Molecular characterization of grapevine from Santa Catarina , Brazil , using microsatellite markers

The objective of this work was to characterize the grape germplasm in Santa Catarina, Brazil, using microsatellite DNA markers (simple sequence repeats – SSR). The DNA samples were collected from leaves and shoots of accessions of public and private collections from the counties Urussanga, Nova Trento, Rodeio, São Joaquim, Campos Novos, Videira, and Água Doce. Ten SSR loci (VVS2, VVMD5, VVMD7, VVMD27, VrZAG62, VrZAG79, VVMD25, VVMD28, VVMD31, and VVMD32) were analysed by capillary electrophoresis. Molecular profiling was conducted for 190 grapevines (European, American, and hybrids), and 67 genotypes were obtained. The data were compared with each other and with those from the literature and from online databases, in order to identify varieties and discover cases of synonymy and homonymy. Forty molecular profiles corresponded to known varieties, while 27 genotypes were described for the first time. The existence of typical germplasm composed mainly of American and hybrid varieties is an important finding for local viticulture. Applications of the results rely on quality control and certification at the nursery level. Increasing precision in the characterization of grapevine genotypes may help breeding programs.


Introduction
The Brazilian viticulture occupies 89.9 thousand hectares and yields about 1.3 million tons of grapes per year, concentrated in the south, southwest and northeast regions.The state of Rio Grande do Sul produces 90% of vines, juices and other grape products (IBGE, 2007).
The state of Santa Catarina produces the second largest crop, with approximately 50,000 tons of grapes per year.Wines are produced mostly from Vitis labrusca, an American species, or from hybrids adapted to the Pesq.agropec.bras., Brasília, v.44, n.5, p.487-495, maio 2009 environmental conditions of the state.Pinheiro Preto, Videira, Tangará, Iomerê, Caçador, Urussanga, Rodeio, and Nova Trento are the main grape-growing counties in Santa Catarina.
Since 1998, viticulture in Santa Catarina has undergone intense transformation, with the establishment of new vineyards of European grapes (V.vinifera).New viticultural areas have been established in the highland regions above 900 m, where climatic conditions determine specific features for grape maturation and wine specificity and quality.The most promising new areas are located in São Joaquim, Bom Retiro, Campos Novos, and Água Doce counties (Schuck et al., 2008).
Unlike the European market, where the "terroir" determines the quality of wines, the Brazilian market is based on the consumption of wines from a single variety.These are classified as regular wines, obtained from American and hybrid grapes, and fine table wines obtained from European white and black grape varieties, mainly Chardonnay, Cabernet Sauvignon, Cabernet Franc, Pinot Noir, and Merlot.The correct identification of grapevine germplasm is important since the expected quality of wines is related to characteristics of the cultivar.
In order to distinguish grape varieties, DNA-based markers offers an advantage over morphological descriptors, as they are less prone to being affected by the environment or the developmental stage (Sefc et al., 2001).
The available markers differ in the methodologies used to detect variability at the DNA level.Since 1990, simple sequence repeats (SSR) markers have been increasingly used as molecular descriptors in grape.Their usefulness has been widely demonstrated in the identification and characterization of stock and rootstock varieties, evaluation of genetic variability, pedigree studies, and genetic mapping (Bowers et al., 1999a;Pollefeys & Bousquet, 2003;Adam-Blondon et al., 2004;Riaz et al., 2004;Costantini et al., 2005).The analysis of grape plants with the aid of SSR markers allows identification profiles to be established and varietal reference data banks to be developed (Dettweiller et al., 1998;Lefort & Roubelakis-Angelakis, 2001;Grando et al., 2002).Access to data banks allows information to be shared and contributes to international cooperation to correctly identify grape germplasm.
The aim of this work was to characterize the diversity of the grape germplasm held in public and private collections in Santa Catarina, Southern Brazil, by means of SSR markers.
DNA extraction was done according to Doyle & Doyle (1990), with the following modifications: freeze-dried material (instead of fresh material) was ground and RNase was added to isopropanol during the DNA precipitation phase.Quantification of the DNA extracts was performed in agarose gel (0.8%), and the samples were then diluted to a concentration of 20 ng μL -1 , at the Laboratório de Fisiologia do Desenvolvimento Genético Vegetal, of the Departamento de Fitotecnia, Universidade Federal de Santa Catarina.
Polymerase chain reaction (PCR) was carried out in a 12.5-µL total volume containing 25 mmol L -1 of each dNTP, 0.5 mmol L -1 of each primer, 0.5 U Taq DNA polymerase (GoldTaq -Applied Biosystem, Foster City, CA, USA or BioTaq -Bioline, London, UK), 1X buffer solution, 1.5 mmol L -1 MgCl 2 , and 20 ng of genomic DNA for each sample.The cycling program used was: denaturation of DNA and activation of Taq DNA polymerase at 95°C for 7 min (GoldTaq) or 3 min (BioTaq); 35 cycles of amplification distributed in 45 s at 94°C, 45 s at 50°C (VVS2, VVMD5, VVMD7, VVMD27, VrZAG62, and VrZAG79) or 56°C (VVMD25, VVMD28, VVMD31, and VVMD32), 1 min and 30 s at 72°C; final extension of 7 min at 72°C; cooling at 4°C.Capillary electrophoresis was used to determine the size of alleles.Separation of the microsatellite fragments was performed using the ABI 3100 sequencer and GeneScan 3.7 software (Applied Biosystems).The availability of primers labeled with different fluorescent dyes (Ned, Hex or Fam) enabled the product of different No. of samples Cultivation region (1) Grape accessions No. of samples Cultivation region  PCR reactions to be loaded in multiplexes.Processing of the data generated by the sequencer was done with the Genotyper 3.7 program (Applied Biosystems).
All tests were performed using DNA of the cultivar Chardonnay as a reference with respect to both the quality of amplification and the size of the amplified alleles, allowing the data to be compared with those available in the literature and in databases.This phase of the work was carried out at the Laboratory of Molecular Genetics of the Fondazione Edmund Mach -Istituto Agrario di San Michele all'Adige, in San Michele all'Adige, TN, Italy.
The Identity program (Wagner & Sefc, 1999) was used to calculate the number of alleles (n), the allele frequencies, expected (He) and observed (Ho) heterozygosity, the estimated frequency of null alleles (r), and the probability of identity (PI) at each locus.This software was also used to detect identical genotypes.Genetic similarity between the genotypes was calculated on the basis of the proportion of alleles in common (DPS), using the Microsat software (Minch et al., 1997).A neighbor-joining dendrogram was constructed from a matrix of genetic divergence (D) between the genotypes, calculated as D = 1-DPS, using Phylip (Felsenstein, 1989) and viewed with Tree View (Page, 1996).
Cases of synonymy could be clones of the same variety, which show phenotypic differences (Vignani et al., 1996;Walker et al., 2006).Despite these evident differences, they are not detectable by a low number of SSR markers, which are located in noncoding regions of the genome (Zulini et al., 2005).This was the case of 'Grano D'Oro', a mutation of the cultivar Bordô, which resulted in changes in vigor, productivity, and rusticity.This was also the case of 'Goethe Primo', a variant with white berries from 'Classic Goethe', which has pinkish berries.
Comparison of the data showed some cases of homonymy, that is, clusters of varieties having identical or similar names but different genotypes.This was the case of 'Isabel-SM' accession and the group of synonyms mentioned above, consisting of 'Isabel', 'Isabel Precoce', Catawba S, and Catawba T.
Comparison between the molecular profiles from the ten SSR markers employed in the present work and those available in data banks allowed the recognition of 40 from the 67 distinct genotypes.They were mainly well-known international wine varieties.This comparison also allowed the identification of additional five cases of synonymy with known international varieties.Moreover, seven accessions without variety name in the collection were identified (Table 2).
The SSR profiles for rootstocks VR 043-43, 101-14 Mgt, Paulsen 1103, and SO4 did not correspond to the molecular characterization of the same rootstocks by Andrés et al. (2007).The possibility of errors in the collecting procedures or in the introduction of these materials to the collections should be considered.
The remaining 27 molecular profiles did not correspond to those found in the literature, nor in the data banks (Table 3).The identification and description of unique molecular profiles is an important aspect of the regional viticulture.Nineteen genotypes were described for the first time in this study.Most of them are interspecific hybrids developed in Brazil.In other cases, are old varieties introduced during the first wave of Italian immigration to Southern Brazil at the end of the 19 th century.Old varieties include the rootstock PESC 2 ("porta-enxerto Santa Catarina") from Rodeio, as well as the well-known 'Goethe' cultivar from Urussanga (Schuck et al., 2008).However, unique SSR profiles were also obtained from accessions thought to belong to international varieties as Catawba, Gros Manseng, and Paulsen 1103, which were determined to not true-to-type.Table 2. Comparison of the microsatellite profiles of the analyzed cultivars with those in the international literature and in online databases.
Table 3. Smaller (SZ) and larger (LZ) allele sizes (bp) at ten microsatellite loci for the accessions analyzed in this study, without a genetic correspondence.

Figure 2 .
Figure 2. Dendrogram of the genetic relationships among the 67 grapevine varieties investigated in this study, calculated with proportional shared alleles (DPS)(Bowcock et al., 1994) genetic distance.Non-European varieties are indicated in italics.(a)Not true-to-type.(b)Without genetic correspondence.

Table 1 .
Grape accessions analyzed in this study.