A comparative analysis of genetic diversity in Portuguese grape germplasm from ampelographic collections fit for quality wine production

Abstract

ance of millions of hectares produced by the attack of that insect. More recently, genetic diversity has suffered another drawback. New plantations with foreign material with low genetic variability have reinforced genetic erosion of native germplasm. Moreover, Euro-pean Union incentives for restructuring and conversion, particularly in Portugal and Spain, have conducted to Abstract Grapevine cultivars diversity is vast and full of synonyms and homonyms. Up to few decades ago characterization of grapevine was based on morphological characters. In the last decades, molecular markers were developed and have been used as tools to study genetic diversity in a range of different plant species. Fifty-six Portuguese accessions representative of 'Vinhos Verdes' and 'Douro' Controlled Designations of Origin (DOC) were analysed through DNA fingerprints generated by Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR). The study aimed to compare the effectiveness of RAPD and ISSR molecular techniques in the detection of synonyms, homonyms and misnames. RAPD and ISSR analysis enabled the detection of 36 different band patterns, reducing in about 36% the initial material. Several accessions grown under different names, between and within collections, were confirmed as the same genotype, namely Gouveio/Verdelho, Sousão Douro/Vinhão and Arinto Oeste/ Pedernã. Similarly, some homonyms/misnames were also identified, namely within Azal Tinto and Rabigato accessions. RAPD and ISSR markers revealed to be adequate molecular techniques for grapevine varieties fingerprinting with advantages over other molecular procedures, contributing for a good management of grapevine collections.

Full text

ance of millions of hectares produced by the attack of
that insect. More recently, genetic diversity has suffered
another drawback. New plantations with foreign mate-
rial with low genetic variability have reinforced ge-
netic erosion of native germplasm. Moreover, Euro-
pean Union incentives for restructuring and conversion,
particularly in Portugal and Spain, have conducted to
RESEARCH ARTICLE OPEN ACCESS
Spanish Journal of Agricultural Research
14(4), e0712, 11 pages (2016)
eISSN: 2171-9292
http://dx.doi.org/10.5424/sjar/2016144-8852
Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)
A comparative analysis of genetic diversity in Portuguese
grape germplasm from ampelographic collections fit for quality
wine production
Isaura Castro1, Olinda Pinto-Carnide1, Jesús M. Ortiz2, Vanessa Ferreira1 and Juan P. Martín2
1 Universidade de Trás-os-Montes e Alto Douro, Centro de Investigação e de Tecnologias Agro-Ambientais e Biológicas (CITAB),
5000-801 Vila Real, Portugal. 2 Universidad Politécnica de Madrid, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria
y de Biosistemas, Departamento de Biotecnología-Biología Vegetal, Avda. Puerta de Hierro, 28040 Madrid, Spain
Abstract
Grapevine cultivars diversity is vast and full of synonyms and homonyms. Up to few decades ago characterization of grapevine
was based on morphological characters. In the last decades, molecular markers were developed and have been used as tools to study
genetic diversity in a range of different plant species. Fifty-six Portuguese accessions representative of ‘Vinhos Verdes’ and ‘Douro’
Controlled Designations of Origin (DOC) were analysed through DNA fingerprints generated by Random Amplified Polymorphic
DNA (RAPD) and Inter Simple Sequence Repeat (ISSR). The study aimed to compare the effectiveness of RAPD and ISSR mo-
lecular techniques in the detection of synonyms, homonyms and misnames. RAPD and ISSR analysis enabled the detection of 36
different band patterns, reducing in about 36% the initial material. Several accessions grown under different names, between and
within collections, were confirmed as the same genotype, namely Gouveio/Verdelho, Sousão Douro/Vinhão and Arinto Oeste/
Pedernã. Similarly, some homonyms/misnames were also identified, namely within Azal Tinto and Rabigato accessions. RAPD and
ISSR markers revealed to be adequate molecular techniques for grapevine varieties fingerprinting with advantages over other mo-
lecular procedures, contributing for a good management of grapevine collections.
Additional key words: Vitis vinifera L.; grapevine germplasm collections; synonyms; homonyms; molecular markers; RAPDs;
ISSRs.
Abbreviations used: DAMD (Directly Amplified Minisatellite DNA); DOC (Controlled Designation of Origin); EVAG (Estação
Vitivinícola Amândio Galhano); ISSR (Inter Simple Sequence Repeat); MI (Marker Index); PIC (Polymorphic Information Content);
RAPD (Random Amplified Polymorphic DNA); Rp (Resolving Power); SM (Simple Matching); SSR (Simple Sequence Repeat);
UTAD (Universidade de Trás-os-Montes e Alto Douro).
Authors’ contributions: Conceived and designed the experiments: IC, OPC, JMO, JPM. Performed the experiments: IC, JPM.
Analysed the data: IC, VF, JPM. Contributed reagents/material/analysis tools: OPC, JMO, JPM. Wrote the paper: IC, OPC, JMO,
VF, JPM.
Citation: Castro, I.; Pinto-Carnide, O.; Ortiz, J. M.; Ferreira, V.; Martín, J. P. (2016). A comparative analysis of genetic diver-
sity in Portuguese grape germplasm from ampelographic collections fit for quality wine production. Spanish Journal of Agricultural
Research, Volume 14, Issue 4, e0712. http://dx.doi.org/10.5424/sjar/2016144-8852.
Supplementary material (Fig. S1) accompanies the paper on SJAR’s website.
Received: 23 Oct 2015. Accepted: 08 Nov 2016.
Copyright © 2016 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution-Non
Commercial (by-nc) Spain 3.0 Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.
Funding: FEDER/COMPETE/POCI – Competitiveness and Internationalization Operational Programme (POCI-01-0145-
FEDER-006958); FCT – Portuguese Foundation for Science and Technology (UID/AGR/04033/2013 and scholarship SFRH/
BD/96400/2013).
Competing interests: The authors have declared that no competing interests exist.
Correspondence should be addressed to Isaura Castro: icastro@utad.pt
Introduction
The first grapevine germplasm banks emerged in the
late nineteenth century, after the appearance of phyl-
loxera in Europe, from North America, in the middle
of that century (Cabello et al., 1999). A significant loss
of native plant material occurred due to the disappear-

Isaura Castro, Olinda Pinto-Carnide, Jesús M. Ortiz, Vanessa Ferreira and Juan P. Martín
Spanish Journal of Agricultural Research December 2016 • Volume 14 • Issue 4 • e0712
2
The objective of the present study was to use RAPD
and ISSR molecular marker systems to evaluate the
content on redundancy, synonymies and homonymies
in a group of 56 Portuguese accessions from two dif-
ferent grapevine germplasm collections, which repre-
sent all the cultivars used for ‘Vinhos Verdes’ quality
wines production and also many cultivars of ‘Douro’
and ‘Porto’ DOC denominations.
Material and methods
Plant material
Fifty-six Portuguese accessions were sampled in two
grapevine germplasm collections from North of Portugal:
(1) the ampelographic collection of the ‘Vinhos Verdes
Region Viticulture Commission’ (CVRVV) ‘Estação
Vitivinícola Amândio Galhano’ (EVAG) in Arcos de
Valdevez, inside ‘Vinhos Verdes’ DOC Region, and (2)
the ampelographic collection of the University ‘Univer-
sidade de Trás-os-Montes e Alto Douro’ (UTAD) in Vila
Real, inside ‘Douro’ DOC Region (Table 1).
DNA isolation, RAPD and ISSR
amplification
Genomic DNA was extracted from leaves using the
‘NucleoSpin® Plant II Kit’ (Macherey-Nagel, Düren,
Germany). DNA was subsequently quantified on aga-
rose gels and working dilutions of 10 ng/μL were made.
Sixty decamers of arbitrary sequence from OPA,
OPE and OPO kits (Operon Technologies Inc., Alam-
eda, CA, USA) and nine from the University of British
Columbia Biotechnology Center (UBC) (Vancouver,
Canada) were tested for the amplification of RAPD
fragments. Eight RAPD primers were selected for this
study, retrieving high number of amplification products,
reproducible and able to be analysed without ambigu-
ity (Table 2).
The amplification was carried out in 25 μL of reac-
tion mixture containing 0.3 μM of the single RAPD
primer, 0.2 mM of each of the four dNTPs, 2.5 mM
MgCl2, 0.85 U of Taq DNA polymerase in 1X manu-
facturer’s buffer (Thermo Scientific, MA, USA), and
50 ng of template DNA. The PCR amplification was
set with an initial denaturation cycle of 6 min at 94 ºC,
followed by 40 cycles of 1 min at 94 ºC, 1 min at 37 ºC,
2 min at 72 ºC, and finally 10 min extension at 72 ºC.
After an initial screening using 36 ISSR primers
provided in the UBC set #9, eight were selected for this
study (Table 2). The amplification was carried out in
20 µL of reaction mixture containing 0.5 µM of the
the loss of hectares of old vineyards and, most proba-
bly, also autochthonous minor cultivars. Germplasm
banks assume a huge importance in the preservation of
local cultivars that, due to their low rentability, have a
reduced area of cultivation or that, inclusive, have no
longer expression in viticulture areas and their exist-
ence is restricted to collections.
The number of different cultivars held in grapevine
germplasm collections around the world is estimated
to be approximately 10,000 (Alleweldt & Dettweiller,
1994). Among them only few hundred are cultivated
for commercial wine production (Truel et al., 1980).
The management of these collections requires attention
to avoid redundancy, to track introductions that were
wrongly assigned to a cultivar and to assist clone selec-
tion (Pelsy et al., 2010). So, identification of the plant
material is crucial and represents the first step in germ-
plasm management (Cipriani et al., 2010; Laucou et
al., 2011).
Morphology has been the most used tool in the char-
acterization of grapevine germplasm in most of the plant
collections (Boursiquot & This, 1996; Ortiz et al., 2004).
International organisations such as the OIV (Office In-
ternational de la Vigne et du Vin) or the ex-IPGRI (In-
ternational Plant Genetic Resources Institute, present
Bioversity International) have published useful descrip-
tors for the ampelography and comparison studies to be
carried out with the germplasm material (OIV, 2009).
However, this process is carried out on adult plants; a
longer period is required before the identification of ac-
cessions and often it is not conclusive on the distinction
of close cultivars. As many synonyms or homonyms
exist for cultivars, passport data are not always sufficient
to certify identities, and errors can arise (Buhner-Zaha-
rieva et al., 2010; Laucou et al., 2011).
In recent decades, DNA-based methodologies were
implemented, enabling easier and more accurate iden-
tification of Vitis germplasm. Pioneer and also recent
research with Random Amplified Polymorphic DNA
(RAPD) and Inter Simple Sequence Repeat (ISSR)
molecular markers has been successful and widely ap-
plied to estimate genetic diversity among cultivated
table and wine grapevine varieties, wild grapes and
also rootstocks and to distinguish grapevine cultivars
and clones, either alone (Moreno et al., 1995, 1998;
Vidal et al., 1999; Ercisli et al., 2008; Karatas &
Ağaoğlu, 2008; Tamhankar et al., 2008; Jing & Wang,
2013), combined both (Herrera et al., 2002) and com-
bined with other markers (Gogorcena et al., 1993:
RAPDs and Restriction Fragment Length Polymor-
phisms (RFLPs); Ulanovsky et al., 2002 and Pinto-
Carnide et al., 2003: RAPDs and Simple Sequence
Repeats (SSRs); Seyedimoradi et al., 2012: ISSRs and
Directly Amplified Minisatellite DNA (DAMD).

Spanish Journal of Agricultural Research December 2016 • Volume 14 • Issue 4 • e0712
3
Genetic diversity of Portuguese grapevine germplasm fit for quality wine production
DNA Ladder Mix., Thermo Scientific, and ISSR - 100
bp Ladder, Pharmacia).
Data analysis
Reproducible and clearly resolved fragments in the
RAPD and ISSR profiles were recorded as present (1)
and absent (0). Genetic similarity matrices among ac-
cessions from RAPD and ISSR data were calculated
using the simple matching (SM) similarity index
(Sneath & Sokal, 1973) and employed to construct
UPGMA dendrograms with the NTSYS-pc version 2.20
software package (Rohlf, 2005).
A cophenetic matrix was produced from each tree
matrix to test the goodness of fit of the cluster analysis
to the similarity matrix on which it was based, by com-
paring the two matrices using the Mantel matrix cor-
respondence test (Mantel, 1967) in the MXCOMP
program of the NTSYS-pc package.
The ability of each primer to differentiate between
genotypes was assessed by calculating their resolving
single ISSR primer, 0.15 mM of each of the four
dNTPs, 2 mM MgCl2, 0.8 U Tth DNA polymerase in
1X manufacturer’s buffer (Biotools, B&M Labs, Ma-
drid, Spain), and 20 ng of template DNA. The PCR
amplification was set with an initial denaturation cycle
of 4 min at 94 ºC, followed by 35 cycles of 30 s at
94 ºC, 45 s at 50 ºC, 52 ºC or 55 ºC (see Table 2), 2 min
at 72 ºC, and finally 5 min extension at 72 ºC.
The RAPD and ISSR amplifications procedure was
always carried out in duplicate. Moreover, in each
amplification run, 10% of samples were duplicated.
Bands were considered to be reproducible when the
same DNA pattern was obtained in, at least, two am-
plification runs.
The RAPD and ISSR products were resolved by
electrophoresis on 2% agarose gels, followed by eth-
idium bromide staining (0.05%). The electrophoretic
patterns of the PCR products were digitally recorded
using the Molecular Image® Gel-Doc™ XRþ with
Image Lab™ Software (BIO RAD, Hercules, CA,
USA). The molecular size of fragments was estimated
by reference to a DNA ladder (RAPDs - GeneRuler
Table 1. List of the 56 Portuguese grapevine accessions analysed in this study.
Accession
code
Name in the
collection
Germplasm
collection[a]
Acession
code
Name in the
collection
Germplasm
collection
1-AlP-U Alfrocheiro Preto UTAD 29-MGR-U Moscatel Galego Roxo UTAD
2-Alv-E Alvarinho EVAG 30-PaB-E Padeiro de Basto EVAG
3-ArB-U Arinto Bairrada UTAD 31-Pen-U Pedernã UTAD
4-ArD-U Arinto Douro UTAD 32-Pen-E Pedernã EVAG
5-ArO-U Arinto Oeste UTAD 33-Pel-U Pedral UTAD
6-Ave-U Avesso UTAD 34-Pel-E Pedral EVAG
7-Ave-E Avesso EVAG 35-Rab-U Rabigato UTAD
8-AzB-U Azal Branco UTAD 36-Rab-E Rabigato EVAG
9-AzB-E Azal Branco EVAG 37-Sou-U Sousão UTAD
10-AzT-U Azal Tinto UTAD 38-Sou-E Sousão EVAG
11-AzT-E Azal Tinto EVAG 39-SoD-E Sousão Douro EVAG
12-Bag-U Baga UTAD 40-SoG-E Sousão Galego EVAG
13-Bat-E Batoca EVAG 41-Tal-U Tália UTAD
14-Bic-E Bical EVAG 42-Tal-E Tália EVAG
15-Bor-U Borraçal UTAD 43-TAm-U Tinta Amarela UTAD
16-Bor-E Borraçal EVAG 44-TBo-U Tinta Barroca UTAD
17-Bra-U Brancelho UTAD 45-TCa-U Tinta Carvalha UTAD
18-BrA-E Brancelho Alvarelhão EVAG 46-TBa-U Tinta da Barca UTAD
19-Esp-U Espadeiro UTAD 47-TFr-U Tinta Francisca UTAD
20-Esp-E Espadeiro EVAG 48-TRo-U Tinta Roriz UTAD
21-Gou-U Gouveio UTAD 49-Tco-U Tinto Cão UTAD
22-GoD-E Gouveio Douro EVAG 50-ToF-U Touriga Franca UTAD
23-Lam-E Lameiro EVAG 51-ToN-U Touriga Nacional UTAD
24-Lou-U Loureiro UTAD 52-Tra-U Trajadura UTAD
25-Lou-E Loureiro EVAG 53-Tra-E Trajadura EVAG
26-MaF-U Malvasia Fina UTAD 54-Ver-U Verdelho UTAD
27-MaF-E Malvasia Fina EVAG 55-Vin-U Vinhão UTAD
28-MGB-U Moscatel Galego Branco UTAD 56-Vin-E Vinhão EVAG
[a] EVAG: Estação Vitivinícola Amândio Galhano (Arcos de Valdevez, Portugal); UTAD: Universidade de Trás-os-Montes e Alto Douro
(Vila Real, Portugal).

Isaura Castro, Olinda Pinto-Carnide, Jesús M. Ortiz, Vanessa Ferreira and Juan P. Martín
Spanish Journal of Agricultural Research December 2016 • Volume 14 • Issue 4 • e0712
4
Misnames, duplications, synonyms and
homonyms
Analysis of the RAPD and ISSR marker systems
individually, allowed detecting 44 and 43 distinct pro-
files, respectively, as can be observed in the respective
dendrograms (Figs. 2 and 3). The group of 56 acces-
sions was reduced, at 0.95 coefficient of similarity, to
37 and 36, considering the different band patterns of
RAPD and ISSR marker systems, respectively (Figs.
2 and 3). The Mantel test revealed a good and signifi-
cant cophenetic correlation for both markers (RAPD:
r = 0.79; p=0.001 and ISSR: r = 0.74; p=0.001), evi-
dencing that dendrograms provide a good fit for the
SM similarity matrices.
The main difference in the clustering of accessions
observed between the two marker systems was that,
with ISSR markers (Fig. 3), the accessions Sousão
(UTAD), Sousão (EVAG), Sousão Douro (EVAG),
Vinhão (UTAD) and Vinhão (EVAG) grouped in the
same cluster with a similarity higher than 0.95, while
RAPD markers (Fig. 2) allowed the separation of these
five accessions in two clusters, one with Sousão
(UTAD) and Sousão (EVAG) and the other with the
remaining three samples, Sousão Douro (EVAG), Vin-
hão (UTAD) and Vinhão (EVAG).
Some synonymies previously identified and regis-
tered at the Vitis International Variety Catalogue (www.
vivc.de) were observed in both collections according
to the RAPD and ISSR markers analysed, namely [Vin-
hão/Sousão Douro], [Gouveio/Gouveio Douro/Ver-
delho] and [Arinto Oeste/Pedernã] (Table 4; Figs. 2
and 3; Fig. S1 [suppl.]) with similarity levels higher
than 0.95.
Given the RAPD and ISSR profiles obtained, sev-
eral supposed misnames and/or homonymies were
detected, namely between the groups of accessions with
different designation [Arinto Bairrada (UTAD)/Baga
power (Rp) according to Prevost & Wilkinson (1999).
The polymorphic information content (PIC) of each
marker was also calculated (Roldán-Ruiz et al., 2000).
Marker Index (MI), defined as the product of the per-
centage of polymorphic bands and PIC, was used to
estimate the overall utility of each marker system
(Sorkheh et al., 2007).
Results
Polymorphism
The sixteen RAPD and ISSR primers selected
(Table 2) allowed amplification of 145 fragments in
the 56 Portuguese accessions studied, of which 116
(80.0%) were polymorphic (Table 3). RAPD and ISSR
marker systems produced a similar average number of
polymorphic bands/primer, 7.6 and 6.9, respectively.
In the RAPDs analysis, all the bands generated by
UBC-561 primer were polymorphic (see Fig. 1A) and
the primer OPO-07 provided the highest absolute num-
ber (14) of polymorphic bands (Table 3). For ISSR, all
the bands produced with the primers UBC-888 (see
Fig. 1B) and UBC-889 were polymorphic and the high-
est number (14) was observed with UBC-888 primer
(Table 3).
The PIC averages were calculated for each marker
system (Table 3) and the highest mean value (0.32)
was observed for RAPD markers. The highest MI
(35.3) was observed in the primer UBC-561 and the
highest mean MI (23.6) was observed for RAPDs
(Table 3). The lowest MI values were observed in the
ISSR UBC-861 (10.3) and RAPD UBC-584 (10.5)
primers. The Rp reached the highest mean values for
RAPDs (3.51), although the highest individual Rp
value (6.46) was observed in the ISSR primer UBC-
888 (Table 3).
Table 2. Sequences of the RAPD and ISSR primers selected for this study, and ISSR primers
annealing temperatures used.
RAPD ISSR
Primer Primer sequence
(5’→3’) Primer Primer sequence
(5’→3’) [a]
Annealing
temp. (ºC)
OPA-15 TTCCGAACCC UBC-811 (GA)8C 52
OPO-03 CTGTTGCTAC UBC-815 (CT)8G 52
OPO-07 CAGCACTGAC UBC-861 (ACC)655
OPO-10 TCAGAGCGCC UBC-868 (GAA)650
OPO-19 GGTGCACGTT UBC-881 (GGGTG)352
UBC-523 ACAGGCAGAC UBC-888 DBD(CA)755
UBC-561 CATAACGACC UBC-889 DBD(AC)755
UBC-584 GCGGGCAGGA UBC-890 VHV(GT)755
[a] B for non-A, D for non-C, H for non-G, V for non-T residue.

Spanish Journal of Agricultural Research December 2016 • Volume 14 • Issue 4 • e0712
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Genetic diversity of Portuguese grapevine germplasm fit for quality wine production
Table 3. Results of the observed genetic diversity in the 56 Portuguese grapevine accessions
studied.
TB PB (%) Rp PIC MI
RAPDs
OPA-15 10 8 (80.0) 3.71 0.33 26.8
OPO-03 10 6 (60.0) 3.75 0.41 24.5
OPO-07 16 14 (87.5) 5.07 0.27 23.7
OPO-10 9 7 (77.8) 3.68 0.36 27.6
OPO-19 11 8 (72.7) 3.82 0.32 23.4
UBC-523 11 7 (63.6) 3.04 0.26 16.6
UBC-561 8 8 (100.0) 3.93 0.35 35.3
UBC-584 7 3 (42.9) 1.07 0.25 10.5
Total 82 61 (74.4)
Mean 10.3 7.6 3.51 0.32 23.6
ISSRs
UBC-811 3 2 (66.7) 1.04 0.31 20.9
UBC-815 4 3 (75.0) 1.89 0.41 30.7
UBC-861 4 2 (50.0) 0.46 0.21 10.3
UBC-868 4 3 (75.0) 1.11 0.21 15.9
UBC-881 8 6 (75.0) 2.04 0.25 18.7
UBC-888 14 14 (100.0) 6.46 0.31 30.8
UBC-889 12 12 (100.0) 5.18 0.29 29.2
UBC-890 14 13 (92.9) 4.29 0.22 20.5
Total 63 55 (87.3)
Mean 7.9 6.9 2.81 0.28 22.1
TB, total of bands; PB, polymorphic bands; Rp, resolving power; PIC, polymorphic information con-
tent; MI, marker index.
Table 4. Groups of accessions with identical patterns in either RAPD, ISSR or in both, at levels of similarity both 1.0, either 1.0
and 0.95 and both 0.95
ISSR 1.0 similarity level
RAPD 1.0 similarity level
ISSR 0.95 similarity level
RAPD 1.0 similarity level
ISSR 1.0 similarity level
RAPD 0.95 similarity level
ISSR 0.95 similarity level
RAPD 0.95 similarity level
6-Avesso (UTAD)
7-Avesso (EVAG)
3-Arinto Bairrada (UTAD)
12-Baga (UTAD)
8-Azal Branco (UTAD)
9-Azal Branco (EVAG)
5-Arinto Oeste (UTAD)
31-Pedernã (UTAD)
32-Pedernã (EVAG)
15-Borraçal (UTAD)
16-Borraçal (EVAG)
17-Brancelho (UTAD)
18-Brancelho Alvarelhão
(EVAG)
19-Espadeiro (UTAD)
20-Espadeiro (EVAG)
27-Malvasia Fina (EVAG)
14-Bical (EVAG)
21-Gouveio (UTAD)
22-Gouveio Douro (EVAG)
54-Verdelho (UTAD)
41-Tália (UTAD)
42-Tália (EVAG)
31-Pedernã (UTAD)
32-Pedernã (EVAG)
39-Sousão Douro (EVAG)
55-Vinhão (UTAD)
56-Vinhão (EVAG)
24-Loureiro (UTAD)
25-Loureiro (EVAG)
55-Vinhão (UTAD)
56-Vinhão (EVAG)
28-Moscatel Galego Branco (UTAD)
29-Moscatel Galego Roxo (UTAD)
33-Pedral (UTAD)
34-Pedral (EVAG)
37-Sousão (UTAD)
38-Sousão (EVAG)
52-Trajadura (UTAD)
53-Trajadura (EVAG)

Isaura Castro, Olinda Pinto-Carnide, Jesús M. Ortiz, Vanessa Ferreira and Juan P. Martín
Spanish Journal of Agricultural Research December 2016 • Volume 14 • Issue 4 • e0712
6
(UTAD)] and [Vinhão (EVAG)/Vinhão (UTAD)],
similarity was slightly below 1.0 in one of the markers
systems (Table 4); therefore, intracultivar variability
was observed with these markers, however, the confir-
mation is still under discussion.
Discussion
In the present study, RAPD and ISSR markers were
used to analyse the variability in 56 Portuguese acces-
sions of V. vinifera cultivars. In several cases, for ac-
(UTAD)] and [Malvasia Fina (EVAG)/Bical (EVAG)],
given their clustering at high level of similarity
(Table 4, Figs. 2 and 3). On the contrary, in the acces-
sions Azal Tinto and Rabigato, sampled both in EVAG
and UTAD, with the same designation, different band
patterns were detected suggesting misnaming or ho-
monymy (Figs. 2 and 3).
The marker systems used showed some potential in
the clonal discrimination. Inside the groups of acces-
sions [Azal Branco (EVAG)/Azal Branco (UTAD)];
[Espadeiro (EVAG)/Espadeiro (UTAD)]; [Pedernã
(EVAG)/Pedernã (UTAD)]; [Tália (EVAG)/Tália
Figure 1. Proles obtained on 2% agarose gels for (A) 17 accessions using the UBC-561 RAPD primer, M – GeneRuler DNA Lad-
der Mix (Thermo Scientic), and (B) 18 accessions using the UBC-888 ISSR primer, M – 100 bp Ladder (Pharmacia). Accessions
code in Table 1.
1-AlP-U
7-Ave-E
2-Alv-E
8-AzB-U
9-AzB-E
3
-ArB-U
10-AzT-U
11-AzT-E
4-ArD-U
1
3
- B
at
-E
1
5
-B
o
r-
U
5
-ArO-U
1
6
-B
o
r-E
6
-Ave-U
17-Bra-U
1
8
-BrA-E
19-Esp-U
M M
24-Lou-U
2
5
-L
ou
-E
2
6
-M
a
F-
U
27-M
a
F-E
M
22-GoD-E
28-MGB-U
2
3
-L
a
m-E
29-MGR-U
30-PaB-E
3
1-Pen-U
36-Rab-E
3
2-Pen-E
37-Sou-U
33-Pel-U
41-Tal-U
34-Pel-E
46-TBa-U
55-Vin-U
M
1500 bp
800 bp
1-AlP-U
7-Ave-E
2-Alv-E
8-AzB-U
9-AzB-E
3
-ArB-U
10-AzT-U
11-AzT-E
4-ArD-U
1
3
- B
at
-E
1
5
-B
o
r-
U
5
-ArO-U
1
6
-B
o
r-E
6
-Ave-U
17-Bra-U
1
8
-BrA-E
19-Esp-U
M M
24-Lou-U
2
5
-L
ou
-E
2
6
-M
a
F-
U
27-M
a
F-E
M
22-GoD-E
28-MGB-U
2
3
-L
a
m-E
29-MGR-U
30-PaB-E
3
1-Pen-U
36-Rab-E
3
2-Pen-E
37-Sou-U
33-Pel-U
41-Tal-U
34-Pel-E
46-TBa-U
55-Vin-U
M
1500 bp
800 bp

Spanish Journal of Agricultural Research December 2016 • Volume 14 • Issue 4 • e0712
7
Genetic diversity of Portuguese grapevine germplasm fit for quality wine production
National Ampelographic Collection and from the Grape-
vine Collection of Terceira (Azores islands) through
microsatellite loci amplification, detected some synonym
cases and only 36 different SSR profiles were found.
Similarly, Laucou et al. (2011) analysed 4,370 accessions
of the INRA grape repository at Vassal with 20 micros-
atellite markers and only found 2,836 SSR single pro-
files. Cipriani et al. (2010) analysed 1,005 grapevine
accessions from CRA-VIT of Conegliano collection by
amplifying 34 microsatellite loci, identified 200 groups
of synonyms and only 745 unique genotypes and Buh-
ner-Zaharieva et al. (2010) in germplasm analysed from
the Movera Grapevine Germplasm Bank in Aragón,
Spain, found in 36 autochthonous accessions only 24
SSR profiles, besides 33 misnamed accessions.
Combination of molecular and morphological char-
acterization methodologies has led to a good manage-
ment of grapevine genetic resources (Ortiz et al., 2004;
Balda et al., 2014; Ferreira et al., 2015; Maul et al.,
2015). In a characterization of V. vinifera L. accessions
from the Spanish gene bank at Alcalá de Henares, Ortiz
et al. (2004) using morphological descriptors, isoen-
zymes and microsatellites, reduced the number of dif-
ferent accessions from 621 to 177, which represents less
than 30% of the initial number. In the scope of the
COST Action FA1003, in 997 accessions of Eastern
European cultivars analysed through ampelography and
nine microsatellite markers amplification, only 659
unique profiles/cultivars were found (Maul et al., 2015).
cessions with the same designation, different samples
collected in two different Portuguese grapevine germ-
plasm collections were analysed (Table 1).
The conservation of band profiles demonstrates the
reproducibility of both techniques used in the study.
RAPD and ISSR molecular techniques accumulate
several advantages, namely, small amount of DNA
required, facility of technical procedure, no fluores-
cence labeling, no need for information on template
DNAs or the synthesis of specific primers and simul-
taneous amplification of several loci. So, their use is
adequate to screen high number of samples in order to
detect duplicated material, suspicious of erroneous
identifications or synonyms and homonyms.
Few RAPD and ISSR primers (see Table 2) were
needed to generate highly diagnostic and reproducible
fingerprint. The RAPD primer OPO-07 and the ISSR
primers UBC-888, UBC-889 and UBC-890 used in this
study, revealed a high capacity for grapevine cultivars
discrimination (Table 3) given the high number of total
and polymorphic bands, which is in agreement with
the results of Zietkiewicz et al. (1994) and Moreno et
al. (1998) for the ISSR amplifications with 5’ three-
anchored primers (UBC-888, UBC-889 and UBC-890).
Using RAPD and ISSR markers 37 and 36 different
molecular profiles were obtained, respectively, within
the 56 accessions analysed, at 0.95 coefficient of similar-
ity (Figs. 2 and 3). Likewise, Lopes et al. (1999), study-
ing 49 supposed different cultivars from the Portuguese
Figure 2. Dendrogram of 56 Portuguese grapevine accessions studied obtained using UPGMA cluster analysis of RAPD marker
data. Accessions code in Table 1. SM: simple matching coefcient of similarity.
SM
0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00
1-AlP-U
52-Tra-U
53-Tra-E
5-ArO-U
32-Pen-E
31-Pen-U
13-Bat-E
2-Alv-E
8-AzB-U
9-AzB-E
50-ToF-U
51-ToN-U
21-Gou-U
22-GoD-E
54-Ver-U
46-TBa-U
44-TBo-U
40-SoG-E
10-AzT-U
24-Lou-U
25-Lou-E
11-AzT-E
39-SoD-E
56-Vin-E
55-Vin-U
30-PaB-E
15-Bor-U
16-Bor-E
37-Sou-U
38-Sou-E
17-Bra-U
18-BrA-E
33-Pel-U
34-Pel-E
23-Lam-E
35-Rab-U
19-Esp-U
20-Esp-E
47-TFr-U
49-Tco-U
28-MGB-U
29-MGR-U
41-Tal-U
42-Tal-E
36-Rab-E
45-TCa-U
43-TAm-U
3-ArB-U
12-Bag-U
26-MaF-U
27-MaF-E
14-Bic-E
4-ArD-U
6-Ave-U
7-Ave-E
48-TRo-U

Isaura Castro, Olinda Pinto-Carnide, Jesús M. Ortiz, Vanessa Ferreira and Juan P. Martín
Spanish Journal of Agricultural Research December 2016 • Volume 14 • Issue 4 • e0712
8
Also Gouveio Douro (EVAG), Gouveio (UTAD) and
Verdelho (UTAD) clustered together in both molecular
markers systems (Table 4; Figs. 2 and 3; Fig. S1
[suppl.]). In this case, the accessions Verdelho and
Gouveio Douro are most probably the cultivar offi-
cially designated Gouveio; in some localities of ‘Douro’
Region, Gouveio is designated as Verdelho (Pereira &
Sousa 1990). Care must be taken to not confuse with
the variety Verdelho, code PRT50317 (DR, 2012), a
different variety, much used for ‘Madeira’ wine produc-
tion with cultivation restricted to the island.
Pedernã is the local designation in ‘Vinhos Verdes’
DOC Region for the official cultivar named Arinto
(Mota & Silva, 1986), and so the accessions Arinto
Oeste (UTAD)/Pedernã (EVAG)/Pedernã (UTAD) have
an identical profile (Table 4; Figs. 2 and 3; Fig. S1
[suppl.]). The designation of UTAD’s accession as
‘Arinto Oeste’ is surely due to the great importance of
Arinto in the Bucelas DOC Region, located in west of
Portugal, near Lisbon, where it is also known as ‘Arin-
to de Bucelas’.
RAPD and ISSR band patterns suggest a few cases
of misidentifications between and within collections.
The accession Arinto Bairrada (UTAD) revealed the
band pattern of Baga (UTAD). This coincidence can
be explained considering that Baga, in ‘Bairrada’ Re-
gion, is frequently designated Tinto Bairrada. The ac-
cessions Malvasia Fina (EVAG) and Bical (EVAG)
were found to be very close but different from the
Of the 56 accessions that were studied, Moscatel
Galego Branco and Moscatel Galego Roxo are consid-
ered to be berry skin-colour mutants and so, ampelo-
graphically well distinguishable. In addition to the
RAPD and ISSR markers used in the present study, as
expected, also nuclear microsatellites amplification
performed in these cultivars failed to distinguish this
pair of cultivars (Ferreira et al., 2016). Other berry
skin–colour mutants have been reported, namely colour
variants of Muscat of Alexandria cultivar, as sharing
the same molecular fingerprint at trueness-to-type es-
tablished loci, and differing in specific genes related
to anthocyanin biosynthesis (De Lorenzis et al., 2015).
Results highlight the genetic proximity between
Sousão and Vinhão accessions. Sousão is the prime name
of a cultivar grown especially in ‘Vinhos Verdes’ Region
whose cultivar designation was modified to Sezão in the
last review of the ‘Portuguese List of Varieties fit for
Wine Production’ (DR, 2012). Vinhão has been reported
as the synonym of the Spanish cultivar Sousón (Martín
et al., 2006). However, a focus of confusion exists in
Douro Region, where, frequently, is given the name
Sousão to the cultivar Vinhão. Though, the observed
separated RAPD clusters for Sousão and Vinhão groups
are correct. Nevertheless, the fact that these accessions
have a miscellaneous of names between the two RAPD
clusters and that they cluster together in the ISSR
marker analysis, suggest that Sousão and Vinhão acces-
sions are genetically close.
Figure 3. Dendrogram of 56 Portuguese grapevine accessions studied obtained using UPGMA cluster analysis of ISSR marker data.
Accessions code in Table 1. SM: simple matching coefcient of similarity.
SM
0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00
1-AlP-U
45-TCa-U
5-ArO-U
31-Pen-U
32-Pen-E
13-Bat-E
23-Lam-E
28-MGB-U
29-MGR-U
19-Esp-U
20-Esp-E
52-Tra-U
53-Tra-E
8-AzB-U
9-AzB-E
49-Tco-U
4-ArD-U
6-Ave-U
7-Ave-E
3-ArB-U
12-Bag-U
27-MaF-E
14-Bic-E
36-Rab-E
26-MaF-U
41-Tal-U
42-Tal-E
43-TAm-U
17-Bra-U
18-BrA-E
21-Gou-U
22-GoD-E
54-Ver-U
44-TBo-U
50-ToF-U
2-Alv-E
24-Lou-U
25-Lou-E
40-SoG-E
35-Rab-U
10-AzT-U
33-Pel-U
34-Pel-E
46-TBa-U
11-AzT-E
37-Sou-U
38-Sou-E
39-SoD-E
55-Vin-U
56-Vin-E
15-Bor-U
16-Bor-E
51-ToN-U
30-PaB-E
47-TFr-U
48-TRo-U

Spanish Journal of Agricultural Research December 2016 • Volume 14 • Issue 4 • e0712
9
Genetic diversity of Portuguese grapevine germplasm fit for quality wine production
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Castro I, Martín JP, Ortiz JM, Pinto-Carnide O, 2011. Vari-
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onymies, homonymies and misnames. Vitis 51: 61-63.
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M, Meneghetti S, Frare E, Vignani R, Cresti M, Morgante
M et al., 2010. The SSR-based molecular profile of 1005
grapevine (Vitis vinifera L.) accessions uncovers new
synonymy and parentages, and reveals a large admixture
amongst varieties of different geographic origin. Theor
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s00122-010-1411-9.
De Lorenzis G, Squadrito M, Brancadoro L, Scienza A, 2015.
Zibibbo Nero characterization, a red-wine grape revertant
of Muscat of Alexandria. Mol Biotechnol 57: 265-274.
http://dx.doi.org/10.1007/s12033-014-9820-7.
Dhanorkar VM, Tamhankar SA, Patil SG, Rao VS, 2005.
ISSR-PCR for assessment of genetic relationships among
grape varieties cultivated in India. Vitis 44: 127-131.
DR, 2012. Ministerial Order 380/2012, of 22 November, that
establishes grapevine varieties fit and authorized for wine
production in Portugal. Diário da República (Portugal),
1ª série – Nº 226, 22/11/2012.
Ercisli S, Orhan E, Hizarci Y, Yildirim N, Agar G, 2008. Ge-
netic diversity in grapevine germplasm resources in the
Coruh Valley revealed by RAPD markers. Biochem Genet
46: 590-597. http://dx.doi.org/10.1007/s10528-008-9173-7.
Ferreira V, Pinto-Carnide O, Mota T, Martín JP, Ortiz JM,
Castro I, 2015. Identification of minority grapevine cul-
tivars from Vinhos Verdes Portuguese DOC Region. Vitis
54: 53-58.
Ferreira V, Fernandes F, Pinto-Carnide O, Valentão P, Falco
V, Martín JP, Ortiz JM, Arroyo-García R, Andrade PB,
accession Malvasia Fina (UTAD) that clustered at a
distant similarity level. That suggests misidentification
of Malvasia Fina in EVAG collection. Also, the acces-
sions Azal Tinto (UTAD)/Azal Tinto (EVAG) and
Rabigato (UTAD)/Rabigato (EVAG) revealed band
patterns quite different (Figs. 2 and 3). Azal Tinto is
the synonym of Amaral (Caño Bravo in Spain, Martín
et al., 2006) and is mentioned as having several de-
scendants (Castro et al., 2012; Lacombe et al., 2013).
One of the Azal Tinto accessions may be in fact other
genotype, eventually its relative. The different Rabi-
gato profiles can be explained considering that there is
a cultivar in ‘Vinhos Verdes’ DOC Region, Rabo de
Ovelha, which is commonly designated Rabigato (Mota
& Silva 1986).
A major obstacle to good management of grapevine
germplasm banks is the persistence of synonyms and
homonyms up to the current days in the viticulture
worldwide. Some germplasm banks accumulate the
responsibility of multiplication and commercialization
of grapevine material. Efforts are being done and have
to proceed in order to find an official name for propa-
gation and distribution. The management of several
grapevine collections has been carried out with the
assistance of molecular markers, namely in Iberian
Peninsula (Lopes et al., 1999; Ibáñez et al., 2003;
Martín et al., 2003, 2006; Santiago et al., 2007; San-
tana et al., 2008; Veloso et al., 2010; Castro et al.,
2011; Balda et al., 2014; Alifragkis et al., 2015) with
great relevance in the correction of identification mis-
takes. In specific, RAPD and ISSR PCR-based finger-
printing are informative for estimating the extent of
genetic diversity and patterns of genetic relationships
among grape accessions in germplasm holdings (Dha-
norkar et al., 2005; Ercisli et al., 2008; Karatas &
Aǧaoǧlu, 2008; Zeinali et al., 2012).
Results evidence the necessity of grapevine mate-
rial characterization in ampelographic collections,
complementing morphological descriptors with mo-
lecular markers for duplicates and synonyms detection.
Acknowledgements
Authors acknowledge Teresa Mota from EVAG for
her help in sampling material.
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