In order to evaluate the genetic distance among 47 grapevine accessions, including major and minor local cultivars grown in Oltrepò pavese (Pavia, Italy), DNA has been analysed with the AFLP approach (VOS et al. 1995). The electrophoretic analysis of the products amplified by 3 primer combinations showed high polymorphism.Furthermore, considering that DNA markers for haploid, uniparentally inherited genomes, such as the chloroplast DNA, are important indicators of pedigree, CpSSR analysis was also performed and this identified cases of maternal common origin among the analysed cultivars. The results show that these molecular tools allow univocal genotype identification and that the analysed germplasm has a wide genetic dissimilarity. Results are coherent with the postulation of a polycentric origin for the Oltrepò pavese cultivars and of a multiple varietal flow from different viticultural regions. The molecular information gathered in this research is essential for the establishment of an appropriate presentation programme of autochthonous varieties

Grassi, F.

Imazio, S.

Labra, M.

Rossoni, M.

Sala, F.

Scienza, A.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 42 (1), 31–34 (2003)Genetic relationships among grapevine cultivars grown in Oltrepò Pavese(Italy)M. ROSSONI1), M. LABRA1), S. IMAZIO1), F. GRASSI1), A. SCIENZA2) and F. SALA1)1) Dipartimento di Biologia, Università di Milano, Milano, Italia2) Dipartimento di Produzione Vegetale, Università di Milano, Milano, ItaliaCorrespondence to: Dr. M. ROSSONI, Dipartimento di Biologia, Università di Milano, Via Celoria 26, I-20133 Milano, Italy.Fax: +39-250-314-764. E-mail: mara.rossoni@unimi.itSummaryIn order to evaluate the genetic distance among 47 grape-vine accessions, including major and minor local cultivarsgrown in Oltrepò pavese (Pavia, Italy), DNA has been ana-lysed with the AFLP approach (VOS et al. 1995). The elec-trophoretic analysis of the products amplified by 3 primercombinations showed high polymorphism.Furthermore, considering that DNA markers for hap-loid, uniparentally inherited genomes, such as the chloro-plast DNA, are important indicators of pedigree, CpSSRanalysis was also performed and this identified cases ofmaternal common origin among the analysed cultivars.The results show that these molecular tools allow uni-vocal genotype identification and that the analysed germ-plasm has a wide genetic dissimilarity. Results are coher-ent with the postulation of a polycentric origin for theOltrepò pavese cultivars and of a multiple varietal flow fromdifferent viticultural regions. The molecular informationgathered in this research is essential for the establish-ment of an appropriate presentation programme of autoch-thonous varieties.K e y    w o r d s :  biodiversity, chloroplast SSR, AFLP, Vitisvinifera ssp sativa.A b b r e v i a t i o n s :  CpSSR = Chloroplast Simple SequenceRepeat; SSR = Nuclear Simple Sequence Repeat; AFLP = Ampli-fied Fragment Length Polymorphism; JC = Jaccard’s coefficient.IntroductionItaly is characterized by a rich, complex and diversifiedviticultural heritage. Oltrepò pavese, located on the North-ern Apennines that rise South of the Po river, gives a rel-evant contribution to this variability since in this area about40 grape varieties occur. Barbera and Croatina, the main col-oured varieties, are the base for the production of local DOCred wines. Other major traditional varieties includeChardonnay, Riesling italico, Riesling renano, Moscatobianco, Cortese, Uva rara, Vespolina and Pinot. In 1884 morethan 200 varieties were listed in Oltrepò pavese by the PaviaAmpelographic Provincial Committee. Due to the economicpressure for a mass market, nowadays, less than 20 of themare extensively grown, while some others are scattered oroccasionally cultivated (SCIENZA et al. 1999).To limit the current loss of intravarietal diversity,germplasm collections have been established. An essentialpre-requisite to this purpose is cultivar genotyping which isalso important for breeding programmes. Nowadays, theavailability of different molecular tools to analyse grapevinegenomes allows to produce data on genetic diversity (KARPet al. 1998), to put in order the large number of synonymsand homonyms and to estimate phylogenetic relationshipsamong different cultivars (THOMAS et al. 1994; SENSI et al.1996; LABRA et al. 1999; LABRA et al. 2001 b).In a previous report (ROSSONI et al. 2001) genotyping ofmajor and minor Oltrepò pavese grapevine cultivars wasachieved by nuclear SSR analysis. This suggested wide ge-netic diversity and some parentage relations.In order to evaluate the genomic distance among theanalysed cultivars, their DNA has been analysed with theAFLP approach (VOS et al. 1995). Better than any otherknown molecular tool this approach offers the possibility toscreen a higher number of anonymous genomic loci andfrequently ensures cultivar distinction even with a singleprimer combination (CERVERA et al. 1998).Furthermore, considering this usefulness of DNA mark-ers for haploid uniparentally inherited genomes to identifycases of maternal common origin among the cultivars CpSSRanalysis was also performed.The aim of this report is to study the genetic relation-ship of the varieties in Oltrepò pavese and its viticulturalhistory.Material and MethodsP l a n t   m a t e r i a l   a n d   D N A   e x t r a c t i o n :  The47 grapevine cultivars (Vitis vinifera L.) listed in Tab. 1 wereobtained from CI.VI.FRU.CE (Voghera, Pavia, Italy). Theyincluded all major cultivated varieties, as well as ancientvarieties from old Oltrepò pavese vineyards and collections.Young leaflets were collected from rooted cuttings, fro-zen in liquid nitrogen and ground to fine powder. GenomicDNA was extracted from this powder as described by LABRAet al. (2001 b).A F L P   a n a l y s i s :  AFLP analysis (VOS et al. 1995)was performed as described in the European Patent 0534858(Keygene, Belgium). Genomic DNA was digested (3 h) withEcoRI (0.5 U) and MseI (0.5 U), and ligated with EcoRI(5 pMol) and MseI adapters (50 pMol). Primer pairs used inthe pre-amplification reaction were M01 and E01 (LABRA etal .2001 a). Amplification was carried out using 3 pairs of selec-tive primers E32-M36, E32-M38, E33-M32, describing in theoriginal protocol (European Patent 0534858, Keygene, Bel-T a b l e  1Grapevine accessions used for the CpSSR and AFLP analyses. Berry colour, status of cultivation, place of origin and haplotype detectedby CpSSR are indicated for each accessionNo. Cultivar Berry color Status Place of origin Haplotype S1 Barbera Black cultivated Piemont 107/116 F2 Barberone Black collection Piemont 107/116 F3 Basgano di Moline Freisa Black collection Lombardy 106/115 B4 Basgano di Oliva Gessi Black collection Lombardy 107/115 E5 Colombaia bianca White collection Lombardy 107/115 E6 Colombaia nera Black collection Lombardy 107/116 F7 Cortese White cultivated Piemont-Lombardy 106/114 A8 Croà acino rosso o Vermiglio Black collection Lombardy 106/115 B9 Croà acino grande Black collection Lombardy 107/116 F10 Croà acino piccolo Black collection Lombardy 107/116 F11 Croatina Black cultivated Lombardy 107/116 F12 Croatina bianca White cultivated Lombardy 107/115 E13 Croatina internodo corto Black collection Lombardy 107/115 E14 Malvasia bianca di Candia White cultivated Lombardy 107/115 E15 Moradella Black cultivated Lombardy 107/115 E16 Moradella di Montalto Black collection Lombardy 107/116 F17 Moretto Black collection Lombardy 107/115 E18 Moscato bianco White cultivated Lombardy 107/116 F19 Moscato nero Black collection Lombardy 107/116 F20 Moscato rosa antico Pink collection Lombardy 107/116 F21 Nebbiolo Black collection Piemont 107/115 E22 Negrara Black collection Trentino-Lombardy 107/116 F23 Pignola Black collection Lombardy 107/116 F24 Pinot bianco White cultivated French 106/114 A25 Pinot grigio Grey cultivated French 106/114 A26 Pinot nero Black cultivated French 106/114 A27 Pollini bianca White collection Lombardy 107/115 E28 Pollini tipo Barbera Black collection Lombardy 107/115 E29 Pulitana Black collection Lombardy 106/114 A30 Riesling italico White cultivated French-Lombardy 107/115 E31 Riesling renano White cultivated Germany 107/115 E32 Rossarone chiuso Black collection Lombardy 107/116 F33 Rossarone gentile Black collection Lombardy 107/116 F34 Rossarone grande Black collection Lombardy 107/115 E35 Rossamia Black collection Lombardy 107/115 E36 Tabernello Black collection Lombardy 107/116 F37 Timoraccio White collection Piemont 107/115 E38 Uva bianca dura invernale White collection Lombardy 106/114 A39 Uva crova Black collection Lombardy 107/115 E40 Uva della cascina Black collection Lombardy 107/115 E41 Uva di Mornico Black collection Lombardy 107/115 E42 Uva di Nigazzo Black collection Lombardy 107/116 F43 Uva dura antica Black collection Lombardy 107/115 E44 Uva rara Black cultivated Lombardy 107/115 E45 Verdea comune White cultivated Lombardy 107/115 E46 Vernassa White collection Liguria-Lombardy 107/116 F47 Vespolina Black cultivated Lombardy 107/116 Fgium). The EcoRI primers were labelled with γ-33P-ATP(Amersham, Italy). Amplification reactions were performedas specified by LABRA et al. (2001 a). Polymorphic bandswere scored by visual inspection of the resulting autora-diograms.C p S S R   a n a l y s i s :  Five primers pairs for chloroplastmicrosatellite amplification, ccmp2, ccmp3, ccmp4, ccmp5,ccmp10 (WEISING and GARDNER 1999) designed for angio-sperms have been used. The PCR reaction was performedby adding 15-20 ng of genomic DNA to a 10 µl PCR mixture32 M. ROSSONI, M. LABRA, S. IMAZIO, F. GRASSI, A. SCIENZA and F. SALAcontaining 0.2 µM of the reverse and forward primers speci-fied for each micro-satellite locus, 0.2 mM of each dNTP,0.4 U Dynazyme (Celbio, Italy) and Dynazyme buffer as speci-fied by the supplier. The PCR amplification programme was:4 min at 94 °C; 32 cycles of denaturation (1 min at 94 °C),annealing (1 min at 50 °C) and extension (1 min at 72 °C);then a final step for 30 min at 72 °C. Loading buffer 6x (2 µl)was added to the 10 µl mixture and 5 ìl of this were analyzedon 8 % acrylamide/bis-acrylamide non-denaturing gel anddeveloped with the silver staining approach as described byECHT et al. (1999). Allele size was defined by visual inspec-tion using a Gel Doc 2000 instrument (Biorad, USA). AFLPand CpSSR fingerprint was confirmed in duplicate.D a t a   a n a l y s i s :  In accordance with numerictaxonomy principles AFLP bands were scored as binary char-acters for absence (0) or presence (1) and the resulting datamatrices analysed using the NTSYSpc 2.1 statistical pro-gramme. Levels of diversity were estimated as the percent-age of polymorphic bands out of the total bands scored.The AFLP similarity-dissimilarity matrices were computedwith the Jaccard’s coefficient (SNEATH and SOKAL 1973). Thefinal products of data processing were dendrograms con-structed by cluster analysis based upon UPGMA (un-weighed pair-group method with arithmetical averages).In the case of CpSSR allele size was obtained by visualinspection. Combining two polymorphic CpSSR loci cultivarhaplotyping was defined.ResultsDNA of 47 grapevine cultivars, including major and mi-nor traditional local cultivars from a local collection was ex-tracted and analysed by AFLP using 3 primer combinations.The electrophoretic analysis of the amplified productsrevealed a total of 156 bands, 90 of which (57.7 %) werepolymorphic. These data were used to compute an UPGMAdendrogram defining genomic relationship among cultivars(Figure).The Jaccard’s similarity index among cultivars variedfrom 1.0 (full genetic similarity) to 0.564 (high genetic dis-similarity), indicating a large genetic variability among theanalysed varieties. In all cases, except for Pinot nero, Pinotbianco and Pinot grigio, the analysis assured full cultivardistinction.In the case of CpSSR analysis, 5 loci were analysed.Only 2 showed DNA polymorphism, with 2 different allelesin the case of ccmp3 (106 and 107 bp) and 3 alleles forccmp10 (114, 115 and 116 bp). Six possible haplotypes weredefined by the combination of allele size in the 2 polymor-phic loci. Of these, 4 haplotypes (named A, B, E, F) weredetected in the analysed grapevine populations. HaplotypeE was the most common, being present in 21 samples (Tabs 1and 2), while haplotype B was rarest; it was present only in2 accessions (Basgano di Oliva Gessi and Croà acino rosso).Discussion and ConclusionThe present work addressed to the definition of geneticrelationships among grapevines that are part of the Oltrepòpavese heritage. The dendrogram obtained from the AFLPanalysis (Figure) showed high genetic variability and re-vealed clear cases of synonymy and homonymy. In the caseof 3 putative synonymous varieties, e.g. Rossarone gentile,Rossarone chiuso and Rossarone grande, results confirmedthe close relationship between the first two but evidencedhigh genetic diversity in the case of Rossarone grande. Thiswas confirmed by CpSSR analysis that grouped the firsttwo in haplotype F and the latter in haplotype E. These conclu-sions are also supported by previous data obtained by nu-clear SSR analysis (ROSSONI et al. 2001). The analysis of thiscase confirms that chloroplast and nuclear SSR are appro-priate markers to investigate genetic relationships amongclosely related accessions (BOWERS et al. 1997).High genetic dissimilarity was recorded by AFLP analy-sis in the case of the other 3 synonymous accessions, Croàacino piccolo, Croà acino rosso and Croà acino grande. Thisconfirms previous suggestions on their independent gene-alogy (ROSSONI et al. 2001).Figure: Dendrogram based on the Jaccard’s similarity index, show-ing the genetic relationship among 47 Oltrepò pavese grapevinecultivars as determined by CpSSR and AFLP analyses.T a b l e  2Possible haplotypes identified by the combination of the ccmp3and ccmp10 chloroplast SSR lociHaplotype CpSSR locusCcmp3 Ccmp10A 106 114B 106 115C. 106 116D. 107 114E 107 115F 107 116Genetic relationships among grapevine cultivars 33Possible common ancestors are suggested in the caseof Croatina and Croatina internodo corto which have closepositions in the dendrogram. On the other hand, these twocultivars showed different haplotypes in the CpSSR analy-sis thus suggesting a different maternal origin.The 3 Pinot accessions were shown to be identical, onthe bases of AFLP and CpSSR analysis as well as on previ-ously reported nuclear SSR data (ROSSONI et al. 2001). BELLINet al. (2000) found few polymorphic AFLP bands within Pinotclones but concluded that these were not reproducible. Thus,new tools are needed for clonal characterization and clonaldiscrimination remains an open issue.Uva bianca dura invernale, Uva di Mornico, Uva dellaCascina and Uva dura antica, known as the oldest varietiescultivated in Oltrepò pavese, were also analysed. From thehigh genetic dissimilarity, evidenced by AFLP and CpSSRanalyses, it was concluded that these old varieties had dif-ferent origin and no common maternal progenitor.In conclusion, in spite of the confirmed synonyms andthe verified genetic similarities, the high genetic diversity ofgrapevines of Oltrepò pavese suggests a polycentric originof today’s grapevine assortment. The Oltrepò pavese re-gion has been an important commercial cross since Romantimes. Thus, it is most likely that cultivars with differentmorphological and genetical traits were introduced by vari-ous colonizing populations turning this region into a rel-evant centre of grapevine diversity. High diversity is stillpreserved in the region and the molecular tools used in thisstudy now offer a concrete basis for its protection and forinsitu  and ex situ preservation programmes.A comparison of results produced by AFLP and SSRsuggests that the AFLP approach, by investigating a largenumber of anonymous loci offers the most appropriate toolto estimate genetic distance among varieties. In the case ofsynonymous varieties (e.g. Rossarone grande and Rossaronechiuso) or hypothetical parental relationship, the use of SSRmarkers is preferable, since they explore highly polymorphicregions and they are co-dominant markers, while AFLP aredominant.We conclude that only the application of both analyti-cal approaches will produce satisfactory data for the screen-ing of intravarietal diversity and for analysing synonymies,homonymies, false attributions and pedigrees. In the caseof hypothetical parental relationships, chloroplast SSR analy-sis has the capacity to elucidate maternal origin.The computational analysis data of this and of a previ-ous report (ROSSONI et al. 2001) is saved on a CD-Rom.AcknowledgementsThis research was supported by the project ”Biodiversity ofthe Italian grapevines and its protection from pathogens” ofFondazione Bussolera Branca, Mairano di Casteggio, Pavia and bythe project ”Monitoraggio dei livelli di resveratrolo nelle varietà divite della Valtellina” of Lombardia region.ReferencesBELLIN, D.; VELASCO, R.; GRANDO, M. S.; 2000: Intravarietal DNApolymorphisms in grapevine (Vitis vinifera L.). Acta Hortic. 546,343-349.BOWERS, J. E.; MEREDITH, C. P.; 1997: The parentage of a classic winegrape, Cabernet Sauvignon. Nat. Genet. 16, 84-87.CERVERA, M. T.; CABEZAS, J. A.; SANCHA, J. C.; MARTINEZ DE TODA, F.;MARTINEZ-ZAPATER, J. 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Ansonica growingin the island of Giglio, Italy, by AFLP and SSR markers.Vitis 38,161-167.LABRA, M.; WINFIELD, M.; GHIANI, A.; GRASSI, F.; SALA, F.; SCIENZA, A.;FAILLA, O.; 2001 b: Genetic studies on Trebbiano and morpho-logically related varieties by SSR and AFLP markers. Vitis 40,187-190.ROSSONI, M.; FASOLI, V.; LABRA, M.; SPINARDI, A.; FAILLA, O.; SCIENZA, A.;SALA, F.; 2001: Exploration of elite grapevine germplasm ofOltrepò pavese (northern Italy) using genetic, chemotaxonomicand morphological markers. Adv. Hort. Sci. 15, 72-78.SCIENZA, A.; VALENTI, L.; MOLINA, M.; (Eds.); 1999: Vitigni antichi dellaLombardia. Ampelografia del germoplasma locale. Provincia diPavia.SEFC, K. M.; LOPES, M. S.; LEFORT, F.; BOTTA, R.; ROUBELAKIS-ANGELAKIS, K.A.; IBANEZ, I.; PEJIÈ, H. W.; WAGNER, J.; GLOSSL, H.; STEINKELLNER, H.;2000: Microsatellite variability in grapevine cultivars from dif-ferent European regions and evaluation of assignment testing toassess the geographic origin of cultivars. Theor. Appl. Genet.100, 489-505.SENSI, E.; VIGNANI, R.; ROHDE, W.; BIRICOLTI, S.; 1996: Characterisation ofgenetic biodiversity with Vitis vinifera L. Sangiovese and Colorinogenotypes by AFLP and ISTR DNA marker technology. Vitis 35,183-188.SNEATH, P. H. A.; SOKAL, R. R.; 1973: Numerical Taxonomy. The Prin-ciples and Practice of Numerical Classification. W H. Freeman,San Francisco, USA.THOMAS, M. R.; SCOTT, N. S.; 1993: Microsatellite repeats in grapevinereveal DNA polymorphisms when analysed as sequenced-taggedsites (STSs). Theor. Appl. Genet. 86, 985-990.THOMAS, M. R.; CAIN, P.; SCOTT, N. S.; 1994: DNA typing of grapevine:A universal methodology and database for describing cultivarsand evaluating genetic relatedness. Plant. Mol. Biol. 25, 939-949.VOS, P.; HOGERS, R.; BLEEKER, M.; REIJANS, M.; VAN DE LEE, T.; HORNES,M.; FRIJTERS, A.; POT, J.; PELEMAN, J.; KUIPER, M.; ZABEAU, M.; 1995:AFLP: a new technique for DNA fingerprinting. Nucl. Acids Res.23, 4407-4414.WEISING, K.; GARDNER, R.; 1999: A set of conserved PCR primers forthe analysis of simple sequence repeat polymorphisms inchloroplast genomes of dicotyledonous angiosperms. Genome42, 9-19.ZABEAU, M., VOS, P.; 1992 : European patent application. Publicationn° 0543858 A1.Received October 14, 200234 M. ROSSONI, M. LABRA, S. IMAZIO, F. GRASSI, A. SCIENZA and F. SALA

Genetic relationships among grapevine cultivars grown in Oltrepò Pavese (Italy)

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Genetic relationships among grapevine cultivars grown in Oltrepò Pavese (Italy)

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