Frisinghelli, C.

Grando, M.S.

Grando, Maria Stella

Archivio istituzionale della ricerca - Fondazione Edmund Mach

Vitis 37 (2),79-82 (1998)Grape microsatellite markers: Sizing of DNA allelesand genotype analysis of somegrapevine cultivarsIstituto Agrario di San Michele all'Adige, SanMichele all'Adige, ItaliaS u m m a r y : Capillary electrophoresis fluorescence-based technology and fragment sizing software were applied in order to typegrape cultivarsat microsatellite loci described by THOMAs and ScoTT (1993) and BOWERS et al. (1996) . Whereas the data set was closelyconsistent with already known genotypes for the same cultivars, estimates of allele length differed remarkably from those reported . Thepedigree of the Riesling crosses Bacchus, Optima, Muller-Thurgau, Scheurebe, Ehrenfelser, Kemer, Rotberger, Rieslaner and IncrocioManzoni 6.0.13 were investigated, and Mendelian inheritance of automatically sized alleles proved to be easily scorable . In addition,ancient cultivars of the vine-growing area of Trentino were characterized at microsatellite loci . Genotypes of Marzemino, Teroldego,Lagrein, Lambrusco a foglia frastagliata and Groppello gentile proved to share more than 50 % ofalleles .K e y w o r d s : DNA typing, automated sizing, microsatellites, parentage analysis, indigenous grapevines .IntroductionDNA markers which are direct characteristics ofthe geno-type and which are independent ofthe phenotype, provideda wealth of polymorphisms, enabling the identification ofcultivars and the construction of saturated genetic maps inmany higher plants .Owing to the relative simplicity ofthe DNA amplifica-tion approach when compared to RFLP (Restriction Frag-ment Length Polymorphism) technology, PCR-based markertools such as RAPD (Random Amplified Polymorphic DNA)and STS (Sequence Tagged Sites) have recently been pre-ferred also by grape research groups for genome studies(THOMAS et al. 1994, LODHi et al. 1995, Xu et al. 1995) .Microsatellite sequences have become the genetic mark-ers of choice in mammalian and plant systems due to theirabundance, high degree of polymorphism and amenabilityto automation (WEBER and MAY 1989) . The co-dominantmode of inheritance of microsatellites not only simplifiesanalysis but also facilitates the transfer of markers betweenmaps derived from different crosses .The development of grapevine genome microsatellitemarkers was first reported by THOMAs and ScoTr (1993) andthen by BOWERS et al . (1996) which referred to STMS (Sequence Tagged Microsatellite Sites) and to SSR (Simple Se-quence Repeats), respectively.These studies described alarge number of alleles, as well as a very high level ofheterozygosity in grapes, which provides a considerableresolving power for the accurate separation of cultivars .Moreover, theprimers for STMS analysis whichderived fromregions flanking the repeated sequences of V. vinifera DNA,successfully amplified polymorphic alleles from other relatedVtis species`.The high level of genetic diversity in grapes which arefound world-wide, the interest in evaluating geneticrelatedness within the Vitis genus, and the need for varietybyMARIA STELLA GRANDO and CLAUDIA FRISINGHELLIrecognizing systems useful for certifying the identity ofgrapecultivars encourage an attempt to integrate and exploit thegenotype data which are revealed at microsatellite loci ingrapes .Microsatellite markers are easily shared by the commu-nication of primer sequences, but results are expressed asrelative allele lengths and allele size estimates are affectedby the different methods of determination (WEBER 1990,SMrrH 1995) . We here compare a first set of grapevine geno-types identified at 9 previously described microsatellite lociusing capillary electrophoresis fluorescence-based technol-ogy and fragment sizing software . Parentage analyzes ofsome Riesling crosses, and the genotypes of ancient grape-vine cultivars still grown in our region (Trentino, Italy), arealso included in this study to verify the identification ofcommon alleles and to increase information for allelic fre-quency estimates in V. vinifera .Material and methodsGrape leaves used for DNA extraction were obtainedfrom the Vitis collection held by the Istituto Agrario at SanMichele all'Adige, Trento, Italy . Studies were carried out onthe wine cultivars listed in Tab . 1 . As a different method forthe sizing ofDNAfragments was used in this study, a numberof varieties were analyzed in order to identify allele classesreported at the same loci by other authors : these wereCabernet franc, Cabernet Sauvignon, Chardonnay, Cheninblanc, Gewurztraminer, Merlot, Muller-Thurgau, Pinotblanc,Riesling, Semillon, Shiraz and Silvaner. In order to followpedigree relationships with microsatellite markers, cultivarsfrom 9 Riesling crosses, including the parents, were exam-ined . Genotype similarity at microsatellite loci was evalu-ated for 9 varieties (e .g ., Lambrusco f.f. (afoglia frastagliata))belonging to local ancient germplasm. After extraction andCorrespondence to : Dr. M . S . GRANDO, Istituto Agrario di San Michele all'Adige, Via E . Mach, I-38010 San Michele all'Adige, Trento,Italy. Fax : +39-461-650872 . E-mail : stella.grando@ismaa .it80Table 1Vitis vinifera cultivars analyzed in this studyRNase A digestion (DOYLE and DOYLE 1991) 100-120 ng ofgenomic DNA was added to a PCRmix containing 0.25 pMofeachprimer, 200 pM ofeach dNTP,1.5 mM MgCl2 (2 mMwith VVMD5 and VVMD8 primers), 0.5 U AmpliTaq Goldand 1X Gene Amp PCR Buffer II (Perkin Elmer), in 25 glvolume . Primer sequences used are those designed byTHOMAS and ScoTT (1993) and BOWERS et al. (1996) . Oneoligonucleotide ofeach primer pair was fluorescently labeledwith Dye Phosphoramidities (Abi Prism) . TET (green) wasattached toVVS1 and VVS3 primers, 6-FAM (blue) was usedto label WS2, VVS5 and VVMD7primers andHEX (yellow)wasattachedtoVVS4, VVMD5, VVMD6 and VVMD8 prim-ers .PCR was performed using a Gene Amp PCR System 9600(Perkin Elmer) under the following conditions: 12 min at 95 °C,then 30 cycles of denaturation (45 s at 94 °C), annealing (30 sat 51 °C ; 54 °C in PCR withWS1 and WS3 primers), exten-sion (1 min 30 s at 72 °C) and a final elongation step at 72 °Cfor 7 min . Amplifications were confirmed by running 7 ltl ofthe PCR product on 2 % agarose gel . DNA fragments werethen analyzed on the ABI Prism 310 Genetic Analyzer run-MARIA STELLA GRANDO and CLAUDIA FRISINGHELLIning GeneScan Software (2.1) through GA 310 POP-4, apre-formulated liquid polymer matrix, heated at 60 °C. Beforeinjection, 0.4-1.5 gl of well defined PCR samples werepooled together and mixed with 12 gl of deionizedformamide and 0.5 gl ofred fluorescently labeled DNA sizestandard (GeneScan 500 TAMRA), and were thendenaturated for 2 min at 95 °C . To allow detection of the400 by peak of the size standard, the data collection timewas set to 24 min. Among analysis parameters, the LocalSouthern Method was chosen for size calling .Results and DiscussionAutomated sizing of microsatellite alle-1 e s : Thegenotypes at VVS 1, VVS2, VVS4, VVS5, VVMD5,VVMD6, VVMD7 and VVMD8 loci proved tobe highly consistent with those reported by THOMAs et al. (1994) andBowERs et al. (1996) forthe same V. vinifera cultivars (Tab . 2).VVS3 alleles were not compared, as difficulties in the ampli-fication of two different DNA products from describedheterozygous genotypes were often observed at this locus .Standard deviations lower than 0.2 by were calculated fordata obtained running the same capillary, whereas differ-ences in size of around 1 by sometimes resulted betweencapillaries (data not shown) . For this reason, the sizing ofalleles at VVS4 and VVMD6 loci required more care, sincemicrosatellite lengths can differ by as little as 1 bp . AssignedDNA fragment sizes differed remarkably from those obtainedin other laboratories for the same alleles detected onacrylamide or agarose gels, the apparent fragment lengthalways proving to be shorter in our case (Tab. 3) . This didnot affect results, as alleles were easily identified by com-paring genotype data sets . Base pairs offset showed a locusdependence, and for some loci this varied in function ofallele sizes . However, a constant difference in size was ob-servedfor all allele classes at VVS1, VVS4 and VVMD5 loci .Such differently estimated sizes of alleles can result by theextreme denaturing electrophoretic conditions we ran asDNA fragments were separated at 60 °C through a polymermatrix containing 8 M urea and additional denaturant agents .Parentage analysis of Riesling crosses :Microsatellite repeats developed in the grapevine genomeshowed Mendelian segregation, indicating that markers areheritable and reliable for genetic analysis or for the examina-tion ofpedigrees. Nine cultivars and theirparental lines werecharacterized at microsatellite loci . Genotypes are reportedin Tab . 2. Three pedigrees showed inconsistencies with theparental genotypes . The lines generating Miiller-Thurgauhave been discussed more than once on the basis of mo-lecular marker findings (BUSCHER et al. 1994, THOMAS et al.1994, REGNERetal . 1996) . Genotypes at 8 microsatellite lociexcluded the cultivar Silvaner as a parent, not onlyfor Miiller-Thurgau but also for Scheurebe and Ehrenfelser, whereasRiesling can be confirmed as a parent in all the crosses in-vestigated in this study . Silvaner was verified in Rieslanerparentage, as well as in that of Optima and Bacchus . It isinteresting to note that both Optima and Bacchus geno-types displayed alleles belonging to two different SilvanerCultivarCabernet francCabernet SauvignonChardonnayParentsChasselas de CourtillierChenin blancGewiirAraminerMerlotPinot blancRieslingSemillonShirazSilvanerMiiller-Thurgau Riesling x Chasselas de CourtillierEhrenfelser Riesling x SilvanerScheurebe Silvaner x RieslingRieslaner Silvaner x RieslingBacchus (Silv. x Riesl .) x Mtiller-ThurgauOptima (Silv. x Riesl .) x Miiller-ThurgauKerner Schiava grossa x RieslingRotberger Schiava grossa x RieslingIncrocioManzoni 6.0.13 Riesling x Pinot blancCorvina veroneseGroppello gentileLagreinLambrusco f.f.MarzeminoNosiolaPortugieserSchiava grossaTeroldegotypes : the "wrong" one related to the Miiller-Thurgau caseand the cultivar currently known as Silvaner. Genotypingpedigree at newly developed microsatellite repeat loci, SEFCetal . (1997) put Chasselas de Courtillier forward as the sec-ond parent ofMiiller-Thurgau . We also defined allele lengthsatWS1, WS2, WS4, WS5, VVMD5, VVA1DD6, VVMD7andVVMD8 loci for the Chasselas de Courtillier genome, andfound results consistent with this suggestion .Kerner and Rotberger genotypes are both explained bythe segregation of Riesling and Schiava grossa alleles, aswell as Incrocio Manzoni 6.0.13, which had alleles in common with the known parents.The pedigree of IncrocioManzoni 6.0.13 has been discussed on a number of occa-sions, and the selfpollination ofRiesling or a cross betweenRiesling and Chardonnay were suggested instead . Micro-satellite and RAPD genotypes verified Incrocio Man-zoni 6.0.13 as a cross ofRiesling x Pinot (GRANDo et al. 1997)but could not identify the male parent Pinot blanc, since themarker system does not show polymorphisms within thePinotfamily.Microsatellite polymorphisms in indi-genous wine cultivars : DNA typing the wine grapevarieties related to our region, a number of different allelelength classes, ranging from 4 at the VVMD6 and VVMD8loci to 7 atthe VVMD7 locus, were detected (Tab. 2) . All loci,except for VVS2, represented more than 60 % of the allelicGrape microsatellite markersTable 2Allele sizes (bp) of cultivars typed at microsatellite loci in order to analyze parentage of Riesling crosses and evaluate geneticdiversity among ancient grapevines related to the Trentino growing areaTable 3Range of allele lengths estimated in this study andbase pairs (bp) offsets when compared to sizes reported by THOMAS and Scoi-r (1994) at VVS lociand BOWERS et al. (1996) at VVMD loci81LocusCultivar VVSl WS2 VVS4 VVS5 VVMD5 VVMD6 VVMD7 VVMD8Riesling 186 - 139 148 166 - 83 98 222 230 204 206 247 255 137 141Silvaner 176 186 148 150 166 - 123 144 222 228 187 204 241 245 135 137M01ler-Thurgau 179 186 139 148 166 171 83 107 222 224 197 204 245 255 137 141Chasselas de C. 179 186 148 152 166 171 107 144 224 232 197 206 241 245 137 152Pinot b . 179 186 133 148 166 171 119 144 224 234 197 - 237 241 135 137Ehrenfelser 186 - 139 148 166 171 98 107 222 224 187 204 237 255 137 141Scheurebe 186 - 139 148 166 - 98 107 222 234 204 206 245 247 141 162Rieslaner 186 - 148 - 166 - 98 144 222 - 204 206 241 247 137 -Bacchus 186 - 148 - 166 171 107 123 222 224 204 206 241 245 137 141Optima 179 186 148 - 166 - 83 123 222 - 197 206 245 247 141 -Kerner 177 186 148 152 166 - 98 107 222 232 204 206 245 255 137 162Rotberger 177 186 131 148 166 - 83 107 230 234 204 - 245 255 137 152Incrocio Manzoni 186 - 148 - 166 - 83 144 222 224 197 206 241 247 137 -Corvina veronese 158 184 148 152 168 173 107 144 228 236 206 - 237 - 135 162Groppello gentile 184 186 129 152 168 173 107 144 222 - 187 197 245 251 135 137Lagrein 177 186 133 152 167 173 107 144 2,24 234 197 206 237 245 135 152Lambrusco ff. 177 186 129 152 168 173 98 144 222 224 197 206 245 247 135 -Marzemino 177 186 129 - 166 173 107 144 222 228 197 206 237 260 135 137Nosiola 184 - 129 152 167 173 92 144 222 224 206 - 245 - 137Portugieser 176 - 139 148 167 173 96 123 222 228 187 204 241 253 137Schiava grossa 177 186 131 152 166 - 107 - 232 234 204 206 245 - 152 162Teroldego 177 179 133 152 171 173 107 144 222 224 197 206 237 245 135Locus Size range (bp) Offset (bp)VVS1 158 - 186 -4VVS2 121 - 5129 - 141 - 4144 - 152 - 3WS4 165 - 175 -2WS5 83 - 85 - 792 - 98 - 5107 - 115 - 3119 - 144 - 2VVMD5 222 -242 -4VVMD6 187 - 7197 -206 - 8VVMD7 231 -255 -2260 - 3WNID8 135 -141 - 6152 - 162 - 582Table 4Similarity values (%) for pairwise comparison ofgrapevine genotypes at 8 microsatellite lociAcknowledgementsvariation drawn by THOMAS and Scorr (1993) and BowERset al. (1996) when surveying international V. viniferacultivarS .Indigenous grapevines tested revealed unique geno-types at microsatellite loci, but also shared many commonalleles . Even if there were enough DNA polymorphisms totype these cultivars, high allele frequencies (28-44 %) wereestimated for one or more length classes at each locus (casesin which only one microsatellite was detected were scoredas heterozygous genotypes having a null allele) .WS2 -152 bp,WS4-173bp, WS5 -144bp, WMD5 -222 bp,WMD6 - 206 bp, WMD7 - 245by andWMD8 -135by werethe most prevalent DNA fragments . Similarity values amonggenotypes were calculated as the percentage of commonalleles shared byeach genotypepair. The wine grapecultivarstypically related to the Trentino growing area, i .e . Lambruscof.f., Teroldego, Marzemino, Lagrein and Groppello gentile,proved to be clustered by a genotype similarity ranging from50 to 75 % (Tab . 4) .We like to thank Dr. M. STEFANINi and Dr. L . DE MICHELI forgrapevinematerial, Dr. F. REGNER for Chasselas de CourtillierDNAand Dr. G.VERSINI for helpful suggestions. This research wassupported in part by the Italian Ministry of Agriculture in the frame-work of the program 'Biotecnologie vegetali, Area 2 - Genomavegetale - mappe molecolari'.MARIA STELLA GRANDo and CLAUDIA FRISINGHELLI9Received January S, 1998ReferencesBOWERS, J. E. ; DANGL, G. S. ; VIGNANI, R. ; MEREDITH, C. P. ; 1996 : Isolationand characterization of new polymorphic simple sequence re-peat loci in grape (Vitis vinifera L.). Genome 39, 628-633.BOSCHER, N. ; ZYPRIAN, E. ; BACHMANN,0.; BLAICH, R. ; 1994: On the originof the grapevine variety Miiller-Thurgau as investigated by theinheritance of random amplified polymorphic DNA (RAPD) .Vitis 33, 15-17.DOYLE, J. J. ; DOYLE , J. L. ; 1990 : Isolation of plant DNA from freshtissue . Focus 12, 13-15.GRANDO,M. S. ; FRISINGHELLI, C. ; MALOSSINI, U. ; 1997 : Analisi molecolaredei parenaggi dell'Incrocio Manzoni 6-0-13 . L'Enotecnico 12,89-92.LODHI, M. A.; DALY,M. J. ; YE,G. N. ; WEEDEN, N. F. ; REISCH, B. I . ; 1995 :A molecular marker based linkage map of Vitis. Genome 38,786-794.REGNER, F. ; STEINKELLNER,H.; TURETSCHEK, E. ; STADLHUBER,A. ; GLOSSL, J. ;1996 : Genetische Charakterisierung von Rebsorten (Vitis vinifera)durch Mikrosatelliten-Analyse . Mitt. Klostemeuburg 46, 52-60.SEFC, K. ; STEINKELLNER, H. ; WAGNER,H. W. ; GLOSSL, J. ; REGNER, F. ; 1997 :Application of microsatellite markers to parentage studies ingrapevine. Vitis 36 (4), 179-183 .SMITH, R. N. ; 1995 : Accurate size comparison of short tandem repeatalleles amplified by PCR. BioTechniques 18, 122-128.THOMAS, M. R. ; CAIN, P. ; SCOTT, N .S . ; 1994 : DNA typing of grape-vines: A universal methodology and database for describingcultivars and evaluating genetic relatedness . Plant Mol. Biol. 25,939-949.- -; SCOTT, N. S. ; 1993 : Microsatellite repeats in grapevine reveal DNApolymorphisms when analysed as sequence-tagged sites (STSs) .Theor. Appl . Genet. 86, 985-990.WEBER, J. L. ; 1990 : Informativeness of human (dC-dA),.(dG-dT)npolymorphisms. Genomics 7, 524-530.- -; MAY, P.E. ; 1989: Abundant class of human DNA polymorphismswhich can be typed using the polymerase chain reaction . Amer.J. Human. Genet. 44, 338-396.XU, H . ; WILSON, D. J. ; 1995 : Sequence-specific polymerase chain re-action markers derived from randomly amplified polymorphicDNA markers for fingerprinting grape (Vitis) rootstocks. J. Amer.Soc. Hort . Sci. 120, 714-720.1 Lambrusco f.f. -2 Teroldego 63 -3 Marzemino 56 564 Lagrein 63 75 56 -5 Groppello gentile 56 50 56 50 -6 Nosiola 50 44 38 44 50 -7 Schiava grossa 31 31 31 50 25 19 -8 Portugieser 13 38 25 13 25 25 6 -9 Corvina veronese 38 44 44 44 44 31 25 191 2 3 4 5 6 7 8

Grape microsatellite markers: sizing of DNA alleles and genotype analysis of some grapevine cultivars

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Grape microsatellite markers: sizing of DNA alleles and genotype analysis of some grapevine cultivars

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Archivio istituzionale della ricerca - Fondazione Edmund Mach