Short communications

Absence of an acid phosphatase isozyme locus as a marker candidate for true to typeness in woodland grape (Vitis vinifera L. ssp. sylvestris Gmelin)

2017: OENO One, 51, 2

DOI: http://dx.doi.org/10.20870/oeno-one.2017.51.1.1620

Abstract

The quest and conservation of existing populations of woodland grape (Vitis vinifera L. ssp. sylvestris Gmelin), the supposed progenitor of the European grapevine (Vitis vinifera L. ssp. sativa) and a significant actor in the evolution of grapevine, has great importance in preserving biodiversity. The proof of true-to-typeness is highly important in ex-situ conservation, because the contamination risk of the woodland grape populations is very high. Some characteristic “sylvestris” simple sequence repeats (SSR) alleles were identified, but they are only characteristic in a specific population.

In our recent study, the SSR profiles of 32 woodland grapes were compared to those of 16 European grapevine varieties and 20 rootstocks. Morphology and SSR analyses suggested that the analysed Vitis vinifera ssp. sylvestris Gmelin accessions were true-to-type. In this report, the results of the acid phosphatase isoenzyme analyses of the same woodland grape accessions are presented and a new marker for true-to-typeness is suggested.

Introduction

The woodland grape (Vitis vinifera L. ssp. sylvestris Gmelin) is supposed to be the progenitor of the European grapevine (Vitis vinifera L. ssp. sativa) (Arroyo‐García et al., 2006). This wild subspecies is endangered, as its populations are destroyed by phylloxera (Daktulosphaira vitifoliae Fitch), fungal diseases (eg. downy mildew, powdery mildew), contamination and human activity (Ocete et al., 2015).

The woodland grape is protected in Hungary (Farkas, 1999). The quest and conservation of its existing populations has great importance in preserving biodiversity, also because of its role in the evolution of grapevine.

The proof of true-to-typeness has high importance in ex-situ conservation (This et al., 2006). The contamination risk of woodland grape populations is very high, as Vitis vinifera L. ssp. sylvestris Gmelin can easily be crossed with other Vitis species, such as the invasive Vitis riparia and other Vitis genotypes used as rootstocks (Bodor et al., 2010; Zoghlami et al., 2013). The seedlings of these crosses have evolutionary benefit, as they can inherit phylloxera resistance from the non-sylvestris parent. Optimal collection sites are far away from commercial vineyards and have never been used for grape production.

The challenge in accession identification is to determine the subspecies to which the accession belongs. The most important morphological difference between the woodland grape and the European grapevine is that the former is dioecious and the latter is monoecious (Ocete et al., 2015).

The molecular analysis of the different populations of woodland grapes began about a decade ago (Arroyo‐García et al., 2006; This et al., 2006). In most of the cases, simple sequence repeats (SSR) markers were used for characterisation (Biagini et al., 2012; Biagini et al., 2014; Bitz et al., 2015). Some characteristic “sylvestris” alleles were identified, but they were only characteristic in a specific population (Doulati Baneh et al., 2015).

In a recent study, we compared the SSR profiles of 32 woodland grapes to those of 16 European grapevine varieties and 20 rootstocks. Morphology and SSR analyses suggested that the analysed Vitis vinifera ssp. sylvestris Gmelin accessions were true-to-type (Jahnke et al., 2016). In this report, the results of the acid phosphatase isoenzyme analyses of the same woodland grape accessions are presented and compared to our previous results.

Material and methods

1. Vitis accessions

68 Vitis accessions ‒ 16 Vitis vinifera ssp. sativa cultivars, 32 Vitis vinifera ssp. sylvestris genotypes and 20 others (mainly used as rootstocks) ‒ were analysed (Table 1).

2. SSR analysis

Dormant canes were collected in January 2016 and subsequently stored in plastic bags at 4 oC until processing, within 2 days. Active enzymes were extracted from the dormant canes of the 68 accessions as described by Arulsekar and Parfitt (1986). Vertical polyacrylamide gel electrophoreses were carried out and gels were stained for acid phosphatase as described by Royo et al. (1997). Results were evaluated visually. Isozyme bands were digitally scored (1-present, 0-absent). Chi-square test of independence and contingency coefficient calculation were carried out using Microsoft Excel.

Table 1. List of the analysed Vitis accessions


No

ID

Accession Name

Genetic Origin

Origin of the Accession

1

Sziren

Szirén

Vitis vinifera ssp. sativa

Kecskemet, Hungary

2

Trilla

Trilla

3

Gesztus

Gesztus

4

Heureka

Heuréka

5

Generosa

Generosa

6

Kecskemet_7

Kecskemét 7

7

Cserszegi_fuszeres

Cserszegi fűszeres

8

Irsai_Oliver

Irsai Olivér

9

Kovidinka

Kövidinka

10

Pinot_gris

Pinot gris

11

Ezerjo

Ezerjó

12

Pozsonyi_feher

Pozsonyi fehér

13

Kadarka

Kadarka

14

Muscat_Lunel

Muscat Lunel

15

Muscat_ottonel

Muscat ottonel

16

Piros_tramini

Piros tramini

17

S1

Sylvestris S1

Vitis vinifera ssp. sylvestris

Badacsony, Hungary (ex-situ collection from Szigetköz, Hungary)

18

S4_1

Sylvestris S4/1

19

S4_2

Sylvestris S4/2

20

S4_3

Sylvestris S4/3

21

S6_1

Sylvestris S6/1

22

S6_2

Sylvestris S6/2

23

S6_4

Sylvestris S6/4

24

S7

Sylvestris S7

25

B1

Sylvestris B1

26

B2

Sylvestris B2

27

B5

Sylvestris B5

28

B10

Sylvestris B10

29

B12

Sylvestris B12

30

B13

Sylvestris B13

31

B16

Sylvestris B16

32

B19

Sylvestris B19

33

B21

Sylvestris B21

34

B24

Sylvestris B24

35

B26

Sylvestris B26

36

B27

Sylvestris B27

37

B30

Sylvestris B30

38

B31

Sylvestris B31

39

B33

Sylvestris B33

40

B34

Sylvestris B34

41

B36

Sylvestris B36

42

B37

Sylvestris B37

43

B41

Sylvestris B41

44

B47

Sylvestris B47

45

B48

Sylvestris B48

46

B49

Sylvestris B49

47

B50

Sylvestris B50

48

B51

Sylvestris B51

49

V._berl._R1

Resseguier N1

V. berlandieri

INRA, Domaine de Vassal, France

50

V._rup._FW3

Fort Worth N3

V. rupestris

51

V._rup._T

Taylor

V. rupestris

52

V._cord.

8029 Mtp2

V. cordifolia

53

V._rip._GdM

Gloire de Montpellier

V. riparia

54

Aramon_rup_G1

Aramon Ganzin N1

V. vinifera x V. rupestris

55

V._vip._Ggb

Riparia Grand glabre

V. riparia

56

V._rup._FW1

Fort Worth N1

V. rupestris

57

Jacquez

Jaquez

V. Bourquina (Vinifera x Aestivalis)

58

Vialla

Vialla

V. labrusca x V. riparia

59

V._cin._Arnold

Cinerea Arnold

V. cinerea

60

V._aest._S.

Sauvage

V. aestivalis

61

V._sol.

Solonis

V. solonis

62

V._rup._FW2

Fort Worth N2

V. rupestris

63

V._berl._R107

Resseguier N107

V. berlandieri

64

Aramon_rup_G2

Aramon Ganzin N2

V. vinifera x V. rupestris

65

N._Mex.

V. Novo Mexicana

V. riparia x V. candicans

66

T5C

Teleki 5C E20

V. berlandieri x V. riparia

Kecskemet, Hungary

67

SO4

Teleki-Fuhr SO4 (133)

V. berlandieri x V. riparia

Cserszegtomaj, Hungary

68

5BB

Teleki-Kober 5BB

V. berlandieri x V. riparia

Results

The isozyme banding patterns of acid phosphatase are presented in Table 2.

Gel photos of Vitis vinifera ssp. sylvestris and Vitis vinifera ssp. sativa accessions are presented in Figures 1 and 2, respectively.

Figure 1. Gel photo of Vitis vinifera ssp. sylvestris accessions (from left to right: sylvestris S4/3, B10, B12, B13, B33, B37, B41, B35, B49, and S6/1).

Figure 2. Gel photo of Vitis vinifera ssp. sativa accessions (from left to right: Pinot gris, Irsai Olivér, Szirén, Pozsonyi fehér, Muscat ottonel, Piros tramini, Kövidinka, Szirén, Heuréka, and Generosa).

Table 2. The banding patterns of acid phosphatase (1-present; 0-absent)


 No.

Accession ID* 

ACP1

ACP2

ACP3

ACP4

ACP5

ACP6

ACP7

ACP8

1

Sziren

1

1

1

1

1

1

1

0

2

Trilla

1

1

1

1

1

1

1

1

3

Gesztus

1

1

1

1

1

1

1

1

4

Heureka

1

1

1

1

1

1

1

0

5

Generosa

1

1

1

1

1

1

1

0

6

Kecskemet_7

1

1

1

1

1

1

1

0

7

Cserszegi_fuszeres

1

1

1

0

1

1

1

1

8

Irsai_Oliver

1

1

1

1

1

1

1

0

9

Kovidinka

1

1

1

0

1

1

1

1

10

Pinot_gris

1

1

1

0

1

1

1

0

11

Ezerjo

1

1

0

1

1

1

1

0

12

Pozsonyi_feher

1

1

1

1

1

1

1

1

13

Kadarka

1

1

1

1

1

1

1

1

14

Muscat_Lunel

1

1

1

1

1

1

1

0

15

Muscat_ottonel

1

1

1

0

1

1

1

0

16

Piros_tramini

1

1

1

0

1

1

1

0

17

S1

1

1

1

0

1

1

1

0

18

S4_1

1

1

1

0

1

1

1

0

19

S4_2

1

1

1

0

1

1

1

0

20

S4_3

1

1

1

1

1

1

1

0

21

S6_1

1

1

1

1

1

1

1

0

22

S6_2

1

1

1

1

1

1

1

0

23

S6_4

1

1

0

1

1

1

1

0

24

S7

1

1

1

1

1

1

1

0

25

B1

1

1

1

1

1

1

1

0

26

B2

1

1

1

1

1

1

1

0

27

B5

1

1

1

1

1

1

1

0

28

B10

1

1

1

0

0

0

0

0

29

B12

1

1

1

1

1

1

1

0

30

B13

1

1

1

1

1

1

1

0

31

B16

1

1

1

0

0

0

0

0

32

B19

1

1

1

0

0

0

0

0

33

B21

1

1

1

1

1

1

1

0

34

B24

1

1

0

1

1

1

1

0

35

B26

1

1

1

0

0

0

0

0

36

B27

1

1

1

0

0

0

0

0

37

B30

1

1

1

0

0

0

0

0

38

B31

1

1

1

0

0

0

0

0

39

B33

1

1

1

0

1

1

1

0

40

B34

1

1

1

0

0

0

0

0

41

B36

1

1

1

0

0

0

0

0

42

B37

1

1

1

0

1

1

1

0

43

B41

1

1

1

0

1

1

1

0

44

B47

1

1

1

1

1

1

1

0

45

B48

1

1

1

1

1

1

1

0

46

B49

1

1

1

0

1

1

1

0

47

B50

1

1

0

1

1

1

1

0

48

B51

1

1

1

0

1

1

1

0

49

V._berl._R1

1

1

1

1

1

1

1

0

50

V._rup._FW3

1

1

1

1

1

1

1

0

51

V._rup._T

1

1

1

1

1

1

1

0

52

V._cord.

1

1

1

1

1

1

1

0

53

V._rip._GdM

1

1

1

1

1

1

1

0

54

Aramon_rup_G1

1

1

1

1

1

1

1

0

55

V._vip._Ggb

1

1

1

1

1

1

1

0

56

V._rup._FW1

1

1

1

1

1

1

1

0

57

Jacquez

1

0

1

0

1

1

1

0

58

Vialla

0

1

1

0

1

1

1

0

59

V._cin._Arnold

0

1

1

0

1

1

1

0

60

V._aest._S.

0

1

0

1

1

1

1

0

61

V._sol.

1

1

1

1

1

1

1

0

62

V._rup._FW2

1

1

1

1

1

1

1

0

63

V._berl._R107

1

1

1

1

1

1

1

0

64

Aramon_rup_G2

1

1

1

1

1

1

1

0

65

N._Mex.

1

1

1

1

1

1

1

0

66

T5C

1

1

1

1

1

1

1

0

67

SO4

1

1

1

1

1

1

1

0

68

5BB

1

1

1

1

1

1

1

0

*For more information about accessions see Table 1

The acid phosphatase isoenzyme patterns consist of 2 zones. The presence of a maximum of 4 bands in the faster migrating region represents a distinct locus. This region consists of 3 or 4 bands in the case of Vitis vinifera ssp. sativa cultivars and 3 bands for all analysed rootstocks and the majority of woodland grapes, but is absent in some Vitis vinifera ssp. sylvestris accessions.

Discussion

Acid phosphatases, which are involved in phosphorus metabolism in plants (Tadano and Sakai, 1991), usually have a high degree of polymorphism. These enzymes are usually glycoproteins, needing other enzymes to add the glycoprotein part. Empirical data suggests that in most cases changes in electrophoretic mobility are caused by changes in the DNA sequence of the structural genes, although it cannot be excluded that some of the polymorphism can be traced back to the polymorphism of the processing enzymes (Weeden and Wendel, 1989).

Acid phosphatases in plants are usually monomeric or dimeric with 2-4 isozymes and different subcellular localisation (de Cherisey et al., 1985 in Weeden and Wendel, 1989). Based on the present results and our previous study (Jahnke et al., 2009), acid phosphatase in grape (Vitis vinifera L.) has 2 zones of activity. The slower migrating zone has a maximum of 4 bands (1-4 in Table 2). The patterns of this zone can be interpreted as two (duplicated) loci with 4 alleles and monomeric enzyme. The faster migrating zone can be interpreted as a single locus coding 2 subunits (dimeric enzyme). In most of the Pontican European grapevine cultivars (Vitis vinifera ssp. sativa proles pontica), this locus is duplicated, which gives a special 4-band pattern in this zone. In about 50 percent of the woodland grape (Vitis vinifera ssp. sylvestris) genotypes, this locus is absent (null allele). This means that this type of acid phosphatase is not essential for the plant.

“The changes in the number and expression of loci in the course of phylogenesis suggest what evolutionary processes may have taken place” (Basaglia, 1989). Taking into account that acid phosphatases are involved in phosphorus metabolism and energy transfer, this extra locus can be advantageous and can play a remarkable role in the domestication of the grape. This phenomenon can be used as a marker in future studies.

Acknowledgements : This research was funded by the Hungarian Scientific Research Fund (project no. PD-109386).

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Authors


Gizella Jahnke

Affiliation : National Agricultural Research and Innovation Center, Research Institute for Viticulture and Enology, Badacsony Research Station, H-8261 Badacsonytomaj, Római út 181., Hungary

gjahnke@mail.iif.hu

Zóra Annamária Nagy

Affiliation : National Agricultural Research and Innovation Center, Research Institute for Viticulture and Enology, Badacsony Research Station, H-8261 Badacsonytomaj, Római út 181., Hungary


Gábor Koltai

Affiliation : Széchenyi István University, Faculty of Agriculture and Food Sciences, H-9200 Mosonmagyaróvár Vár 2., Hungary


Edit Hajdu

Affiliation : National Agricultural Research and Innovation Center, Research Institute for Viticulture and Enology, Kecskemét Research Station, H- 6000 Kecskemét, Katona Zsigmond u. 5., Hungary


János Májer

Affiliation : National Agricultural Research and Innovation Center, Research Institute for Viticulture and Enology, Badacsony Research Station, H-8261 Badacsonytomaj, Római út 181., Hungary

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