phloem extracts of woody stems also suitable for cultivar identification (Stefanovits-Bányai et al., 1999; Stefanovits-Bányai et al, 2002).

Sixty-four Muscat flavoured grapevine accessions were analysed in the work of Crespan and Milani (2001). An analysis was performed at two isozymes and 25 microsatellite loci. The 64 accessions were reduced to 20, which were easily distinguishable from each other at the molecular level by as few as two microsatellite loci. The remaining 44 were found to be synonyms. Three mutants with red and pink coloured berries were identified in the Moscato bianco group. Moscato nero encompasses at least two, Moscato rosa three different varieties. It seems that only two of the analysed Muscats are the main progenitors of the Muscat family: Moscato bianco and Muscat of Alexandria, which in turn are joined by a direct parent-offspring link.

Sànchez-Escribano et al. (1998) analysed 43 table grape varieties by 6 isoenzyme systems (PER, CO, GOT SOD, EST, AcP). The last 2 enzymes were found unsuitable for identification, by the combination of the zymogram of the other 4 enzymes, they were able to identify 31 cultivars, as the remaining 12 were clustered to 5 groups.

Protein and esterase isozyme patterns of authentic grapes and wines of 13 white wine cultivars were determined by means of isoelectric focussing (range of pI: 2.5-10) by Paar et al (1999). Esterase staining with grapes showed active zones mainly in the alkaline pI-range, with most of the cultivars, however indicating no qualitative, but only quantitative differences. Staining of the protein patterns of grapes and wines with Coomassie Brilliant Blue proved to be well suitable for the differentiation of cultivars. With grapes as well as wines the most predicative bandings focussed in the acid pI-range of 4. With the cultivars Grüner Veltliner, Rotgipfler and Riesling Italico the protein banding patterns were so characteristic, that these cultivars were easily identified, whereas with the other cultivars detailed comparisons of the phenogrammes were necessary.

Isozyme and RAPD markers were used for the characterization of Hungarian grapevine varieties and their parents (HAJÓS-NOVÁK And HAJDÚ 2003). The cathecol-oxidase system was found the most suitable for identification proposes.

pattern defined by 'fixed' bands that were always present at both localities and during the

An evaluation of the genetic diversity of 'Albariño' (*Vitis vinifera* L.) was carried out by Vidal et al. (1998). The 73 isozyme and 308 RAPD markers were common in the samples tested. The results show the existence of a genetic homogeneity within 'Albariño'cultivated in Galicia. Minor ampelographic differences among samples could be due to external factors

DNA and isoenzyme analyses were used to characterize 20 table grape cultivars including Moscato d'Amburgo, Italia, Sultanina, Bicane and some recently released new cultivars (Crespan et al., 1999). GPI and PGM isoenzyme systems were able to separate the cultivars into 9 groups whereas the 8 microsatellite loci that were analysed revealed a higher discriminating power. Parentage analysis confirmed that the cultivar Italia was obtained

Hungarian researchers used isoelectric focusing for the peroxidase and esterase isozymes of some grapevine cultivars. Samples were gathered at different times of the year. The leaf samples after blooming were found the best for the identification of varieties, but they found the phloem extracts of woody stems also suitable for cultivar identification (Stefanovits-

Sixty-four Muscat flavoured grapevine accessions were analysed in the work of Crespan and Milani (2001). An analysis was performed at two isozymes and 25 microsatellite loci. The 64 accessions were reduced to 20, which were easily distinguishable from each other at the molecular level by as few as two microsatellite loci. The remaining 44 were found to be synonyms. Three mutants with red and pink coloured berries were identified in the Moscato bianco group. Moscato nero encompasses at least two, Moscato rosa three different varieties. It seems that only two of the analysed Muscats are the main progenitors of the Muscat family: Moscato bianco and Muscat of Alexandria, which in turn are joined by a direct

Sànchez-Escribano et al. (1998) analysed 43 table grape varieties by 6 isoenzyme systems (PER, CO, GOT SOD, EST, AcP). The last 2 enzymes were found unsuitable for identification, by the combination of the zymogram of the other 4 enzymes, they were able

Protein and esterase isozyme patterns of authentic grapes and wines of 13 white wine cultivars were determined by means of isoelectric focussing (range of pI: 2.5-10) by Paar et al (1999). Esterase staining with grapes showed active zones mainly in the alkaline pI-range, with most of the cultivars, however indicating no qualitative, but only quantitative differences. Staining of the protein patterns of grapes and wines with Coomassie Brilliant Blue proved to be well suitable for the differentiation of cultivars. With grapes as well as wines the most predicative bandings focussed in the acid pI-range of 4. With the cultivars Grüner Veltliner, Rotgipfler and Riesling Italico the protein banding patterns were so characteristic, that these cultivars were easily identified, whereas with the other cultivars

Isozyme and RAPD markers were used for the characterization of Hungarian grapevine varieties and their parents (HAJÓS-NOVÁK And HAJDÚ 2003). The cathecol-oxidase

to identify 31 cultivars, as the remaining 12 were clustered to 5 groups.

detailed comparisons of the phenogrammes were necessary.

system was found the most suitable for identification proposes.

resting period of the plant (autumn —winter).

from the crossing Bicane x Moscato d'Amburgo.

Bányai et al., 1999; Stefanovits-Bányai et al, 2002).

rather than to genetic differences.

parent-offspring link.

Isozyme and SSR analysis were carried out for the differentiation of the grapevine cultivars Kéknyelű and Picolit. The name of the grapevine cultivar 'Kéknyelű' has become inseparable from the name of the Badacsony vine region (Hungary), whose fame is also well known beyond the Hungarian frontier. In the Vitis International Variety Catalogue (http://www.genres.de/idb/vitis/) 'Kéknyelű' is reported, as the synonym of the Italian grapevine cultivar 'Picolit'. Vertical poliacrylamide-gel electrophoresis was used for the investigation isoenzymes of catechol-oxidase (CO) and acid phosphatase (AcP). Microsatellite analyses were carried out at 6 loci (VVS2, VVS16, VrZag79, VVMD7, VMC4A1, VMC4G6). The results of the isoenzymatic and microsatellite analyses confirmed, that this two cultivars are different (Jahnke et al., 2007).

Jahnke et al. (2009) investigated the genetic diversity of Hungarian grapevine cultivars with biochemical and molecular markers (isoenzyme and SSR). The isoenzyme patterns of 4 enzyme systems (catechol-oxidase, glutamate-oxalacetate-transaminase, acid phosphatase and peroxidase) and the microsatellite profile in 6 loci (VVS2, VVS16, VVMD7, VMC4A1, VMC4G6, VrZag79) of 48 grapevine varieties were analysed.

The results with CO, GOT, AcP and PER enzymes were reproducible and the zymograms obtained from the woody stems were independent from the time of sampling during the dormant period of the grape (Fig 5.).

Based on the isoenzyme patterns of these 4 enzymes most of the investigated varieties (40/48) were identified. A correlation was found between the isoenzyme patterns and the classification to convarietas of the varieties.

Fig. 5. Characteristic interpretative zymograms observed for CO, GOT, AcP and PER enzymes. The letters mark the different types of isoenzyme patterns, while numbers refer to the number of different isoenzyme bands (Jahnke et al. 2009.)

It was established, that while the varieties of the convarietas pontica differed from those of the convarietas orientalis and occidentalis, the two latter groups could have not been differentiated from each other. Based on the SSR (simple sequence repeat) analyses 46 of the

Gel Electrophoresis of Grapevine (*Vitis vinifera* L.) Isozymes - A Review 79

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Fig. 6. Isoenzyme gel photos for CO (A) and AcP (B) respectively. The numbers show the band numbers, and the capital letters the banding pattern types shown in Figure 5. (Jahnke et al. 2009)

#### **7. References**

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Fig. 6. Isoenzyme gel photos for CO (A) and AcP (B) respectively. The numbers show the band numbers, and the capital letters the banding pattern types shown in Figure 5. (Jahnke

Bachmann O., Blaich R. (1988): Isoelectric focusing of grapevine peroxidases as a tool for

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Grape Breeding ; Vitis Special Issue: 134-141

23-3, Phoenix, Arizona, USA, May 17-22, 2010


UPOV, Geneva, Switzerland TG/36/6


**5** 

*1,3Taiwan 2USA* 

**Molecular Electrophoretic Technique for** 

**Authentication of the Fish Genetic Diversity** 

Tsai-Hsin Chiu, Yi-Cheng Su, Hui-Chiu Lin and Chung-Kang Hsu *1Department of Food Science, National PengHu University of Science and Technology* 

*3Penghu Marine Biology Research Center, Fisheries Research Institute, COA, EY* 

Cobia (*Rachycentron canadum*) is the sole representative of their family, the Rachycentridae They are distributed worldwide in tropical and subtropical seas, as the Atlantic and Pacific Oceans (Miao, et al., 2009). There are several species, including cobia, seabream, red progy, snappers, scads and groupers that are raised by cage culture in Taiwan. Among these cagecultured fishes, cobia certainly takes a leading distribution in both annual total production (81.9%) and total value production (75.4%) as compared to the rest in Taiwan (Fisheries

Giant grouper (*Epinephelus lanceolatus*) are also found in tropical and subtropical waters from the Indo-Western Pacific Ocean. It is one of the two largest species of groupers in the world. Due to its fast growth and high price, giant grouper currently is regarded as a

Red coral trout (*Plectropomus leopardus*) a reef-associated fish in Western Pacific, distributed from southern Japan to Australia and eastward to the Caroline Islands (Zhang, et al., 2010). Only few studies concerning population genetics of *Plectropomus leopardus* has been

All of cobia, giant grouper, and red coral trout are high-valued fish market in Taiwan and neighboring countries, including China, Japan, and Vietnam. For the globalization of the seafood industry, seafood authentication and food safety are very important. We must know that the source of fish or accurately species of the fish. Traditional method to distinguish the fish species was observed the external traits. It can cause the error judgment. Today, DNAbased methods are also more frequently employed for food authentication (Lockley and Bardsley, 2000). It has proven to be reliable, sensitive and fast for many aspects of fish species and food authentication. Asensio et al. (2009) were suggesting that the species-specific PCR method could be potentially used by regulatory agencies as routine control assay for the commercial grouper fillets authentication. PCR-based methods commonly used for fish species identification include PCR-sequencing, random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR). Those methods are simplicity, specificity and sensitivity.

favorite species for marine culture in Taiwan (Hseu, et al., 2004).

**1. Introduction** 

Agency 2006).

reported.

*2Seafood Research and Education Center, Oregon State University* 

