COBISS Code 1.01
DOI: 10.2478/v10014-012-0014-3
Agrovoc descriptors: grapevines, vitis vinifera, genotypes, microsatellites, genetic markers, genetic variation, varieties, land varieties, genetic parameters, designation of origin, quality controls, standardizing, genetic distance, polymorphism, genetic parameters
Agris category code: f30, f70
Collection and genetic characterization of Vitis vinifera ' Žilavka' by microsatellites and AFLP markers
Lidija TOMIČ1, Nataša ŠTAJNER2, Tatjana JOVANOVIČ CVETKOVIC1, Miljan CVETKOVIC1 and Branka
JAVORNIK2
Received May 23, 2012; accepted September 3, 2012. Delo je prispelo 23. maja 2012, sprejeto 3. septembra 2012.
ABSTRACT
'Žilavka' has been grown in Bosnia and Herzegovina since the XlVth century and is exploited for wine production. Although not sufficiently studied, this grapevine cultivar has high economic potential for the country. Five survey missions resulted in the collection of eighty 'Žilavka' accessions that varied in terms of names and characteristics. Because of the unknown origin of the cultivar, these accessions were analyzed with microsatellites markers in order to obtain a standard 'Žilavka' genotype. AFLP markers were used to investigate the genetic basis of variability within the cultivar. 'Žilavka' grapevines were screened on 14 microsatellite loci, thus revealing 4 different genotypes arising from mutations observed at 10 polymorphic loci. AFLP analysis of 52 'Žilavka' accessions revealed 35 different genotypes, with an average polymorphism of 57 %. Cluster analysis showed no grouping of different Žilavka accessions according to their names, characteristics or collection locations. The standard 'Žilavka' genotype was further compared to 211 cultivars from Slovenia (49), Austria/Germany (20), France (13), Portugal (27), Croatia (19), Greece (32), Spain (21) and Italy (30) in order to assess their genetic relationships. In pairwise comparisons, the highest genetic similarity was found with Slovenian cultivars 'Glera' and 'Briška Glera' (64 %) and the highest genetic dissimilarity (100 %) with two Italian cultivars, 'Nebbiolo Lampia' and 'Vespolina'. Inventory, collection and genetic characterization of 'Žilavka' accessions are important steps towards cultivar standardization, identification of parental cultivars and investigation of cultivar origin, required for its sustainable use.
Key words: grapevine, microsatellite, standard genotype, variability, AFLP
IZVLEČEK
ZBIRANJE VINSKE TRTE SORTE ŽILAVKA (Vitis vinifera L.) IN GENETSKA KARAKTERIZACIJA Z MIKROSATELITNIMI IN AFLP MARKERJ
'Žilavka' je pomembna vinska sorta z območja Bosne in Hercegovine kjer jo gojijo že od XIV stoletja. V preteklosti ni bila dovolj proučevana vendar ima velik ekonomski potencial za omenjeno območje. Akcesije žilavke (80), ki imajo različna imena in se razlikujejo po nekaterih osnovnih karakteristikah so bile nabrane na različnih področjih Bosne in Hercegovine. Za pridobitev standardnega genotipa žilavke smo uporabili mikrosatelitne markerje in z analizo 14 lokusov odkrili mutacije na 10 lokusih in določili 4 različne genotipe. Vse akcesije (52) katerim smo na osnovi mikrosatelitov določili standardni genotip smo nadalje analizirali z AFLP markerji in tako določili 35 različnih genotipov. Povprečni polimorfizem pri AFLP analizi je bil 57 %. S klastrsko analizo nismo odkrili skupin povezanih z različnimi karakteristikami, poimenovanjem ali izvorom akcesij žilavke. Standardni genotip žilavke smo nadalje primerjali z genotipi 211 kultivarjev iz Slovenije (49), Avstrije/Nemčije (20), Francije (13), Portugalske (27), Hrvaške (19), Grčije (32), Španije (21) in Italije (30), da bi ugotovili kakšna je sorodnost z njimi. V analizi primerjave parov smo ugotovili, da je žilavka najbolj sorodna (64 %) s slovenskima kultivarjema 'Glera' in 'Briška Glera' ter najbolj genetsko oddaljena od italijanskih kultivarjev 'Nebbiolo Lampia' in 'Vespolina'. Zbiranje, vrednotenje in genetska karakterizacija akcesij žilavke je pomembna za standardizacijo kultivarjev, analizo starševstva in izvora sorte.
Ključne besede: vinska trta, mikrosateliti, standardni genotip, variabilnost, AFLP
1	University of Banjaluka, Faculty of Agriculture, Bulevar vojvode Petra Bojovica 1A, 78 000 Banjaluka, Bosnia and Herzegovina, lidija.tomic@agrofabl.org
2	University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, SI-1111 Ljubljana, Slovenia
1 INTRODUCTION
Vitis vinifera 'Zilavka' is a white grapevine cultivar and is used traditionally for wine production. In the region of Herzegovina (Bosnia and Herzegovina), this cultivar has been grown for more than 600 years, the Bosnian King Tvrtko having been mentioned as drinking Zilavka wine in the 14th century (Vuksanovic and Kovacina 1984). In the 19th century, due to the grape quality and, particularly, to its resistance to bunch rot (Botrytis cinerea Pers.), the Austro-Hungarians exploited this cultivar by the production of a special dessert wine of the Malaga type (Mijatovic 1988). Apart from its historic importance and long tradition, Zilavka wine has high economic potential for Bosnia and Herzegovina, since it is exported and is popular in neighboring countries. It is also grown in adjacent regions of Croatia, Macedonia, Montenegro and Serbia. Vuksanovic and Kovacina (1984), Mijatovic (1988), Tarailo (1991) and Cindric et al. (2000) have provided an ampelographic description of this cultivar, which is characterized by high morphological heterogeneity of various traits: the shape, size and compactness of the cluster, the color, shape and size of the berry, vegetative potential, grape and wine quality, and resistance to bunch rot (Botrytis cinerea Pers.). In 1978 and 1991, clonal selection was performed at various locations and different types of 'Zilavka' were mentioned. Tarailo (1991), who described the clonal selection of 'Zilavka', also reported four different 'Zilavka' types and claimed that they are
different genotypes. 'Zilavka' heterogeneity might be a result of ecological/growth factors or genetic mutations. Many authors designate 'Zilavka' as an autochthonous cultivar to Herzegovina (Cindric et al. 2000; Milosavljevic 1998; Buric 1985; Vuksanovic and Kovacina 1984; Licul and Premuzic 1979; Tarailo et al. 1978; Avramov 1974), where it can be found under various synonymic names: 'Zilavka Mostarska' (Cindric et al. 2000; Milosavljevic 1998; Mijatovic 1988), 'Zilavka Hercegovacka' (Cindric et al. 2000; Avramov 1974), 'Zilavka Bijela' ^ (Mijatovic ^ 1988; Avramov 1974), 'Zilavka Zutka' and 'Zilavka Zelenka' (Anicic op cit. Mijatovic 1988). In our study, two different marker systems, microsatellites (SSR - short sequence repeats) and AFLP (amplified fragment length polymorphism), were applied for genetic analysis, thus providing a complementary tool to ampelographic descriptions. Microsatellites are among the most frequently used DNA markers for cultivar identification, revealing synonyms and homonyms and geographical origin, studying genetic relationships within large groups of cultivars and for clonal variability characterization. AFLP has been found to be an efficient and sensitive method for establishing genetic intra-varietal variability. A combination of SSR and AFLP molecular markers is used to obtain a more accurate and complete framework for genomic identity of different accessions within the cultivar (Vignani et al. 2002; Labra et al. 2001).
2 MATERIALS AND METHODS
2.1 Plant material and DNA extraction
Survey expeditions were organized to the region of Herzegovina as the traditional centre of 'Zilavka' cultivation. 'Zilavka' was collected from 5 different locations, which
were inventoried according to the information received from local people. Four locations belong to the Mostar area (43°20'58''N; 17°48'45''E) and one is from the Trebinje area (42°42'40''N; 18°20'44''E). Three of them are
old vineyards established during the seventies, one is a collection vineyard and one is a modern vineyard. Fresh leaves were collected from each of 80 chosen accessions. Plant material of accessions with different characteristics and names ('Starinska Zilavka', 'Zilavka', 'Stara Zilavka', 'Zuta Zilavka', 'Zelena Zilavka') were included in the study (data available upon request).
Total genomic DNA was extracted from fresh leaf	tissue	by	CTAB
(cetyltrimethylammonium bromide) extraction buffer (Kump and Javornik 1996), re-suspended in TE buffer (1 M Tris-HCl, 0,5 M EDTA, pH 8.0) and stored at 4 °C.
2.2 Microsatellite analysis
Eighty 'Zilavka' accessions were analyzed at 14 microsatellite (SSR) loci: VVS2 (Thomas and Scott 1993), VVMD5 and VVMD6 (Bowers et al. 1996), VVMD24 24, VVMD25 25 and VVMD27 27 (Bowers et al. 1999), VrZAG21, VrZAG47, VrZAG62, VrZAG64, VrZAG67 and VrZAG79 (Sefc et al. 1999), and Uch11 and Uch29 (Lefort et al. 2001). The PCR reaction in a total volume of 10 ^l contained 20 ng of extracted DNA, 10 x PCR buffer (Fermentas), 0.2 mM dNTP's (Fermentas), 2 mM MgCl2 (Fermentas), 0.5 ^M of each primer and 0.5 U of Taq polymerase (Fermentas). One of each of the primer pairs was labeled with fluorescent Cy-5 dye. Amplification of SSR loci was done in a Whatman Biometra T-Gradient thermocycler with the following steps: hot start for 5 minutes at 95 °C; 26-40 cycles of: denaturation at 94 °C for 30-45 seconds, annealing at 50-56 °C for 30-45 seconds and an extension step at 72 °C for 90 seconds. Amplification of loci Uch11 and Uch29 was done by a tailing protocol using three different primers: 0.2 ^M of each of unlabelled Uch primers and 0.075 ^M of 18 bp M13 tail sequence attached to the forward primer for subsequent fluorescent labeling (5' TGT A A A AC GACGGC C AGT '3). An optimized touchdown protocol was
used for PCR of these two loci: initial denaturation of 94 °C for 5 minutes: 5 cycles of 94 °C for 45 seconds, 60 °C for 30 seconds with a decrease of -1 °C per cycle and 72 °C for 1.5 minutes; followed by 25 cycles at the annealing temperature of 55 °C. PCR reactions were completed at 72 °C for 8 minutes (incubation). Amplified SSR fragments were denatured for 4 minutes at 95 °C and separated on 6 % polyacrylamide gel electrophoresis containing 7 M urea and detected by an ALFexpress DNA automated sequencer (GE Healthcare). The allele sizes were analyzed with AlleleLocator version 1.03 software (Amersham Pharmacia Biotech, 1998). Alleles were precisely sized against ALFexpress sizer 50 - 500 bp (GE Healthcare) and by internal DNA standards of various sizes amplified from plasmid.
2.3 AFLP Analysis
Fifty-two 'Zilavka' accessions were analyzed by 6 different AFLP primer combinations: Msel-CAA/Pstl-ACA, Msel-CAA/Pstl-AAC, Msel-CAT/Pstl-ACA, Msel-AG/Pstl-AGA, Msel-CT/Pstl-ACA and Msel-AG/Pstl-ACA. Isolated DNA (250 ng) was digested by 2.5 U of PstI and 2.5 U of Msel restriction endonucl eases (New England Biolabs) in a reaction volume of 40 ^l at 37 oC for 3 h. After digestion, 5 pmol of Pst and 50 pmol of Mse double stranded adaptors were ligated (1 Weiss U of T4 DNA ligase) to the sticky ends of the genomic fragments in a final volume of 50 ^l at 37 oC for 3 h. Adaptors were obtained by mixing equimolar amounts of primers PstRI linker1 and PstRI linker2 for Pst adapters and MseI linker1 and MseI linker2 for Mse adapters. The pre-amplification procedure was done in a total reaction volume of 50 ^l containing: 5 ^l of ligation reaction serving as template, 1x PCR buffer, 1.5 mM MgCh, 200 ^M each dNTP's, 1.25 U Taq polymerase, 50 ng of both PstI and Msel primers with one selective nucleotide (Pstl+A and Msel+C). Reactions were amplified in a Whatman Biometra T-Gradient thermocycler, repeating 20 cycles of: 94 °C for 30 seconds, 56 °C for 60 seconds and
72 °C for 105 seconds. The PCR products from the pre-amplification step were diluted 10-fold and 5 ^l of the dilution was used for selective amplification. Selective amplification was done by 15 ng of both Pstl and MseI primers with two or three selective nucleotides at the 3' end and Pstl primers were Cy5 labeled at their 5' end to allow automated laser fluorescence analysis. Selective reactions were performed in a 10 ^l PCR reaction mix with the same reaction components as for the pre-amplification step, except that 0.3 U of Taq polymerase was used. The reaction was amplified with an initial touch down protocol: 94 °C for 30 seconds, 65 °C for 30 seconds and 72 °C for 60 seconds with a touchdown of - 0.7 °C of annealing temperature per cycle, followed by 23 cycles at 94 oC 30 s, 56 oC 30 s and 72 oC 1 minutes, and the samples were immediately transferred onto ice. The amplification reaction was mixed with an equal volume (10 ^l) of formamide loading buffer (5 mg dextrane blue in 1 ml of formamide), denaturated by heating to 94 oC for 4 minutes. Five ^l of sample was loaded onto 5% polyacrilamide denaturating gel (5% acrylamide-bisacrylamide 19:1, 1*TBE, 7 M urea) and electrophoresed on an ALFexpress DNA automated sequencer (GE Healthcare). An external size standard ranging from 50 to 500 bp was run together with samples to allow further sizing of the fragments. AFLP bands ranging from 50 to 500 base pairs were analyzed using AlleleLocator version 1.03 software, (Amersham
Pharmacia Biotech, 1998). In the first instance, all 80 accessions were included in AFLP testing but only 52 produced scorable fingerprints and were included in the AFLP analysis.
2.4 Data analysis
Microsatellite and AFLP amplified bands across all analyzed loci were scored by state as present and given numerical value 1 or absent 0, making a presence-absence matrix of bands. This matrix served as a basis for clustering and assessing overall similarity among the analyzed accessions. A similarity index was calculated from band sharing data of each pair of accessions using the Jaccard (1908) coefficient of similarity. A dendrogram was constructed from the matrix of pairwise distances using an unweighted pair group method (UPGMA) for clustering in the NTSYS-PC software package, version 1.80.
The obtained microsatellite profile of 'Zilavka' was further compared to 211 already genotyped cultivars in Europe in order to assess their genetic relationships.
For AFLP analysis, the percentage of polymorphism was calculated for each primer pair combination from the total number of amplified bands and the number of polymorphic bands.
3 RESULTS AND DISCUSSION
3.1 Microsatellite analysis
Eighty 'Zilavka' accessions were screened on 14 microsatellite loci. These analyses resulted in 4 different genotypes arising from mutations observed at 10 polymorphic loci (Tab. 1). Seventy-seven accessions had the same allelic
profile across all 14 loci and could be considered as a standard genotype for 'Zilavka'. Three accessions (9/2, 10/2 and 11/2), which were thought to be 'Zilavka', resulted in polymorphism at 5 to 6 loci (Tab. 1) and could not be considered to be 'Zilavka'.
Table 1: Standard 'Zilavka' genotype profiled at 14 microsatellite loci with three distinguished genotypes and 'Chardonnay' as reference cultivar.
Genotype	'Zilavka'	9/2	10/2	11/2	'Chardonnay'
VVS2	132:152	n.a.	132:152	132:152	137:152
VVMD5	226:238	226:238	226:238	226:238	232:236
VVMD6	208:208	208:208	190a:208	208:208	198:208
VVMD24	208:208	208:208	208:208	208:208	208:216
VVMD25	240:242	250a:250a	n.a.	240:242	240:256
VVMD27	179:194	179:194	181a: 194	179:194	n.a.
VrZAG21	201:203	201:206b	n.a.	201:206b	200:206
VrZAG47	158:173	158:173	158:173	158:173	160:168
VrZAG62	189:189	189:189	189:189	189:205a	188:196
VrZAG64	144:144	160b:164b	144:144	160b:164b	160:164
VrZAG67	151:161	141b: 151	141b:141b	141b: 151	140:153
VrZAG79	249:249	237b:249	242a:258a	237b:249	244:246
Uch11	235:235	235:235	235:235	235:249a	248:263
Uch29	208:295	208:295	295:306a	208:295	289:297
"new alleles different from standard 'Zilavka' genotype,
balleles shared between distinguished 'Zilavka' genotypes, n.a. no amplification
However, detailed morphological description of these accessions should be performed for clear definition. Grapevine cultivars are vegetatively propagated and individuals of one cultivar are expected to be genetically identical to each other, but some cultivars may comprise several different genotypes due to a polyclonal origin (Kozjak et al. 2003) or somatic mutations can be accumulated over years of clonal propagation.
Comparison of standard 'Zilavka' from our analyses with 'Zilavka' from Croatia, genotyped by Maletic et al. (1999) resulted in the same allelic profiles at all 7 comparable loci, which confirmed the trueness to type of the 'Zilavka' genotype.
The standard 'Zilavka' genotype was further compared to 211 cultivars, from Slovenia (49), Austria/Germany (20), France (13), Portugal (27), Croatia (19), Greece (32), Spain (21) and Italy (30) in order to assess their genetic relationships. Comparison was enabled by using one reference cultivar, 'Chardonnay', for allele size standardization. The proportion of
shared alleles was used as the basis for distance measurement among analyzed varieties. In pairwise comparisons of 'Žilavka' with cultivars from the various European regions, the highest genetic similarity was found with a Slovenian cultivar 'Glera' (64 %) and the highest genetic dissimilarity (100 %) with two Italian cultivars, 'Nebbiolo Lampia' and 'Vespolina'. A phenogram of all 211 genotypes was constructed (data available on request) in order to illustrate the genetic relationships between 'Žilavka' and other European cultivars. 'Žilavka' clustered together with Slovenian old cultivars 'Glera', 'Briška Glera', 'Vitovska', 'Zunek', 'Duranja' and 'Popetre'.
3.2 AFLP analysis
Fifty-two accessions of standard 'Žilavka' genotype analyzed at 14 microsatellite loci were included in further AFLP analysis. Six primer pair combinations generated 163 scorable bands, of which 87 (57%) were polymorphic, expressing intra-varietal variability (Tab. 2).
Table 2: Characteristics of analyzed AFLP loci expressed by number of amplified bands, polymorphic bands and % of polymorphism.
Primer pair combination	Number of amplified bands	Number of polymorphic bands	Polymorphism (%)
MseI-CAA / PstI-ACA	15	13	87
MseI-CAA / PstI-AAC	20	18	90
MseI-CAT / PstI-ACA	13	5	38
MseI-AG / PstI-AGA	44	27	61
MseI-CT / PstI-ACA	36	11	31
MseI-AG / PstI-ACA	35	13	37
Total	163	87	57 (Average)
The degree of polymorphism was higher than reported previously for some cultivars, e.g., 'Carmenere' (2.3%) (Moncada and Hinrichsen 2007), 'Cabernet Sauvignon' (5.9%) (Moncada et al, 2005) or 'Traminer' (34%) (Imazio et al. 2002). The high level of detected polymorphism might be explained by the accumulation of somatic mutations over
years of cultivation. 'Zilavka' was spontaneously propagated for many years without formal selection of clones, which might also have resulted in higher heterogeneity within the cultivar.
Based on AFLP data, the 52 'Zilavka' accessions revealed 35 genotypes, which are clustered on the dendrogram (Fig. 1).
Main AFLP cluster
■ i i i
0.85
Gsnetic Similarity

_1 5/2 -P 7/3
^ 733 : 1325
- 18/5
m
J
rC
1/3
3/4
//55
15/3
■ 19/5
12/4 14/1
i i i i
-3/5
1.00
Figure 1: Dendrogram showing genetic similarity among 'Zilavka' accessions generated from AFLP data
The clustering of accessions did not correlate locations. The main AFLP cluster consists of with their names, characteristics or sampling 16 non-differentiated accessions of the
5
5
0.90
standard 'Zilavka' genotype, which had previously been differentiated by their different names, characteristics or sampling locations (data available upon request), which means that AFLP analysis revealed no type specific marker.
Three out of 52 accessions had an AFLP genetic similarity lower than 0.90 (33/5, 6/5 and 2/1). On the basis of some reports (e.g.,
Cervera et al. 2000, 1998), accessions showing similarities > 0.90 can be considered to belong to the same cultivar, while the cultivars that are different show similarities between 0.65 -0.90). These three accessions all belong to the standard 'Zilavka' genotype according to the SSR analysis, but further detailed morphological analyses should be done before reaching any final conclusions.
4 DISCUSSION
Identification and distinction of 'Zilavka' accessions was possible on the basis of two marker systems. Microsatellite markers enabled determination of a standard 'Zilavka' genotype and, together with AFLP markers, revealed intra-varietal polymorphism. The two molecular markers, SSR and AFLP, explore different parts of the genome and have a different structure in terms of primer sequences and amplified motifs, so they are both valuable for obtaining reliable results on accession variability.
Molecular analysis has shown that 'Zilavka' is not a highly homogenous cultivar (high polymorphism is observed), which can be explained by non-formal clonal selection of Zilavka in Bosnia and Herzegovina and also by the long cultivation period, resulting in an accumulation of mutations. Microsatellite genotyping enabled us to exclude the accessions that are different from the true-to-type 'Zilavka', since it is known that different cultivars show at least four allelic differences, while clones show fewer differences but can
also be distinguished in some cases by SSR analysis (Laucou et al. 2011). In our study, 80 different accessions of 'Zilavka' were sampled from 5 locations (data available on request) but molecular analysis revealed no clustering of 'Zilavka' accessions according to the observed characteristics, names or locations. Detailed research on ampelographic data needs to be performed to explain the high AFLP polymorphism obtained for three accessions (33/5, 6/5 and 2/1) and to confirm their different origin. The remaining accessions analysed with AFLP markers expressed lower AFLP polymorphism (< 0.90) explaining their intra-varietal variability and true-to-type identity, which was also confirmed by SSR genotyping.
On the basis of the results obtained by molecular analysis, we can clearly distinguish accessions genotypically different from the true-to-type 'Zilavka', which is a step toward cultivar identification, standardization and collection.
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