Grassi, F.

Imazio, S.

Labra, M.

Scienza, A.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 41 (3), 157158 (2002)Research NoteChloroplast SSR markers to assessDNA diversity in wild and cultivatedgrapevinesF. GRASSI1), M. LABRA2), A. SCIENZA3) and S. IMAZIO3)1) Department of Biology, University of Milan, Milano, Italia2) Department of Environmental Sciences, University of Milano-Bicocca, Milano, Italia3) Department of Crop Science, University of Milan, Milano,ItaliaIntroduction: Simple sequence repeats (SSRs) are abun-dant and well-distributed throughout the nuclear genomesof Vitis vinifera L. Simple sequences length polymorphism(SSLP), caused by variation in the number of repeat units,can be detected easily by PCR using pairs of primers de-signed from unique sequences bordering the SSR motifs. Inthe last years SSLP analysis was used for cultivar genotypingand for defining genetic relationships among different vari-eties. Recently simple sequence repeats of several plantspecies were found in chloroplast DNA (PROVAN et al. 1999;BRYAN et al. 1999). CpSSRs result a powerful tool for manyaspects of evolutionary population biology (PROVAN et al.2001), including population histories (ECHT et al. 1998) andthe level of differentiation (MCCAULEY 1995) because or-ganelle genomes are typically non-recombinant, uniparen-tally inherited and effectively haploid. Universal angiospermchloroplast microsatellite primers were developed byWEISING and GARDNER (1999) on the bordering sequences of10 cpSSRs. In this study 8 of these primer pairs were testedon wild and cultivated grapevines and the degree of varia-tion was evaluated.Material and Methods: A total of 12 cultivated and 12 wildItalian grapevines were analysed in this study (Table). Cul-tivated grapevines were collected from the grapevine col-lection of C.I.VI.FRU.CE. (Regional Centre for Agriculture),Riccagioia, Pavia, Italy. Wild accessions were obtained fromseveral populations in Italy: 2 plants from Rome, 4 fromGrosseto, 1 from Isernia, 1 from Potenza, 2 from Cosenza,and 2 from Nuoro.Genomic DNA was extracted in 5 ml of CTAB buffer(2 % CTAB, 100 mM Tris-HCl, pH 8.0, 20 mM EDTA pH 8.0,1.4 M NaCl, 1% w/v polyvinylpyrrolidone, 0.1 % v/v(b-mercaptoethanol) as described by LABRA (2001).C p S S R   a n a l y s i s :  DNA was analysed at thefollowing 8 microsatellite loci: ccmp2, ccmp3, ccmp4, ccmp5,ccmp6, ccmp8, ccmp9, ccmp10 (WEISING and GARDNER 1999).The analysis was performed by adding 10 ng of total DNAto a 20 µl PCR mixture containing, 3 ng of labelled forwardprimer, 5 ng of reverse primer, 200 ng of each dNTP, 0.5 UDynazyme II (Celbio, Italy) and 2 ml of Dynazyme buffer. Theforward primer was end-labelled with 33P-ATP (Amersham,Italy). PCR amplification was performed with the followingthermal cycles: 3 min at 94 °C; 35 cycles of denaturation(45 s at 94  °C), annealing (30 s at 50 °C) and extension (1 minat 72 °C); then a final step for 7 min at 72 °C. In the case ofccmp2 the annealing temperature was 53 °C. A total of 1.5 µlof the PCR-amplified mixture was added to an equal volumeof loading buffer (80 % formamide, 1 mg ml-1 xylene cyanolFF, 1 mg ml-1 bromophenol blue, 10 M EDTA, pH 8.0), dena-tured for 5 min at 92 °C, loaded onto a 6 % denaturing poly-acrylamide gel and electrophoresed in TBE electrophoresisbuffer for 3 h at 80 W. The gel was finally fixed in 10 % aceticacid and exposed to an X-ray film for 24 h. Polymorphicbands were scored by visual inspection of the resultingautoradiograms.Results and Discussion:  DNA amplification with the8 primer pairs used for chloroplast SSR analysis showedthat 2 (ccmp3 and ccmp10) out of the 8 analysed loci werepolymorphic in a total of 24 individuals. Two (106 and 107 bp)and 3 (114, 115, and 116 bp) different size variants were foundat locus ccmp3 and at locus ccmp10, respectively. Size vari-ants of both combined loci defines a total of 5 differenthaplotypes (Table). The most frequent haplotypes, both incultivated and in wild grapes, were I and IV. Any privateCorrespondence to: Dr. M. LABRA, Department of Environmental Sciences, University of Milano-Bicocca, Piazza della Scienza,I-20126 Milano, Italy. Fax: +39-2-5031-4818. E-mail: massimo.labra@unimib.itT a b l eHaplotypes and allele sizes (bp) of ccmp3 and ccmp10 SSR markers, found in Vitis vinifera ssp. sativa and silvestriscpSSR loci Vitis vinifera ssp. sativa Vitis vinifera ssp. silvestris (numberHaplotype ccmp3 ccmp10 of accessions and location of populationI 107 115 Sangiovese, Cesanese, Girò, Ciliegiolo, 2, Rome    Lambrusco graspa rossaII 106 115 Fiudedda 1, Grosseto; 1, IserniaIII 107 114 *** 1, PotenzaIV 106 114 Lambrusco foglia frastagliata, Lambrusco 2, Cosenza; 2 Nuoro; 2 Grosseto    Maestri, Pinot Nero, Canaiolo, Aglianico,    Grechetto nero SardoV 106 116 *** 1, Matera158 F. GRASSI, M. LABRA, A. SCIENZA and S. IMAZIOallele was detected in wild or cultivated accession. Thesepreliminary results show that all analysed loci had a clearreproducible amplification product (Figure). Detectedpolymorphisms were not sufficient to early distinguish aclear distinction between the two subspecies or to definegenetic relationships.Figure: An example of cpSSR polymorphism (locus ccmp3) in12 samples of Vitis vinifera ssp. sativa and silvestris.The good results obtained in amplification and the highdegree of reproducible allele bands allow us to considercpSSRs as a powerful tool to investigate many aspects ofgrapevine domestication and diffusion (GRASSI et al. 2002).BRYAN, G. J.; MCBICOLL, J.; RAMSAY, G.; MEYER, R. C.; DE JONG, W. S.;1999: Polymorphic simple sequence repeat markers in chloroplastgenomes of Solanaceous plants. Theor. Appl. Genet. 99, 859-867.ECHT, C. S.; DEVERNO, L. L.; ANZIDEI, M.; VENDRAMIN, G. G.; 1998:Chloroplast microsatellites reveal population genetic diversityin red pine, Pinus resinosa Ait. Mol. Ecol. 7, 307-317.GRASSI, F.; IMAZIO, S.; FAILLA, O.; SCIENZA, A.; OCETE RUBIO, R.; SALA, F.;LABRA, M.; 2002: Vitis vinifera ssp. silvestris: Genetic isolationand post-glacial recolonisation of wild Italian and Spanishpopulations as estimated by nuclear and chloroplast microsatelliteanalysis. Mol. Ecol. (submitted).LABRA, M.; CARRENO-SANCHEZ, E.; BARDINI, M.; BASSO, B.; SALA, F.; SCIENZA,A.; 2001: Extraction and purification of DNA from grapevineleaves. Vitis  40, 101-102.MCCAULEY D. E.; 1995: The use of chloroplast DNA polymorphism instudies of gene flow in plants. Trends Ecol. Evol. 10, 198-202.PROVAN, J.; POWELL, W.; HOLLINGSWORTH, P. M.; 2001: Chloroplastmicrosatellites: new tools for studies in plant ecology and evolu-tion. Trends Ecol. Evol. 16, 142-147.PROVAN, J.; SORANZO, N.; WILSON, N. J.; GOLDSTEIN, D. B.; POWELL, W.;1999: A low mutation rate for chloroplast microsatellites. Ge-netics 153, 943-947.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.

Chloroplast SSR markers to assess DNA diversity in wild and cultivated grapevines

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Chloroplast SSR markers to assess DNA diversity in wild and cultivated grapevines

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