Genetic Resources and Crop Evolution

, Volume 56, Issue 8, pp 1171–1181

Genotypes and phenotypes of an ex situVitis vinifera ssp. sylvestris (Gmel.) Beger germplasm collection from the Upper Rhine Valley

Authors

  • Susanne Barth
    • Department of Special Crop Cultivation and Crop Physiology, Viticulture GroupUniversity of Hohenheim
    • Teagasc Crops Research Centre
    • Department of Special Crop Cultivation and Crop Physiology, Viticulture GroupUniversity of Hohenheim
    • Department of Applied Plant Sciences and Plant Biotechnology, Institute of Horticulture, Fruit-Growing and ViticultureUniversity of Natural Resources and Applied Life Sciences
  • Fabienne Verzeletti
    • Department of Special Crop Cultivation and Crop Physiology, Viticulture GroupUniversity of Hohenheim
  • Rolf Blaich
    • Department of Special Crop Cultivation and Crop Physiology, Viticulture GroupUniversity of Hohenheim
  • Fritz Schumann
Research Article

DOI: 10.1007/s10722-009-9443-1

Cite this article as:
Barth, S., Forneck, A., Verzeletti, F. et al. Genet Resour Crop Evol (2009) 56: 1171. doi:10.1007/s10722-009-9443-1

Abstract

Wild grapevine Vitis vinifera ssp. sylvestris species are endangered in their natural habitats by modern landscape use and thus there is need of further collection and preservation of these species in ex situ collections or the preservation in situ. In this study 34 wild Vitis vinifera ssp. sylvestris accessions from the Upper Rhine Valley including a defined subpopulation are described and compared with six accessions from the former Yugoslavia stored in an ex situ collection. The accessions are described by means of ampelographic descriptors and genotyped at six SSR loci. Both marker types were helpful to characterize the V. vinifera ssp. sylvestrisex situ collection. Differentiation in accession groups was found based on genotypic variation. Ampelographic traits split the accession in two major groups, where one group holds mostly accessions from the Ketsch area and the Upper Rhine Valley. Preservation of V. vinifera ssp. sylvestris genotypes is essential for the maintenance of genetic diversity and the resistance of genetic erosion. More accessions of this species should be collected and conserved for conversation and future breeding applications.

Keywords

Ampelografic descriptorsEx situ collectionMorphologySSRVitis vinifera ssp. sylvestris

Introduction

Vitis vinifera is the only species among the Vitaceae that is extensively used in viticulture and the only species indigenous to Eurasia and is suggested to have first appeared approximately 65 millions yeas ago. Two forms co-exist in Eurasia and North Africa: the cultivated form, V. vinifera L. ssp. vinifera and the wild form V. vinifera ssp. sylvestris (Gmel.) Beger (This et al. 2006).

Vitis vinifera ssp. sylvestris is considered to be an autochthonous and dioecious relative of European cultivated vines. Populations of wild grapevines exist in diverse natural ecosystems of Central and Northern Europe, the Middle East, Northern Africa and Western Asia (Ocete et al. 2002) and have been utilized by human kind more than 790,000 years ago (Goren-Inbar et al. 2004). Wild grapes are predominantly forest climbers and occur in disjunct populations. They occasionally form complex introgressive hybrid groups in transition zones nearby vineyards with cultivated grapes (Aradhya et al. 2003). While wild grapevine is dioecious with anemophilous pollination domesticated grapevine is self-pollinating (hermaphrodite) and both differ in several traits (e.g., sugar content, berry- and bunch size and form) (Di Vecchi-Staraz et al. 2009; Grassi et al. 2008; This et al. 2006). Knowledge on the allelic diversity for genes involved in these traits is of great benefit for the analysis of domestication and for genetic resources issues.

Wild grapevines V. vinifera ssp. sylvestris have been threatened in the past by introduced pests, and are today endangered in their natural European habitats by modern land use practices. Therefore, a high priority should be given to the collection and preservation of wild Vitis germplasm. The genetic diversity of this species has been recognized in Germany more than a century ago. Wild grapevine individuals originating in the Upper Rhine Valley have been continuously collected and morphologically studied (e.g., Bronner 1857; Scheu 1937; Schumann 1974) and these results suggest that a genetically diverse population of Vitis vinifera ssp. sylvestris existed within this riverine landscape. Today only few individual plants can be found on their native range and an important collection is preserved in an ex situ collection at the DLR Neustadt, Germany. Germplasm protection relies on both in situ or ex situ conservation, including in vitro techniques. A major advantage of in situ conservation is the protection of complete ecosystems. Because wild grapevine is a liana-like woody perennial crop whose seeds are highly heterozygous, the long-term conservation of V. vinifera ssp. sylvestris genotypes must be realized by ex situ techniques. Grapevines propagate vegetatively by suckering in their natural habitats and are easily propagated on appropriate rootstocks for ex situ purposes, because otherwise they cannot withstand endemic root-feeding grape phylloxera (Daktulosphaira vitifoliae Fitch). Characterizing genetic diversity and its distribution throughout the species range is important for our understanding about the adaptation and survival of wild species to ensure that genetic resources are available for use in research and breeding programs (Forneck 2005; Pavek et al. 2001). SSR markers, being abundant, multi-allelic and polymorphic, provide a means of detecting genetic polymorphism. Due to their co-dominant structure this marker system enables studies on population genetic analysis, assessment of genetic structures and differentiation in germplasm collections and natural populations. Information on the amount and pattern of distribution of genetic variation is essential for effective conservation strategies. Several studies successfully used microsatellite markers to fingerprint and genotype V. vinifera ssp. sylvestris and V. vinifera ssp. vinifera germplasm (e.g., Aradhya et al. 2003; Dangl et al. 2001; Imazio et al. 2003; Lacombe et al. 2003; Pavek et al. 2003; Perret et al. 2000; Regner et al. 2000; This and Dettweiler 2003).

The objectives of this study are (1) to evaluate the genetic variation, structure and differentiation in an ex situ grape germplasm collection using polymorphisms at six microsatellite loci and (2) to examine the phenotypic diversity applying a set of 12 ampelographic descriptors (IPGRI, UPOV, OIV 1997). (3) To draw conclusions for future collection and conservation management.

Materials and methods

Plant material

Vitis vinifera ssp. sylvestris accessions (Table 1) propagated in the ex situ collection in the DLR Neustadt, Germany, were analyzed. The ex situ collection was assembled by F. Schumann from clones of the earlier collections of (1) Scheu, (2) Schumann and (3) Turkovic and contains vegetative propagated genotypes collected directly from the native habitats. The largest proportion of the samples was derived from the habitat of the Upper Rhine Valley including a sub population within the Upper Rhine Valley population defined by recent recombined genotypes of the ‘Ketsch’ area. Ketsch is a protected island within the river Rhine. Seven accessions hold in the collection originated from a geographically distinct area in the former Yugoslavia/Anatolia and were included in the study for comparative reasons. Due to sampling structure and cloning of genotypes by vegetative propagation the age of the plants may vary considerably. Hybrids with V. vinifera ssp. vinifera cultivars in the collection were excluded from the analysis based on ampelographic traits and SSR analysis.
Table 1

Vitis vinifera ssp. sylvestris accessions used in the study including geographical origin, region grouping, plant derived from seed or ramet, origin of collection, and scoring results for OIV descriptors

Code

Origin

Region grouping

Seed/ramet

Origin

OIV descriptors

7

8

53

67

68

70

75

76

79

N01

Neckarau (25/477)

Upper Rhine Valley

NA

Ex situa

2

1

7

3

1

1

3

4

3

N03

Hoerdt (5/59)

Upper Rhine Valley

NA

Ex situa

3

1

3

3

3

1

3

4

2

N04

Hoerdt (24/57)

Upper Rhine Valley

NA

Ex situa

2

1

7

3

4

1

7

4

3

N05

Dirmstein (2)

Upper Rhine Valley

NA

Ex situa

2

1

7

3

3

3

7

5

4

N06

Sponeck (21)

Upper Rhine Valley

NA

Ex situa

3

1

1

3

1

3

3

2

4

N10

Colmar (2)

Upper Rhine Valley

Ramet/river wood

Native habitatb

3

2

1

2

3

3

1

3

2

N11

Mannhein (3)

Upper Rhine Valley

Ramet/river wood

Native habitatb

2

2

1

3

3

3

1

2

3

N13

Colmar (2)/France

Upper Rhine Valley

Ramet/river wood

Native habitatb

3

2

1

2

3

3

3

4

2

N14

Colmar (5)/France

Upper Rhine Valley

Ramet/river wood

Native habitatb

3

2

1

2

4

3

3

4

2

N31

Ungstein

Upper Rhine Valley

Seed

Ex situb

3

2

1

3

3

3

5

2

2

N32

Ungstein

Upper Rhine Valley

Seed

Ex situb

3

2

1

3

3

3

3

2

2

N07

Ketsch (2)

Rhine, Ketsch

Ramet/river wood

Native habitatb

3

2

1

3

4

3

7

5

2

N09

Ketsch (32)

Rhine, Ketsch

NA

Ex situa

2

1

3

2

4

1

5

2

2

N23

Ketsch

Rhine, Ketsch

NA

 

3

2

1

3

4

5

3

4

2

N25

Ketsch

Rhine, Ketsch

Seed, “Ketsch 34”

Native habitatb

3

2

1

3

3

3

3

4

2

N26

Ketsch

Rhine, Ketsch

Seed, “Ketsch 34”

Native habitatb

2

1

1

3

3

3

3

2

2

N27

Ketsch

Rhine, Ketsch

Seed, “Ketsch 34”

Native habitatb

2

1

3

2

2

1

7

4

2

N28

Ketsch

Rhine, Ketsch

Seed, “Ketsch 34”

Native habitatb

2

1

1

3

4

3

5

2

3

N29

Ketsch

Rhine, Ketsch

NA

Native habitatb

3

2

3

2

2

3

7

2

3

N30

Ketsch

Rhine, Ketsch

NA

Native habitatb

2

1

9

2

2

1

5

3

3

N36

Ketsch (7)

Rhine, Ketsch

Seed

Native habitatb

2

2

1

2

3

1

3

4

2

N37

Ketsch (10)

Rhine, Ketsch

Seed

Native habitatb

3

3

1

2

4

3

3

2

4

N38

Ketsch (18)

Rhine, Ketsch

Seed

Native habitatb

3

2

1

2

3

5

3

2

3

N40

Ketsch (31)

Rhine, Ketsch

Seed

Native habitatb

2

1

1

2

2

1

5

2

2

N41

Ketsch (34)

Rhine, Ketsch

Seed

Native habitatb

2

1

7

2

4

3

3

2

4

N42

Ketsch (36)

Rhine, Ketsch

Seed

Native habitatb

3

2

3

3

4

3

3

2

3

N44

Ketsch (46)

Rhine, Ketsch

Seed

Native habitatb

3

2

1

2

4

5

3

2

3

N45

Ketsch (53)

Rhine, Ketsch

Seed

Native habitatb

3

1

1

3

1

3

3

2

1

N46

Ketsch (55)

Rhine, Ketsch

Seed

Native habitatb

3

3

7

2

2

3

7

4

1

N47

Ketsch (58)

Rhine, Ketsch

Seed

Native habitatb

2

3

7

2

2

3

5

4

1

N48

Ketsch (60)

Rhine, Ketsch

Seed

Native habitatb

3

3

7

2

3

1

5

4

1

N49

Ketsch (21)

Rhine, Ketsch

Seed

Native habitatb

2

3

7

2

3

3

3

2

5

N50

Ketsch (44)

Rhine, Ketsch

Seed

Native habitatb

2

3

9

2

3

3

3

2

5

N51

Ketsch (48)

Rhine, Ketsch

Seed

Native habitatb

2

2

1

2

4

3

5

2

5

N12

NA

Yugoslavia/Anatolia

NA

NA

2

1

1

3

4

1

1

3

3

N17

Anatolia/Yenikatha

Anatolia

Seed

Native habitatb

3

2

1

3

2

5

3

3

1

N18

Anatolia/Yenikatha

Anatolia

Seed

Native habitatb

2

1

1

2

3

1

1

4

1

N19

NA

Yugoslavia/Anatolia

NA

Ex situc

2

1

3

2

2

1

3

4

3

N20

NA

Yugoslavia/Anatolia

NA

Ex situc

3

2

1

2

1

3

1

4

3

N22

NA

Yugoslavia/Anatolia

NA

Ex situc

1

1

1

3

3

1

1

2

3

Code

Origin

Region grouping

Seed/ramet

Origin

OIV descriptors

81–2

84

87

202

204

220

223

225

 

N01

Neckarau (25/477)

Upper Rhine Valley

NA

Ex situa

2

7

5

1

5

2

1

5

 

N03

Hoerdt (5/59)

Upper Rhine Valley

NA

Ex situa

1

3

3

      

N04

Hoerdt (24/57)

Upper Rhine Valley

NA

Ex situa

1

3

5

      

N05

Dirmstein (2)

Upper Rhine Valley

NA

Ex situa

1

7

5

1

3

2

2

5

 

N06

Sponeck (21)

Upper Rhine Valley

NA

Ex situa

1

1

3

1

9

2

2

5

 

N10

Colmar (2)

Upper Rhine Valley

Ramet/river wood

Native habitatb

1

3

3

      

N11

Mannhein (3)

Upper Rhine Valley

Ramet/river wood

Native habitatb

1

1

3

1

3

2

2

5

 

N13

Colmar (2)/France

Upper Rhine Valley

Ramet/river wood

Native habitatb

1

3

3

1

3

1

2

5

 

N14

Colmar (5)/France

Upper Rhine Valley

Ramet/river wood

Native habitatb

1

5

1

      

N31

Ungstein

Upper Rhine Valley

Seed

Ex situb

2

3

3

      

N32

Ungstein

Upper Rhine Valley

Seed

Ex situb

1

3

3

1

5

1

2

5

 

N07

Ketsch (2)

Rhine, Ketsch

Ramet/river wood

Native habitatb

1

5

5

      

N09

Ketsch (32)

Rhine, Ketsch

NA

Ex situa

1

5

3

1

7

2

1

5

 

N23

Ketsch

Rhine, Ketsch

NA

 

2

5

5

      

N25

Ketsch

Rhine, Ketsch

Seed, “Ketsch 34”

Native habitatb

1

5

3

      

N26

Ketsch

Rhine, Ketsch

Seed, “Ketsch 34”

Native habitatb

1

5

3

      

N27

Ketsch

Rhine, Ketsch

Seed, “Ketsch 34”

Native habitatb

1

5

5

1

5

2

2

5

 

N28

Ketsch

Rhine, Ketsch

Seed, “Ketsch 34”

Native habitatb

1

3

3

      

N29

Ketsch

Rhine, Ketsch

NA

Native habitatb

1

5

3

      

N30

Ketsch

Rhine, Ketsch

NA

Native habitatb

1

5

5

      

N36

Ketsch (7)

Rhine, Ketsch

Seed

Native habitatb

1

3

3

      

N37

Ketsch (10)

Rhine, Ketsch

Seed

Native habitatb

1

3

1

      

N38

Ketsch (18)

Rhine, Ketsch

Seed

Native habitatb

1

3

5

      

N40

Ketsch (31)

Rhine, Ketsch

Seed

Native habitatb

1

5

5

1

5

2

2

5

 

N41

Ketsch (34)

Rhine, Ketsch

Seed

Native habitatb

1

5

5

      

N42

Ketsch (36)

Rhine, Ketsch

Seed

Native habitatb

1

3

3

      

N44

Ketsch (46)

Rhine, Ketsch

Seed

Native habitatb

1

3

3

      

N45

Ketsch (53)

Rhine, Ketsch

Seed

Native habitatb

1

1

3

      

N46

Ketsch (55)

Rhine, Ketsch

Seed

Native habitatb

1

7

5

      

N47

Ketsch (58)

Rhine, Ketsch

Seed

Native habitatb

1

5

5

      

N48

Ketsch (60)

Rhine, Ketsch

Seed

Native habitatb

1

7

3

      

N49

Ketsch (21)

Rhine, Ketsch

Seed

Native habitatb

1

5

5

      

N50

Ketsch (44)

Rhine, Ketsch

Seed

Native habitatb

1

5

5

      

N51

Ketsch (48)

Rhine, Ketsch

Seed

Native habitatb

1

5

3

      

N12

NA

Yugoslavia/Anatolia

NA

NA

1

3

5

      

N17

Anatolia/Yenikatha

Anatolia

Seed

Native habitatb

1

1

1

      

N18

Anatolia/Yenikatha

Anatolia

Seed

Native habitatb

1

1

1

      

N19

NA

Yugoslavia/Anatolia

NA

Ex situc

2

3

3

3

3

2

2

5

 

N20

NA

Yugoslavia/Anatolia

NA

Ex situc

1

3

3

1

9

2

2

5

 

N22

NA

Yugoslavia/Anatolia

NA

Ex situc

2

3

3

1

9

2

1

5

 

NA information not available

aCollector/collection: Scheu (1937)

bCollector/collection: Schumann (1974)

cCollector/collection: Turkovic (1953)

Phenotype analysis

Morphological characters were selected for their importance to germplasm management and specificity to V. vinifera ssp. sylvestris. Twelve standardized morphological descriptors were selected for phenotypic analysis and measured on each genotype (IPGRI, UPOV, OIV 1997) according to (Jung 2003, personal communication). Vegetative characters scored or measured were: (7) color of dorsal site of internodes, (8) color or ventral site of internodes, (53) young leaf: density of prostrate hairs between veins, (67) shape of blade of mature leaf, (68) number of lobes, (70) anthocyanin coloration of main veins on upper side of the blade, (75) blistering of the upperside of the blade, (76) shape of teeth, (79) degree of petiole sinus opening, (81–2) petiole sinus limited by veins, (84) density of prostrate hairs between the main veins, (87) density of erect hairs on the main veins. The following generative characteristics were measured using twelve female plants: (202) bunch length, (204) bunch density, (220) berry length, (223) berry shape and (225) skin color of berries.

Three clones per genotype were available within this collection for the analysis on morphological descriptors. These morphological descriptors were measured in five replicates per vine.

Genotype analysis

DNA extraction was performed using freshly sampled meristematic leaves. Leaves were shock frozen in liquid nitrogen and ground to obtain a fine powder. Total genomic DNA was extracted using a commercial kit following the manufacturer’s instructions (Dneasy Plant; Qiagen, Hilden, Germany). SSR analysis was performed with six SSR markers, which have been previously used for fingerprinting within the framework of the European Vitis database (This and Dettweiler 2003): VVS2 (Thomas and Scott 1993), VVMD7, VVMD27, VVMD32 (Bowers et al. 1996, 1999), VVZAG64 and VVZAG79 (Sefc et al. 1999) following the protocol of Dangl et al. (2001) modified by the employment of Cy-5 labeled sense primers (Amersham, Freiburg). Samples were electrophoresed with internal sizing standards on an ALF-express automated sequencer (Amersham, Freiburg) according to the manufactorer’s protocol.

Data analysis

Phenotypes

The phenotypes were compared by discriminant analysis using ten leaf descriptors. Data from bunch and berry descriptors were analyzed separately for female individuals. Analysis was carried out with NTSYS-PC software V2.2 performing a linear transformation of the data matrices eliminating the effects of different scales of measurement, followed by computing similarity indices for interval measure data applying Euclidian square distances.

Genotypes

A genetic distance matrix was calculated based on Nei’s unbiased measures of genetic identity and distance (Nei 1978), using allele data (characters) without size information for the three populations as partitioned in Tables 1 and 2 for the total of the Rhine population and the population from former Yugoslavia and Anatolia. Calculations were carried out as implemented in POPGENE V3.2 (Yeh and Boyle 1997). A genetic distance matrix was calculated for single accessions based on the average taxonomic distance using the presence or absence of each allele. The similarity matrix was subjected to sequential agglomerative hierarchical nested (SAHN) clustering using unweighted pair-group method analysis (UPGMA) as implemented in NTSYS V2.2 software. F-Statistics measures were calculated for the six SSRs as implemented in POPGENE after Nei (1987). FIS and FST were calculated. Expected homozygosity and heterozygosity were calculated after Levene (1949). Inferential statistics were estimated with these data to estimate population structure, even if the collection is based on a mixture of ex situ and in situ accessions. The genotypic data from each of the six SSR markers were summarized by the following measures of genetic variation: number of alleles per locus and polymorphic information content (PIC). PIC is defined as 1 minus the sum of squares of all gene frequencies at one locus. Model-based clustering of the SSR data was performed using the Structure algorithm (Falush et al. 2003; Pritchard et al. 2000), which clusters individuals into groups. Analysis was performed with prior information about the origin of accessions. The admixture model was used. Each run consisted of 10,000 repetitions after 20,000 burn-in steps.
Table 2

Basic information on SSR markers across all accessions: polymorphic information content (PIC), observed number of alleles, Wrights’s fixation index (FIS), degree of population differentiation (FST), and observed and expected heterozygosities

SSR

Observed number of alleles

PIC

FIS

FST

Observed heterozygosity

Expected heterozygosity

VVS2

12

0.5710

0.0181

0.0639

0.5526

0.5786

VVMD7

7

0.8870

0.1881

0.0224

0.5789

0.6979

VVMD27

6

0.3774

0.4560

0.1467

0.1842

0.2888

VVMD32

6

0.5585

0.0850

0.0730

0.4615

0.5658

Vripzag62

4

0.2801

−0.0565

0.0716

0.2895

0.2839

Vripzag79

9

0.5779

0.0789

0.1712

0.4286

0.5863

Overall

  

0.1600

0.0915

0.42

0.50

Results

Wild phenotypic diversity

The morphological traits varied widely among the accessions (Table 1). Twelve of the accessions were female, twenty-eight were male plants. Among the female plants, the trait bunch density (OIV 204) varied from loose to dense bunches (Table 1) exhibiting diversity for this important agronomic character. Berry shapes (OIV 223) were either narrow elliptic, elliptic or round and thus less variable. The skin color of the berries (OIV 225) was always dark red violet. The other two reproductive OIV characters bunch length (OIV 202) and berry length (OIV 220) varied little among the accessions studied (Table 1).

The color of the dorsal side of internodes was either green and red striped or completely red in all accessions with one exception of one Yugoslavia accession (N22), whereas the color of ventral sides of internodes varied among all accessions (Table 1). The density of prostrate hairs between veins on the young leaf varied between very sparse and dense. There were no leaves with absent hairs and two accessions (N30, N50) showed very dense hairs. The shape of leaves was either wedge-shaped or pentagonal in all accessions and had mainly three-seven lobes. Only four accessions (N1, N6, N20 and N45) had entire leaves. The anthocyan coloration of main veins on the upper sides of blades varied among the accession from very weak—medium and strong coloration in three accessions (N11, N42, N17). The blistering of blade ranged from very weak to strong with the strong proportion of accessions showing weak blistering. The shape of teeth of leaves showed high variability (Table 1). The general shape of the petiole ranged between wide open, half open and slightly open. Only one accession (N12) had a closed petiole sinus. The density of prostrate hairs between veins ranged from very sparse—dense, whereby three accessions showed very dense hairs (N5, N46, N47). The density of erect hairs on main veins on the lower side of the blade varied from very sparse—medium over all accessions as did the density of erect hairs on main veins.

The accessions could be clustered into two major groups basing on morphological descriptors (Fig. 1a). One group contained three accessions (N01: Neckarau, N19 and N22: Yugoslavia/Anatolia), the second group contained all other remaining accessions from the former Yugoslavia/Anatolia and ‘Ketsch’ and Upper Rhine Valley accessions.
https://static-content.springer.com/image/art%3A10.1007%2Fs10722-009-9443-1/MediaObjects/10722_2009_9443_Fig1_HTML.gif
Fig. 1

a Unrooted dendrogram showing similarities between accessions based on ten leaf OIV descriptors, constructed using the unweighted pair group method with arithmetic means (UPGMA) method as implemented in NTSYS based on squared Euclidean distance measures. b Unrooted dendrogram showing similarities between accessions based on six SSR marker loci, constructed using the unweighted pair group method with arithmetic means (UPGMA) method as implemented in NTSYS based on the simple matching coefficient distance measure

Genetic diversity of the ex situ collection

Extensive genetic polymorphisms were observed among the accessions. The total number of different alleles for the six SSR loci ranged from four to twelve (Table 2). Some of these alleles were more frequent in specific accession groups, however, due to sample size and sampling structure assumptions on the specificity of alleles for V. vinifera L. ssp. sylvestris (Gmel.) Beger have to be made with caution. The most variable loci over all samples were VVS2 and VVMD7 for which a majority of accessions were heterozygous. Gene diversity or PIC values varied from 0.2801 (Vripzag62) to 0.8870 (VVMD7). These comparatively high PIC values may be explained by the broad array of genotypes collected. Generally a deficiency in heterozygotes was found (H observed 0.42, H expected 0.50; Table 2). Overall accessions only little inbreeding was detected (FIS = 0.16; Table 2). Severe inbreeding depression (FIS) was noticed only at locus VVMD27 (0.4560). Overall population differentiation (FST = 0.0915) was small.

Structure and differentiation of the ex situ collection

Neither the groups nor the clusters exhibited appreciable levels of genetic divergence for statistical significance, however the composition of groups displayed by the dendrogram clustering allowed for some generalizations about their genetic structuring (Fig. 1). The upper Rhine accessions and the accessions sampled form the Ketsch island were rather identical as measured with Nei’s identity index (Nei 1978) (0.9765). The total Rhine population (upper Rhine accessions combined with Ketsch island accessions) was more distinct from the population from the former Yugoslavia and Anatolia (0.8479).

Within the population two identical genotypes could be assessed: N32 and N26 as well as N36 and N38. Three of the four accessions are seedlings from the Ketsch island one is from a near by location. The two most distant genotypes are N17 and N18, the two accessions sampled at Yenikatha by Schumann and obviously representing a distinct V. sylvestris group. Together with the four remaining accessions sampled in the former Yugoslavia area by Turkovic (1953) a loose cluster can be found. Some “population structure” can be assessed among accessions from the Ketsch area, accessions from the Upper Rhine Valley and the six accessions from the former Yugoslavia (Fig. 2). The six accessions, sampled originally by Scheu (1937) did not cluster, but included the most distant Upper Rhine Valley accessions (N01, N06, N11) in this analysis (Fig. 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs10722-009-9443-1/MediaObjects/10722_2009_9443_Fig2_HTML.gif
Fig. 2

Population structure of V. vinifera ssp. sylvestris accessions displayed in triangle plots partitioned in three populations: Upper Rhine Valley (red), sub-population Ketsch (green) and Anatolia/Yugoslavia (blue). Calculations were done in Structure using 30,000 reps and 20,000 burnins using the admixture model. (Color figure online)

Discussion

European wild grapevine Vitis vinifera L. ssp. sylvestris (Gmel.) Beger has dramatically declined and in central Europe only few remaining genotypes can be found (Perret et al. 2000). For the conservation of germplasm for viticulture production and grapevine breeding as well as for usages in restoration of alluvial and colluvial forests, the analysis and documentation of genetic diversity are essential.

Considerable variation has been found among the accessions studied. Since all genotypes studied originated from ex situ or in situ collections, which rarely reflect the genetic structuring of naturally existing populations general conclusions on a population genetic level, based on the presented data, should not be drawn. But show a collection of frequently occurring phenotypes and diverging off-type phenotypes and genotypes.

Collection of high phenotypic variation

Phenotypic variation in natural populations reflects both genetic and environmental effects on different populations. Our data shows descriptors to differentiate among morphological characters measured among groups of accessions. The plasticity of morphological parameters, especially pertaining leaves, varied among one vine, possibly due to environmental influences and the age of tissues.

Among the commonly used leaf descriptors the opening of the petiolar sinus is significant to differentiate between ssp. sylvestris with a generally more open sinus than ssp. vinifera (Levadoux 1956) however, a general distinction could not be drawn. We did not observe foliar dimorphisms as been discussed in ssp. sylvestris (Levadoux 1956) although it is not regarded to be a common character of wild European grapevine (Martinez de Toda and Sancha 1999). We found the traits of blistering of mature leaf, density of erect hairs on main veins to be most suitable for the analysis of these accessions. These traits seem to differ in there efficiency among populations/collections. Other traits proposed to discriminate Spanish V. vinifera ssp. sylvestris populations were length of teeth/width at end of the base and the density of erect hairs on the main veins (lower side). Other than previous morphological description of V. vinifera ssp. sylvestris populations or accessions we did not consider the size of leaf to be a good trait, since these were not evenly pruned and did not suit for comparison studies. We have also observed high differences in cluster densities among the genotypes assessed ranging from loose to dense clustered fruits. The latter occurred in three genotypes is not typically described for V. vinifera ssp. sylvestris and thus indicates a potential hybrid with a V. vinifera ssp. vinifera cultivar. Furthermore we would like to draw attention on the artificial growth habits of wild European grapevine accessions in ex situ collection which may likely lead to changes in morphological measures among the accessions.

Genetic variation through recombination or mutation?

The genetic variation detected within groups of accessions is considerable and may be explained by several reasons. Generally, molecular DNA markers often provide weak discrimination between populations of outbreeding highly heterozygous perennials, such as wild grapevine and the majority of variation resides within population variation (Aradhya et al. 2003). We employed six nuclear SSR markers based on their resolution (Jung 2003, personal communication) and found efficient amplification for the detection of V. vinifera ssp. sylvestris based polymorphisms. Previous studies used up to ten (Perret et al. 2000) or 13 (Lacombe et al. 2003) SSR markers. By using a small set of very well known markers we confirmed allelic data and were able to draw comparisons by database search. Other studies (e.g., Pavek et al. 2003) identified specific SSR-alleles for individual populations of rock grape (V. rupestris Scheele), which suggests that SSR—polymorphism extent the species range. They occur on the population level, indicating a high level of genomic variation.

Besides genetic diversity, driven by recombination and detected by SSR markers, somatic mutations may have a major role in long-term vegetatively propagated V. vinifera ssp. sylvestris genotypes. Identification and maintenance of these individuals in germplasm collections is essential and further studies may provide insight on microevolution of this species, given the possibility to elucidate measures on the ages of these vines.

Further information on the genetic background of accessions studied here could be obtained analysing their haplotypes employing cp-SSR markers as previously done by e.g., Grassi et al. (2006) or Imazio et al. (2006). This data on plastid lineages could provide further insight into the distribution and/or domestication of V. vinifera ssp. sylvestris and the role of the Upper Rhine valley population. Analyses of variation in germplasm collections provides an added value for gene banks that make the research investment. Well-documented analysis of the number and types of useful polymorphism allow gene bank curators to offer specific accessions with the desired characteristics to research geneticists or applied plant breeders, who can then select material tailored to their objectives. Assessments of variation in gene banks rely on plant morphological characters. These characters are limited because many of the descriptions are more or less subjective, which makes comparisons difficult. Furthermore they are influenced by the environment leading to variation in characters (Ortiz et al. 2004).

In contrast, regional in situ conservation can help preserve the co-evolutionary dynamics between crops and wild relatives, and the pathogen populations of each species (Frankel et al. 1995). Dynamic genetic interactions exist for micro-evolutionary changes in this whole host-disease system. These changes however can be a response to the introduction of new cultivars containing introgressed wild resistance genes. Likewise, new pathogens or pathogen biotypes from the wild alternative host can invade crops, eliciting a response reaction by wild resistance genes.

Although these ex situ collections provide convenient, rapid access to germplasm, they are expensive to maintain and do not evolve under natural forces and processes. Conservation of genetic resources under in situ conditions would ensure that evolutionary dynamic forces continue to influence plant adaptation and survival. In situ conservation provides a practical way to complement the ex situ collections and meets the need of expanding germplasm collections (Pavek et al. 2003).

Future considerations

The magnitude of genetic and phenotypic plasticity shows the importance to conserve wild grapevines for purposes of breeding, gene conservation and demonstration of cultivated grapevine.

Previous pledges have proposed to protecting of the existing autochthonous and planted wild grapevine plants through the propagation of wild grapevines in ex situ collections and the propagation of ramets in nurseries or on-farm for reintroduction in native riverine habitats (Schumann 1996).

Extensive molecular data from wild individuals and cultivars from different regions is needed to analyse the existence of wild grapevine genotypes in Europe. Further studies are underway to elucidate the genetic divergence of the gene pool of wild grapevines of the upper Rhine valley. Research is required to determine accurately the extent and origin of the existing genetic diversity and cross breeding of V. vinifera ssp. sylvestris to exhibit new phenotypes is of great value for the sustaining of this genepools.

Acknowledgments

This work has been supported by a grant of the Geschwister Stauder Schenkung, Stuttgart and the Stiftung Natur und Umwelt of the Landesbank Baden Württemberg. We are grateful to the excellent technical assistance of Ms Sonja Havrda. This work contributes in part to the Bachelor thesis of FV.

Copyright information

© Springer Science+Business Media B.V. 2009