Studio sulla variabilità genetica inter- e intra-varietale in Vitis vinifera. Study of inter-and intra-varietal genetic variability in Vitis vinifera

Transcription

1 OIV GRANT 2013 Final Report Scientific Coordinator: Prof. BAVARESCO Luigi (CRA-VIT Conegliano) Boursier: PhD MENEGHETTI Stefano Original title in Italian: Studio sulla variabilità genetica inter- e intra-varietale in Vitis vinifera Title in English: Study of inter-and intra-varietal genetic variability in Vitis vinifera 1

2 1. ABSTRACT The Vitis genus is characterized by a large number of cultivars, ecotypes, biotypes and clones. This great morphological and genetic variability is causing confusions and ambiguity for biotypes and clones identification, in particular considering varieties that are widely distributed and cultivated for centuries. Biotype identification in viticulture has a long tradition and it became science with the ampelography, ampelometry, chemical and biochemical traits. All these approaches have limitations and some variants were erroneously included in a cultivar when they underwent changes to their phenotype under different environmental conditions. Simple sequence repeat (SSR) markers are universally used for the identification of the grape varieties. The molecular approaches are also essential for internationally accepted grapevine identification and to investigate the genetic interand intra-varietal variability. In this study, 63 biotypes of Sangiovese from different Italian areas including clones registered in the Italian National Catalog were analyzed in order to identify the cultivar and the potential molecular differences between genotypes, as we have studied and observed for other Italian varieties of Vitis vinifera; in addition to these materials 15 accessions of Montepulciano were investigated using AFLP, SAMPL, MAFLP and ISSR molecular markers. Microsatellite analysis showed two main varieties of Sangiovese in Italy, the Sangiovese grosso (Sangiovese classic) and the Sangiovese piccolo (Sanvicetro); these two cultivars co-exist in the Sangiovese DOC or DOCG zones. Molecular markers were able to distinguish all Sangiovese (S. grosso and S. piccolo) and Montepulciano accessions. Intra-varietal results have highlighted the existence of genetic variability among the accessions of the three cultivars from different geographic cultivation areas and suggesting the need for the preservation of autochthonous grapevine biotypes found on different zones to approve the correct choice and selection of the grape multiplication materials. 2

3 2. INTRODUCTION The Sangiovese is an important Italian variety grown in many Italian regions (Galet, 2000) and it shows a strong morphological variability due to its ancient origin and includes a rich clonal population, as the many local denominations are witnessing (Di Rovasenda, 1877; Molon, 1906; Brevigleri & Casini, 1965; Silvestroni & Intrieri, 1995; Calò et al., 2000; Vignani et al., 2002). In Italy, there are mainly two different varieties of Sangiovese named Sangiovese grosso (with large berries, the Sangiovese classic ) and Sangiovese piccolo (with small berries, therefore with a different berry morfology) (Falchini, 1970; Calò et al., 2000; Calò & Costacurta, 2004). These two varieties showed obviously two different SSR profiles. Microsatellite analysis of the last decades showed the existence of many cultivars unknown but very similar to these two Sangiovese main varieties (Calò et al., 2000). In Italy the Sangiovese piccolo cultivar is known also as Sanvicetro while Sanvicetrino variety is a different variety of Sangiovese grosso and Sanvicetro (Calò et al., 2000) but for the Italian Sangiovese there are several local synonyms in all Italian peninsula that create confusion about the exact identification of Sangiovese materials (Villifranchi, 1773; Di Rovasenda, 1877; Molon, 1906; Calò et al, 2000). Biotype identification in viticulture has a long tradition (Odart, 1874, Meneghetti et al., 2012a) and ampelography, ampelometry, chemical and biochemical traits have limitations when the biotypes undergo changes to their phenotypes, under different environmental conditions (Meneghetti et al, 2012b). The molecular approaches are essential for internationally accepted grapevine identification (SSR) and to investigate the genetic inter- and intra-varietal variability. Many molecular markers have been used to study the molecular variability in Vitis (Böhm & Zyprian, 1998; Regner et al., 2000; Imazio et al., 2002; Pelsey et al., 2002; Owens, 2003; Labra et al., 2004; Blaich et al., 2007; Cipriani et al., 2008; D Onofrio et al., 2009; Cretazzo et al., 2010; Meneghetti et al., 2011). In this study, genetic variability of Sangiovese grape was investigated using in particular AFLP, ISSR, SAMPL, SSR and M-AFLP molecular markers (Meneghetti et al., 2012c). To simplify the Sangiovese study and analysis in this work we call the two varieties of Sangiovese as Sangiovese grosso (this is the Sangiovese classic ) and Sangiovese piccolo (the Sanvicetro variety) (Calò et al., 2000). The biotypes of Sangiovese from many Italian regions for both Sangiovese varieties (i.e., Sangiovese grosso and Sangiovese piccolo) were sampled. In Tuscany, where Sangiovese produces the best wines (e.g., Sangiovese classico, Sangiovese Montepulciano, Chianti, Brunello, Morellino, etc ) were selected a greater number of biotypes in reference to the different geographical areas of this Italian region. 3

4 For further clarity, were also analyzed biotypes of Montepulciano variety from two adjacent regions, Tuscany and Marche, because Sangiovese Montepulciano is identified as Sangiovese grosso variety but the Montepulciano is not a Sangiovese cultivar but a different variety (Calò et al., 2000; Calò & Costacurta, 2004). The Italian synonyms of Sangiovese grosso are: Sangiovese classico, Sangiovese tipico, Chianti, Chianti Classico, Brunello, Brunellino, Morellino, Nielluccio, Prúgnolo, Prugnólo, Sangiovese Montepulciano, Sangiovese piccolo di Pitigliano (is not the Sangiovese piccolo variety), while those of Sangiovese piccolo are: Sangiovese forte, Sangiovese montanino, Sanvicetro, Sanvicetro casentino, etc. In the vineyards of Sangiovese grosso, one may find some genotypes of Sangiovese piccolo and in Sangiovese piccolo vineyards, one may also find some genotypes of Sangiovese grosso (Calò et al., 2000). There are also many varieties morphologically similar to one of the two Sangiovese varieties but still unknown (not recorded in the Italian Catalog of Grapevine Varieties). The purpose of this study was to analyze the various biotypes of Sangiovese and Montepulciano cultivars present in different geographic areas of Italy in order to quantify the genetic variability (inter- and intra-varietal) highlighting differences at the genomic level and to characterize and preserve the genetic resources that may be related to the typicality of Italian Sangiovese grapes. In particular, the Sangiovese biotypes from Tuscany, from Northern Italy (i.e., Veneto, Trentino Alto Adige and Friuli Venezia Giulia regions), from Central Italy (i.e., Marche, Emilia-Romagna, Abruzzo, Umbria, Latium regions near Tuscany), from Southern Italy (Campania, Basilicata, Calabria and Apulia regions) with some Sangiovese clones from different Institutes were analyzed; also Montepulciano biotypes were analyzed from Marche and Tuscany regions. 4

5 3. MATERIALS & METHOD 3.1 PLANT MATERIAL a) Sangiovese (= S. grosso and S. piccolo): The plant materials were collected in different geographical areas along the Italian peninsula as reported in Figure 1. With Sangiovese we mean all the samples named Sangiovese in Table 1, including those named Morellino, Brunello, Sanvicetro and Brunellino. 63 accessions of Sangiovese from different Italian areas were analyzed (Table 1), in particular 27 Sangiovese biotypes from Tuscany (i.e., Firenze, Siena, Arezzo, Grosseto, Pistoia) with 7 reference clones, and 29 Sangiovese biotypes from other Italian Regions (i.e., 6 accessions from Veneto region, 4 from Emilia-Romagna region, 1 from Umbria region, 1 from Abruzzo region, 3 from Marche region, 6 from Latium region, 2 from Campania region, 2 from Friuli Venezia Giulia region, 1 from Basilicata region, 1 from Calabria region, 1 from Apulia region, 1 from Trentino Alto Adige region) (Table 1). These Sangiovese accessions are reported in Table 1 with different names: Sangiovese, Sanvicetro, Brunello, Brunellino and Morellino; in fact these names given by the winegrowers are not significant for the varietal identity because often there are samples of Sangiovese grosso and samples of Sangiovese piccolo mixed in the vineyards. Only the molecular analysis (SSR) can clarify the exact name for these Sangiovese accessions. The Sangiovese (i.e., Sangiovese grosso variety) is a grape variety typical of Tuscany region where is named by different local synonyms (e.g., Chianti, Brunello, Morellino, Sangiovese del Chianti, Sangiovese Montepulciano, Prùgnolo, etc.), but its cultivation was spread to many zones of Italy, where it was called by different names (Prugnòlo, Pergola, Sangiovese delle Marche, Sangiovese rosso, Gentile Sangiovese, Sangiovese trevigiano, Sangiovese Trentino, Frascati Brunellino, etc); for this reason we collected other 18 Sangiovese biotypes (i.e., Sangiovese grosso and Sangiovese piccolo varieties) from Northern to Southern Italy to understand the Genetic Relationship between Tuscan Sangiovese and that from other Italian regions (i.e., Trentino Alto Adige, Friuli Venezia Giulia, Veneto, Emilia-Romagna, Marche, Abruzzo, Umbria, Latium, Campania, Calabria, Basilicata and Apulia Italian Regions). The abbreviations for the Italian provinces and their regions (from north to south Italy) are respectively: TN = Trento (Trentino Alto Adige region), UD = Udine (Friuli Venezia Giulia region), TS = Trieste (Friuli Venezia Giulia region), VR = Verona (Veneto region), VI = Vicenza (Veneto region), TV = Treviso (Veneto region), RO = Rovigo (Veneto region), FC = Forlì-Cesena (Emilia-Romagna region), RE = Reggio Emilia (Emilia-Romagna region), PR = Parma (Emilia- 5

6 Romagna region), MO = Modena (Emilia-Romagna region), FI = Firenze (Tuscany region), PT = Pistoia (Tuscany region), AR = Arezzo (Tuscany region), SI = Siena (Tuscany region), GR = Grosseto (Tuscany region), PG = Perugia (Umbria region), AP = Ascoli Piceno (Marche region), MC = Macerata (Marche region), CH = Chieti (Abruzzo region), TE = Teramo (Abruzzo region), PE = Pescara (Abruzzo region), VT = Viterbo (Latium region), RI = Rieti (Latium region), FR = Frosinone (Latium region), RM = Roma (Latium region), SA = Salerno (Campania region), BN = Benevento (Campania region), PZ = Potenza (Basilicata region), CS = Cosenza (Calabria region), BA = Bari (Apulia region) (Figure 1, Table 1). Figure 1: Geographical map of the thirteen Italian regions (i.e., Trentino Alto Adige, Friuli Venezia Giulia, Veneto, Emilia-Romagna, Tuscany, Marche, Abruzzo, Umbria, Latium, Campania, Basilicata, Calabria, Apulia) where the Sangiovese biotypes (S. grosso and S. piccolo varieties) were collected. In particular (Figure 1), the following biotypes were analyzed: 27 Sangiovese biotypes (S. Grosso or S. Piccolo) from Tuscany region with 15 of Siena (SI), 10 of Firenze (FI), 1 of Arezzo (AR), 5 of Grosseto (GR) and 1 of Pistoia (PT); 2 biotypes from Friuli Venezia Giulia (i.e., UD and TS); 1 biotype from Trentino Alto Adige region (i.e., TN); 6 biotypes from Veneto region (i.e., 1 of VR, 2 of TV, 2 of VI and 1 of RO; 5 biotypes from Emilia-Romagna region with 1 of RE, 1 of PR, 1 of MO and 2 of FC; 1 biotype from Umbria region (i.e., PG); 4 biotypes from Marche region (i.e., 1 of AP and 1 of MC); 2 biotypes from Abruzzo region (i.e., CH); 6 biotypes from Latium region (i.e., 1 of VT, 1 of RI, 2 of FR, 1 of RM; 2 biotypes from Campania region (i.e., 1 of SA and 1 of BN); 1 biotype from Basilicata region (i.e., PZ); 1 biotype from Calabria region (i.e., CS); 1 biotype from Apulia region (i.e., BA). 6

7 Table 1: Number (No.), name, geographical origin with reference (REF) of the 78 genotypes analyzed (i.e., 15 Montepulciano and 63 Sangiovese accessions with local names). No. name accession/genotype REF No. name accession/genotype REF 1 Sangiovese Montepulciano (SI) 01TB Sangiovese campano (BN) 85GJK77 2 Montepulciano grosso (FI) MM0Z65 41 Montepulciano teramano (TE) 6TTZY78 3 Sangiovese Gaiole (SI) 96LF Sangiovese trevigiano (TV) 36KZ571 4 Sanvicetro di Toscana (SI) GSBI Brunellino rosso (FR) 58JL966 5 Sangiovese lungo (SI) 856FG12 44 Sangiovese giove (MO) 11VV155 6 Sangiovese carsico (TS) 26QW Sangiovese Chianti (AR) 67MM540 7 Sangiovese toscano (FI) 20KJN74 46 Sangiovese Susegana (TV) 40SS333 8 Sangiovese Scansano (GR) 325RR67 47 Sangiovese Morellino (SI) 31LX867 9 Montepulciano glabro (PE) 3ZMK Sangiovese marchigiano (AP) 75PP Sangiovese Brunello (SA) 398UU00 49 Sanvicetro laziale (VT) 41LLL01 11 Morellino di Toscana (FI) 1FG Sangiovese trentino (TN) 08NN Sanvicetro Morellino (FI) KL4567P 51 Sanvicetro emiliano (FC) 02SPM40 13 Sangiovese Scansano (GR) 325NM45 52 Brunello Montalcino (RI) 56WWE4 14 Montepulciano di toscana (AR) 12ZMO4 53 Montepulciano nobile (TE) GHZ Brunello abruzzese (CH) 145KL55 54 Sanvicetro toscano (SI) 333ZA21 16 Sangiovese classico (FI) 09YH Sangiovese nostrano (BA) 14PP Sanvicetro fiorentino (FI) 210SGR5 56 Brunello di Frascati (RM) 55GH Sangiovese emiliano (RE) 83FB Montepulciano (FI) 67TZU55 19 Montepulciano classico (CH) 54JZ4RZ 58 Morellino romano (FR) 59OS Sangiovese Montalcino (PR) 45RR Montepulciano Marche (MC) BMO43Z 21 Montalcino Prugnolo (SI) 30PP Sangiovese Prugnolo (PT) 23LD Montepulciano abruzzese (CH) GZ7U Brunello Montalcino (SI) 96DF Brunello di Montalcino (SI) 49ZX Sangiovese senese (SI) NN Sangiovese rosso (GR) 60PP Sangiovese perugino (PG) 05LG Sangiovese toscano (SI) 21UF Sanvicetro laziale (FR) GSD Montepulciano toscano (SI) Z8818RI 65 Sangiovese Brunello (FI) 55DP Sangiovese Chianti (FI) 73LA Sanvicetro piccolo (VI) 23JD Sanvicetro Marche (MC) ASG Sangiovese grosso (SI) 45TY Sangiovese Chianti (FI) 99KG Montepulciano sangiovese (FI) V0Z523W 30 Morellino di Potenza (PZ) 08HI Morellino berico (VI) 69XBO23 31 Montepulciano Marche (AP) MCZ Montepulciano rotondo (CH) RFZ66PL 32 Sangiovese Rovigo (RO) 28UJ Clone Sangiovese I (SI) UMI BF10 33 Morellino di Scansano (GR) 15TG Clone Sangiovese II (SI) MI BF50 34 Montepulciano classico (PE) 1PZ987Z 73 Clone Sangiovese III (SI) VCR Sangiovese calabro (CS) 364TG61 74 Clone Sangiovese IV (FI) VCR 4 36 Morellino gentile (VR) 09TL Clone Sangiovese V (GR) VCR Sanvicetro abruzzese (CH) TSG Clone Sangiovese VI (FC) VCR Sangiovese friulano (UD) 44TN Clone Sanvicetro I (CRA-VIT) gf59p6c2 39 Morellino precoce (FI) 22LK Clone Montepulciano I (TE) VCR R7 7

8 b) Montepulciano: 15 accessions of Montepulciano variety (i.e., 14 biotypes and 1 clone) from different Italian areas were analyzed: 5 biotypes from Tuscany region (i.e., Firenze, Arezzo, Siena) and 9 from Marche region (i.e., Ascoli, Macerata, Chieti, Teramo, Pescara) with a reference clone from Chieti (Abruzzo region) (Table 1). 3.2 DNA EXTRACTION The DNA was extracted automatically by a Microlab StarLET Liquid Handling Workstation (Hamilton Robotics) from young leaves (stored at -20 C) using the NucleoSpin 8 Plant kit (Macherey-Nagel) with a liquid handling workstations. 3.3 DNA QUANTIFICATION The quantification of DNA was obtained by FLx800 TBI Fluorometer (BioTek Instruments) and PicoGreen dsdna quantification assay (Invitrogen). 3.4 CULTIVAR IDENTIFICATION Cultivars identification was performed by microsatellite analysis using eleven SSR loci (i.e., VVS2, VVMD5, VVMD7, VVMD27, VVMD28, VMC6E1, VMC6F1, VMC6G1, VMCNG4b9, VrZAG62 et VrZag79) (Meneghetti et al., 2012c). The microsatellite PCR reaction mixture at 11 loci was performed by a workstations using SSR forward (F) labeled primers with 6FAM (VVS2, VVMD27, VVMD7), VIC (ISV2, VrZAG62, VVMD5), NED (VMCNG4b9, VrZAG79) and PET (ISV3, ISV4, VVMD28), and SSR reverse (R) primers unlabeled each at 5 pmol/µl (Lifetech) as reported by Meneghetti et al., 2012a. SSR polymorphisms were resolved on an ABI-3130XL capillary sequencer (Applied Biosystems) using GeneMapper version 4.1 (Applied Biosystems) with a Vitis vinifera microsatellite BinSet of 11 standard loci. 3.5 GENETIC ANALYSIS The genetic variability of accessions from the same grape cultivar was investigated by means of AFLP (Amplified Fragment Length Polymorphism), SAMPL (Selective Amplification of Microsatellite Polymorphic Loci), ISSR (Inter Simple Sequence Repeat), M-AFLP (Microsatellites Amplified Fragment Length Polymorphism) and ISSR (Inter-SSR) molecular markers (Albertini et 8

9 al., 2003; Barcaccia et al., 2003; Meneghetti et al., 2012c). The Restriction of DNA were performed by EcoRI, PstI, and MseI enzymes with EcoRI, PstI, and MseI adapters (a and b); ligation enzyme was the T4 ligase. Restriction and ligation enzymes, as well as primers and adapters, were provided by New England Biolabs. Pre-amplification were performed using 5 µl of sevenfold diluted digested and ligated DNAs in 20 µl of reaction mixture containing 75 ng of EcoRI+1 (or PstI+1) and MseI+1 primers (one selective nucleotide), 1x PCR buffer (50 mm KCl, 1.5 mm MgCl2, 10 mm Tris HCl), 10 mm dntps and 1 U of Taq DNA polymerase (Sigma-Aldrich Corp.). The AFLP-based and ISSR analysis was performed using a 6FAM/VIC/NED/PET labelled EcoRI+3 (or PstI+2) primer and an unlabeled MseI+3 primer (three selective nucleotides). Primer and primer combinations were reported in Table 2. Table 2: Primers and primer combinations: AFLP, SAMPL and M-AFLP primer combinations and ISSR primers used to study the intra-varietal genetic variability of Sangiovese, Sanvicetro and Montepulciano accessions. E, P and M are the Eco-RI, Pst-I and Mse-I AFLP primers respectively with the two or three selective nucleotides (A, G, C and T), As1 and As2 are the SAMPL primers, while M-AFLPs are the SSRs/ISSRs and AFLP (P or E or M) primer combinations. n AFLP SAMPL M-AFLP ISSR 1 P+AC/M+AAG As1/M+AGC E+CGT/ISSR#06 (CA) 7 GATC 2 P+AG/M+CGG As1/M+AGT E+AAT/ISSR#08 (CA) 7 ATCT 3 P+AT/M+CAA As1/M+AGG E+CAA/ISSR#13 (CA) 7 AGTC 4 P+AA/M+CGA As1/M+ACC ISSR#02/M+AGG (TC) 7 ACAT 5 P+AG/M+CAA As1/M+CTC E+TGA/ISSR#19 (TC) 7 AGTC 6 E+CTT/M+ATG As1/M+CGT P+AC/ISSR#05 (TC) 7 ACGG 7 E+ATG/M+CAG As2/M+AGA P+AT/ISSR#22 GGTC(AC) 7 8 E+CGT/M+CTG As2/M+TGG VVMD7f/M+ATC CGTC(AC) 7 9 E+CAT/M+CCT As2/M+CAG VVS2f/M+CGT AGAT(TC) 7 10 E+AGA/M+ATA As2/M+CGA ISSR#07/M+ACG GTGC(TC) 7 11 E+TCA/M+CAG As2/M+CGT ISSR#11/M+ACC CAGC(TA) 7 A binary presence or absence (1 vs. 0) matrix was created for AFLP, SAMPL, ISSR and M-AFLP markers and for each genotype. Molecular markers were defined by a standard ladder using the GeneMapper software with some reference DNA genotypes and visualized automatically by the software of ABI-3130XL capillary sequencer. Genetic similarity (GS) estimates among individuals were calculated in all possible pair-wise comparisons using Dice s genetic coefficient (1945) by NTSYS software (Rohlf, 2000) and UPGMA algorithm (Meneghetti et al., 2011, 2012b). GS was 9

10 calculated within (GS W ) and between (GS B ) accession groups and marker systems (AFLP, SAMPL, M-AFLP, ISSR). The cluster analysis of GS (Dendrograms, Centroids) was performed according to the UPGMA algorithm using the NTSYS software (Sneath & Sokal, 1973). The biotypes of Sangiovese, Sanvicetro and Montepulciano sampled from different geographical areas were identified by SSR markers for subsequent studies about the Sangiovese molecular diversity (Intra- and Inter- varietal Genetic Similarity). The molecular profile of the Sangiovese, Sanvicetro and Montepulciano clones (SSR, AFLP, ISSR, MAFLP, SAMPL) was used only as references. 10

11 4. RESULTS & DISCUSSION SSR molecular identification of Sangiovese and Montepulciano materials are shown in Table 3 where SG is Sangiovese grosso, SV is Sangiovese piccolo (or Sanvicetro) and M is Montepulciano grape varieties. 51 Sangiovese accessions were identified as Sangiovese grosso (i.e., 21 from Tuscany, 6 from Veneto, 2 from Emilia-Romagna, 1 from Umbria, 1 from Abruzzo, 1 from Marche, 5 from Latium, 2 from Campania, 1 from Basilicata, 1 from Calabria, 1 from Apulia, 2 from Friuli Venezia Giulia and 2 from Trentino Alto Adige regions). It is necessary to clarify that this SSR profile (Table 4) is that of Sangiovese classic (i.e., Sangiovese grosso variety, with its synonyms as Sangiovese del Chianti, Morellino di Scansano, Brunello di Montalcino) and not this of Sangiovese piccolo variety (= Sanvicetro). The presence of these two Sangiovese cultivars with a similar name increases the problem about the confusion for this important Italian grape variety. The wines Chianti, Morellino di Scansano, Sangiovese Montepulciano and Brunello di Montalcino are produced with the variety Sangiovese grosso (Calò et al., 2000). 12 Sangiovese accessions were identified as Sangiovese piccolo (or Sanvicetro) grape variety (i.e., 6 from Tuscany, 2 from Emilia-Romagna, 3 from Marche and 1 from Latium regions). In particular, 5 Sangiovese accessions (Table 1) were identified as Sanvicetro (= Sangiovese piccolo) and not as Sangiovese grosso (i.e., samples No. 7, 24, 44, 47 and 62; Table 1 and 3) while 4 Sanvicetro accessions (Table 1) were identified as Sangiovese grosso and not as Sanvicetro or Sangiovese piccolo (i.e., samples No. 12, 49, 54 and 66; Table 1 and 3). This fact (i.e., wrong name given by the winemakers) confirms the great confusion around the Italian Sangiovese grapes. The 6 Sangiovese clones as reference (i.e., samples numbered from 71 to 76, Table 1 and 3) showed the microsatellite profile of Sangiovese grosso variety (Table 4). The Sanvicetro clone from CRA- VIT collection showed the microsatellite profile of the Sangiovese piccolo (or Sanvicetro) variety (Table 5). 15 Moltepulciano accessions were identified as Montepulciano by microsatellites markers (i.e., 5 from Tuscany, 10 from Marche regions). The Montepulciano clone (i.e., sample No. 78; Table 1 and 3) showed the microsatellite profile of this grape variety (Table 6). 11

12 Table 3: Cultivar identification by SSR of the 78 accessions analyzed (Code) where SG is Sangiovese grosso, SV is Sangiovese piccolo (=Sanvicetro) and M is Montepulciano variety. No. name accession/genotype Code No. name accession/genotype Code 1 Sangiovese Montepulciano (SI) SG01 40 Sangiovese campano (BN) SG26 2 Montepulciano grosso (FI) M01 41 Montepulciano teramano (TE) M09 3 Sangiovese Gaiole (SI) SG02 42 Sangiovese trevigiano (TV) SG27 4 Sanvicetro di Toscana (SI) SV01 43 Brunellino rosso (FR) SG28 5 Sangiovese lungo (SI) SG03 44 Sangiovese giove (MO) SV07 6 Sangiovese carsico (TS) SG04 45 Sangiovese Chianti (AR) SG29 7 Sangiovese toscano (FI) SV02 46 Sangiovese Susegana (TV) SG30 8 Sangiovese Scansano (GR) SG05 47 Sangiovese Morellino (SI) SV08 9 Montepulciano glabro (PE) M02 48 Sangiovese marchigiano (AP) SG31 10 Sangiovese Brunello (SA) SG06 49 Sanvicetro laziale (VT) SG32 11 Morellino di Toscana (FI) SG07 50 Sangiovese trentino (TN) SG33 12 Sanvicetro Morellino (FI) SG08 51 Sanvicetro emiliano (FC) SV09 13 Sangiovese Scansano (GR) SG09 52 Brunello Montalcino (RI) SG34 14 Montepulciano di toscana (AR) M03 53 Montepulciano nobile (TE) M10 15 Brunello abruzzese (CH) SG10 54 Sanvicetro toscano (SI) SG35 16 Sangiovese classico (FI) SG11 55 Sangiovese nostrano (BA) SG36 17 Sanvicetro fiorentino (FI) SV03 56 Brunello di Frascati (RM) SG37 18 Sangiovese emiliano (RE) SG12 57 Montepulciano (FI) M11 19 Montepulciano classico (CH) M04 58 Morellino romano (FR) SG38 20 Sangiovese Montalcino (PR) SG13 59 Montepulciano Marche (MC) M12 21 Montalcino Prugnolo (SI) SG14 60 Sangiovese Prugnolo (PT) SG39 22 Montepulciano abruzzese (CH) M05 61 Brunello Montalcino (SI) SG40 23 Brunello di Montalcino (SI) SG15 62 Sangiovese senese (SI) SV10 24 Sangiovese rosso (GR) SV04 63 Sangiovese perugino (PG) SG41 25 Sangiovese toscano (SI) SG16 64 Sanvicetro laziale (FR) SV11 26 Montepulciano toscano (SI) M06 65 Sangiovese Brunello (FI) SG42 27 Sangiovese Chianti (FI) SG17 66 Sanvicetro piccolo (VI) SG43 28 Sanvicetro Marche (MC) SV05 67 Sangiovese grosso (SI) SG44 29 Sangiovese Chianti (FI) SG18 68 Montepulciano sangiovese (FI) M13 30 Morellino di Potenza (PZ) SG19 69 Morellino berico (VI) SG45 31 Montepulciano Marche (AP) M07 70 Montepulciano rotondo (CH) M14 32 Sangiovese Rovigo (RO) SG20 71 Clone Sangiovese I (SI) C Morellino di Scansano (GR) SG21 72 Clone Sangiovese II (SI) C Montepulciano classico (PE) M08 73 Clone Sangiovese III (SI) C Sangiovese calabro (CS) SG22 74 Clone Sangiovese IV (FI) C Morellino gentile (VR) SG23 75 Clone Sangiovese V (GR) C Sanvicetro abruzzese (CH) SV06 76 Clone Sangiovese VI (FC) C Sangiovese friulano (UD) SG24 77 Clone Sanvicetro I (CRA-VIT) C Morellino precoce (FI) SG25 78 Clone Montepulciano I (TE) C-08 12

13 Each genotype analyzed (Table 1) was identified by the letters SG (=Sangiovese grosso variety) or SV (=Sangiovese piccolo or Sanvicetro variety) or M (=Montepulciano variety) or C (=Clones of these three varieties) followed by a progressive number (i.e., SG01/SG45 for Sangiovese grosso; SV01/SV11 for Sangiovese piccolo or Sanvicetro; M01/M14 for Montepulciano and C-01/C-08 for reference clones) (Table 3). These codes were used for the cluster analysis. In Table 4 and 5 were reported the two SSR profiles at 11 loci microsatellites for Sangiovese grosso and Sangiovese piccolo (=Sanvicetro) varieties. Table 4: SSR profile of Sangiovese or Sangiovese grosso (11 Loci). VVS2 VVMD5 VVMD7 VVMD27 VVMD VrZAG62 VrZAG79 VMC6E1 VMC6F1 VMC6G1 VMCNG4b Table 5: SSR profile of Sanvicetro or Sangiovese piccolo (11 Loci). VVS2 VVMD5 VVMD7 VVMD27 VVMD VrZAG62 VrZAG79 VMC6E1 VMC6F1 VMC6G1 VMCNG4b The two microsatellites profiles of Sangiovese varieties (i.e., Sangiovese grosso and Sangiovese piccolo / Sanvicetro) are different and only 2 SSR loci showed the identical microsatellite alleles (i.e., VVMD5 and VVMD27). 9 SSR loci are different between the two Sangiovese varieties (i.e., VVS2, VVMD7, VVMD28, VrZAG62, VrZAG79, VMC6E1, VMC6F1, VMC6G1 and VMCNG4b9) but 8 of these shared 1 SSR allele per locus (i.e., VVS2, VVMD7, VrZAG62, VrZAG79, VMC6E1, VMC6F1, VMC6G1 and VMCNG4b9), but a first degree relationship between them can be discarded because of one excluding loci (i.e., VVMD28) (Table 4 and 5). In Table 6 was reported the SSR profiles at 11 loci microsatellites for Montepulciano variety. 13

14 Table 6: SSR profile of Montepulciano (11 Loci). VVS2 VVMD5 VVMD7 VVMD27 VVMD VrZAG62 VrZAG79 VMC6E1 VMC6F1 VMC6G1 VMCNG4b Biotypes belonging the two different varieties of Sangiovese (S. grosso and S. piccolo) were then analyzed separately (intra-varietal analysis) and together (inter-varietal analysis) with also Montepulciano cultivar materials using AFLP, SAMPL, ISSR and MAFLP molecular markers. The most important results is that this molecular methodology allowed to differentiate very efficiently the biotypes belonging to the same variety of Vitis vinifera, but there is another important aspect that has emerged from the molecular analysis about the different geographic origins of these materials (Figure 2). A total of 1,905 reproducible amplification products were obtained with the four molecular marker systems: 689 AFLPs, 650 SAMPLs, 503 M-AFLPs and 63 ISSRs. Of these, 972 (51.2%) were polymorphic: 326 (47.3%) AFLPs, 355 (54.6%) SAMPLs, 268 (53.2%) M-AFLPs and 23 (36.5%) ISSRs. Molecular experiments were repeated twice or more: duplicate DNA samples analyzed in every experiment confirmed the molecular approach reproducibility. The mean number of marker loci assayed per single experiment was 62.6, 59.1, 45.7 and 5.7 for AFLP, SAMPL, M-AFLP and ISSR markers, respectively. Dice s GS matrix was constructed for all possible pairwise comparisons of 51 Sangiovese grosso (i.e., 45 biotypes and 6 clones), 12 Sangiovese piccolo (i.e., 11 biotypes and 1 clone) and 15 Montepulciano accessions (14 biotypes and 1 clone). The observed GS TOT was : the GS W within the 51 Sangiovese grosso accessions was , GS W within the 12 Sangiovese piccolo accessions was and GS W within the 15 Montepulciano accessions was Therefore the variety more homogeneous inside was the Sangiovese piccolo or Sanvicetro cultivar. GS B was between Sangiovese grosso (= Sangiovese) and Sangiovese piccolo (= Sanvicetro) varieties, between the Sangiovese grosso and Montepulciano varieties and between Sangiovese piccolo and Montepulciano varieties. These results showed that the two Sangiovese cultivars (i.e., Sangiovese grosso and Sangiovese piccolo) were genetically similar but can be 14

15 discriminated molecularly by the four marker systems used (i.e., AFLP, SAMPL, MAFLP and ISSR). Finally Montepulciano variety is more similar genetically to the Sangiovese grosso (=Sangiovese) that Sangiovese piccolo (= Sanvicetro). The UPGMA Dendrogram in Figure 2 displayed the 78 genotypes clustered into 2 distinct groups: Group 1 with Sangiovese piccolo and Sangiovese grosso accessions, Group 2 with Montepulciano accessions. The three grape varieties were separated using this molecular approach according to microsatellite analysis (Table 3). The first group (Sangiovese varieties) show 2 distinct subgroups, the Sangiovese grosso accessions (i.e., Sangiovese of Tuscany, Sangiovese of Emilia-Romagna & Latium, Sangiovese of Southern Italy, Sangiovese of Northern Italy) and the Sangiovese piccolo (= Sanvicetro) accessions. Therefore, all Sangiovese grosso accessions were hence separated on the basis of their geographic origins (i.e., Tuscany region, Emilia-Romagna & Latium regions with also accession from Marche and Abruzzo regions, Southern Italy as Campania, Basilicata, Calabria and Apulia regions, Northern Italy as Veneto, Trentino Alto Adige and Friuli Venezia Giulia regions). The dendrogram based of genetic diversity reported in Figure 2 show that the biotypes of Sangiovese grosso are really different at DNA level: these Sangiovese grosso accessions were grouped into four main groups: Sangiovese form Tuscany, Sangiovese from central Italy, Sangiovese from Southern Italy and Sangiovese from Northern Italy. For the genotypes of Sangiovese from Tuscany it was also possible to discriminate the material into different subgroups. GS matrices estimates and Dendrogram results were in agreement with Principal Component Analysis reported in Figure 3. The 3-dimentional plotting of centroids showed seven different groups: 1) Sangiovese of Tuscany; 2) Sangiovese of Emilia-Romagna & Latium with Abruzzo and Marche accessions; 3) Sangiovese of Southern Italy; 4) Sangiovese of Northern Italy; 5) Sanvicetro (=Sangiovese piccolo) from Tuscany; 6) Sanvicetro (=Sangiovese piccolo) from Latium, Emilia- Romagna and Marche; 7) Montepulciano. Considering these molecular results, genotypes of same cultivar can be discriminated using these molecular approach and the molecular characterization has yielded excellent results because it was possible to notice that Sangiovese grosso and Sangiovese piccolo (=Sanvicetro) biotypes differed also their different geographic origins (Figure 2 and 3). Similar results were shown for the Montepulciano accessions from Tuscany and Abruzzo. 15

16 Figure 2: Dendrogram of the 78 genotypes (Sangiovese grosso, Sangiovese piccolo / Sanvicetro and Montepulciano varieties, Table 1 and 3) studied based on AFLP, ISSR and SAMPL molecular polymorphisms. 16

17 Figure 3: Centroids 3D of the 78 genotypes (Sangiovese grosso, Sangiovese piccolo / Sanvicetro and Montepulciano varieties, Table 1 and 3) based of AFLP, SAMPL, M-AFLP and I-SSR polymorphisms. These results are very important because they are in agreement with the results of other analyzes conducted with the same molecular method on other grape varieties as Malvasia nera di Brindisi/Lecce, Malvasia di Candia, Negroamaro, Primitivo/Zinfandel, Grenache noir/garnacha tinta/cannonau, Malvasia Istriana (Meneghetti et al., 2011; Meneghetti et al., 2012a & b). The study of these cultivars highlighted the possibility to discriminate at molecular level the genotypes according with their different geographical origins; genotypes from neighboring areas showed a greater genetic similarity respect to these materials of the same variety but from regions most geographically distant. These results were also confirmed by the 8 clones taken as a reference (i.e., 6 Sangiovese grosso, 1 Sangiovese piccolo or Sanvicetro and 1 Montepulciano clones, Table 3). 17

18 4. CONCLUSIONS The SSR analysis confirmed that there are two different Sangiovese varieties (i.e., Sangiovese grosso and Sangiovese piccolo) in Italy: these two cultivars co-exist and are often grown together in the same vineyards of Sangiovese DOC or DOCG zones. Markers (i.e., AFLPs, SAMPLs, M-AFLPs and ISSRs) were able to clearly distinguish all the Sangiovese (=Sangiovese grosso), Sanvicetro (=Sangiovese piccolo) and Montepulciano accessions also in relation to their different geographic origins: Tuscany region, Emilia-Romagna & Latium regions, Southern Italy (i.e., Campania, Basilicata, Calabria and Apulia regions), Northern Italy (i.e., Veneto, Trentino Alto Adige and Friuli Venezia Giulia regions). Results confirm the ability of M-AFLPs and SAMPLs to discriminate the Vitis accessions by plantspecific molecular markers. This molecular approach provides an excellent means of Sangiovese (i.e., Sangiovese grosso and Sangiovese piccolo) and Montepulciano biotypes identification and fingerprinting. Intra-varietal molecular results have highlighted the existence of genetic variability among the Sangiovese, Sanvicetro and Montepulciano accessions from different geographic cultivation areas and suggesting the need for the preservation of autochthonous grapevine biotypes found on different zones to approve the correct choice and selection of the grape multiplication materials. 5. REFERENCES 1. Albertini, E., Porceddu, A., Marconi, G., Barcaccia, G., Pallottini, L. & Falcinelli, M. (2003). Microsatellite-AFLP for genetic mapping of complex polyploids. Genome, 46: Barcaccia, G., Meneghetti, S., Albertini, E., Triest, L., & Lucchin, M. (2003). Linkage mapping in tetraploid willows: Segregation of molecular markers and estimation of linkage phases support an allotetraploid structure for Salix alba x Salix fragilis interspecific hybrids. Heredity, 90, Blaich, R., Konradi, J., Rühl, E., Forneck, A. (2007). Assessing Genetic Variation among Pinot noir (Vitis vinifera L.) Clones with AFLP Markers. American Journal of Enology and Viticulture, 58-4: Böhm, A. & Zyprian, E. (1998). RAPD marker in grapevine (Vitis spp.) similar to plant retrotransposons. Plant Cell Reports, 17-5: Brevigleri, N. & Casini, E. (1965). Sangiovese. I principali vitigni da vino in Italia. Ed. MAF, Rome, Italy. 6. Calò, A. & Costacurta, A. (2004). Dei vitigni italici. Ed. Matteo, Treviso, Italy. 18

19 7. Calò, A., Costacurta, A., Egger, E., Storchi, P., Crespan, M., Milani, N., sensi, E., Carraro, R. (2000): Caratterizzazione molecolare, ampelografia e ampelometrica di 30 accessioni di Vitis vinifera L. riferibili al Sangiovese. International Symposium "Il Sangiovese", Florence, Palazzo dei Congressi, February Cipriani, G., Marrazzo, M. T., Di Gaspero, G., Pfeiffer, A., Morgante, M. & Testolin R. (2008). A set of microsatellite markers with long core repeat optimized for grape (Vitis spp.) genotyping. BMC Plant Biology, 8, Cretazzo, E., Meneghetti, S., De Andrés, M. T., Frare, E., Gaforio, L., & Cifre, J. (2010). Clone differentiation and varietal identification by means of SSR, AFLP, SAMPL and M- AFLP in order to assist the clonal selection of grapevine. The case of study of Manto Negro, Callet and Moll, autochthonous cultivar of Majorca. Annals of Applied Biology, 157-2, Dice, L. R. (1945). Measures of the amount of ecological association between species. Ecology, 26, Di Rovasenda, (1877). Saggio per un ampelografia universale. Ed. Tipografia Subalpina, Turin, Italy. 12. D Onofrio, C., De Lorenzis, G., Giordani, T., Natali, L., Scalabrelli, G., Cavallini, A. (2009). Retrotransposon-based molecular markers in grapevine species and cultivars identification and phylogenetic analysis. Acta Horticulturae (ISHS), 827: Falchini, D. (1720). Trattato di agricoltura, manuscript. Ed. Provincia di Firenze, Archivio Storico, Florence, Italy. 14. Galet, P. (2000). Dictionnaire encyclopédique des cépages. Ed. Hachette, Paris. 15. Imazio, S., Labra, M., Grassi, F., Winfield, M., Bardini, M., Scienza, A. (2002). Molecular tools (SSR, AFLP, MSAP) for clone identification: the case of the grapevine cultivar Traminer. Plant Breeding, 121-6, Labra, M., Imazio, S., Grassi, F., Rossoni, M., Sala, F. (2004). Vine-1 retrotransposon-based sequence-specific amplified polymorphism for Vitis vinifera L. genotyping. Plant Breeding, 123-2: Meneghetti, S., Costacurta, A., Morreale, G., & Calò, A. (2012b). Study of intravarietal genetic variability in grapevine cultivars by PCR-derived molecular markers and correlations with the geographic origin. Molecular Biothecnology, 50-1, Meneghetti, S., Poljuha, D., Frare, E., Costacurta, A., Morreale, G., Bavaresco, L. & Calò A. (2012a). Inter- and intra-varietal genetic variability in Malvasia cultivars. Molecular Biothecnology, 50-3, Meneghetti, S., Calò, A. & Bavaresco, L. (2012c). A Strategy to investigate the Intravarietal Genetic Variability in Vitis vinifera L. for clones and biotypes identification and to correlate molecular profiles with morphological traits or geographic origins. Molecular Biothecnology, 52-1, Meneghetti, S., Costacurta A., Frare, E., Da Rold, G., Migliaro, D., Morreale, G., Crespan, 19

20 M., Sotés, V., Calò, A. (2011). Clones Identification and Genetic Characterization of Garnacha Grapevine by Means of Different PCR-Derived Marker Systems. Molecular Biotechnology, 48-3: Molon, (1906). Ampelografia. Vol. II. Ed. Hoepli, Milan, Italy. 22. Odart, G. (1874). Traité de cépages. Paris: Librairie agricole. 23. Owens, C.L. (2003). SNP detection and genotyping in Vitis. Acta Horticulturae, 603: Pelsey, F., Schehrer, L., Merdinoglu, D. (2002). Development of grapevine molecular markers based on retrotransposons. Acta Horticulturae, 603: Regner, F., Wiedeck, E. & Stadlbauer, A. (2000). Differentiation and identification of White Riesling clones by genetic markers. Vitis, 39-3, Rohlf, F.J. (2000): Numerical taxonomy and multivariate analysis system. Version 2.1. Stony Brook, NY: State University of new York. 27. Silvestroni, O., & Intrieri, C. (1995). Ampelometric assessment of clonal variability in the Sangiovese winegrape cultivar. International Symposium on Clonal Selection, June, Portland, OR, USA. American Society for Enology and Viticulture, Villifranchi, (1773). Oenologia toscana, ossia memoria sopra i vini e in specie toscani. Ed. Accademia dei Georgofili, Vol. No. XV-XVI, Florence, Italy. 29. Vignani, R., Scali, M., Masi, E., & Cresti, M. (2002): Genomic variability in Vitis vinifera L. "Sangiovese" assessed by microsatellite and non-radioactive AFLP test. Electronic Journal of Biotechnology, 5-1,