Use of microsatellite markers to assess the identity and genetic diversity of vitis labrusca and vitis rotundifolia cultivars/Uso de marcadores microssatelites para acessar a identidade e diversidade genetica de cultivares de vitis labrusca e vitis rotundifolia.
Grapevine belongs to the Vitaceae family, comprising 19 genera. Vitis is the only genus that produces edible fruits and is divided into two subgenera or sections, namely Euvitis (bunch grapes) and Muscadinia (muscadine grapes). Muscadine grapes are genetically and morphologically distinct from the species within the subgenera Euvitis, and the most obvious genetic difference between these two taxa is the number of somatic chromosomes: Muscadinia species have 40, and Euvitis species have 38. Muscadinia species also differ from Euvitis species in their seeds, bark, tendrils and cluster morphology (OLIEN, 2001).
Euvitis species are divided into three geographical groups: American, Asian and European. Vitis vinifera, the main species currently cultivated worldwide, is the only member of the European group and has produced thousands of cultivars. The American group has approximately 30 species. Among these, V. labrusca, known for its 'foxy' fruit flavor, was widely used in the 18 and 19th centuries directly as rootstock for V. vinifera cultivars due to its phylloxera tolerance and in-crossings originating the so-called 'direct producer' vines. These hybrids were planted ungrafted and were bred to incorporate the resistance to the foliar diseases of the North American grapes with the fruit characteristics of the European grapevines (POMMER, 2003). Muscadinia is endemic to the southeastern United States of America, and, among the three species known, only Vitis rotundifolia Michx. is of commercial value, possessing a very strong resistance to grape pests and diseases, and is employed worldwide in breeding programs (OLIEN, 2001).
The knowledge of the genetic diversity of species has two advantages in a breeding program: the first concerns the genetic heterogeneity that limits the vulnerability of a species to pests and diseases; and the second is related to providing a large supply of allelic variation, which can be used for creating new combinations of favorable genes. The feasibility of using genetic diversity as a criterion for the selection of the parents for hybridization has been demonstrated in several species, including grapevine (OLIVEIRA et al., 2005).
DNA-based molecular markers offer an advantage over morphological descriptors, as they are less prone to being affected by the environment or the developmental stage (CARIMI et al., 2010). Simple sequence repeat (SSR) markers have been increasingly used as molecular descriptors in grape. The usefulness of SSRs has been widely demonstrated in the identification and characterization of stock and rootstock varieties (DZHAMBAZOVA et al., 2007), evaluation of genetic variability (RIAZ et al., 2008; SCHUCK et al., 2009), pedigree studies (ORTIZ et al., 2004; VARGAS et al., 2009), and genetic mapping (RIAZ et al., 2004).
The development of a standard set of microsatellite markers for the identification of grape cultivars was proposed by the European Project GENRES 081 using six microsatellite loci (THIS et al., 2004). This set of markers, VVS2 (THOMAS; SCOTT, 1993), VVMD5, VVMD7 (BOWERS et al., 1996), VVMD27 (BOWERS et al., 1999), VrZAG62 and VrZAG79 (SEFC et al., 1999), was used by this project to evaluate 13 reference cultivars in Europe and are widely recommended by the International Organization of Vine and Wine for grapevine genotyping (OIV, 2007).
Considering that the knowledge about the genetic diversity of a specie movides subsidies to a breeding program, this study aimed to confirm the identity and estimate the genetic diversity among ten grapevine cultivars using eight microsatellite markers.
Material and methods
Vitis rotundifolia ('Bountiful', 'Carlos', 'Magnolia', 'Magoon' and 'Regale') and Vitis labrusca ('Bordo', 'Goethe', 'Isabel', 'Marta' and 'Niagara Rosada') cultivars (Table 1) from the grapevine germplasm collection of Estacao Experimental do Canguiri, Universidade Federal do Parana, Pinhais, Parana State, Brazil, Estacao Experimental de Videira, Videira, Santa Catarina State, Brazil, and Estacao Experimental de Campos Novos, Campos Novos, Santa Catarina, State, Empresa de Pesquisa Agropecuaria e Extensao Rural de Santa Catarina (Epagri) were evaluated. These cultivars were selected due to their desirable agronomic characteristics and their potential for use as the parents in a breeding program.
The DNA of the Vitis labrusca cultivars was analyzed in a previous study by Schuck et al. (2009).
The Genomic DNA of the Vitis rotundifolia cultivars was extracted using a modified CTAB (hexadecyltrimethylammonium bromide) procedure (RIAZ et al., 2004). In the final step, the DNA pellets were suspended in 100 [micro]L 1X Tris-EDTA buffer and stored at -20[degrees]C. The DNA quality was visualized on 1.2% agarose gels stained with ethidium bromide (10 mg m[L.sup.-1]), and the samples were then diluted in ultrapure water to a final concentration of 10 ng [micro][L.sup.-1].
The eight microsatellite loci most frequently used by the international scientific community, VVS2 (THOMAS; SCOTT; 1993), VVMD27 (BOWERS et al., 1996), VVMD5, VVMD7, VVMD31, VVMD32 (BOWERS et al., 1999), VrZAG62 and VrZAG79 (SEFC et al., 1999), were used to genotype the samples.
The PCR reactions were performed in 10 [micro]L reaction mixtures containing 5 pmol each primer, 2.5 mmol [L.sup.-1] each dNTP, 1 [micro]L 10X gold PCR buffer (Perkin Elmer Inc., Wellesley, MA, USA), 0.5 unit AmpliTaq Gold DNA polymerase (Perkin Elmer Inc., Wellesley, MA, USA), 2 mmol [L.sup.-1] Mg[Cl.sub.2] and 10 ng genomic DNA. The temperature cycling for the PCR was performed using either a Peltier Thermal Cycler-200 (MJ Research, Inc., Waltham, MA, USA) or a Bio"Rad iCycler (Bio"Rad Laboratories, Hercules, CA, USA). The following cycling program was used: denaturation of DNA and activation of Taq DNA polymerase at 95[degrees]C for 10 min.; 35 cycles of amplification consisting of 45 s at 94[degrees]C, 45 s at 56[degrees]C and 1 min. at 72[degrees]C; a final extension of 10 min. at 72[degrees]C; and cooling at 4[degrees]C. To separate the amplification products, the PCR reactions were mixed with denaturing dye (98% formamide, 10 mmol [L.sup.-1] EDTA, 0.05% bromophenol blue and xylene cyanol) and heated at 94[degrees]C for 2 min. before loading onto 5% polyacrylamide sequencing gels. The gels were electrophoresed at a constant 70 W for 2-3 hours, depending on the allele sizes. The samples were visualized by silver staining using a commercial kit (Promega, Madison, WI, USA). All of the gels were visually examined and scored on a light box and were then digitally scanned to preserve the images.
To determine the size of the DNA fragments obtained by the amplification of the microsatellite products of the V. rotundifolia samples, aliquots of the V. rotundifolia DNA already diluted and available in the laboratory were loaded on the gels. These samples are part of the Grape Germplasm Collection of the University of California--Department of Viticulture and Enology, and the genetic profile in base pairs (bp) is in the reference database for the identification of grapevine that is maintained by the Foundation Plant Service, University of California, Davis, CA, USA. The genetic profile of V. labrusca was obtained in a previous study by Schuck et al. (2009) using the same microsatellite markers used in the present study (VVS2, VVMD5, VVMD7, VVMD27, VrZag62 and VrZag79).
Various genetic parameters for the ten cultivars over the eight SSR loci were calculated. The GDA v1.0 program package software (LEWIS; ZAYKIN, 2001) was used to calculate the allelic frequencies, number of alleles per locus (N), percentage of polymorphic loci (P), observed heterozygosity ([H.sub.o]) and expected heterozygosity (He). The He values were estimated using the following formula (NEI, 1972):
[H.sub.e] = 1 [a.summation over (i=1)] [p.sup.2.sub.1],
where [p.sub.i] is the frequency of the ith allele.
The GENES v2007.0.0 program package software (CRUZ, 2007) was used to determine the polymorphic information content (PIC). The PIC values were calculated according to Botstein et al. (1980), as follows:
PIC = [a.summation over (i=1)] [p.sup.2.sub.i] - [a.summation over (i,j=1)] [a.summation over (i [not equal to] 1)] [p.sup.2.sub.i] [p.sup.2.sub.j],
where [p.sub.i] and [p.sub.j] are the frequencies of the ith and jth allele. The probability of null alleles (r) was calculated for each microsatellite, as described by Brookfield (1996):
r = ([H.sub.e] - [H.sub.0])/(1 + [H.sub.e]),
where [H.sub.e] is the expected heterozygosity and [H.sub.o] is the observed heterozygosity.
The genetic distance between the cultivars were estimated as follows (NEI, 1972):
D = - ln(I),
where ln is the natural logarithm and (I) is the measure of the genetic identity, as calculated using the following formula:
I = [J.sub.PQ]/[square root of ([J.sub.P][J.sub.Q])],
where [J.sub.P] = [SIGMA] [p.sup.2.sub.i], [J.sub.Q] = [SIGMA] [q.sup.2.sub.i] and [J.sub.PQ] = [SIGMA][p.sub.i][q.sub.i], considering [p.sub.i] and [q.sub.i] as the frequencies of the i allele in the P and Q cultivars. Based on the matrix of Nei's (1972) genetic distance, a dendrogram was constructed using the Unweighted Pair Group Method Arithmetic Average (UPGMA). To verify the adjustment between the distance matrix and the dendrogram, a cophenetic correlation coefficient (r) was applied (SOKAL; ROHLF, 1962) using the GENES program package software (CRUZ, 2007).
Results and discussion
Among the five V. rotundifolia cultivars analyzed based on eight SSR loci, four different SSR profiles were detected (Table 2). 'Magoon' matched the same genetic profile of 'Regale' in the present study and the 'Regale' present in the Grape DNA Identification Reference Database, which is maintained by the Foundation Plant Service (University of California, Davis), indicating that the genotype was misnamed at the time of introduction and that the same genotype was planted under different names. The occurrence of misidentification is common, particularly for such old clonal species as Vitis, and it has been observed that 5 to 10% of the grape cultivars maintained in grape collections are incorrectly annotated (ANDRES et al., 2007). The identification of the V. labrusca cultivars was part of a previous study (SCHUCK et al., 2009), and the SSR profiles of 'Bordo', 'Goethe', 'Isabel', 'Niagara Rosada' and 'Marta' were the same as those available in the database for the same cultivars using the same SSR markers used in the present study. Therefore, these cultivars were correctly identified (Table 2).
Table 3 shows the alleles and their frequencies obtained for the different loci, with the allele sizes ranging from 118 bp (VVS2) to 301 bp (VVMD32). There were found important differences in the allelic composition, reflecting the existence of different sets of alleles for V. rotundifolia and V. labrusca and the existence of private alleles in each set. There were 23 alleles specific for the V. rotundifolia cultivars, 32 specific to V. labrusca and three alleles (246, 249 and 259 bp) common to the two groups. Private alleles were found in the V. rotundifolia cultivars Bountiful (4) and Carlos (3) and in the V. labrusca cultivars Niagara Rosada (2), Goethe (7) and Marta (1). The most frequent alleles were VVS2-147 and -149, VVMD5-230 and -234, VVMD7-235 and -243, VVMD31-201, VVMD32-249 and -301, and VrZAG62-199, showing frequencies greater than 20%. In contrast, 18 alleles were relatively infrequent (a frequency less than 10%). It is also worth noting that, of the alleles with a frequency higher than 20%, seven were unique to V. rotundifolia, further emphasizing the differences in the allelic composition of this species.
The alleles found in this study and in another study (RIAZ et al., 2008) indicate that the V. rotundifolia alleles are very different in size and frequency from those of Euvitis species. Riaz et al. (2008) reported the presence of specific alleles in a genetic diversity study of 57 accessions of V. vinifera and V. rotundifolia and a VR hybrid (V. vinifera x V. rotundifolia), including the loci VVMD7-245, VVMD27-197, -199 and -215, and VrZAG62-199 also used in our study. These authors also found that, of the total 184 alleles of 14 microsatellites loci used, 88 were specific to V. rotundifolia. In the present study, there was a relatively high number of alleles (39%) specific to the V. rotundifolia cultivars (Table 4); however, these alleles were common among the group of V. rotundifolia cultivars, suggesting that the genetic base of these cultivars is limited.
The main genetic parameters, such as the number of alleles per locus (N), percentage of polymorphic loci (P), observed ([H.sub.o]) and expected ([H.sub.e]) heterozygosity, estimated frequencies of null alleles (r) and polymorphic information content (PIC) for the eight SSR loci are shown in Table 4.
A total of 58 alleles were amplified, ranging from five (VVM5) to nine (VVMD31), with an average of 7.25 alleles per locus. These SSR loci have previously been used in the genetic characterization of other grape cultivars (LEAO et al., 2009). Moreno-Sanz et al. (2008) analyzed the diversity among 46 accessions of V. vinifera and obtained a total of 37 alleles, with an average of 6.2 alleles per locus using six of the eight loci used in the present study. A larger number of alleles per SSR loci were observed in the V. vinifera cultivars (11) (MARTIN et al., 2003), French-American hybrid cultivars (11) (POLLEFEYS; BOUSQUET, 2003) and table-grape cultivars (18) (IBANEZ et al., 2009). In the case of the V. vinifera and table-grape cultivars, it is likely that the large number of cultivars analyzed (176 and 376, respectively) could account for the marginally higher number of alleles detected.
The expected heterozygosity ([H.sub.e]) averaged 79.95%, ranging from 71.47% (VVMD5) to 85.59% (VVMD31). These values are higher than those reported by Salmaso et al. (2008) for 13 local northeastern Italian cultivars (20-79.6%) and Riaz et al. (2008) for V. vinifera, V. rotundifolia and V. vinifera x V. rotundifolia hybrids cultivars (5-85%) but similar to those found in the grapevine collections of Santa Catarina State, which are composed mainly of European, American and hybrid cultivars (SCHUCK et al., 2009).
The observed heterozygosity varied between 37.14% (VVMD5) to 97.14% (VvMd27), with a mean value of 79.44%, slightly higher than the corresponding expected heterozygosity for all of the loci, with the exception of the VVS2, VVMD5 and VVMD32 loci. The estimated frequency of null alleles (r) was negative for five out of eight loci and positive for each of those loci that showed [H.sub.o] < [H.sub.e] and only high for VVMD5. For the VVMD5 locus, the [H.sub.e] was high (71.47%), as with the other loci, but a very low [H.sub.o] was observed (37.14%). For the other loci (VVS2 and VVMD32), the He value was slightly higher than the [H.sub.o] value, and the frequency of null alleles was very low (Table 4). Nevertheless, the [H.sub.e] and [H.sub.o] values were high for these two loci. Such null alleles can arise when mutations prevent the primers from binding to the region. Cipriani et al. (2008) reported that a locus with a positive null allele value indicates an excess of homozygotes, but this does not necessarily imply that a null allele is present. Hence, the assumption of homozygosity rather than heterozygosity for a null allele could be considered adequate in the present study. Indeed, the highest number of homozygous cultivars in relation to the others was found for the VVMD5 locus (Table 2), a trend also observed by Martin et al. (2003). In the present study, the average value of observed heterozygosity (79.44%) was higher than that obtained for the V. vinifera (70.7%) (IBANEZ et al., 2003) and V. rotundifolia (60%) (RIAZ et al., 2008) cultivars but is in the same range as that obtained when rootstock cultivars are included in the analyses (ANDRES et al., 2007; THIS et al., 2004). Additionally, it was observed in the present work that the values of the expected and observed heterozygosity were greater than 70% for the majority of the loci (93.75%), indicating a high genetic variability of the markers analyzed. A high heterozygosity is commonly found in vegetatively propagated perennial species, such as grape (ARADHYA et al., 2003).
To characterize the usefulness of the eight SSR markers, the polymorphic information content (PIC) of each locus was assessed (Table 4). The PIC values were higher than 0.25 for more than 85% of the loci analyzed. The loci VVS2, VVMD27 and VrZAG79, which exhibited eight, seven and seven alleles, respectively, were also the most informative loci in other studies (MARTIN et al., 2003). The number of alleles per locus, expected heterozygosity and polymorphic information content indicate VVMD31 as the locus with the highest information content (nine alleles, [H.sub.e] = 85.59% and PIC = 0.34), whereas VVMD5 (PIC = 0,19) is the least informative. According to Botstein et al. (1980), markers with PIC values between 0.25 and 0.50 are considered moderately informative, and the markers chosen in this study are consistent with this criterion. The mean value of PIC (0,2825) and the number of alleles per locus (7,25) obtained in our study were lower than in previous studies: 0,782 and 11 (MARTIN et al., 2003) and 0,777 and 7,8 (GONZALEZ-ANDRES et al., 2007), respectively. The authors of each study used six microsatellite loci, five (VVS2, VVMD5, VVMD7, VrZAG62 and VrZAG79) equal to those used in our study, to characterize the genetic diversity of 318 and 79 Spanish cultivars, respectively. The lowest number of alleles per locus and the PIC value for the loci used in this study can be attributed to the smaller number of cultivars analyzed (9).
A dendrogram depicting the genetic distance among the ten cultivars analyzed at eight loci was constructed using the UPGMA method (Figure 1). The cophenetic correlation coefficient (r) was 0.97, revealing a correlation between the dendrogram and the distance matrix. According to Sokal and Rohlf (1962), values of r [greater than or equal to] 0.8 indicate a good fit between the original matrix and the cophenetic matrix values.
[FIGURE 1 OMITTED]
The dendrogram analysis identified two main groups: group I, containing the five V. rotundifolia cultivars ('Bountiful', 'Carlos', 'Magnolia', 'Regale' and 'Magoon'), and group II, with the five V. labrusca cultivars ('Bordo', 'Goethe', 'Isabel', 'Martha' and 'Niagara Rosada') (Figure 1). This result was expected because, in addition to the chromosomal differences, there are important distinguishing anatomical and morphological features between these species. For example, Euvitis species, such as V.labrusca, have a haploid number of 19 chromosomes, longitudinally shredding bark, lack lenticels on the stems, and possess a non-continuous pith that is segmented by diaphragms. In contrast, V. rotundifolia has 20 haploid chromosomes, adherent bark, prominent lenticels on the shoots and fruit, and a continuous pith lacking diaphragms (POMMER, 2003).
As expected, 'Regale' and 'Magoon' in the V. rotundifolia group presented a distance equal to zero (Figure 1). 'Magoon' showed the same genetic profile as the 'Regale' in this study and the 'Regale' present in the grape reference database (Table 2). The distance from 'Magnolia' to 'Regale' was 26%, and the distance from 'Carlos' to this group was 38.12% (Figure 1). 'Magnolia' shared 74% of its alleles with 'Regale', whereas 'Carlos' shared 67 and 62% of its alleles with 'Magnolia' and 'Regale', respectively (Table 2). These three cultivars have their geographic origin in North Carolina, and 'Magnolia' is also reported to be in the pedigree of 'Regale', showing the genetic similarity between these cultivars (RIAZ et al., 2008). 'Bountiful' was the most distant cultivar from all of the others in the same group, with a distance of 85.76% (Figure 1). Moreover, 'Bountiful' showed the largest number of private alleles within the V. rotundifolia group (Table 3).
In the V. labrusca group, 'Bordo' and 'Marta' were separated by a distance of 20.8% (Figure 1) and differed genetically only at four alleles (Table 2). 'Niagara Rosada' was separated from 'Bordo' and 'Marta' by a distance of 41.58%, and 'Isabel' was separated from this group by 48.51%. 'Goethe' was the cultivar most distant from all of the others of the same group, with a distance of 74.1% (Figure 1) and, within the V. labrusca group, was the most divergent cultivar and with the largest number of private alleles (Table 3).
The knowledge of the most divergent cultivars is essential to guide breeders in choosing the best combination for a cross. However, when selecting the parents, the agronomic characteristics of the genotypes and the purpose of the breeding program should be considered in addition to the genetic divergence. In a breeding program aimed at creating rootstocks, it is essential to choose cultivars with resistance and/or tolerance to biotic and abiotic stress. Regarding the cultivars characterized in this study, it is known that the V. rotundifolia cultivars are resistant to almost all of the pests and diseases of grapevine. It is worth highlighting the cultivars with resistance ('Bountiful' and 'Magnolia') and tolerance ('Regale') to the main soil pest of southern Brazil, ground pearl (BOTTON; COLLETA, 2010). The cultivars belonging to the V. labrusca group are well adapted to the vineyards of southern Brazil and display tolerance to Fusarium oxysporum f.sp. herbemontis (GARRIDO et al., 2004), making them an important genetic source for grapevine breeding programs.
SSR markers are efficient tools to confirm the identity and to assess the genetic diversity of Vitis cultivars.
The dendrogram clustered the ten grapevine cultivars in two groups, proving to be efficient in the separation of the cultivars based on their genealogy and botanical classification, showing relationships among cultivars of the same group.
The V. rotundifolia and V. labrusca groups have a high genetic diversity and can be used to support breeding programs.
ANDRES, M. T.; CABEZAS, J. A.; CERVERA, M. T.; BORREGO, J.; MARTINEZ-ZAPATER, J. M.; JOUVE, N. Molecular characterization of grapevine rootstocks maintained in germplasm collections. American Journal of Enology and Viticulture, v. 58, n. 1, p. 75-86, 2007.
ARADHYA, M. K.; DANGL, G. S.; PRINS, B. H.; BOURSIQUOT, J. M.; WALKER, M. A.; MEREDITH, C. P.; SIMON, C. J. Genetic structure and differentiation in cultivated grape, Vitis vinifera L. Genetics Research, v. 81, n. 3, p. 179-192, 2003.
BOTSTEIN, D.; WHITE, R. L.; SKOLNICK, M.; DAVIS, R. V. Construction of a genetic linkage map in man using restriction fragment lenght polymorphisms. American Journal of Human Genetics, v. 32, n. 3, p. 314-331, 1980.
BOTTON, M.; COLLETA, D. Avaliacao da resistencia de cultivares de Vitis rotundifolia a perola-da-terra (Hemiptera: Margarodidae) na regiao sul do Brasil. Acta Scientiarum. Agronomy, v. 32, n. 2, p. 213-216, 2010.
BOWERS, J. E.; DANGL, G. S.; VIGNANI, R.; MEREDITH, C. P. Isolation and characterization of new polymorphic simple sequence repeat loci in grape (Vitis vinifera L.). Genome, v. 39, n. 4, p. 628-633, 1996.
BOWERS, J. E.; DANGL, G. S.; MEREDITH, C. P. Development and characterization of additional microsatellite DNA markers for grape. American Journal of Enology and Viticulture, v. 50, n. 3, p. 243-246, 1999.
BROOKFIELD, J. F. Y. A simple new method for estimating null allele frequency from heterozygote deficiency. Molecular Ecology, v. 5, n. 3, p. 453-455, 1996.
CARIMI, F.; MERCATI, F.; ABBATE, L.; SUNSERI, F. Microsatellite analyses for evaluation of genetic diversity among Sicilian grapevine cultivars. Genetic Resources and Crop Evolution, v. 57, n. 1, p. 703-719, 2010.
CIPRIANI, G.; MARRAZZO, M. T.; DI GASPERO, G.; PFEIFFER, A.; MORGANTE, M.; TESTOLIN, R. A set of microsatellite markers with long core repeat optimized for grape (Vitis spp.) genotyping. BMC Plant Biology, v. 8, n. 127, p. 1-13, 2008.
CRUZ, C. D. Programa Genes versao 2007.0.0 Aplicativo computacional em genetica e estatistica. Vicosa: Universidade Federal de Vicosa, Departamento de Biologia Geral, 2007. Available from: <http://www.ufv.br/ dbg/genes/gdown1.htm>. Access on: Sept. 10, 2010.
DZHAMBAZOVA, T.; HVARLEVA, T.; HADJINICOLI, A.; TSVETKOV, I.; ATANASSOV, A.; ATANASSOV, I. Characterization of grapevine rootstocks using microsatellite markers. Biotechnology and Biotechnological Equipment, v. 21, n. 1, p. 58-62, 2007.
GARRIDO, L. R.; SONEGO, O. R.; GOMES, V. N. Fungos associados com o declinio e morte de videiras no Estado do Rio Grande do Sul. Fitopatologia Brasileira, v. 29, n. 3, p. 322-324, 2004.
GONZALEZ-ANDRES, F.; MARTIN, J. P.; YUSTE, J.; RUBIO, J. A.; ARRANZ, C.; ORTIZ, J. M. Identification and molecular biodiversity of autochthonous grapevine cultivars in the 'Comarca del Bierzo', Leon, Spain. Vitis, v. 46, n. 2, p. 71-76, 2007.
IBANEZ, J.; ANDRES, M. T.; MOLINO, A.; BORREGO, J. Genetic study of key Spanish grapevine varieties through microsatellite analysis. American Journal of Enology and Viticulture, v. 54, n. 1, p. 22-30, 2003.
IBANEZ, J.; VARGAS, A. M.; PALANCAR, M.; BORREGO, J.; ANDRES, M. T. Genetic relationships among table-grape varieties. American Journal of Enology and Viticulture, v. 60, n. 1, p. 35-42, 2009.
LEAO, C. S.; RIAZ, S.; GRAZIANI, R.; DANGL, G. S.; MOTOIKE, S. Y.; WALKER, M. W. Characterization of a brazilian grape germplasm collection using microsatellite markers. American Journal of Enology and Viticulture, v. 60, n. 4, p. 517-524, 2009.
LEWIS, P.; ZAYKIN, D. Genetic data analysis: computer program for the analyses of allelic data (software). Version I. 0. 2001. Available from: <http://alleyn.eeg.uconn.edu/ gda>. Access on: Sept. 3, 2011.
MARTIN, J. P.; BORREGO, J.; CABELLO, F.; ORTIZ, J. M. Characterization of the Spanish diversity grapevine cultivars using sequence-tagged microsatellite site markers. Genome, v. 46, n. 1, p. 10-18, 2003.
MORENO-SANZ, P.; SUAREZ, B.; LOUREIRO, M. D. Identification of synonyms and homonyms in grapevine cultivars (Vitis vinifera L.) from Asturias (Spain). Journal of Horticultural Science and Biotechnology, v. 83, n. 6, p. 683-688, 2008.
NEI, M. Genetic distance between populations. American Naturalist, v. 106, n. 949, p. 283-292, 1972. OIV-Organizacion Internacional de
la Vina y el Vino. Estadistica: informe del director general sobre la situacion de la vitivinicultura em 2007. Available from: <http://www.oiv.int/oiv/info/esstatistiquessecteurvitivinic ole>. Access on: June 5, 2011.
OLIVEIRA, P. R. D.; SCOTTON, D. C.; NISHIMURA, D. S.; FIGUEIRA, A. Analise da diversidade genetica por AFLP e identificacao de marcadores associados a resistencia a doencas em videira. Revista Brasileira de Fruticultura, v. 27, n. 3, 454-457, 2005.
OLIEN, W. Introduction to the Muscadines. In: BASIOUNY, F. M.; HIMELRICK, D. G. (Ed.). Muscadine grapes. Alexandria: ASHS Press, 2001. p. 1-13.
ORTIZ, J. M.; MARTIN, J. P.; BORREGO, J.; CHAVEZ, J.; RODRIGUES, I.; MUNOZ, G.; CABELLO, F. Molecular and morphological characterization of a Vitis gene bank for the estabilishment of a base collection. Genetic Resources and Crop Evolution, v. 51, n. 4, p. 403-409, 2004.
POLLEFEYS, P.; BOUSQUET, J. Molecular genetic diversity of the French-American grapevine hybrids cultivated in North America. Genome, v. 46, n. 6, p. 1037-1048, 2003.
POMMER, C. V. Uva tecnologia de producao, poscolheita, mercado. In: POMMER, C. V.; TERRA, M. M.; PIRES, E. J. P. Cultivares, Melhoramento e Fisiologia Porto Alegre: Cinco Continentes, 2003. p. 109-294.
RIAZ, S.; DANGL, G. S.; EDWARDS, K. J.; MEREDITH, C. P. A microsatellite based framework linkage map of Vitis vinifera L. Theoretical and Applied Genetics, v. 108, n. 5, p. 864-872, 2004.
RIAZ, S.; TENSCHER, A. C.; SMITH, B. P.; NG, D. A.; WALKER, M. A. Use of SSR markers to assess identity, pedigree, and diversity of cultivated muscadine grapes. Journal of the American Society for Horticultural Science, v. 133, n. 4, p. 559-568, 2008.
SALMASO, M.; VALLE, R. D.; LUCCHIN, L. Gene pool variation and phylogenetic relationships of an indigenous northeast Italian grapevine collection revealed by nuclear and chloroplast SSRs. Genome, v. 51, n. 10, p. 838-855, 2008.
SCHUCK, M. R.; MOREIRA, F. M.; GUERRA, M. P.; VOLTOLINI, J. A.; GRANDO, M. S.; LIMA DA SILVA, A. Molecular characterization of grapevine from Santa Catarina State using microsatellite markers. Pesquisa Agropecuaria Brasileira, v. 44, n. 5, p. 487-495, 2009.
SEFC, K. M.; REGNER, F.; TURETSCEIK, E.; GLOSSL, J.; STEINKELLNER, H. Identification of microsatellite sequences in Vitis riparia and their applicability for genotyping of different Vitis species. Genome, v. 42, n. 3, p. 367-373, 1999.
SOKAL, R. R.; ROHLF, F. J. The comparison of dendograms by objective methods. Taxon, v. 11, n. 2, p. 33-40, 1962.
THIS, P.; JUNG, A.; BOCCACCI, P.; BORREGO, J.; BOTTA, J.; COSTANTINI, L.; CRESPAN, M.; DANGL, G. S.; EISENHELD, C.; FERREIRAMONTEIRO, F.; GRANDO, S.; IBANEZ, J.; LACOMBE, T.; LAUCOU, V.; MAGALHAES, R.; MEREDITH, C. P.; MILANI, N.; PETERLUNGER, E.; REGNER, F.; ZULINI, L.; MAUL, E. Development of a standard set of microsatellite reference alleles for identification of grape cultivars. Theoretical and Applied Genetics, v. 109, n. 7, p. 1448-1458, 2004.
THOMAS, M. R.; SCOTT, N. S. Microsatellite repeats in grapevine reveal DNA polymorphisms when analysed as sequence-tagged sites (STSs). Theoretical and Applied Genetics, v. 86, n. 8, p. 985-990, 1993.
VARGAS, A. M.; ANDRES, T. M.; BORREGO, J.; IBANEZ, J. Pedigrees of fifty table-grape cultivars. American Journal of Enolology and Viticulture, v. 60, n. 4, p. 525-532, 2009.
Received on July 9, 2012. Accepted on September 17, 2012.
Mariane Ruzza Schuck (1) *, Luiz Antonio Biasi (1), Flavia Maia Moreira (2), Aparecido Lima da Silva (3), Summaira Riaz (4) and Michael Andrew Walker (4)
(1) Departamento de Fitotecnia e Fitossanitarismo, Setor de Ciencias Agrarias, Universidade Federal do Parana, Rua dos Funcionarios, 1540, Cx. Postal 19061, 80035-050, Curitiba, Parana, Brazil. (2) Instituto Federal de Educacao, Ciencia e Tecnologia de Santa Catarina, Sao Jose, Santa Catarina, Brazil. (3) Departamento de Fitotecnia, Centro de Ciencias Agrarias, Universidade Federal de Santa Catarina, Florianopolis, Santa Catarina, Brazil. (4) Department of Viticulture and Enology, University of California, Davis, California, United States of America. * Author for correspondence. E-mail: email@example.com
Table 1. Description of the ten grapevine cultivars, type of flower, fruit color, species or parentage and geographical origin. Cultivar Flower Fruit Species or Geographical type (1) color (2) parentage origin Vitis rotundifolia 'Bountiful' H B Vitis USA rotundifolia 'Carlos' H Br Howard' x USA ('Topsail' x Tarheel') 'Magnolia' H Br ('Thomas' x North Caroline 'Scuppernong') x ('Topsail' x Tarheel') 'Magoon' H Thomas'x Mississippi 'Burgaw' 'Regale' H Bp Hunt' x North Caroline Magnolia' Vitis labrusca 'Bordo' H B V. labrusca x Ohio V. aestivalis x V. vinifera 'Goethe' H PR Muscat of USA Hamburg (Black Muscat) (V. vinifera) x Carter' 'Isabel' H B V. labrusca x South Caroline V. vinifera 'Marta' H PR V. labrusca x USA V. vinifera 'Niagara H PR Somatic Tundiai (SP) Rosada' mutation of Niagara branca' ('Concord' and Cassady' V. labrusca x V. vinifera) (1) Hermaphrodite. (2) B=black; Br=bronze; Bp=black to dark purple; PR=pink-red. Table 2. Genetic profile of cultivars of Vitis spp. with the observed base par (bp). Vitis rotundifolia Locos 'Bontiful' 'Carlos' 'Magnolia' 'Magoon' VVS2 153-153 147-149 147-147 147-147 VVMD5 230-246 230-246 230-230 226-230 VVMD7 235-235 243-245 243-245 243-243 VVMD27 199-199 197-215 185-215 185-197 VVMD31 246-250 168-170 170-210 170-210 VVMD32 249-301 249-301 249-301 249-301 VrZag62 215-223 199-215 199-215 199-199 VrZag79 255-255 257-287 259-287 255-259 Vitis rotundifolia Vitis labrusca (1) Locos 'Regale' 'Bordo' 'Goethe' 'Isabel' VVS2 147-147 118-130 120-130 118-146 VVMD5 226-230 234-234 230-236 236-236 VVMD7 243-243 235-239 235-247 235-249 VVMD27 185-197 179-181 181-183 175-179 VVMD31 170-210 201-213 203-209 201-213 VVMD32 249-301 248-248 246-252 248-272 VrZag62 199-199 200-202 190-204 200-202 VrZag79 255-259 247-247 239-247 237-247 Vitis labrusca (1) Locos 'Marta' 'Niagara Rosada' VVS2 120-146 118-128 VVMD5 234-234 234-234 VVMD7 235-249 235-241 VVMD27 179-181 175-181 VVMD31 201-203 201-201 VVMD32 248-248 248-272 VrZag62 200-202 200-202 VrZag79 247-264 237-259 (1) Cultivars analyzed by Schuck et al. (2009). Table 3. Allele size (AS) and allele frequencies (AF) for 8 SSR markers evaluated among V. rotundifolia and V. labrusca cultivars. VVS2 VVS2 VVMD5 VVMD7 VVMD27 AS AF FA AS TA AF FA AS TA AF FA AS TA AF FA 118 0.15 226 0.10 235 0.35 175 0.10 120 0.10 230 0.35 239 0.05 179 0.15 1283 0.05 234 0.30 2413 0.05 181 0.20 130 0.10 236 0.05 243 0.30 1834 0.05 146 0.10 246 0.10 245 0.10 185 0.15 147 0.35 2474 0.05 197 0.15 1492 0.25 249 0.10 199 0.10 1531 0.10 215 0.10 VVS2 VVMD31 VVMD32 VrZag62 VrZag79 AS AS TA AF FA AS TA AF FA AS TA AF FA AS TA AF FA 118 1682 0.05 246 0.05 1904 0.05 237 0.10 120 170 0.20 248 0.20 1991 0.30 2394 0.05 1283 201 0.25 249 0.25 200 0.20 247 0.15 130 203 0.10 2524 0.05 202 0.20 255 0.20 146 2094 0.05 272 0.10 2044 0.05 2572 0.05 147 210 0.15 301 0.25 215 0.15 259 0.20 1492 213 0.10 2231 0.05 2645 0.05 1531 246 0.05 287 0.10 2501 0.05 Alleles in italic are specific to V. labrusca, in bold are specific to V. rotundifolia and underlined are common between the two groups. Private alleles: 1-Bountiful, 2-Carlos, 3-Niagara Rosada, 4-Goethe, 5-Marta. Table 4. Genetic parameters at the eight microsatellite loci, number of alleles per locus (N), percentage of polymorphic loci (P), observed ([H.sub.o]) and expected ([H.sub.e]) heterozygosity, estimated frequencies of null alleles (r) and polymorphic information content (PIC), calculated by grouping all of the cultivars into one group. Loci N p (%) [H.sub.o] [H.sub.e] r PIC (%) (%) (%) VVS2 8 100 0.8128 0.8286 0.0086 0.23 VVMD5 5 100 0.3714 (1) 0.7147 (1) 0.2002 0.19 VVMD7 7 100 0.8857 0.7901 -0.0529 0.26 VVMD27 8 100 0.9714(2) 0.8356 -0.0739 0.34 VVMD31 9 100 0.8571 0.8559 (2) -0.0006 0.34 VVMD32 6 100 0.7143 0.7557 0.0236 0.30 VrZag62 7 100 0.9143 0.7983 -0.0645 0.30 VrZag79 8 100 0.8286 0.8174 -0.0062 0.30 Total 58 Mean 7.25 100 0.7944 0.7995 0.2825 (1) low value; (2) high value.