DIFFERENTIAL GENETIC STABILITY IN VINEYARDS OF THE CULTIVAR ‘ITALY’ ( VITIS VINIFERA L.) CULTIVATED IN DIFFERENT REGIONS OF SOUTHERN AND SOUTHWESTERN BRAZIL

During more than 50 years the vegetative propagation has been the form of maintaining and multiplication of the cv. ‘Italy’ vineyards, a ‘Bicane’ x ‘Muscat Hamburg’ hybrid. In the current study, polymorphism in 17 microsatellite loci was used to evaluate the genetic stability at DNA level in vineyards of cv. ‘Italy’ planted in different regions of the states of Paraná and São Paulo, Brazil. Unchanged and equal allele frequency indicating genetic stability was reported in 47% of the microsatellite loci in vineyards of six localities, while allele frequency variation has been observed in Scu15vv , Udv44 , Udv74 , Udv96 , Udv107 , Udv108 , Vvmd5 , Vvmd6 and Vvs3 microsatellite loci . Alleles Udv96 140 and Vvs3 448 , detected in vines in only one of the vineyards, evidenced somatic mutations at


INTRODUCTION
In the vegetative propagation of grape cultivars, there are several natural mutations at gene level which are linked to skin coloration and which play a significant role in the diversification of grape cultivars (Walker et al., 2007). A somatic mutation occurring on one side branch of the cv. 'Italy' (VIVC 5582), grown in Santa Mariana, northeastern region of the state of Paraná, Brazil, originated the cv. 'Rubi' (VIVC 22689;Kishino and Mashima, 1980). Another somatic mutation occurring on one side branch of cv.
The cv. 'Italy' ('Piróvano 65'), which gave rise to three different cultivars ('Rubi', 'Benitaka' and 'Redmeire'), is a hybrid from the cross between 'Bicane' x 'Muscat Hamburg'. It was introduced in the state of São Paulo, Brazil, in 1927 and its culture started in the northeastern region of the state of Paraná (Marialva) in 1962 (Camargo, 1998). For more than fifty years the vegetative propagation has been the form of maintaining and multiplication of cv. 'Italy' vineyards. Consequently, somatic variation has been the primary source of genetic variation employed to produce new phenotypic features. At molecular level, isozymes were used to measure the genetic stability of the introduced cv. 'Italy' (Oliveira-Collet et al., 2005). The lack of allele variation in cv. 'Italy' and in the 'Rubi', 'Benitaka', and 'Brasil' cultivars would indicate apparent genetic stability during the cultivation period. However, high frequency of the null EST-3 carboxylesterase phenotype (61.7%) suggested the occurrence of genetic variations within cv. 'Italy' and their derivatives (Orasmo et al., 2007). High level of polymorphism within and between the cultivars 'Italy', 'Rubi','Benitaka' and 'Brasil' (65.1%) were detected by random amplified polymorphic DNA (RAPD) markers, contrasting the hypothesis that the four cultivars were genetically identical (Maia et al., 2009).
Results described by Orasmo et al. (2007) and Maia et al. (2009) indicate that the genetic diversity of cv. 'Italy' must be high for a cultivar that has been maintained by vegetative propagation, in contrast to genetic uniformity preliminarily described by Oliveira-Collet et al. (2005). The simple sequence repeats (SSR) of DNA in current study were employed to investigate the genetic stability at DNA level in vineyards of cv. 'Italy' maintained in different regions of the states of Paraná and São Paulo, Brazil. SSR markers, also known as microsatellites, proved to be efficient to characterize the genetic diversity between varieties of grapes since the 1990s (Thomas and Scott, 1993;Thomas et al., 1994;Sefc et al., 1999). Although several studies have demonstrated the ineffectiveness of microsatellites to detect genetic diversity within clone collections (Aradhya et al., 2003;Martínez et al., 2006;Pelsy et al., 2010;Emanuelli et al., 2013), microsatellite markers may reveal the level of somatic mutations occurring in vines of cv. 'Italy'.
Although mutations that generated the cultivars 'Rubi', 'Benitaka' and 'Redmeire' were described only in the region of Santa Mariana, Florai, and Marialva in the state of Paraná, Brazil, it is possible that not easily identified mutations by morphological characteristics may be occurring in vines of cv. 'Italy' grown in different regions of the country. A high occurrence of somatic mutations may indicate low genetic stability in vineyards of cv. 'Italy', while a low incidence of somatic mutations may indicate greater genetic stability. In addition, information on greater or lesser genetic identity of the vineyards of cv. 'Italy' may be useful for agricultural and cultural practices (nutrition, fertilization, pruning, thinning, among others) of vineyards since plants genetically divergent in a vineyard may respond differently to the same agricultural and cultural practices. Moreover, lesser genetic stability of the vineyards may stimulate greater investment in the search of promising somatic mutations to generate new cultivars. Current hypothesis analyzes whether polymorphism in microsatellite loci may reveal a differential genetic stability in vineyards of cv. 'Italy' cultivated in different regions of the states of Paraná and São Paulo, Brazil.

Samples of the cv. 'Italy' of Vitis vinifera
DNA was extracted from 69 plants obtained from vineyards in two Brazilian states, namely, Marialva, Paiçandu and Urai in the state of Paraná, and in Jales, Pilar do Sul and São Miguel Arcanjo in the state of São Paulo (Figure 1). Partially expanded leaves without phytosanitary contaminants were collected in 7 to 15 plants in each vineyard. The samples were 68 individually stored in labelled plastic screen bags to avoid the mixture of vineyards, maintained on ice (4 °C) and transferred to the laboratory. The samples were frozen in liquid nitrogen and stored at -80 °C until DNA extraction.

DNA extraction
DNA was extracted from leaf tissues following Thomas et al. (1994) with minor modifications. The modifications included the use of 100 mg of leaves from individual plants replacing the 2.0 g of leaves used in the Thomas and Scott (1993) protocol. After DNA extraction, DNA quantity and quality were determined by 0.8% agar gel electrophoresis buffered with 1x TAE (0.04 M Tris-Acetate and 0.001 M EDTA). A standard DNA ladder (λ phage, 50, 100 and 150 ng) was used as a marker. The gel was stained with 0.5 μg·m/L ethidium bromide, and the image was visualized with a Molecular Image LOCCUS L-PIX -HE (LOCCUS DO BRASIL LTDA., São Paulo SP Brazil) with Picasa 3 software. UV quantification by visual comparison with known quantities of lambda DNA (Invitrogen) averaged between 15 ng/μL and 200 ng/μL per sample. After quantification, DNA samples were diluted in a 10 ng/μL concentration.

Primer selection and amplification reactions
Thirty-eight SSR primers previously developed for Vitis vinifera were used with DNA samples to define the polymorphic primers. After screening the 38 SSR primers, a set of 17 polymorphic SSR primers, designed VVS (Thomas and Scott, 1993), VVMD (Bowers et al., 1996(Bowers et al., , 1999, SCU (Scott et al., 2000), and UDV (Di Gaspero et al., 2005) (Table I), were selected for further analysis. Only 17 primers were polymorphic and used for all 69 plants.
Polymerase chain reaction (PCR) was performed with Techne TC-512 thermal cycler. Amplifications were performed by Touchdown PCR program (Don et al., 1991) using volumes of 20 µL containing 25 ng of genomic DNA, buffer reaction (10 mM Tris-HCl pH 8.8), 2.0 mM of MgCl 2, 0.1 mM each of dATP, dGTP, dCTP, dTTP, 0.3 µM of each primer (F and R primers), and 1 unit of Taq DNA polymerase (Invitrogen) and Milli-Q water to bring the reaction to the final volume. Electrophoresis was performed in a 4% MS-8 agar gel using 0.5 x TBE buffer (44.5 mM Tris-borate and 1 mM EDTA) at 60 V, for 4 h. After electrophoresis, the gels were stained with ethidium bromide at 0.5 μg·m/L and images were taken with a Molecular Image LOCCUS L-PIX -HE using Picasa 3 software. The size of PCR fragments was determined with a 100 bp DNA Ladder (Invitrogen).

SM27
The dendrogram generated by Nei´s coefficient from the analysis of individual vines of cv. 'Italy' (constructed from 17 microsatellite primers data using adegenet package from R Development Core Team, 2013) identified two well-defined larger groups and two smaller groups formed by vines from six cv. 'Italy' vineyards ( Figure 2). Dendrogram showed several homogeneous groups formed by vines from São Miguel Arcanjo (80%), Jales (60%), Urai (53%), and Paiçandu (80%) vineyards. Heterogeneous groups were also formed by mixture of vines from different vineyards. Genetic identity in the dendrogram was evident between 60% of the vines of the same or of different vineyards. Vines from Jales, Pilar do Sul, Urai, Marialva, and São Miguel Arcanjo vineyards showed genetic identity, so that the six 'Italy' vineyards represented a moderate genetic divergence.  (Nei, 1978)  The extra alleles observed as having an intensity comparable with the two original alleles defined a triallelic pattern at Udv107 and Scu10vvw microsatellite loci. The tri-allelic phenotype was not considered at analysis of polymorphism in the SSR loci of cv. 'Italy', but is important to highlight a third allele extra at SSR locus since the detection of three alleles at the same locus could be the result of a chimeric structure in which genotypes of different layers of cells displayed different alleles. The tri-allelic pattern in SSR loci have been considered an evidence of chimeras, a mechanism of genetic diversification well characterized by Hocquigny et al. (2004) occurring in grapevines clones (Crespan, 2004;Moncada et al., 2006). In cv. 'Italy' vines the chimeras were evident in 13%, 46%, 85%, and 20% of the plants from Urai, São Miguel Arcanjo, Marialva, and Paiçandu, respectively. High level of mean observed heterozygosity in microsatellite loci is expected for cv. 'Italy' as a product of the cross between the 'Bicane' and 'Moscat Hamburg' cultivars (Sousa, 1996). Most grape cultivars are highly heterozygous (Di Vecchi-Staraz et al., 2009;This et al., 2011) due to vegetative (clonal) propagation to maintain the selected genotypes. High Ho rates have been reported in cultivars of Central Europe (Ho = 0.776 -0.904; Sefc et al., 2000) and Spain (Ho = 0.756 -0.909; Martín et al., 2003, and Ho = 0.744 -0.949;Fernández-González et al., 2007), in cultivars of Tunisia (Ho = 0.71 -0.98; Snoussi et al., 2004) and also in different species of Vitis (Ho = 0.68 -0.92; Fernández et al., 2008). High rates of observed heterozygosity (Ho = 0.743 -0.857; Carimi et al., 2010, and Ho = 0.78 -0.92;Alba et al., 2014, respectively) were reported in genotypes of Italian grapevines and Italian table grapes (cultivars and candidate cultivars). Lower rates with greater interval were reported in cultivars in Portugal (Ho = 0.60 -0.97 and 0.55 -0.91; Lopes et al., 1999Lopes et al., , 2006, in North America (Ho = 0.30 -1.0; Pollefeys and Bousquet, 2003), in hybrids of France (Ho = 0.0 -1.0; Merdinoglu et al., 2005), in Peruvian and Argentine Criollas varieties (Ho = 0.615 -1.000; Martínez et al., 2006), and in Iranian grape cultivars (Ho = 0.49 -0.97; Doulati-Baneha et al., 2013). High rates of observed heterozygosity in the cv. 'Italy' (Ho = 0.7000 -0.9647; Table III) agrees to the highest Ho values reported for cultivars of Central Europe, Spain, Tunisia, and different Italian genotypes and species of Vitis.
Somatic mutations are also expected in the vegetative propagation of cv. 'Italy'. Alleles Udv96 140 and Vvs3 448 detected only in the vines of Paiçandu (Table  II) evidence somatic mutations at molecular level in cv. 'Italy'. Somatic mutations in microsatellite loci of clones are more often detected by screening large numbers of loci and clones. Riaz et al. (2002) detected intra-varietal polymorphism in 17 microsatellite markers when 100 markers were genotyped in more than 20 clones each for 'Pinot noir' and 'Chardonnay'. Higher polymorphism was detected in 49 microsatellites of 145 clones of 'Pinot' cultivar (22.4%;Hocquigny et al., 2004) and in 84 microsatellite loci of 59 clones of 'Cabernet Sauvignon' (21.4%; Moncada et al., 2006). Current authors detected somatic mutations in two microsatellite loci in cv. 'Italy' after screening of only 17 loci in six clones: a polymorphism of 11.8%, which was consistent with rate described by Moncada and Hinrichsen (2007) in the analysis of 20 loci in 25 clones of 'Carmenére' (10%) from Chile, France and Italy.
On the other hand, the polymorphism at microsatellite loci of the cv. 'Italy' may be higher than 11.8% if the chimeras pattern is considered since the tri-allelic pattern at microsatellite loci have been proposed as a mechanism of genetic diversification in grapevines clones (Crespan, 2004;Hocquigny et al., 2004;Moncada et al., 2006), which have vegetative propagation. The tri-allelic pattern at Udv107 and Scu10vv loci has been observed in 24% of vines in cv. 'Italy' from Uraí (13%), São Miguel Arcanjo (46%), Marialva (85%), and Paiçandu (20% Collet, personal information). The oldest vineyards of cv. 'Italy' in Marialva showed greater genetic stability than the most recently vineyards established in Paiçandu. In contrast, the chimeras were more evident in vines from Marialva (85%). It may be supposed that the older varieties were exposed for a longer time to environmental stress and therefore they had a great chance to accumulate more mutations (Pelsy, 2010).
The genetic potential of Paiçandu vineyards should be underscored. Somatic variants potentially represent a commercial interest if they improve an important agronomical trait (e.g., height, yield, pathogen susceptibility and others) or provide innovation of the product (This et al., 2011). The vineyards of Paiçandu with low genetic stability (greater genetic variability) should be more carefully investigated as a source of somatic mutants with favorable agronomic traits. Somatic mutations contribute towards the generation of genetic variation which may be relevant for improvements. They may eventually give rise to plant variants without going through the gametophytic phase. Somatic variation has a strong relevance in the generation of the cultivars 'Rubi', 'Benitaka', 'Brasil', 'Redmeire' and 'Black Star' (Kishino and Mashima, 1980;Sousa, 1996;Pires et al., 2003;Roberto et al., 2012) (Fernández et al., 2008) and associated to the emergence of null alleles. High frequency of null EST-3 carboxylesterase phenotype (52.3%) has been suggested in cvs. 'Italy' (Orasmo et al., 2007). Null alleles in microsatellite loci have also been reported in varieties and cultivars of grapes from Portugal (Lopes et al., 1999), Spain (Martín et al., 2003) and in North American hybrids and in cultivars from France (Pollefeys and Bousquet, 2003).
The occurrence of somatic crossing-over events is another factor that may lead to changes in the frequency of alleles in microsatellite locus with the emergence of homozygous phenotypes. The somatic recombination induced by retrotransposons was used to explain changes in frequency for berry skin color in the colored ('Benitaka' and 'Brasil') cv. 'Italy' (Oliveira-Collet et al., 2005) and in the high frequency of the null EST-3 carboxylesterase phenotype within cv. 'Italy' (Orasmo et al., 2007). For the 'Benitaka' cultivar, Azuma et al. (2009) (Table II).
Double-strand breaks in DNA leading to the occurrence of somatic recombination may be caused by external factors in response of pathogen attack and/or poisons used to their control, or by internal factors during replication and transposon excision and even in response to intrinsic metabolic activities induced by external factors (Schuermann et al., 2005). Geminivirus infection in experimental conditions also promoted somatic homologous recombination (Richter et al., 2014). Consequently, the exposition of leaves of cv. 'Italy' in different types and concentrations of fungicides and poisons (frequently applied for disease control; M. Collet, personal information) may induce point mutations and/or chromosomal breaks that contribute towards the enhancement of different frequencies of alleles in microsatellite loci of cvs 'Italy' in different regions. The reaction of fungicides in inhibiting seven carboxylesterases from different loci in the leaves of cv. 'Italy' was described by Orasmo et al. (2015).
The emergence of the new alleles Udv96 140 and Vvs3 448 beside of changes in allele frequencies detected at the loci Scu15vv, Udv44, Udv74, Udv96, Udv107, Udv108, Vvmd5, Vvmd6, and Vvs3 have leading to a moderate genetic divergence among the six vineyards of cv. 'Italy' maintained by vegetative propagation. Low rates between cultivated grapevine populations have been commonly related with F ST pairwise rates ranging between 0.0224 (Eastern-Western Europe) and 0.0374 (Western Europe-North Africa). Results showed higher F ST pairwise rates only when the wild population was compared to other populations of cultivated grapevines. The highest genetic differentiation rate (0.0747) was recorded between wild accessions and North African cultivars (Riahi et al., 2012). A moderate genetic divergence among the vineyards of Italy coupled to the somatic variation may be a very powerful tool to improve the classical cv. 'Italy' and a good insight for producers of new and different varieties. The preservation of the inter-and intra-vineyards diversity must be a goal for producers interested in the selection and use of somatic variation.
Although several studies have demonstrated the ineffectiveness of some microsatellites when genetic diversity within clone collections is addressed (Aradhya et al., 2003;Martínez et al., 2006;Pelsy et al., 2010;Emanuelli et al., 2013), current study proved the effectiveness of microsatellites for clonal polymorphism in vegetative propagation of the cv. Italy, due to the somatic variation leading to new alleles and changes in allele frequencies.
Polymorphism in microsatellite loci revealed different genetic stability in vineyards of cv. 'Italy' cultivated in six different regions of Brazil. It actually indicates vineyards with low genetic stability as a possible source of somatic mutants revealing traits of agronomic interest capable of generating new cultivars. Furthermore, the microsatellite primers selected in the present study could be used to investigate the genetic stability of somatic mutants derived from the cultivar 'Italy': 'Rubi', 'Benitaka', 'Brasil'', 'Black Star' and 'Redmeire'.

CONCLUSIONS
The microsatellite markers were effective to evidence somatic mutations and alterations in allele frequencies in the SSR loci that led to a moderate genetic divergence among the vineyards of the cultivar 'Italy' maintained by vegetative propagation in the south and southeast regions of Brasil. Polymorphism in microsatellite loci was also useful to identify the genetically more stable vineyards (from Jales and Pilar do Sul) and to estimate the genetic identity among the vineyards of the cv. 'Italy' cultivated in the six localities of the south and southeast regions of Brasil. A prospect for evolution due to somatic mutations in 'Italy' cultivar was more evident in the vineyard of Paiçandu due to the appearance of two new alleles with defined frequencies. Also, evidence of chimeras in the vineyards of Urai, São Miguel Arcanjo, Marialva, and Paiçandu may be highlighted as potential mechanisms of genetic diversification acting as a probable evolutionary force in 66.6% of the investigated vines.