Study of Genetic Relationships Between Wild and ... - ZooPlantLab

Rendiconti Lincei 19, 223 – 240 (2008) DOI: 10.1007/s12210-008-0016-6

Fabrizio De Mattia · Serena Imazio · Fabrizio Grassi · Hamed Doulati Baneh · Attilio Scienza · Massimo Labra

Study of Genetic Relationships Between Wild and Domesticated Grapevine Distributed from Middle East Regions to European Countries

Received: 19 October 2007/ Accepted: 21 January 2008 – © Springer-Verlag 2008

Abstract Archaeobotanical-archaeological, cultural and historical data indicate that grapevine domestication can be dated back from 6000 to 7000 years ago and that it took place in the Caucasian and Middle East Regions. However, events leading to the domestication of this crop species are still an open issue. In this paper, 6 chloroplast microsatellites have been used to assess genetic similarities among, and within, domesticated and wild grapevine acM. Labra (B) Dipartimento di Biologia e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, I-20126 Milano, Italy Tel.: +390264483472, Fax: +390264483450, E-mail: [email protected] F. De Mattia Dipartimento di ProduzioneVegetale, Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy Tel.: +390264483472, Fax: +390264483450, E-mail: [email protected] S. Imazio Dipartimento di ProduzioneVegetale, Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy Tel.: +390250316563, E-mail: [email protected] A. Scienza Dipartimento di ProduzioneVegetale, Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy Tel.: +390250316559, Fax: 022365302, E-mail: [email protected] F. Grassi Orto Botanico di Cascina Rosa, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy Tel.: +390250320891, E-mail: [email protected] H. Doulati-Baneh Department of Seed and Plant breeding, Agricultural and Natural Resource Research Center of West Azerbaijan, P.O.Box 365, Urmia, Iran Tel.: +9844126222226, Fax: +984412622221, E-mail: [email protected]

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cessions representative of 7 distinct geographical regions from the Middle-East to Western Europe. Results show that 2 out of the 6 analyzed chloroplast loci are polymorphic within the 193 domesticated individuals and the 387 samples of 69 wild populations. Allele variants of the Cp-SSR loci combine in a total of 6 different haplotypes. The data show that the haplotype distribution is not homogeneous: the 6 haplotypes are present in the domesticated varieties, but only 5 (haplotype VI is absent) are observed in wild populations. The analysis of haplotype distribution allows discussion of the relationships between the two grape subspecies. The contribution of the wild grape germplasm to the domesticated gene pool still growing in different geographical regions can be, in cases, made evident, suggesting that beside domestication, gene introgression has also played a role in shaping the current varietal landscape of the European viticulture. Keywords Chloroplast microsatellite, Crop domestication, Grapevine, Vitis vinifera L Subject codes L11006, L19031, L19090, L24000, L24027, L24051 Abbreviations Cp-SSR = Chloroplast Simple Sequence Repeat 1 Introduction The Eurasian grapevine (Vitis vinifera L.) is the most widely cultivated and economically important fruit crop in the world (Vivier and Pretorius 2002). Vitis vinifera L. includes: V. vinifera ssp. silvestris (wild grapevine), distributed in the native vegetation of West-Asia, the Mediterranean basin and in Central and Southern Europe (Hegi 1925), and V. vinifera ssp. vinifera (domesticated grapevine), which has been domesticated from wild populations of V. vinifera ssp. silvestris (Levadoux 1956). Wild grapevine prefers wet soils while domesticated accessions grows best in dry habitats. The most relevant difference among the two subspecies is the mating system: wild grapevine is dioecious while the domesticated relative is self-compatible (hermaphrodite). McGovern (2003) suggests that humans developed interest in wild grapes during the Paleolithic period in the upland regions of eastern Turkey, northern Syria, or/and in north-western Iran. The earliest evidence of grape cultivation dates back to the fourth millennium in the Middle East (Zohary and Hopf 2000), and wine production was already present in Iran during the second half of the sixth millennium B.C. (McGovern 2003). Starting from the primary domestication center, the grapevine spread to the South-eastern Mediterranean regions, to Palestine, Southern Lebanon and Jordan (Zohary and Spiegel-Roy 1975). Later, during the 3rd millennium B.C., domesticated grapevines appeared in

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the Near East, Southern Greece, Crete, Cyprus and Egypt. In the beginning of 2nd millennium B.C., domesticated grapevines were found in the Southern Balkans (Logothetis 1970; Kroll 1991). In the second half of the 2nd millennium B.C. they appeared in Southern and Northern Italy, Southern France, Spain, and Portugal in the second part of the first millennium (Levadoux 1956; Forni 1990; Hopf et al. 1991). The history of grapevine domestication is still a matter of debate: there is no agreement on the localization of the area where grapevine was first domesticated and on the existence of secondary centers. McGovern and Olmo (1976) suggested a monocentric origin of viticulture where domestication started from a restricted pool of wild plants. However, molecular investigations combined with archaeo-botanical and historical evidence support and testify domestication experiences in the Mediterranean area (Dedet 1980; Rivera-Nunez and Walker 1989; Grassi et al. 2003; Sefc et al. 2003). In recent years, the availability of molecular markers offered new possibilities in defining genetic relationships between wild and domesticated grapevines. Particularly, chloroplast DNA markers are a good tool in the reconstruction of species history (King and Ferris 1998; Palmé and Vendramin 2002), due to the non-recombining nature of chloroplast DNAs and to its low genome mutation rate (Ferris et al. 1998; Marchelli et al. 1998; Fineschi et al. 2002). Indeed, organelle genomes are maternally inherited in most angiosperms (Rajora and Dancik 1992; Dumolin et al. 1995) and cytoplasmic analysis assumes particular relevance in the study of plant diffusion mediated by seeds. The objective of the present study is to use the chloroplast microsatellites (Cp-SSRs) to study genetic variability in wild and domesticated grapevine A total of 193 domesticated varieties and 387 wild grapevine accessions distributed from Iran to Spain-Portugal, were sampled and grouped in 7 different geographical clusters. The haplotype group constitution allows to test the hypothesis of the existence of different routes for grapevine origin and diffusion. 2 Materials and methods 2.1 Plant material One-hundred and ninety-three grapevine cultivars have been selected as representatives of the distribution area of domesticated grapevine. Plant material was from different germplasm collections and was subdivided into 7 groups based on the area of cultivation (Table 1). Group 1 – Iran: 55 accessions; Group 2 – Near East: 18 accessions mainly cultivated in the Turkish and Caucasian Region;

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Haplotype II

Haplotype III

Agh melhi∗ , At ouzum∗, Dizmari∗ , Fakhri∗, Ghara ouzum∗, Ghara shira∗ , Ghara melhi∗ , Lal bidaneh∗, Lal siah*, Maiemo∗ , Makaii∗ , Mosli∗ , Rezghi∗ , Rishbaba sefid∗ , Sorav∗ .

–

GROUP 1

Agh shani∗ , Akuz guzi∗ , Angotka∗, Abi balo∗ , Bol mazo∗ , Chava ga∗ , Garmian∗ , Goi maleki∗ , Gonka∗, Hosaini∗ , Inak amjaii∗ , Khoshnav∗, Klaka revi∗ , Lal ghermez∗, Mam braima∗ , Rasha∗ , Sachakh∗, Sayani∗ , Sarghola∗ , Shirazi∗ , Siah mamoli∗ , Taiefi∗ , Zardka∗. Agialesci∗∗ , Aladasturi∗∗ , Alexandrouli∗∗, Avassirkhva∗∗ , Brola∗∗ , Cetcipesci∗∗ , Charistvala∗∗ , Chichaveri∗∗, Dondglabi∗∗, Giani∗∗ , Gorula∗∗ , Jgya∗∗ , Kamouri∗∗, Kapistoni Tetri-1∗∗ , Kapistoni Tetri-2∗∗ , Mteva∗∗ .

Armenia∗∗∗.

Kisi∗∗ .

Agria Sultanina§ , Akominato§, Dafnia§, Fraoula kokkini§, Kakotrygis§, Kritiko mavro§, Liatiko§, Mavrodafni§, Moemvasia§ , Moschardina§, Moschato amvourgou§, Moschato aspro§ , Moschato chondro§, Moschato kerkyras§, Moschato mazas§ , Moschopatata§, Mouska§, Razaki lefkos§, Rome§ .

Aetonychi§, Dermatas§ , Eftakoilo§, Korithi mavro§, Psarosyriko§.

–

Aglianico precoce∗∗, Cesanese∗∗∗ , Prunesta∗∗∗ , Grego di Tufo∗∗∗, Montepulciano∗∗∗, Mammolo∗∗∗, Trebbiano toscano∗∗∗, Vernaccia di S. Giminiano∗∗∗, Cigliegiolo∗∗∗, Grechetto di Todi∗∗∗, Bellone∗∗∗ , Monsonico∗∗∗.

Aleatico∗∗∗ , Croatina∗∗∗ , Dolcetto∗∗∗ , Fiano∗∗∗ , Lambrusco marani∗∗, Malvasia bianca∗∗∗ , Malvasia del Chianti∗∗∗ , Sangiovese∗∗∗, Moscato terracina∗∗∗ , Teroldego†, Verdicchio∗∗∗, Vespolina∗∗∗.

Corvinone∗∗∗.

Traminer∗∗, Silvaner∗∗ .

–

S. Laurent∗∗, Furmint∗∗.

Cabernet franc†, Cabernet sauvignon†, Gamay∗∗ , Mazuela∗∗∗ , Sauvignon blanc† , Semillon∗∗ .

–

Clairette∗∗ .

Albillo† , Beba∗∗ , Beba de Huelva∗∗ , Beba de Jaen∗∗ , Beba de Jerez∗∗ , Blanca Temprana Almeria# .

Arinto de Bucelas# .

Doradilla∗∗ .

GROUP 7

GROUP 6

GROUP 5

GROUP 4

GROUP 3

Haplotype I

GROUP 2

Table 1 List of grapevine cultivar groups to their site of sampling with indications of their haplotypes at the marker loci ccmp3 and ccmp10.

∗ – Agricultural Research Center of West Azarbaijan,Grapevine Collection of Kahriz, Ourmia-Iran; ∗∗ – Regional Agency for Agriculture and Forests (ERSAF), Pavia, Italy; ∗∗∗ – Regional Agency for Agriculture (ERSA), Gorizia, Italy; § – http://oldweb.biology.uoc.gr/gvd/contents; † – Istituto Agrario di San Michele all’Adige (ISMA), Trento, Italy; †† – Experimental Institute for Viticulture (ISV), Treviso, Italy; # – Authoctonous Grapevine Germplasm Collection of Jerez de la Frontera (XF), Jerez de la Frontera, Spain.


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Haplotype V

Haplotype VI

–

Alhaghi∗ , Askari∗ , Dastarchin∗, Galin barmaghi∗, Ghara gandoma∗, Ghara shani∗ , Ghzl ouzum∗, Gazandaii∗ , Kazhav∗ , Keshmesh sefid∗ , Keshmesh ghermez∗, Khalili*, Kalati∗ , Tabarza ghermez∗, Rejin∗ , Rishbaba ghermez∗, Sahebi∗ .

–

–

–

–

Fokiano§, Kotsifali§ , Krystalli§ , Moschato spinas§ , Petrachladi§ , Razaki chakidikis§, Razaki kavalas§ , Sefka§, Tressallier§ .

Karatsova naousis§ , Kolokythas lefkos§ , Nychato§, Perle de Csaba§ , Razaki moschato§, Roditis kokkinos§, Roditis lefkos§ , Tsaousi§ .

–

Colorino∗∗∗, Canaiolo∗∗∗ , Ucelut∗∗∗ .

Aglianico nero∗∗ , Cesanese comune∗∗∗.

Aglianico∗∗, Canaiolo∗∗∗ , Cannonau∗∗∗, Frappato∗∗∗, Lattuario∗∗, Nasco∗∗∗ .

Riesling∗∗ .

Harslevelue∗∗ , Coarnaalba∗∗ , Limberger∗∗.

–

Aligotè∗∗ , Pansè∗∗ , Shiraz∗∗ .

Chardonnay∗∗, Cot∗∗ , Merlot∗∗ .

Altesse∗∗ , Parellada∗∗, Pinot noir†† , Syrah∗∗ .

Albino de Souza# .

Arinto de Dao# , Bobal# .

Acheria# , Ahmed∗∗∗ , Baladi Verdejo#, Boal de Madere# , Brancelho#, Ciguentes∗∗ , Corrazon de Cabrito∗∗ .

GROUP 7

GROUP 6

GROUP 5

GROUP 4

GROUP 3

GROUP 2

GROUP 1

Haplotype IV

∗ - Agricultural Research Center of West Azarbaijan,Grapevine Collection of Kahriz, Ourmia- Iran; ∗∗ - Regional Agency for Agriculture and Forests (ERSAF), Pavia, Italy; ∗∗∗ - Regional Agency for Agriculture (ERSA), Gorizia, Italy; §− http://oldweb.biology.uoc.gr/gvd/contents; †− Istituto Agrario di San Michele all’Adige (ISMA), Trento, Italy; †† - Experimental Institute for Viticulture (ISV), Treviso, Italy; # - Authoctonous Grapevine Germplasm Collection of Jerez de la Frontera (XF), Jerez de la Frontera, Spain.

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Fig. 1 Distribution of wild grape populations considered in the paper. Table 2 describes the characteristics of each population and the clustering strategy based on geographical coordinates.

Group 3 – Greece: data of 41 accessions were obtained from the Greek database (http://oldweb.biology.uoc.gr/gvd/contents/); Group 4 – Italy: 36 accessions representing the Italian traditional grapevine germplasm; Group 5 – Central Europe: 8 accessions from Austria and Germany; Group 6 – France: 17 traditional varieties; Group 7 – Iberian Peninsula: 18 cultivars of Western European countries such as Spain and Portugal. The collection of cultivated accessions consisted only of traditional varieties growing in clarified area. This is why some regions, such as Central Europe, are represented by a low number of cultivars. A total of 387 individuals from 69 wild grapevine populations belonging to the same area of the cultivated accessions were collected (Figure 1) and clustered in groups corresponding to those of the cultivated germplasm. Some of these populations were previously analyzed by Grassi and co-workers (2006). Table 2 indicates names, identification code, number of individuals and geographical coordinates of populations belonging to each group.


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Table 2 List of wild grapevine populations, code, geographic coordinates of sites of sampling (* = Approx ±3 ), number of samples analyzed in each population (N) and their haplotypes at the marker loci ccmp3 and ccmp10. The 69 wild populations were divided into 6 geographical groups. No wild accessions from Greece were considered. POPULATION NAME

GROUP 1

GROUP 2

GROUP 4

GROUP 5

GROUP 6

GROUP 7

Sardasht – Ghasm rash Sardasht – Ghasm rash Sardasht – Shalmash Sardasht – Shivakan Sardasht – Shakhiju Piranshahr – Prdanan Piranshahr – Bradinaveh Banah – Tazhan Armenia Armenia Armenia Armenia – Talin Russia – Kabardino Russia – Dagestan Russia – Majkop Azerbaijan Georgia – Kisi Liguria – Valgrande Liguria – Castiglione Toscana – Grosseto Toscana – Parco Uccellina Toscana – Sticciano Toscana – Ringhiere Toscana – Muro Toscana – La Sterza; Era river Lombardia – Po river Lombardia – Bosco S. Negri Emilia Romagna – Mesola Roma – Tolfa Frosinone Campania Calabria – Cosenza Calabria – Rossano Calabria – San Anargia Calabria – San Apollinare Calabria – San Zaccaria Molise – Isernia Basilicata – Sinni Basilicata – Tempa Martina Austria Èeská Rep. – Bøeclav Deutschland – Ketsch Deutschland – Dirmstein Deutschland – Koller Deutschland – Hoerdit Hendave Hendave Hendave Valence Rio Esca – Huesca Rio Ver – Huesca Navarra Guipuzcoa Cadiz Ciudad Real Menorca La Mirilla Rivera del Hueznar Siviglia Malaga PN. Doñana PN. Doñana a Rocinas PN. Doñana Acebròn Rio Murtiga Arrovo Peguera Arrovo Nateruela El Chorreadero Pantano de los Hurones Jaen

CODE SG1 SG2 SHA SS SHAK PR PB BT AR1 AR2 AR3 AR4 R1 R2 R3 AZ1 G1 I1 I2 I3 I4 I5 I6 I7 I8 I9 I10 I11 I12 113 I14 I15 I16 I17 I18 I19 I20 I21 I22 A1 C1 D1 D2 D3 D4 F1 F2 F3 F4 F5 F6 F7 F8 E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 E13 E14 E15 E16

COORDINATES 36◦ 12 ; 45◦ 22 36◦ 13 ; 45◦ 23 36◦ 05 ; 45◦ 30 36◦ 25 ; 45◦ 22 36◦ 09 ; 45◦ 28 36◦ 14 ; 45◦ 05 36◦ 34 ; 45◦ 11 35◦ 59 ; 45◦ 53 41◦ 03 ; 44◦ 54 41◦ 05 ; 45◦ 07 40◦ 55 ; 45◦ 11 – – – – – – 44◦ 21 ; 09◦ 43 – 42◦ 81 ; 10◦ 95 42◦ 55 ; 11◦ 21 42◦ 91 ; 11◦ 18 – – – – 45◦ 12 ; 09◦ 03 – 42◦ 09 ; 11◦ 90 41◦ 60 ; 12◦ 82 41◦ 41 ; 13◦ 92 39◦ 21 ; 16◦ 28 39◦ 58 ; 16◦ 70 – – – 41◦ 62 ; 14◦ 20 40◦ 25 ; 16◦ 52 40◦ 20 ; 15◦ 96 48◦ 10 ; 16◦ 30 – – 49◦ 61 ; 08◦ 23 – 49◦ 03 ; 08◦ 21 43◦ 10 ; 01◦ 22 43◦ 10 ; 01◦ 31 43◦ 19 ; 01◦ 22 – 42◦ 31 ; 00◦ 90 42◦ 30 ; 00◦ 70 – 42◦ 75 ; −02◦ 74 – – – 37◦ 39 ; −06◦ 09 37◦ 55 ; −05◦ 42 – – 36◦ 52 ; −06◦ 23 36◦ 07 ; −06◦ 30 37◦ 08 ; −06◦ 32 38◦ 01 ; −06◦ 47 36◦ 22 ; −05◦ 38 36◦ 20 ; −05◦ 37 36◦ 49 ; −05◦ 29 36◦ 46 ; −05◦ 33 –

N◦ 10 10 8 3 8 12 5 10 8 4 8 2 2 4 4 4 2 3 2 7 12 6 6 3 3 2 3 8 13 7 2 2 7 3 8 9 6 7 10 16 6 8 2 2 2 2 7 2 3 12 12 4 2 4 9 5 5 6 3 3 6 4 2 6 6 6 4 3 2

I 6 1 1 – 7 11 5 2 2 1 – – – 1 – 1 – – – 2 4 – – – – – – – 7 – 1 – – – – – 2 1 1 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

HAPLOTYPES II III IV 4 – – 9 – – 7 – – 3 – – 1 – – 1 – – 0 – – 8 – – 6 – – 2 – 1 – – 8 – – – – – 1 3 – – – 1 3 – 1 2 2 – – – – 3 – – 2 1 – 4 1 – 6 – 2 4 2 – 4 – – 3 – 1 2 – – 1 1 – 3 – – 8 3 1 2 – 1 6 – – 1 – – 2 1 – 6 – – 3 – – 8 – – 9 3 – 1 1 1 4 – – 7 4 – 12 – – 6 – – 8 – – 2 – – 2 – – 2 – – 2 – – 7 – – 2 – – 3 – – 12 – – 12 – – 4 – – 2 – – 1 1 – 8 – – 5 5 – – – – 6 3 – – – – 3 6 – – 4 – – 2 – – 5 – 1 – – 6 1 – 5 – – 4 – – 2 2 – –

V◦ – – – – – – – – – – – 2 1 – – – – – – – 1 – – – – – – – – – – – – – – – – – 2 – – – – – – – – – – – – – – 3 – – – – – – – – – – – – – 1 –

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No wild grapevine sample of Greek origin was considered in this work; the reason is the loss of sampled accessions due to Phylloxera attacks, (information from Angelakis-Roubelakis; personal e-mail communication). In populations, the number of individuals ranged from 2 to 12. More details on wild grape population size and locations of sampling are found in Grassi et al. (2006). 2.2 DNA extraction and Cp-SSR analysis DNA was extracted as described by Labra et al. (2001) avoiding the purification steps. DNA was analyzed at the following 6 microsatellite loci: ccmp2, ccmp3, ccmp4, ccmp6, ccmp7, and ccmp10 (Weising and Gardner 1999; Grassi et al. 2002). The amplification was performed using the PCR-beads Ready-to-go KIT (Amersham- Bioscience, Italy) starting from 10 ng of total DNA and 5 ng of forward and reverse primers. The forward primer was labelled with 33 P-ATP (Amersham, Italy). PCR amplification was performed with the following thermal cycles: 3 min at 94 ◦ C; 35 cycles of denaturation (45 s at 94 ◦ C), annealing (30 s at 50 ◦ C) and extension (1 min at 72 ◦ C); then a final step for 7 min at 72 ◦ C. In the case of ccmp2, the annealing temperature was 51.5 ◦C. A total of 5 µl of the PCR-amplified mixture was added to an equal volume of loading buffer (80 % formamide, 1 mg ml−1 xylene cyanol FF, 1 mg ml−1 bromophenol blue, 10 mM EDTA, pH 8.0). A volume of 1.5 µl was loaded onto a 6 % denaturing polyacrylamide gel, and electrophoresed in TBE buffer for 3 h at 80 W. The gel was fixed in 10 % acetic acid and exposed to an X-ray film for 24 h. The Cp-SSR allele size was scored by visual inspection of the resulting autoradiograms. 2.3 Statistical analysis Haplotype frequencies were measured as the percentage of accessions or individuals sharing the same haplotype in each group, both for domesticated and wild grapevines. Values for haplotype diversity (h), considered a measure of gene diversity (Nei, 1987), were estimated as GD = (n/(n − 1) (1 − p i 2) where n is the number of sampled individuals and p the frequencies of the different alleles. The n/n − 1 value is negligibly small when n > 50. Therefore, it is recommended that this equation be used unless sampling is particularly numerous. The haplotype frequency distribution among different geographical groups and variation of haplotype frequences from east to west regions and from south to north regions were also defined. The correlation between haplotype frequency and geographical distances (R and P) was calculated by Statistica 6.0 (2001).


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3 Results 3.1 Cp-SSR alleles and haplotype definition Genetic diversity at the microsatellite cpDNA level was analyzed starting from 580 individuals, 193 were domesticated accessions and 387 wild grapevine. Results showed that, out of the 6 SSR loci tested, two (ccmp3 and ccmp10) were polymorphic. Two (106 and 107 bp) and three (114, 115, and 116 bp) were the polymorphic bands found at ccmp3 and ccmp10 respectively (Table 3). To confirm the alleles’ size, defined by gel electrophoresis analysis, some alleles obtained from the analyzed samples (allele 106 from E1 and I12 populations of Table 2; allele 107 from the I3 and AR1 populations; allele 114 from the I12, and D1 populations; allele 115 from the E8 and A1 populations; allele 116 from the AR4 and E4 populations) were sequenced (data not shown). In all cases, results confirm data obtained from gel electrophoresis analysis, and suggest that all loci showed unimodal distribution, with alleles differing by 1 bp from each other. Thus the observed variation conforms to the stepwise mutation model (SMM), which can be explained by replication slippage at SSR loci (Freimer and Slatkin 1996). Table 3 Sizes (bp) of alleles at the ccmp3 and ccmp10 polymorphic chloroplast loci and definition of the six haplotypes based on allele combinations. Haplotype I II III

ccmp3 (bp) 107 106 107

ccmp10 (bp) 115 115 114

Haplotype IV V VI

ccmp3 (bp) 106 106 107

ccmp10 (bp) 114 116 116

At ccmp3, the most frequent fragment had a size of 106 bp in the wild populations and of 107 bp in the cultivars. At ccmp10, the most represented fragment in the wild was of 114 bp, while that of 116 bp was present only in 9 samples. In the domesticated accessions, a 115 bp fragment was the most frequent, followed by one of 116 bp. Allele variants at both polymorphic loci ccmp3 and ccmp10 generated a total of 6 different haplotypes, as shown in Table 3. 3.2 Haplotype variation in the geographical groups Tables 1 and 2 list the haplotype variants for each domesticated and wild accession, respectively. The distribution of the six haplotypes in the geographical groups was not homogeneous (Figure 2a and 2b). The first important observation is the existence of haplotype VI only in the domesticated accessions, specifically in

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Fig. 2 (a): Domesticated (Vitis vinifera L. ssp sativa) and (b) Wild grape (Vitis vinifera L. ssp silvestris) haplotype distribution in the analyzed geographical groups.

the cultivars from Italia, France and the Iberian germplasm. No group-specific haplotype, however, was found. Figure 3 shows the variation of each haplotype from East to West and from South to North respectively for domesticated and wild accessions. Data suggest a clearly different distribution of haplotypes in the two subspecies among the analyzed groups. In the domesticated accessions, haplotype I was the most frequent and ubiquitous across all groups, but differences in frequencies were noted, as shown in Figure 2a. Haplotype V was also largely distributed in the cultivars of all groups, with the exception of group 2. Haplotype II was mainly present in the first four groups and absent in Central European and French cultivars. Haplotype III was the rarest among the domesticated accessions (Table 1). Haplotype IV was found almost exclusively in the Mediterranean basin (Figure 2a). Generally, the haplotypes richness increases from East to West (Figure 3) as also indicated by the analysis of gene diversity (GD) values calculated for each group, reported in Table 4. Gene diversity values were high within each group with the exception of the Near East, where almost all samples shared haplotype I. Based on the number of haplotypes and on gene diversity, Mediterranean Countries (from Group 3 to 7) showed the highest level of genetic richness when compared to Eastern Countries (Groups 1, 2). The analysis of haplotype distribution from South to North regions in the domesticated grapevine do not show relevant variation as also suggested from gene diversity values (Table 4). In the case of the wild grape, haplotype IV turned out to be the most frequent (more than 50 % of accessions) and it was present in all analyzed groups, with the exception of those of Iranian origin (Table 2 and Figure 2b). Haplotype II was widespread with the exclusion of group 6. Only 9 wild individuals showed haplotypes III and V, and haplotype VI was completely absent. Moreover, Italian and Near Eastern groups were defined by all 5 haplotypes, while French and Iranian accessions showed only one or two haplotypes, respectively (Figure


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Fig. 3 Haplotype frequency distribution among different geographical groups (from 1 to 7). The analysis was conduced from East to West and from North to South regions. For each group the following meridians and parallels were considered: G1 (53◦ , 32◦ ); G2 (43◦ , 42◦ ); G3 (22◦ , 39◦ ); G4 (12◦ , 43◦ ); G5 (10◦ , 49◦ ); G6 (2◦ , 46◦ ); G7 (−3◦ , 40◦ ). R values indicate the correlation between haplotype frequency and geographical distances for wild and domesticated grape, respectively. P indicates the significance of R values ( 0,05).

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Table 4 Value of the haplotype parameter (Nei, 1987) diversity calculated for each germoplasm group of domesticated and wild accessions. G.D. = Gene Diversity; S.D. = Standard Deviation.

Group1 Group2 Group3 Group4 Group5 Group6 Group7

Domesticated G.D. S.D. 0.4675 0.0177 0.0977 0.0424 0.4952 0.0065 0.5059 0.0103 0.6428 0.0884 0.5228 0.0262 0.4232 0.1718

Wild G.D. S.D. 0.2500 0.3536 0.4523 0.1528 – – 0.3511 0.0682 0.1016 0.1397 0 0 0.2683 0.3794

Wild and Domesticated G.D. S.D. 0.3866 0.1539 0.4708 0.0388 – – 0.4550 0.1006 0.2707 0.1490 0.3758 0.1024 0.5436 0.1628

2B). Thus, wild grape haplotype diversity resulted higher in Near Eastern and Italian groups than in the other regions (Table 4). The analysis of haplotype distribution from South to North regions showed that the groups in the middle (2, 4 and 7) were characterized by greater haplotype richness than the samples collected in the Northern (1 and 3) and Southern (5 and 6) regions (Figure 3). Finally, the analysis of gene diversity values calculated for each group (Table 4) showed a clear correlation for the haplotype frequences detected in the domesticated and wild accessions collected in the same regions: GD values calculated for wild and domesticated accession of the same region resulted to be lower than the sum of the GD values detected for the domesticated and wild accession, respectively. These data were detected for all analyzed groups with the exception of the samples of Near East regions (group 2). 4 Discussion In this paper, Cp-SSR analysis allows evaluation of the genetic relationships among domesticated and wild grapevine accessions sampled in seven geographical regions. Due to the maternal inheritance of the chloroplast genome (Dumolin et al. 1995), Cp-SSR variants accumulate in uniparental lineages providing information on the level and distribution of genetic diversity at the regional level. Organelle genomes help to define events governing population and evolutionary processes (Provan et al. 2001). Our analysis shows that two chloroplast SSR regions are polymorphic and that the resulting haplotypes are not similarly distributed in Eastern and Western regions as well as in Southern and Northern regions, both in wild and domesticated grapevine, thus supporting the results of previous experiments of the species distribution (Imazio et al. 2006; Grassi et al. 2006; Arroyo-Garcia 2006). Wild grape haplotype richness in the Mediterranean basin strongly suggests the relevance of its refugia of wild grapevine in Near East, Italy and also in


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Spanish regions. The presence and diffusion of all detected haplotypes in Near East regions suggests a possible center of origin of the species (Levadoux 1956; Grassi et al. 2006). At the same time, higher haplotype diversity in Southern Europe (Italy and Spain) is most likely the result of refugial persistence and accumulation of variation over several ice ages, while lack of refugia and rapid postglacial colonization is probably responsible for Northern purity (Ferris et al. 1998; Hewitt 2001). The analysis of the genetic relationships among domesticated grapevine based on molecular markers, allows the breeding history of grapevine cultivars to be presented with a new prospective. Nuclear SSR and AFLP techniques indicate the existence of a high degree of genetic diversity in domesticated varieties collected from different Mediterranean regions (Sefc et al. 2000; Fossati et al. 2001; Aradhya et al. 2003; Martin et al. 2003). Grapevine nuclear SSRs are a valuable tools in pedigree reconstructions or in the definition of the relationships between genetically close varieties (Bowers et al. 1999; Vouillamoz et al. 2006; Vouillamoz et al. 2007). However, the allele richness detected in the nuclear SSR loci and the high heterozygosis of grapevine clones render this analyses less efficient when the aim is the definition of genetic relatedness among large groups of cultivars (Labra et al. 2002; Imazio et al. 2006). Cp-SSR markers, on the contrary, proved to be particularly useful in the study of cultivar origin (Arroyo et al 2002; Palmé and Vendramin 2002; Snoussi et al. 2004). A general conclusion was that the use of Cp-SSRs in the study of domesticated grapevine revealed an increase in haplotype number from East to West, suggesting that grapevine diversity is higher in the Mediterranean basin. In the case of Vitis vinifera ssp silvestris, our data show two major hot spots of genetic diversity located in the Italian peninsula and in Near East regions. It is also clear that the Eastern wild grapevine germplasm is different from that of Western countries: haplotypes I and II, present in the Iranian germplasm are uncommon in other populations where haplotype IV is the most important. In this paper we provide evidence that domesticated accessions collected in the Near and Middle East regions (group 1 and 2) have a lower number of haplotypes compared to accessions collected from Western groups. These data are very interesting because they may contradict a common belief: based on archaeological and historical studies (Zohary and Spiegel-Roy 1975; Zohary and Hopf 2000), the primary center of grape domestication is most frequently assigned to the Near East regions, allowing the conclusion that all grapevine cultivars derived from this area. In the last years, several authors have opened a debate regarding the existence of minor domestication centers spread along the natural dispersion habitat of wild grapevine. This hypothesis became stronger when molecular data provided evidence of a close relationship between cultivars and local wild plants (Grassi et al. 2003). It is commonly assumed that at least 6000 years of diffusion of viticulture and oenological

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knowledge (Mc Govern 1986) – including variety breeding, selection, maintenance and distribution of cultivars and cultivar substitution – are still not sufficient to know details of this long and extremely complicated history. The reconstruction of part of the history can be approached analyzing the recent cultivar spectrum. The absence of three haplotypes in the domesticated accessions of Near East and Iranian samples, could be explained by two different hypotheses: i) the genetic erosion of several autochthonous cultivars growing in the these regions generated a loss of haplotypes diversity; ii) the existence of secondary domestication centers which have contribuited to the genetic variability of modern day European cultivars. The first hypothesis would be supported by a cultural explanation: the haplotypes absent in the Eastern grapevine germplasm could have been lost due to religious reasons, forbidding winemaking and wine consumption due to Muslims rules. Growers of grapes living in those areas could have concentrated their attention on edible usage of local varieties as fresh or conserved fruit (Tafazzoli et al. 1993). All this could have lead to the loss of genetic diversity, thus of haplotypes. The second hypothesis implies that modern day cultivars derive not only from a primary domestication center located in the Near and Middle East regions. Some varieties may infact derive from secondary domestication events or from local selection of the progenies of hybrids between wild and cultivated accessions (Grassi et al. 2003). Archaeological investigations show that in the Iberian peninsula, grapevine cultivation and wine consuming was already known during the 3rd millennium B.C. (Rivera-Nunez and Walker 1989; Hopf 1991). In the same period of time, in Gailhan (France), grapevine seeds dating back to the Iron age were discovered (Dedet 1980), information supporting the production and consumption of wine in the Mediterranean basin before contacts with Eastern cultures, thus before the arrival of grapevine cultivars from the Near and Middle East. The distribution of haplotypes detected in our analysis allow us to hypothesize that modern day grapevine germplasm is the result of both primary and secondary domestication events. As an example, a large proportion of genotypes included in groups 1 and 2 share haplotype I, which is present at large in wild grapevines sampled in Iran. The finding is compatible with haplotype data from wild and domesticated accessions and with historical and archaeological conclusions supporting the existence of primary domestication events in the Near and Middle East (Olmo 1976; Forni 1990). Probably, domesticated accessions were spread to the West from the Middle East by population movements and cultural exchanges (Labra et al. 2002). Several cultivars sampled in the Mediterranean basin (group 3 to 7) are characterized by haplotype I, while this marker is completely absent in the wild pools of central Europe,


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France, Spain and Portugal; this supports the extra-regional origin of european domesticated varieties characterized by haplotype I. On the other hand, Western domesticated accessions (group 3 to 7) show a high level of haplotype diversity, with six haplotypes recovered in Spain and Italy, five in France and four in central Europe and Greece. These data are compatible with the hypothesis of local events of domestication, or local interbreeding with wild grapes living in the same area. For example, haplotype IV is the most represented in the wild European germplasm and is also present in all Mediterranean groups of domesticated varieties. The haplotype distribution in the 5 Mediterranean region samples supports the existence of a strong varietal exchange throughout Europe (Labra et al. 2002). This germplasm circulation is confirmed by archaeological findings, historical documentation and molecular genetics (Bowers et al. 1999; Labra et al. 2003; McGovern 2003). The exclusive presence of haplotype six in Italian, French and Iberic cultivars excludes the origin of these accessions from the wild samples collected in the same areas. However, we can not exclude that these cultivars derive from domestication of rare local wild accessions extinct or not sampled. In conclusion, we underline the usefulness of chloroplast genome markers in the study of grape origin and diffusion. The haplotype distribution helps to trace geographical sites of grapevine origin, making it easier to pinpoint cultivars as targets for future investigations. In addition, the data allows the separation of cultivars within the area of cultivation into more homogeneous clusters in order to better investigate their pedigree. Acknowledgements. We would like to thank ERSAF (Italy), Dr. Anzani, Prof. Ocete Rubio, Ranger Brodolini from Tolfa (Roma), Ranger Congiu from Tonara (Nuoro), Ranger Giura and Tommaso from Macchia di Isernia (Isernia) and Ranger Guarnini from Bella (Potenza) for providing plant material; Fabio Renzi, Ermete Realacci (Symbola Foundation), Franco Bonanini (President of the ‘Cinque Terre’ National Park) for believing in the importance of biodiversity of grapevine and in helping us in the safeguard of wild grapevine populations. Research was supported by INGENIO- Finlombarda and by the Italian Ministry of Environment – Ministero dell’Ambiente e della Tulela del Territorio e del mare, within the research projects: ‘Creazione di una collezione ex-situ atta a preservare la biodiversità della specie Vitis vinifera’ and ‘Application of biotechnology to the protection of the environment’, in collaboration with China peoples Republic.

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