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

1 National Agricultural Research and Innovation Center, Research Institute for Viticulture and Enology, Badacsony Research Station, H-8261 Badacsonytomaj, Római út 181, Hungary 2 Széchenyi István University, Faculty of Agriculture and Food Sciences, H-9200 Mosonmagyaróvár Vár 2, Hungary 3 National Agricultural Research and Innovation Center, Research Institute for Viticulture and Enology, Kecskemét Research Station, H-6000 Kecskemét, Katona Zsigmond u. 5, Hungary

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.

SSR analysis
Dormant canes were collected in January 2016 and subsequently stored in plastic bags at 4 °C 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.

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.
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 andWendel, 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.