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ANALYSIS OF GRAPE ROOTSTOCKS BY SSR MARKERS Gizella JAHNKE1*, Gitta KOCSISNÉ MOLNÁR2, János MÁJER1, Barna SZÖKE1, Erik TARCZAL2, Péter VARGA1 and László KOCSIS2 1 : University of Pannonia, Centre of Agricultural Sciences, Research Institute for Viticulture and Oenology, Badacsony H-8261 Badacsonytomaj, Római út 181, Hungary 2 : University of Pannonia, Georgikon Faculty, Department of Horticulture, Keszthely, Hungary

Abstract

Résumé

Aims: The aim of this work was to determine the SSR profile of 96 Vitis accessions (mainly rootstocks) at 19 loci to find genetic relatedness between them.

But: Le but de notre travail a été de déterminer des profils SSR de 96 lots Vitis (majoritairement des porte-greffes) dans 19 « loci » pour trouver des relations génétiques entre eux.

Methods and results: The molecular markers used in the last 30 years are not or less affected by the environment, making them a valuable tool for the determination of genetic distances. Based on our former experiences, an SSR analysis was carried out on 96 grape accretions, mainly rootstocks. DNA was extracted from young leaves and analyses were carried out at 19 microsatellite loci. The loci were selected to cover all of the linkage groups to get a more accurate genotyping of each grape rootstock. Results of the SSR analysis and the resulting dendrogram showing genetic relatedness are presented.

Méthodes et résultats: Étant donné que les marqueurs moléculaires utilisés il y a 30 ans étaient moins influencés par le milieu, ils représentent de bons outils pour déterminer les distances génétiques. En utilisant nos précédentes expériences, nous avons réalisé des recherches pour les 96 lots de la vigne qui étaient pour la plupart des porte-greffes. L'ADN a été extrait des jeunes feuilles. Nous avons réalisé des analyses dans 19 microsatellites loci. Les loci ont été choisis pour couvrir chacun des groupes de liens, pour obtenir une image plus précise du génome des porte-greffes. Les résultats de l'analyse SSR et des relations génétiques sont présentés par dendrogramme.

Conclusions: Based on the results, it can be established, that most of the selected loci showed appropriate polymorphism for the assessment of genetic relatedness. The rootstocks derived from Teleki’s seedlings showed high similarity. A low genetic distance was also detected between the accessions originated from the cross between Börner and Georgikon 28. Finally, the Vitis sylvestris accessions showed close relatedness with the Vitis vinifera L. varieties.

Conclusion : Nous avons pu constater que les loci ont montré un polymorphisme convenable. Les porte-greffes des plants de semis de Teleki ont montré une grande similitude. De plus, nous avons trouvé quelques différences entre les lots du croisement de Börner et de Georgicon 28. Les lots de Vitis sylvestris ont montré une forte similitude avec ceux de Vitis vinifera. Importance et impact de l’étude : Nos résultats montrent un polymorphisme certain entre les différents porte-greffes. Nous avons cependant pu constater une importante différence entre les clones de T5C et de TK5BB. En conséquence, ce ne sont pas des clones réels, mais ils ont des génotypes différents avec des propriétés morphologiques relativement similaires.

Significance and impact of the study: Our results indicate a high level of polymorphism between grape rootstock accessions. Strong differences were detected between the so-called Teleki 5C (T5C) and Teleki-Kober 5BB (T5KBB) “clones”, suggesting that they are not real clones but different genotypes with highly similar morphological features. Key words: simple sequence repeats (SSR), microsatellite, grape, rootstock

Key words: répétitions simples (SSR), microsatellite, vigne, porte-greffes

manuscript received 18th February 2010 - revised manuscript received 24th May 2011 *Corresponding author : [email protected]

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The correct identification of rootstocks is possible by isoenzymes (Boursiquot and Parra, 1992), if the time of sampling and the followed procedure is standardized; however, the discrimination of similar rootstocks by isoenzyme analysis can fail because of the limited polymorphism (Walker and Liu, 1995)

INTRODUCTION Rootstocks are used in most grape growing areas of the world to control grape phylloxera and to adapt to soil characteristics. The rootstock variety, similarly to the scion variety, has become a « production tool » for grape growing, determining the phylloxera resistance, nutrient uptake, lifetime, soil requirements, drought-, salt- and lime tolerance of the grafts, and the primary growth and lifetime of the stocks. It also influences the quality and quantity of crop, and the economic viability of grape growing. Although the main features, such as phylloxera resistance and lime- or drought tolerance, differ among rootstocks, rootstock identification is very difficult because many of them are closely related and have similar morphological characters.

Microsatellite markers (also known as SSRs, VNTRs, STMSs or STRs) are highly polymorphic DNA sequences comprised of mononucleotides, dinucleotides, trinucleotides or tetranucleotides that are repeated in tandem arrays and distributed throughout the genome (Jeffreys et al., 1985). They can provide a suitable tool for grape rootstock identification, phylogenetic studies or markerassisted selection (MAS). Here are a few examples of successful application of microsatellite markers to rootstock identification: (1) A representative group of rootstock accessions and cultivars of the Vitis species commonly used in rootstock breeding (V. vinifera, V. berlandieri, V. riparia, and V. rupestris) and conserved in the largest European germplasm banks of Vitis have been analyzed using sequence-tagged microsatellite sites (STMS) and amplified fragment length polymorphism (AFLP) markers (de Andrés et al., 2007). The STMS analysis allowed assigning a microsatellite genotype to most of the rootstock cultivars, highlighting numerous misclassified accessions in the studied collections, and the AFLP analysis provided information on the genetic relationships within and between hybrid groups (de Andrés et al., 2007).

The clone selection and cross breeding of grape rootstocks dates back more than a hundred years. Nevertheless, none of the rootstocks has all of the features required to meet production demands. Zsigmond Teleki, a vintner in the 1880s, experienced this in his own vineyards, which led him to search for new rootstock types (Bakonyi, Kocsis, 2004). Because of the embargo on import of vine-stocks, he bought grape seeds from France, from a famous Vitis berlandieri breeder named Euryale Rességuier. His aim was to breed rootstocks with high lime tolerance (Németh, 1975). Up until now, the origin of the 40 000 Teleki’s seedlings derived from the seeds brought from France has not been completely established. So, the species from which the world most widespread Berlandieri x Riparia rootstocks (Teleki 5C, Teleki Kober 5BB, and Teleki Fuhr SO4) originated is still unknown. Teleki sorted his seedlings into 10 groups (Table 1).

(2) The screening of a Vitis riparia genomic library for the presence of (GA)n SSRs has allowed the identification of 18 new markers applicable to the genotyping of a range of Vitis species (Sefc et al., 1999).

Table 1. The origin and classification of Teleki’s varieties based on morphological characters (after Bakonyi and Kocsis, 2004).

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Figure 1. Dendrogram based on the microsatellite analysis results of 96 varieties at 19 loci.

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Table 2. List of the analyzed Vitis accessions.

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Table 3 - List of the analyzed SSR loci.

a

Linkage groups are numbered according to Adam-Blondon et al. (2004)

Figure 2. Genotype and allele number of microsatellite loci based on the microsatellite analysis results.

(3) Eleven microsatellites isolated from grapevine (Vitis vinifera L.) have been used to study the degree of conservation of these sequences across different Vitis species. The results demonstrate the possibility of extending the use of microsatellite markers to wild germplasm and inter-specific hybrids (Di Gaspero et al., 2000).

from a cross of Ramsey (V. champinii) x Riparia Gloire (V. riparia) (Lowe and Walker, 2006).

The DNA extracted from cambium tissues of grape rootstocks (Vitis spp., Muscadinia rotundifolia SMALL) has been found to be suitable for molecular analysis (Lin and Walker, 1997). Its quality is equivalent to that of DNA extracted from leaf tissues, although the yield is higher from leaves. In addition, the use of cambium tissue allows DNA extractions during dormancy or from grafted rootstocks where leaves are not available.

1. Plant material and DNA extraction

The aim of this work was to determine the SSR profile of 96 Vitis accessions (mainly rootstocks) at 19 loci.

MATERIALS AND METHODS

Young leaves of 96 Vitis accessions (listed in Table 2) were collected in 2008 in the collection of the University of Pannonia in Cserszegtomaj (Hungary). Total genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen), following the manufacturer’s instructions. The amount and quality of DNA was determined spectrophotometrically. The DNA was diluted to a concentration of 10 ng/µl for use in polymerase chain reaction (PCR) assays.

Microsatellites can provide data for mapping as well. The first genetic linkage map of grape derived from rootstock parents was constructed using 188 progeny

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Table 4. Results of the microsatellite analysis of 96 cultivars at 19 loci. Data in base pairs. 0 = null allele/no amplification. 1/4

2. DNA amplification

One primer of each primer pair was fluorescently labeled with 6-FAM (6-carboxyfluorescein) on the 5’ end of the DNA chain. The PCR products were run on a PEApplied Biosystems 3100 Automated Capillary DNA Sequencer, and the size of the DNA fragments was determined using the GeneScan 2.0 software (Applied Biosystems).

Microsatellite (SSR) analysis was performed at 19 loci (Table 3). The primers were chosen from each chromosome to give well-defined heterozygosis (Costantini et al., 2007). PCRs were carried out in a total volume of 25 µl containing 12,5 µl of Hot Start Master Mix (Qiagen), 0,2 µM of each primer, and 50 ng of template DNA, using the following thermal profile: (1) 94 °C for 45 min; (2) 35 cycles of 94 °C for 1 min, annealing temperature (Table 3) for 1 min, 73 °C for 1 min; (3) 73 °C for 7 min.

3. SSR analysis and dendrogram The PowerMarker v. 3.25 was used for the calculation of summary statistics of the microsatellite marker data and the creation of the dendrogram (Liu and Muse, 2005).

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Table 4. Results of the microsatellite analysis of 96 cultivars at 19 loci. Data in base pairs. 0 = null allele/no amplification. 2/4

RESULTS AND DISCUSSION

The heterozygosity of the loci in the analyzed population ranged from 0.1771 to 0.9032. The lowest heterozygosity (0.1771) was detected at the VVIM10 locus and the highest heterozygosity (0.9032) was detected at the VMC5G8 locus (Figure 3).

The results of microsatellite analysis are summarized in table 4. Based on these results, it can be established that most of the loci showed appropriate polymorphism (Table 5). The total number of alleles per locus ranged from 12 to 25, and the number of genotypes ranged from 15 to 40 (Figure 2). The VVIM10 locus showed the worst result, presenting 12 and 15 alleles per locus and genotypes, respectively. The VVMD28 and VVS2 loci both showed good results with 40 genotypes and 23 and 21 alleles per locus, respectively. The highest allele number (25) was detected in VMC8A7.

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The distance matrix (not presented) was calculated using the shared alleles method (distance is defined as one minus half the average number of shared alleles per locus) and the dendrogram (Figure 1) was constructed using the neighbor-joining method (Saitou and Nei, 1987). In this figure, three clusters can be easily distinguished. All of the Vitis sylvestris (ALHAROS and DORGO) and Vitis vinifera (Cabernet-sauvignon, Pinot noir and Chardonnay) accessions are in the upper cluster with different rootstocks. In the middle cluster, we can find

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Table 4. Results of the microsatellite analysis of 96 cultivars at 19 loci. Data in base pairs. 0 = null allele/no amplification. 3/4

The history and origin of the rootstocks derived from the seedlings of Zsigmond Teleki is described in the introduction. The explained common origin may be the reason that in this study quite high similarity was detected between the Teleki 5C and the Teleki Kober 5BB accessions. The distance ranged from 0.0556 to 0.7692 with an average of 0.4031. On the other hand, strong differences were detected between the so-called « clones » of Teleki 5C and Teleki Kober 5BB, suggesting that they are not real clones Similarly, the inability of the SSR method to detect clonal variability between these

Georgikon 28 (G28) and Börner (BORNER) with different crossbred accessions (F10_3, G241, G236, G254, G251, G235, G243, F11_1, F10_1, G255, F9_1, and F9_2). In the pedigree of these hybrids both Georgikon 28 and Börner are present. Most of the rootstocks derived from Teleki’s seedlings are found in the lower cluster, with the Vitis riparia Gloire de Montpellier rootstock, which is supposed to be a parent of Teleki 5C E20 (this study).

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Table 4. Results of the microsatellite analysis of 96 cultivars at 19 loci. Data in base pairs. 0 = null allele/no amplification. 4/4

studied. Another explanation could be related to (2) problems with passport data or bank management, such as those reported by de Andrés et al. (2007), or with mistakes in DNA analysis if they show phenotypic differences that cannot be related with somatic mutation. There may be differences at the two loci (VrZAG79 and VMC8A7) for which SSR analysis was not possible (no amplification in Teleki Kober 5BB GK14 - either failed or corresponded to null allele). Unless genetic differences between both of these accessions can be identified by increasing the number of genotyped loci, which would

« clones » has been reported several times (This et al., 2006; Sefc et al., 1998; Moncada et al., 2006; Moncada and Hinrichsen, 2007; Regner et al., 2006; Wolf et al., 2003). It is surprising that two of the analyzed Teleki rootstocks (Teleki Kober 5BB GK14 and Teleki Kober 8B GK69) showed identical genotypes. One possible explanation for this can be (1) an identical genetic origin (being derived from the same cross), but the likelihood of this is very low because of the high number of loci

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Table 5. Statistical results of the analyzed SSR loci.

for these accessions. On the other hand, recent microsatellite (SSR) analyses have shown clear distinctions between wild and cultivated individuals (This et al., 2006), so this aspect needs further analysis.

confirm our first hypothesis, the most probable explanation is the second one. In this study, the rootstock variety Paulsen 1103 (V. berlandieri x V. rupestris) is very close to the Teleki Kober 5BB rootstocks, which are supposed to derived from crosses between V. berlandieri and V. riparia. There may be similarity because of the similar origin. The origin of the rootstocks bred by Zsigmond Teleki are not clear, but they were selected for good lime and drought tolerance, and Paulsen 1103 was selected in southern Italy (by Paulsen in 1895) for its strong drought tolerance and its ability to grow well on lime-based soils as well. The similar economical features can be traced back to the similar genetic background.

The possible parent-offspring combinations were considered when two genotypes shared at least one allele per locus. A possible parent for the following five rootstocks was found: T5C GK42, T5C E20, T5C GK46, T5C WED, and SZILÁGYI 157. This possible parent is the Riparia Gloire de Montpellier rootstock, which was selected in Montpellier, France by Viala in 1880. This rootstock is male flowered and was one of the first to be used after the phylloxera crisis in Europe. It also provides enough iron to its scions in limestone-based soils (Németh, 1975). The first four possible offsprings are the Teleki rootstocks (T5C GK42, T5C E20, T5C GK46, and T5C WED), and the fifth (SZILÁGYI 157) is a rootstock selected in Pécs, Hungary by János Szilágyi, from V. berlandieri x V. riparia seedlings from France (Bakonyi and Kocsis, 2004). All of the base material for selection in these five accessions were originated from France and were selected later, as Riparia Gloire de Montpellier occurred, which make it more possible to be a parent.

A low distance was also detected between accessions with complex gene pool (V. berlandieri x V. riparia x V. vinifera x V. cinerea). Most of them originated from the cross between Börner and Georgikon 28. The parentoffspring relation cannot be justified in any cases, probably because they went through a long selection period, when mutations can have occurred. The Vitis sylvestris accessions (ALHAROS and DORGO) showed closer relatedness with the Vitis vinifera varieties (Figure 3). This rejects the possibility of a V. sylvestris x V. riparia hybrid origin, but does not exclude the possibility of a V. vinifera x V. sylvestris hybrid origin

Acknowledgements : This research was funded by the Hungarian Scientific Research Fund (project no. K 73708).

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