IntroductionTop

Grapevine (Vitis vinifera L.) is a species that presents a wide genetic diversity and is widely conserved in germplasm banks. The existence of synonyms, homonyms and misnomers are one of the major problems for viticulture worldwide (Veloso et al., 2010) and are obstacles for an international network on the conservation of Vitis germplasm in Europe (Maul, 2008). In order to clarify the misidentification and confusion in grapevine variety designations caused by the morphological characterization subjectivity, many collections have already been characterized using Sample Sequence Repeats (SSR) markers (Lopes et al., 1999; Martín et al., 2003, 2011; Ortiz et al., 2004; De Mattia et al., 2007; Dzhambazova et al., 2009; Ibáñez et al., 2009; Vargas et al., 2009; Cipriani et al., 2010; Laucou et al., 2011; Lacombe et al., 2013; Milla-Tapia et al., 2013; Aliquó et al., 2017). Although, this problematic is collected also in The Vitis International Variety Catalogue (VIVC, www.vivc.de), which is currently the source of reference to help group the varieties under a common and consensual identifying label (Lacombe et al., 2011). A clear example of mistakes is registered for ‘Romé’ cultivar. The VIVC database (Vitis International Variety Catalogue [VIVC, www.vivc.de]) includes two varieties of Spanish origin called ‘Rome’: a white-berry cultivar (VICV 16035), which only is conserved in two Spanish collections [Finca El Encín (Institute Code VIVC ESP080) and Rancho de la Merced Germplasm Bank (Institute Code VIVC ESP074)], and a black-berry cultivar (VIVC 10181), whose conservation location is unknown. This European database also includes a variety with the prime name ‘Rome Tinto’ (VIVC 40905) that only is conserved in the Rancho de la Merced and its origin is uncertain.

Ibáñez et al. (2009) characterized a ‘Rome’ conserved in the Finca El Encín using 20 SSR loci. Results showed identical genotype with ‘Muscat of Alexandria’. They proposed that it could be an unknown synonym for this cultivar. Similar results were obtained by García de Luján et al. (1990) with 128 OIV ampelographic descriptors by ‘Rome’ conserved in the Rancho de la Merced collection. However, the genotype of ‘Rome Tinto’ is now unpublished.

The main objective of this work was to identify different ‘Romé’ and ‘Rome Tinto’ grapevine accessions that are conserved in the Rancho de la Merced Germplasm Bank with difference origin. Their genetic and morphologic characterization could help to detect synonyms, homonyms and false attributions. This research is necessary to provide a solid basis to develop a germplasm collection to protect grapevine diversity and to recover cultivars that may be in danger of extinction.

Material and methodsTop

A total of eight accessions, six ‘Romé’ and two ‘Rome Tinto’, were analyzed using 22 SSR loci. Six of the ‘Romé’ accessions were collected during different prospecting trips carried out from 1999-2001 in vineyards situated at the localities of Cómpeta, Torrox and Ronda (province of Málaga, Andalusia community, South Spain). All accessions analyzed are conserved in the Rancho de la Merced Germplasm Bank. Furthermore, four reference cultivars (‘Cabernet Sauvignon’, ‘Chardonnay’, ‘Muscat a Petits Grains Blancs’ and ‘Pinot Noir’) were also included to test for genetic profiles obtained with the different databases published. In order to identify the geographical origin of this ‘Romé’ accessions, the genetic analyses were complemented with other cultivars sampled at the Rancho de la Merced Germplasm Bank that originally came from different geographical areas and included Vitis vinifera varieties (‘Airén’, ‘Flame Seedless’, ‘Garrido Fino’, ‘Graciano’, ‘Mantúo de Pilas’, ‘Michele Palieri’, ‘Muscat of Alexandria’, ‘Muscat Hamburg’, ‘Ohanes’, ‘Palomino Fino’, ‘Pedro Ximenes’, ‘Syrah’, ‘Tempranillo’, ‘Vijiriega Común’, ‘Viura’ and ‘Zalema’ ‘Cabernet Sauvignon’, ‘Chardonnay’, ‘Muscat a Petits Grains Blancs’ and ‘Pinot Noir’) and interspecific hybrids (‘RM2’ and ‘Jacquez’), that were used as an outgroup in the genetic relationship analysis.

DNA extraction from plant material was performed using young leaves collected in spring. Genomic DNAs were isolated from 100 mg of frozen leaf tissue using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). A total 22 SSR loci were analysed in order to verify the varietal identity. A first set of 20 microsatellite loci located in the 19 linkage groups of grapevine genome [VMC1B11 (Zyprian & Topfer, 2005), VMC4F3-1 (Di Gaspero et al., 2000); VVMD5, VVMD7, VVMD21, VVMD24, VVMD25, VVMD27, VVMD28, VVMD32 (Bowers et al., 1996, 1999); VVS2 (Thomas & Scott, 1993); VVIB01, VVIH54, VVIN16, VVIN73, VVIP31, VVIP60, VVIQ52, VVIV37, VVIV67 (Merdinoglu et al., 2005)] were analysed using two multiplex PCRs as described in a previous study (Vargas et al., 2007). Another set of 2 microsatellite loci [VrZAG62 and VrZAG79 (Sefc et al., 1999)] were analysed to complete the list of loci authorized by the International Organisation of Vine and Wine (OIV, 2009). These last two loci were used under the conditions detailed in a previous work (Jiménez-Cantizano et al., 2006).

PCR amplifications were carried out in an Applied Biosystems 9700 thermocycler. Amplified products were separated by capillary electrophoresis using an automated sequencer (ABI Prism 3130, Appl. Biosyst.). Fluorescently labelled fragments were detected and sized using GeneMapper v. 3.7 software (Appl. Biosyst.) and fragment lengths were determined with the help of internal size standards (GeneScan-500 LIZTM, Appl. Biosyst.). SSR profile comparisons were carried out using the Microsatellite toolkit v. 9.0 software package (Park, 2001). Genetic distances between grapevine genotypes were calculated as [-ln (proportion shared alleles)] using Microsat (Minch et al., 1997). The obtained data were used for the construction of a dendrogram using the program EXE from the PHYLIP software package (Felsenstein, 1989) and Treeview (Page, 1996).

Ampelographic analyses were carried out during three years (2012-2015) and using the descriptor list for grapevine cultivars and Vitis species from the OIV (2009) using 5 plants per accession.

Results and discussionTop

All accessions ‘Romé’ and ‘Rome Tinto’ characterized showed identical genotype at 22 SSR loci (Table 1). The genotype obtained was not included in published databases (Ibáñez et al., 2009; Vargas et al., 2009; De Andrés et al., 2012; Lacombe et al., 2013; Jiménez-Cantizano, 2014; and VIVC (www.vivc.de)), thus, this new genotype corresponds to a new grape cultivar. According to the results obtained in this study, the ‘Rome Tinto’ accession registered in the VIVC database with the variety number “40905”, should be considered a synonym of ‘Rome’ “10181”, which could be the true-to-type of the variety prime name. This variety is conserved only in the Rancho de la Merced Germplasm Bank. On the other hand, morphological characterization showed the same phenotypes in all ‘Romé’ and ‘Rome Tinto’ accessions (Table 2). This variety could be ‘Romé’ (black-berry cultivar) described in Andalusia by several authors in the 16th (Herrera, 1513) and 19th century (Clemente-Rubio, 1807; Abela & de Andino, 1885). In this way, ‘Romé’ (white-berry) characterized by García de Luján et al. (1990) and Ibáñez et al. (2009) identified as ‘Muscat of Alexandria’ could be a misnamed.

Table 1. Genetic profle of ‘Rome’ accessions, reference varieties and other cultivars analyzed at 22 SSR loci. Allele sizes are given in base pairs.

Table 2. Mean values for the OIV (2009) ampelografc and morphologic descriptors observed during three years (2012- 2015).

The resulting dendrogram using the UPGMA method (Fig. 1) defines the genomic relationships among the analyzed cultivars and shows the existence of six groups, denoted A-F. The formation of these groups may be related to the use of the cultivars and regions of origin. According to the results, ‘Romé’ cultivar is grouped (Group A) with white cultivars (‘Zalema’, ‘Pedro Ximenes’, ‘Viura’, ‘Palomino Fino’ and ‘Garrido Fino’) that are of Spanish origin and used in winemaking. Within this group A ‘Romé’ has a greater affinity with ‘Zalema’, ‘Pedro Ximenez’ and ‘Viura’ (Fig. 1). These three varieties share first-degree relationships with ‘Hebén’ (Lacombe et al., 2013; Zinelabidine et al., 2015). In addition, ‘Romé’ presented the same genotype as the ‘Viura’ cultivar for seven SSR loci (Table 1): VVIB01, VVMD7, VVMD28, VVIH54, VVIN16, VVIN73 and VVIP60. These results could indicate that ‘Romé’ variety have ‘Hebén’ as a female parent. 'Hebén' is a variety, already described in the 16th century (Herrera, 1513) as a white variety of grapevine and it was grown in the Andalusian region (García de los Salmones, 1914). The white female ‘Hebén’ proved to be a key genitor in the Iberian Peninsula (Lacombe et al., 2013; Zinelabidine et al., 2015). This variety seems to originate from North Africa (Galet, 2000), which would be consistent with the relationships between Spanish and North African grape gene pools (El Oualkadi et al., 2011). In grapevine numerous changes have occurred as a result of human selection, including the emergence of hermaphroditism and greatly increased variation in berry color (This et al., 2007). Previous research works showed that approximately 800 bp insertion is present in a few red-skinned varieties that are somatic mutants of white-skinned varieties (Kobayashi et al., 2004). In addition, according to Lijavetzky et al. (2006) VvmybA1 gene could be a major determinant of berry colour variation in grapes. The results obtained in a study of the examined allelic variation in VvmybA1 in over 200 accessions of cultivated grapevine including several well characterized fruit color mutants, indicated that the white fruited-allele of VvmybA1 most likely arose a limited number of times and that variation in this gene is likely responsible for the majority of the fruit color variation present in modern grapevine cultivars (This et al., 2007). In this sense, this same effect may have occurred with the ‘Romé’ variety.

Autochthonous cultivars are a genetic resource which could play an important role in the future, to study their warm climatic conditions adaptation capacity and their oenological potential into new wines. According to Fraga et al. (2016), adapting to future climates may comprise a selection of more resilient varieties to warming and drying. In this regard, studies carried out with Portuguese varieties (Lopes et al., 2008; Fraga et al., 2016) shows that genetic, morphological and physiological differences between each variety allows a better adaptation to different climates resulting in of singular wines production. According to the EU and Spanish normative only registered plant material is possible to be used in new plantations. As a conclusion, ‘Romé’ cultivar could be considered as an Andalusian autochthonous black grape cultivar and its correct identification could facilitate ‘Romé’ inclusion in the Official Register of Spanish grapevine varieties.

AcknowledgmentsTop

The authors thank José Antonio Pérez Ortiz and Mª José Serrano Albarrán for technical assistance with ampelographic description.