Mol Biotechnol DOI 10.1007/s12033-012-9600-1

RESEARCH

Sangiovese and Its Offspring in Southern Italy Marica Gasparro • Angelo Raffaele Caputo • Carlo Bergamini Pasquale Crupi • Maria Francesca Cardone • Rocco Perniola • Donato Antonacci

•

Ó Springer Science+Business Media, LLC 2012

Abstract This paper demonstrates the importance of different approaches such as ampelography, historical researches, and molecular analysis to reveal direct parent– child relationship. The aim of this paper was to highlight the degree of relationship to five varieties spread in southern Italy, through ampelographic and molecular characterization: Sangiovese, Mantonico di Bianco, Gaglioppo di Ciro`, Mantonicone, and Nerello Mascalese. Molecular characterization was carried out through 52 SSR molecular markers, showing that Sangiovese and Mantonico di Bianco are the parents of Gaglioppo di Ciro`, Mantonicone, and Nerello Mascalese. Ampelographic description was performed using the method developed by the Organisation Internationale de la Vigne et du Vin. This analysis identifies three distinct groups: the first brings together Sangiovese and the two offspring Nerello Mascalese and Gaglioppo di Ciro`, while Mantonico di Bianco and Mantonicone are positioned at a distance from the first and between them. Using molecular characterization, supported by the ampelographic one, we showed that Gaglioppo di Ciro`, Mantonicone, and Nerello Mascalese, three varieties recovered in the southern regions of Italy, such as Calabria and Sicily, originated by the cross between a nationally spread grape variety as Sangiovese and a Calabria autochthonous vine as Mantonico di Bianco.

Marica Gasparro and Angelo Raffaele Caputo contributed equally to this study. M. Gasparro  A. R. Caputo  C. Bergamini  P. Crupi  M. F. Cardone  R. Perniola  D. Antonacci (&) CRA-UTV Research Unit for Viticulture and Enology in Southern Italy, Via Casamassima 148, 70010 Turi, BA, Italy e-mail: [email protected]; [email protected]

Keywords Vitis vinifera L.  Autochthonous vine  Ampelography  Molecular markers  SSR  Kinship

Introduction The grapevine (Vitis vinifera L.) is one of the most important crop plants of the world. Grapes were grown in Europe even before the Roman age, and there are many traditional and newly bred cultivars all over the world. From the area of first domestication, most likely at the southern shores of the Black and Caspian seas and nearby, the domesticated forms spread westward and arrived at the Mediterranean basin following major civilization and colonization events [1]. The spread of viticulture from Greece to Western Europe crossed the southern part of Italy. There is a growing interest in understanding the origin and genetic diversity of germplasm rescued in different geographical areas, as well as resolving the intricacy of relationships among that germplasm and the most widely known international varieties. In fact, researches on grapevine varieties pedigree determination have been increased in the last years, allowing to clarify the evolution of the present ampelography and to reconsider vines that have historically played a role in the emergence of varieties currently grown. Sangiovese is an ancient and renowned wine variety, widely cultivated throughout Italy; it was mentioned for the first time by SODERINI (1590) as ‘‘Sangiogheto’’ [2]. Actually it is the most commonly cultivated black grape variety in Italy and it is the basis to product famous vines, such as Chianti and Brunello di Montalcino. Several studies have identified the Sangiovese as the potential progenitor of a number of vineyards in central and southern Italy [3–5], including some old and autochthonous vines of

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Sicily and Calabria. Furthermore, a recent work has identified two previously unreported candidate parents for Sangiovese [6]. The first putative parent is Ciliegiolo, a well know variety already addressed as relative of Sangiovese; the second putative parent is Negrodolce, an old local variety recovered in the southern Apulia region and considered lost during the last century. This latest proposed parentage provides a new strong evidence for a south Italian origin of Sangiovese. Historical evidence combined with morphological data (ampelography) has been useful for the identification of well-known grape varieties and to define phenological relationships among them. However, conclusions based on these data are often questioned, because the morphological characteristics can be affected by the environment. This could lead to mistakes in cultivar identification, thus suggesting the need of confirmation at the genetic level. Several studies have been conducted on the ampelographic characterization of autochthonous vines of southern Italy. They have provided an extensive territory scouting for the recovery of local vines biodiversity, but these studies were not fully comprehensive for germplasm characterization, as reported above [7, 8]. The advent of molecular markers offered a powerful tool to address these issues; indeed they were largely used by ampelographers and grape geneticists. Individual fingerprinting based on molecular markers has become commonly used for population genetic studies and analysis of genetic diversity in germplasm collections, including the solution of synonymy/homonymy and the analysis of paternity and kinship [9]. Genetic profiling of individuals is nowadays based on simple sequence repeat (SSR) markers, which have a number of positive features that make them superior to any other type of molecular marker developed so far for DNA fingerprinting [10]. These markers are co-dominant, with a Mendelian inheritance, frequently and evenly distributed throughout the genome, highly reproducible and polymorphic. Several sets of SSR markers have been proposed in grape. The best known is the set suggested by the European group working within the grape GENRES projects that is based on six highly reproducible microsatellites with di-nucleotide repeats [11]. Recently, the list of di-nucleotide SSR markers, mainly developed by the Vitis Microsatellite Consortium, was extended [4]. The use of di-nucleotide repeats remain problematic due to the high amount of stuttering, which make the interpretation of electropherograms and the call of true alleles less reliable, and a narrow distance between adjacent alleles that complicates binning [12, 13]. For this reason, they have been discarded in animal and human fingerprinting in favor of microsatellites with longer core repeats [14], namely tetra- and penta-nucleotides, for which neighbor alleles are more easily separated and identified

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from each other. Microsatellites with a longer core repeat are less frequent in the genome and difficult to isolate through enriched libraries, but in 2007, the complete grape genome sequence became available [15, 16] and thousands of SSRs with 3-nt or longer core repeat size could be retrieved from the NCBI genebank [17]. In the present paper, we report the degree of relationship to five varieties spread in the wine distribution areas of southern Italy: Sangiovese, Mantonico di Bianco, Gaglioppo di Ciro`, Mantonicone, and Nerello Mascalese. The analysis was carried out with 34 di-nucleotide SSR markers and 18 tri-, tetra-, and penta-nucleotide SSR markers, supported by historical data and ampelographic characterization.

Materials and Methods Plant Material The Agricultural Research Council (CRA), Research Unit for Viticulture and Enology in southern Italy of Turi (Bari, Italy) has collected more than 2,000 accessions directly from private vineyards of southern Italy, in relation to the Vitivin-Valut project that involves among its research lines the recovery and the exploitation of the autochthonous vines in the southern Italian regions (Basilicata, Calabria, Campania, Apulia and Sicily). All accessions were grafted to 1103 Paulsen (Vitis berlandieri 9 Vitis rupestris), spaced 2.5 m between rows 9 1 m on the row, in the experimental field collection of the CRA, Research Unit of Turi (Bari, Italy). As regards the varieties analyzed in this study, we have recovered in the different regions 13 accessions of Sangiovese, 12 of Mantonico di Bianco, four of Gaglioppo di Ciro`, three of Mantonicone, and 41 of Nerello Mascalese (Table 1). Microsatellite Analysis Total genomic DNA was extracted from young leaves using DNAeasyÒ Plant Mini Kit (Qiagen, Hilden, Table 1 Varieties analyzed in this study Variety

Place of sampling

Number of accessions

Sangiovese

Apulia

13

Mantonico di Bianco Gaglioppo di Ciro`

Calabria Calabria

12 4

Mantonicone

Calabria

Nerello Mascalese

Sicily

3 38

Calabria

2

Apulia

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Germany), following the manufacturer’s instructions, and the DNA was checked in terms of quality and quantity by 0.8 % agarose gel electrophoresis and a spectrophotometer at 260 nm. DNA was used as template in a PCR amplification for genotyping using a first set of thirteen di-nucleotide SSR loci, six of them required by the EU project Genres CT96 N° 81: VVS2, VVMD5, VVMD7, VVMD27, VrZAG62 and VrZAG79 [11], VVMD28, VVMD25 and VVMD32 [18], ISV2, ISV3, ISV4 and VMCNG4b9 [19]. The cycling profile was: an initial heat activation step at 95 °C for 5 min, 35 cycles of denaturation at 98 °C for 5 s, annealing at 55 °C for 30 s, extension at 68 °C for 9 s, and a final extension at 72 °C for 1 min. Eighteen long core repeat SSR loci were additionally assayed to improve likelihood ratios (LRs) for the proposed parentages: VChr-1b, VChr-2b, VChr-2c, VChr-4a, VChr5b, VChr-5c, VChr-6a, VChr-7b, VChr-9a, VChr-10b, VChr-11b, VChr-12a, VChr-13a, VChr-15a, VChr-16a, VChr-17a, VChr-18a, and VChr-19a [9]; PCR amplifications for this set of SSR loci were performed as described in Cipriani et al. [9]. Finally, to confirm parental relations found, additional genetic characterization was performed using 21 more di-nucleotide SSR loci: ssrVrZAG21 [20] and VVI-b01, VVI-b63, VVI-b94, VVI-f52, VVI-h54, VVI-i51, VVI-m10, VVI-m11, VVI-m25, VVI-n61, VVIn94, VVI-p25b, VVI-p37, VVI-p77, VVI-r09, VVI-s21, VVI-s58, VVI-s63, VVI-v37, and VVI-v69 [21]. PCR thermal profile was the following: one cycle at 95 °C for 3 min, followed by eight touch down cycles at 94 °C for 10 s, 59–0.5 °C/cycle for 20 s, 68 °C for 25 s, followed by 24 cycles at 94 °C for 10 s, 55 °C for 20 s, 68 °C for 25 s, and a final extension at 72 °C for 3 min. All the above reported PCR reactions were conducted in 10 ll final volume containing 25 ng of genomic DNA, 5 pmol of each forward and reverse primer and 5 ll of QIAGEN Fast Cycling PCR Master Mix 2X (Qiagen, Hilden, Germany). Three or more primer pairs were carefully combined to co-amplify in a single reaction and each forward primer was labeled with WellRED dyes, D2-PA (black), D3-PA (green), or D4-PA (blue), at the 50 end. Amplicons were analyzed on a CEQTM 8000 Series Genetic Analysis System, automatically sized using a CEQ DNA Size Standard Kit 400 (Beckman Coulter S.p.A., Milan, Italy) and then visually inspected and manually recorded. Ampelographic Characterization Ampelographic descriptions of the five varieties under study were performed during the biennium 2008–2009, using the method developed by the Organisation Internationale de la Vigne et du Vin [22], drafted by the Panel of the Genres 081 project (http://www.genres.de/eccdb/vitis).

A first rapid and accurate description was conducted using 14 primary descriptors, including the preliminary minimal traits relative to shoots, leaves, bunches, and berries. Additional 27 characteristic were chosen in our study to improve the discriminating ability. Twenty readings per shoot and leaf descriptors were taken on ten plants, while bunch and berry measurements were made at harvest using 50 berries from 20 bunches. The 41 ampelographic characters evaluated in the present study corresponding to six characters that described the young shoot, two for the young leaf, 17 for the mature leaf, one for the flower, seven for the bunch and eight for the berry. Data Analysis The SSR profiles obtained were identified by comparing with the database of the CRA, Research Unit of Turi (Bari, Italy), which contains the molecular profiles of more than 2,000 accessions, representing over 350 different wine and table grapes varieties. The molecular profiles were also analyzed by the software Identity version 1.0 [23] to identify pedigree relationships and calculate their significance. Amplicons sizes were rounded, according to the length of the core repeat of each analysed SSR, with an Excel (Microsoft, Redmond, WA) computational sheet. The program Identity was used to calculate cumulative LRs for the proposed parentage. Ampelographic data were subjected to Principal Components Analysis (PCA) with STATISTICA v6.0 software (StatSoft Inc., Tulxa, OK) to value phenotypic relationship between the varieties described.

Results and Discussion We genotyped more than 2,000 grape accessions from southern Italy, using a first set of 13 di-nucleotide SSR loci. Redundant genotypes and accessions already existing were discarded thus allowing the identification of over than 350 different genotypes of wine and table grapes varieties. All of them were reported in our own custom database. Molecular profiles were then analyzed by the software Identity to discover significant pedigree relationships. Genotyping results with the basic set of 13 SSR markers suggested that Sangiovese and Mantonico di Bianco are the parents of Gaglioppo di Ciro`, Mantonicone, and Nerello Mascalese. To confirm this hypothetical pedigree relationship and to improve LRs for the proposed parentages, we extended genotype analysis using additional 18 long core repeat SSR loci and 21 di-nucleotide microsatellites. The proposed offspring vines exhibited one allele derived from each of the presumed parents at 50 SSR loci for Mantonicone and at 51 SSR loci for Gaglioppo di Ciro`. As

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regards Nerello Mascalese, all 52 SSR loci were in agreement with the preliminary hypothesis about a parentprogeny relationship among these cultivars. Specifically Mantonicone showed discrepancy at two SSR loci, VChr6a and VChr-17a, and Gaglioppo di Ciro` only at VChr-6a (Table 2). Genotypes showed a single peak for these two loci in Sangiovese, Gaglioppo di Ciro`, and Mantonicone, so they were recorded as homozygous. If so, for VChr-6a Mantonicone and Gaglioppo di Ciro` exhibited only one allele derived from the presumed parent Mantonico di Bianco and none from Sangiovese, moreover for VChr-17a only Mantonicone presented one allele derived from Mantonico di Bianco and none from Sangiovese, while Gaglioppo di Ciro` exhibited one allele derived from each of the presumed parents. The program Identity was used also to calculate for each locus in our database the expected (He) and observed (Ho) heterozygosity and the estimated frequency of null alleles (null alleles). Statistical analysis of VChr-6a and VChr-17a allele frequencies showed that the estimated frequency of null allele was positive for these two loci (Table 3). The existence of null alleles for these two loci in Sangiovese could explain the discrepancy found in Gaglioppo di Ciro` and Mantonicone: the apparently homozigosity, in fact, could be explained as the result of inherited null alleles in the offspring. For this reason, VChr-6a and VChr-17a were excluded from further analysis. Cumulative LRs analysis, performed on 50 SSR loci, were computed using the observed allele frequencies and the 95 % upper confidence limits (Table 4). LRs of the proposed parentage versus any other pair of parents were extremely high: 1.51 9 1038 for Gaglioppo di Ciro`, 1.94 9 1037 for Mantonicone and 2.55 9 1036 for Nerello Mascalese. These ratios remained far higher also compared to the values calculated when one of the suggested parents was assumed and the other parent was a close relative of the second suggested parent. These data strongly supported that Sangiovese and Mantonico di Bianco are the parents of Gaglioppo di Ciro`, Mantonicone, and Nerello Mascalese. The expression levels of the ampelographic characteristics are shown in Table 5. They showed that the five varieties shared only six characteristics: the number of shoot consecutive tendrils (two), the color of the fourth young leaf upper side of blade, the absence of teeth in the upper lateral sinuses of mature leaf, the flower perfectly hermaphrodite and the berry with colorless flesh and welldeveloped seeds. The offspring vines, Gaglioppo di Ciro`, Nerello Mascalese and Mantonicone, shared 13 characteristics (OIV Codes 008, 016, 051, 073, 078, 079, 081-1, 083-2, 151, 228, 231, 235, and 241); only six of them, as mentioned before, are shared with both parents, Mantonico di Bianco and Sangiovese, instead the others come from each

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parent separately: three from Mantonico di Bianco (OIV Codes 078, 081-1, and 235) and four from Sangiovese (OIV Codes 008, 073, 079, and 228). A principal component analysis was developed (Fig. 1) using the ampelographic characteristics shown in Table 5 as variables and the five varieties (Sangiovese, Mantonico di Bianco, Gaglioppo di Ciro`, Nerello Mascalese, and Mantonicone) under study as case. Three factors showed eigenvalues higher than one (Kaiser’s rule) and accounted for about 84 % of the total variation. The projection on a two dimensional plane defined by the first two axes (Factor 1 and Factor 2) explained for about 65 % of the total variation. The variables with the highest contribution to the first component, accounting for about 38 % of total variation, were OIV 072 (Factor loading 0.95), OIV 053 (0.93), OIV 084 (0.93), OIV 202 (0.93), and OIV 204 (0.93); while the variables OIV 001 (0.93) and OIV 225 (0.91) weighted more on the second component that represents about 27 % of the total variation (Fig. 1). This analysis identified three distinct groups: the first brings together Sangiovese and the two offspring black berry vines (Nerello Mascalese and Gaglioppo di Ciro`), mainly characterized by similar opening of the shoot tip (OIV 001) and color of skin (OIV 225), together with bunch width (OIV 203) and length of peduncle and number of wings of primary bunch (OIV 206 and OIV 209). Conversely, the two white varieties, were separated from each other according to Factor 1; Mantonico di Bianco was distinguished by the mature leaf characteristics goffering of blade (OIV 072), undulation of blade (OIV 073) and opening degree of petiole sinus (OIV 079) and by the color of the dorsal and ventral side of shoot internodes (OIV 007, OIV 008), while Mantonicone was primarily characterized by herbaceous flavor of berry (OIV 236). This finding revealed greater morphological similarity between the black grapes than the white one. By combining ampelographic description and molecular analysis with 52 microsatellite markers, in the present paper, we are able to confirm and further validate the existence of a strong kinship between the five varieties Sangiovese, Montonico di Bianco, Mantonicone, Gaglioppo di Ciro`, and Nerello Mascalese. In fact, as previously reported by Cipriani and colleagues [17], Sangiovese and Mantonico di Bianco are the parents of Gaglioppo di Ciro` and Nerello Mascalese. In addition, we are able to identify Mantonicone as another offspring of this cross. In this study, we use 18 long core SSR loci of the 34 reported by Cipriani et al. [17] in their genotyping work, while the other 34 di-nucleotide microsatellite are added to have a different and more informative set of markers to realize the genetic characterization. These additional microsatellite data demonstrate clearly that the putative parents of Gaglioppo di Ciro`, Mantonicone, and Nerello

Mol Biotechnol Table 2 Molecular data at 52 SSR loci of the five varieties analyzed. Allele lengths are in bp

First set of 13 di-nucleotide SSR loci

18 long core repeat SSR loci

21 additional di-nucleotide SSR loci

SSR loci

Sangiovese

Mantonico di Bianco

Nerello Mascalese

Gaglioppodi Ciro`

Mantonicone

VVS2

133–133

143–151

133–143

133–151

133–151

VVMD5

224–234

224–238

224–234

224–224

224–238

VVMD7

238–262

238–248

238–248

238–262

248–262

VVMD25

241–241

241–255

241–241

241–241

241–255

VVMD27

178–184

178–178

178–178

178–184

178–184

VVMD28

235–245

229–237

229–245

229–235

229–245

VVMD32

252–256

252–252

252–256

252–256

252–256

VrZAG62

193–195

201–201

195–201

193–201

193–201

VrZAG79

242–258

250–250

250–258

242–250

242–250

ISV2

144–166

142–170

142–144

166–170

142–144

ISV3 ISV4

140–140 178–198

134–146 170–192

134–140 170–178

134–140 178–192

134–140 178–192

VMCNG4b9

158–168

150–174

150–168

150–168

150–158

VChr-1b

100–108

96–108

100–108

108–108

96–108

VChr-2b

120–124

124–124

124–124

120–124

124–124

VChr-2c

151–153

153–165

153–153

153–153

151–153

VChr-4a

199–199

199–199

199–199

199–199

199–199

VChr-5b

202–202

202–210

202–202

202–202

202–210

VChr-5c

88–104

104–104

104–104

88–104

88–104

VChr-6a

182–//

182–186

182–182

186–//

186–//

VChr-7b

189–189

185–185

185–189

185–189

185–189

VChr-9a

90–112

112–112

112–112

90–112

90–112

VChr-10b

138–144

138–144

138–144

138–138

144–144

VChr-11b

158–160

156–164

158–164

158–164

158–164

VChr-12a

144–144

137–144

144–144

144–144

144–144

VChr-13a

157–157

146–149

149–157

146–157

146–157

VChr-15a VChr-16a

149–157 114–114

149–157 114–114

149–157 114–114

149–149 114–114

149–157 114–114

VChr-17a

189–//

181–189

181–189

189–189

181–//

VChr-18a

164–168

164–164

164–168

164–164

164–168

VChr-19a

123–141

138–138

123–138

123–138

123–138

VrZAG21

203–205

191–201

191–205

201–205

191–205

VVI-b01

292–294

298–302

294–302

292–298

294–302

VVI-b63

148–148

154–156

148–156

148–154

148–156

VVI-b94

294–306

294–294

294–294

294–294

294–294

VVI-f52

272–292

260–260

260–292

260–292

260–272

VVI-h54

166–176

164–166

164–166

166–166

166–166

VVI-i51

253–265

253–265

253–265

253–265

253–265

VVI-m10

369–371

369–369

369–369

369–369

369–371

VVI-m11

278–289

278–292

289–292

278–278

278–289

VVI-m25

168–178

168–168

168–178

168–168

168–168

VVI-n61 VVI-n94

375–377 278–290

351–375 278–290

375–375 278–290

375–375 278–290

375–375 278–290

VVI-p25b

342–362

362–362

342–362

342–362

342–362

VVI-p37

123–140

123–123

123–140

123–140

123–140

VVI-p77

172–180

180–190

172–190

172–180

172–190

VVI-r09

258–263

254–256

256–258

254–263

256–258

VVI-s21

278–284

284–284

278–284

284–284

284–284

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Mol Biotechnol Table 2 continued SSR loci

Sangiovese

Mantonico di Bianco

Nerello Mascalese

Gaglioppodi Ciro`

Mantonicone

VVI-s58

304–304

304–308

304–304

304–308

304–304

VVI-s63

174–174

190–190

174–190

174–190

174–190

VVI-v37

157–173

157–157

157–157

157–173

157–173

VVI-v69

257–257

257–277

257–277

257–277

257–277

// supposed null alleles Table 3 Genetic parameters of VChr-6a and VChr-17a SSR loci Locus

He

Ho

Null alleles

VChr-6a

0.494

0.411

0.055

VChr-17a

0.520

0.308

0.139

He expected heterozygosity, Ho observed heterozygosity, Null alleles estimated frequency of null alleles

Mascalese are Sangiovese and Mantonico di Bianco, since other possible cross combinations in our database are far less probable. These data are based on a cumulative LRs analysis realized on 50 over 52 SSR loci, because two loci are not in agreement with the proposed kinship. Pedigrees with incompatible profiles at one or two SSR loci are usually manually checked. In most cases, the incompatible profiles occur at loci with a high frequency of null alleles, and thus can be solved with the hypothesis of the occurrence of a null allele in the parent and in its offspring. In our analysis, VChr-6a and VChr-17a loci show a positive value of the estimated frequency of null alleles (0.055 for

VChr-6a and 0.139 for VChr-17a) as already reported by Cipriani et al. [17] in their work (0.072 for VChr-6a and 0.184 for VChr-17a). Consequently, it is very likely that Sangiovese has null alleles in both SSR loci and Mantonicone and Gaglioppo di Ciro` inherited these null alleles, therefore, we propose that the hypothesized pedigree relationship is validated at all 52 SSR loci. From an historical point of view, while Sangiovese is widely famous and spread in Italy, Mantonico di Bianco is less known. In Calabria, Mantonico di Bianco is considered a variety of very ancient introduction because it is localized mainly in Locride, an historical province of Magna Graecia, so it might have been imported by the ancient Greek colonists [7]. In Locride, it is also called ‘‘Mantonacu viru,’’ which translates in ‘‘the real Mantonico’’ [24]. This cultivar should not be confused with Montonico, a white grape variety grown in central Italy. Similarly, Gaglioppo di Ciro` and Mantonicone are autochthonous vine of Calabria [7, 25] while Nerello Mascalese is a typically production of Sicily [26].

Table 4 Cumulative likelihood ratio (LR) values for the proposed relationships versus other possibilities, calculated over 50 microsatellite loci Combined over all loci

X 9 Ya

(1) 9 Xb

(2) 9 Xd

(1) 9 (2) relativec

(2) 9 (1) relativee

Proposed parentsa of Gaglioppo of Ciro`: (1) Mantonico di Bianco, (2) Sangiovese LRs including calculated allele frequencies LRs including 95 % upper confidence limits of observed allele frequencies

1.51 9 1038

8.32 9 1024

5.59 9 107

1.44 9 1020

1.02 9 107

28

19

6

15

4.74 9 10

5.86 9 105

1.71 9 10

2.17 9 10

3.57 9 10

1.94 9 1037

7.08 9 1022

2.51 9 107

2.64 9 1022

2.14 9 107

27

17

6

17

2.72 9 10

1.27 9 106

2.55 9 1036

2.74 9 1022

1.13 9 107

2.86 9 1021

1.61 9 107

26

17

5

16

1.01 9 106

Proposed parentsa of Mantonicone: (1) Mantonico di Bianco, (2) Sangiovese LRs including calculated allele frequencies

LRs including 95 % upper confidence limits of observed allele 1.90 9 10 4.56 9 10 frequencies Proposed parents of Nerello Mascalese: (1) Mantonico di Bianco, (2) Sangiovese LRs including calculated allele frequencies LRs including 95 % upper confidence limits of observed allele frequencies

5.42 9 10

1.74 9 10

1.43 9 10

7.03 9 10

6.61 9 10

a The ratio of the probability that the proposed parents gave rise to the offspring’s genotype versus the probability that two random individuals give rise to the offspring’s genotype: (proposed parents) versus (two random cultivars) b LR for: (proposed parents) versus (random individual 9 proposed parent (1)) c

LR for: (proposed parents) versus (close relative of proposed parent (2) 9 proposed parent (1))

d

LR for: (proposed parents) versus (proposed parent (2) 9 random cultivar)

e

LR for: (proposed parents) versus (proposed parent (2) 9 close relative of proposed parent (1))

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Mol Biotechnol Table 5 Ampelographic descriptions of the five varieties under study [22] OIV code

Characteristic

Mantonico di Bianco B.

Sangiovese N.

Gaglioppo N.

Nerello Mascalese N.

Mantonicone B.

OIV 001

Young shoot: opening of the shoot tip

3

5

5

5

3

OIV 003

Young shoot: intensity of anthocyanin coloration on prostrate hairs of the shoot tip

1

3

1

3

1

OIV 004

Young shoot: density of prostrate hairs on the shoot tip

5

3

5

5

3

OIV 007

Shoot: color of the dorsal side of internodes

2

1

1

2

1

OIV 008

Shoot: color of the ventral side of internodes

2

1

1

1

1

OIV 016

Shoot: number of consecutive tendrils

1

1

1

1

1

th

OIV 051

Young leaf: color of upper side of blade (4 leaf)

1

1

1

1

1

OIV 053

Young leaf: density of prostrate hairs between main veins on lower side of blade (4th leaf)

7

3

5

5

3

OIV 065

Mature leaf: size of blade

7

5

5

5

5

OIV 067 OIV 068

Mature leaf: shape of blade Mature leaf: number of lobes

2 3

2 3

3 3

2 2

2 2

OIV 070

Mature leaf: area of anthocyanin coloration of main veins on upper side of blade

2

1

2

3

1

OIV 072

Mature leaf: goffering of blade

5

1

3

1

1

OIV 073

Mature leaf: undulation of blade between main or lateral veins

9

1

1

1

1

OIV 074

Mature leaf: profile of blade in cross section

1

1

1

3

1

OIV 075

Mature leaf: blistering of upper side of blade

1

1

3

1

1

OIV 076 OIV 078

Mature leaf: shape of teeth Mature leaf: length of teeth compared with their width

5 5

5 7

4 5

5 5

5 5

OIV 079

Mature leaf: degree of opening/overlapping of petiole sinus

7

3

3

3

3

OIV 080

Mature leaf: shape of base of petiole sinus

3

1

1

1

3

OIV 081-1

Mature leaf: teeth in the petiole sinus

1

2

1

1

1

OIV 081-2

Mature leaf: petiole sinus base limited by vein

1

1

2

1

1

OIV 083-2

Mature leaf: teeth in the upper lateral sinuses

1

1

1

1

1

OIV 084

Mature leaf: density of prostrate hairs between main veins on lower side of blade

7

3

5

5

3

OIV 087

Mature leaf: density of erect hairs on main veins on lower side of blade

3

3

3

5

3

OIV 151

Flower: sexual organs

3

3

3

3

3

OIV 202

Bunch: length (peduncle excluded)

7

5

5

5

3

OIV 203

Bunch: width

5

5

5

5

3

OIV 204

Bunch: density

7

5

5

5

3

OIV 206

Bunch: length of peduncle of primary bunch

5

5

5

5

3

OIV 208 OIV 209

Bunch: shape Bunch: number of wings of the primary bunch

3 2

2 2

2 2

1 2

1 1

OIV 223

Berry: shape

3

3

3

2

2

OIV 225

Berry: color of skin

1

6

5

6

1

OIV 228

Berry: thickness of skin

3

5

5

5

5

OIV 231

Berry: intensity of flesh anthocyanin coloration

1

1

1

1

1

OIV 235

Berry: firmness of flesh

2

3

2

2

2

OIV 236

Berry: particular flavor

1

1

1

1

4

OIV 241

Berry: formation of seeds

3

3

3

3

3

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Mol Biotechnol Table 5 continued OIV code

Characteristic

Mantonico di Bianco B.

Sangiovese N.

Gaglioppo N.

Nerello Mascalese N.

Mantonicone B.

OIV 502

Bunch: single bunch weight

5

5

5

3

3

OIV 503

Berry: single berry weight

5

1

3

3

5

1–9 = expression levels of the characteristics

OIV 225

OIV 001

OIV 203209 - OIV 206 – OIV 209 OIV 203 206

5

OIV 078 OIV 081-1 OIV 003 OIV 003 OIV 235 OIV OIV081-1 078 235

4 3

OIV 081-2 OIV 067 OIV 075 Sangiovese Sangiovese OIV OIV081-2 067 075 Gaglioppo diCirò Cirò Gaglioppo di

OIV 228

2

OIV 074–OIV 087 OIV 087 074 Nerello Nerello Mascalese Mascalese

1

Factor 2: 27,29%

Fig. 1 Principal component diagram of ampelographic characteristics in the five grape varieties (Sangiovese, Mantonico di Bianco, Gaglioppo, Nerello Mascalese, and Mantonicone). Factor score plot 1–2: axes 1 and 2 account for 65.42 % of the total variance explained. Variables correspond to the 41 ampelographic characters analyzed in grapes

OIV 068 – OIV 223 – OIV 502 OIV 502 068 223

OIV 202 – OIV 204 OIV 204 202

OIV 070

0

OIV 004 OIV 208

OIV 084 053 OIV 053 – OIV 084 OIV 072 OIV 007 OIV 072 OIV 007

-1

Mantonico Mantonico di Bianco

-2

OIV 079 008 065 073

OIV 076

-3

OIV 008 OIV 073 OIV 079

OIV 076

-4

Mantonicone Mantonicone

-5

OIV 236 OIV 503 OIV 503 OIVOIV 080080

OIV 236

-6 -7 -8 -8,00

-6,00

-4,00

-2,00

0,00

2,00

4,00

6,00

8,00

10,00

Factor 1: 38,13%

This study confirms and corroborates, on one side, some of the indications given by Cipriani et al. [17], also using a larger and mostly different set of SSR markers with the support of historical data and ampelographic characterization. On the other side, our work adds another offspring to Sangiovese in southern Italy, indicating that Sangiovese is crucial in the evolution of the Italian ampelographic assortment and particularly that Sangiovese has clear and dated relationships with southern Italian varieties, in keeping with the latest kinship proposed for Sangiovese [6]. In conclusion, in this work, we show by means of molecular analysis with 52 microsatellite markers and ampelographic characterization a degree of direct parent– child relationship.

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This work further demonstrates the importance of molecular analysis for the varietal characterization especially to solve ambiguity and discrepancy derived by morphological descriptions. Furthermore, it emerges clearly that the combination of different approaches such as ampelography, historical researches and molecular analysis is fundamental to reveal direct parent–child relationship. Indeed our data show that Gaglioppo di Ciro`, Mantonicone, and Nerello Mascalese, these recovered in the Southern regions of Italy, such as Calabria and Sicily originate by the cross between a nationally spread grape variety as Sangiovese and a Calabrian autochthonous vine as Mantonico di Bianco. This strongly suggests that Sangiovese has been cultivated in southern Italy for a long time

Mol Biotechnol

and has played an important role in the development of southern Italian autochthonous vines. Acknowledgments This research was supported by the project Vitivin-valut, ‘‘Progetto per il miglioramento qualitativo delle produzioni vitivinicole e dell’uva da tavola nel Mezzogiorno d’Italia,’’ funded by Ministry of Agricultural and Forestry Policy (MIPAF), Italy.

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