Ana Raquel Santos1, António Graça2,3, Ana Catarina Gomes1
1 BIOCANT,
Centro de Inovação em Biotecnologia, BIOCANT PARK – Parque Tecnológico de Cantanhede, Núcleo 04, Lote 8, 3060-197 Cantanhede, Portugal, 2 Sogrape Vinhos, S.A., 4430-852 Avintes, Portugal
3
PORVID- Associação Portuguesa para a Diversidade da Videira, Tapada da Ajuda, 1349-017 Lisboa
Abstract and objectives
Plant Material
Worldwide there are about 1400 grapevine varieties used for wine production, however, and
despite such high biodiversity, about 80 percent of wine is produced from only 20 of these varieties
[1]. In result, such important heritage is facing extinction, endangering grapevine biodiversity, in a
process that must be reverted as genetic resources are valuable reservoirs of new traits,
indispensable to meet future demands.
In Portugal, most genetic variability was stored in a network of commercial and public vineyards
most of which currently approaching their lifetime limit, which has urged for actions to safeguard
and to organize Portuguese biodiversity. Indeed, the Portuguese Association for Grapevine
Diversity (PORVID) has led a prospection and conservation project, whose principal objective is to
build a full-diversity in vivo library of the Portuguese grapevine heritage.
The great interest and urgency of this problem led to the molecular
analysis to be regarded as a strategy for prompt detection of putative
new varieties. Herein, we will describe how the use of molecular
genotyping system that we have developed, has resolved unknown V.
vinifera identities, thus resulting in the identification of possible new
varieties and their genitors.
ADVID –
Associação para o
Desenvolvimento
da Viticultura
Duriense, Peso da
Régua
SDASM Serviços de
Desenvolviment
o Agrário de São
Miguel, Ponta
Delgada
ATEVA –
Associação
Técnica dos
Viticultores do
Alentejo,
Évora
AVIPE Associação de
Viticultores do
Concelho de
Palmela,
Palmela
PORVID –
Associação
Portuguesa para
a Diversidade da
Videira, Lisboa
Prospection,
conservation
and clonal
material
evaluation
ISA – Instituto
Superior de
Agronomia,
Lisboa
DRAPALG Direcção
Regional de
Agricultura e
Pescas do
Algarve, Faro
DRAPC Direção Regional
de Agricultura e
Pescas do
Centro, Castelo
Branco
INIAV - Instituto
Nacional de
Investigação
Agrária e
Veterinária, IP,
Oeiras
DRAPN - Direção
Regional de
Agricultura e Pescas
do Norte, Mirandela
 A total of 72 Grapevine
samples were collected from
seven
Portuguese
wine
regions, after a 4-year
prospection work, led by
several entities (Fig. 1).
 Fresh young leaves were
collected and DNA was
extracted using Nucleo-Spin
Plant II Kit (Macherey-Nagel.)
DRAPLVT Direção Regional
de Agricultura e
Pescas de Lisboa
e Vale do Tejo,
Santarém
Fig. 1- List of participating entities on the prospection, conservation and clonal material evaluation work.
Methods
Data Analysis
SSR Amplification
 National and International Databases consulted for matching purposes.
 Improved set of 14 markers amplified in 3 Multiplex reactions (Fig. 2)[2].
GrapeGen 9 SSR
6 chromosomes
Set of 14 SSR
11 chromosomes
Database
Links
Number of
compared
SSR
Vitis International Variety Catalogue
www.vivc.de
6
630
Sistema de Identificación de Variedades
de Vid Españolas
mediante Microsatélites
http://sivvem.monbyte.com
6
280
Projet Pl@ntGrape
http://plantgrape.plantnet-project.org/
9
313
European Vitis Database
http://www.eu-vitis.de/index.php
9
4359
US National Grape Registry
http://www.ars.usda.gov/Main/docs.htm?docid
=13743
6
224
Lacombe et al. 2013
http://link.springer.com/article/10.1007%2Fs0
0122-012-1988-2
12
1130
Portuguese Consortium
-
6
617
Biocant Database
-
14
480
Number of
accessions
Fig. 2- Illustration of number of chromosomes covered by GrapeGen06 Project and by our improved set.
Increase in genomic resolution
Total
8033
Costs of analisys reduced
Results
 Matching
 Interestingly, 10 of the 72 grapevine samples did not obtain any match in the databases consulted and
consequently were considered as being previously undescribed varieties. Thus, 18% of the analyzed vine
samples are new varieties, not described elsewhere. Such abundant and increasing biodiversity, further
strengthens the scenario of the Iberian Peninsula, as a Center of Grapevine domestication [3].
13 samples
10 putative new
varieties
72
41 samples
21 native
varieties
 More than half of the identified varieties are native, nevertheless, varieties of neighbour countries like Spain or
France were also found. Interestingly, in Algarve Region many origins were revealed, such as Greece, Italy, or
Algeria. This may be due to the fact that this region has always been on the itinerary of merchant ships of the
Mediterranean .
18 samples
13 foreign
varieties
 Clear evidence of the importance of the wide on-going program of prospection, conservation and
characterization of endangered varieties is provided.
 Results show the full
progenies of four samples
and in six samples one
parent is revealed (Tab.I).
 We found one more progeny
of Touriga Nacional X
Marufo.
 Paternity analysis
Table I –Progenies obtained with Cervus3.0 Software for the new genotypes [4].
Sample
1001-40
1003-08
5083-16
ML0501
Parent 1
Branco Escola B
Mourisco Branco B
Marufo N
Camarate N
Parent 2
Azal Tinto N
Tinta Miúda N
Touriga Nacional N
Moreto N (Alentejo)
LOD Score
2,20E+15
2,26E+15
1,94E+15
2,03E+15
Confidence
95%
95%
95%
95%
Unknown
Esganinho B
Na
7,25E+14
95%
2043-67b
Espadeiro N
Na
1,13E+15
95%
2043-80
30011-04
2022-59
30015-06
Donzelinho Branco B
Ferral R
Azal B
Diagalves B
Na
Na
Na
Na
5,66E+14
1,08E+15
8,94E+14
1,45E+15
80%
95%
95%
95%
Conclusions
 10 putative new native grape varieties, that enrich the already
abundant Portuguese heritage of autochthonous grape varieties
were discovered.
BIBLIOGRAPHY
[1] E. Freire, Enovitis , Vol July, August,
September (2013).
http://www.winemosaic.org/
[2] AR. Santos, D. Duarte, S. Sousa, J. EirasDias, AC. Gomes, Proceedings of the 36th
world congress of vine and wine OIV,
Bucharest, Romania (2013).
[3] I.Castro, O. Pinto-Carnide, JM . Ortiz, JP .
Martín, Mol Biotech 54, p 528–540 (2013).
[4] S.T. Kalinowski, M.L. Taper, T.C. Marshall
Mol Ecol 16, p 1099-1006 (2007).
This work was carried out under the InovWine project funded by the FEDER, through the Programa Operacional Fatores de Competitividade – COMPETE, and “Quadro de
Referência Estratégico Nacional” – QREN, reference FCOMP-01-0202-FEDER-030272. The authors wish to express their appreciation to Professors Antero Martins and Elsa
Gonçalves for their support, as well as to all involved technical staff of institutions mentioned in Figure 1 for their commitment to the PORVID diversity prospection projects
currently under way, which provided the material basis for this work.
 Native varieties are particularly promising in terms of
uniqueness and provide a new niche in the competitive market.
 This work sheds a new light on the grape biodiversity that might
still be uncovered.
 Genotype’s conservation efforts as these may be of
strategic importance for future needs.