Absence of an acid phosphatase isozyme locus as a marker candidate for true to typeness in woodland grape (Vitis vinifera L. ssp. sylvestris Gmelin)
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).
References
- Arroyo‐García R., Ruiz‐García L., Bolling L., Ocete R., Lopez M. A., Arnold C. ... and Martinez-Zapater J.M., 2006. Multiple origins of cultivated grapevine (Vitis vinifera L. ssp. sativa) based on chloroplast DNA polymorphisms. Mol. Ecol. 15, 3707-3714. doi:10.1111/j.1365-294X.2006.03049.x
- Arulsekar S. and Parfitt D.E., 1986. Isozyme analysis procedures for stone fruits, almond, grape, walnut, pistachio, and fig. HortScience 21, 928-933.
- Basaglia F., 1989. Some aspects of isozymes of lactate dehydrogenase, malate dehydrogenase and glucosephosphate isomerase in fish. Comp. Biochem. Physiol. B 92, 213-226. doi:10.1016/0305-0491(89)90269-1
- Biagini B., De Lorenzis G., Scienza A., Failla O., Imazio S. and Maghradze D., 2012. Wild grapevine (Vitis vinifera L. subsp. sylvestris (Gmelin) Hegi) in Italy: distribution and preliminary genetic analysis. Acta Hortic. 948, 211-216. doi:10.17660/ActaHortic.2012.948.23
- Biagini B., De Lorenzis G., Imazio S., Failla O. and Scienza A., 2014. Italian wild grapevine (Vitis vinifera L. subsp. sylvestris) population: insights into eco-geographical aspects and genetic structure. Tree Genet. Genomes 10, 1369-1385. doi:10.1007/s11295-014-0767-4
- Bitz L., Zinelabidine L. H., Rühl E. H. and Bitz O., 2015. Microsatellite analysis of traditional eastern grapevine varieties and wild accessions from Geisenheim collection in Germany. Vitis 54, 17-21.
- Bodor P., Höhn M., Pedryc A., Deák T., Dücső I., Uzun I., Cseke K., Böhm É. I. and Bisztray G. D., 2010. Conservation value of the native Hungarian wild grape (Vitis sylvestris Gmel.) evaluated by microsatellite markers. Vitis 49, 23-27.
- de Cherisey H., Barreneche M. T., Jusuf M., Ouin C. and Pernes J., 1985. Inheritance of some marker genes in Setaria italica (L.) P. Beauv. Theor. Appl. Genet. 71, 57-60. doi:10.1007/BF00278254
- Doulati Baneh H., Mohammadi S.A., Labra M. and Shafaie F., 2015. Genetic diversity of wild grape (Vitis vinifera ssp. sylvestris) populations in Zagros forests as revealed by SSR markers. Acta Hortic. 1082, 189-194. doi:10.17660/ActaHortic.2015.1082.25
- Farkas S., 1999. Magyarország Védett Növényei (Protected plants of Hungary - in Hungarian). Budapest: Mezőgazda Kiadó.
- Jahnke G., Májer J., Varga Z., Deák E. and Varga P., 2009. An acid phosphatase isoenzyme pattern is characteristic for the Pontican cultivars. Cereal Res. Commun. 37, 205-208.
- Jahnke G., Nagy Z. A., Koltai G., Hajdu E. and Májer J., 2016. Preservation and characterization of woodland grape (Vitis vinifera ssp. sylvestris GMEL.) genotypes of the Szigetköz, Hungary. In Walton M. (ed.) Germplasm: Characteristics, Diversity and Preservation. Nova Science Publishers, Hauppauge, NY, USA., 27-45.
- Ocete R., Gallardo A., Guerra M. T., Vidal-Aragón M. C., Riveira D., Obón C. and López M.Á., 2015. Relic wild grapevines in Extremadura (Spain). Vitis 54, 217-221.
- Royo J.B., Cabello F., Miranda S., Gogorcena Y., Gonzalez J., Moreno S., Itoiz R. and Ortiz J.M., 1997. The use of isoenzymes in characterization of grapevines (Vitis vinifera L.). Influence of the environment and time of sampling. Sci. Hortic. 69, 145-155. doi:10.1016/S0304-4238(97)00007-1
- Tadano T. and Sakai H., 1991. Secretion of acid phosphatase by the roots of several crop species under phosphorus-deficient conditions. Soil Sci. Plant Nutr. 37, 129-140. doi:10.1080/00380768.1991.10415018
- This P., Lacombe T. and Thomas M.R., 2006. Historical origins and genetic diversity of wine grapes. Trends Genet. 22, 511-519. doi:10.1016/j.tig.2006.07.008
- Weeden N.F. and Wendel J.F., 1989. Genetics of plant isozymes. In Soltis D. E., Soltis P. S. and Dudley T. R. (eds.) Isozymes in Plant Biology. Springer Netherlands. doi:10.1007/978-94-009-1840-5_3
- Zoghlami N., Riahi L., Laucou V., Mliki A., Ghorbel A. and This P., 2013. Genetic structure of endangered wild grapevine Vitis vinifera ssp. sylvestris populations from Tunisia: implications for conservation and management. For. Ecol. Manage. 310, 896-902. doi:10.1016/j.foreco.2013.09.039
Views: 1079
XML: 106
PDF: 304