The forgotten, ancient olive trees of the Spanish northwest: A first molecular and botanical analysis

Abstract

No country has a larger area under olive (Olea europaea subs. europaea var. europaea) cultivation than Spain. In the Spanish northwest, however, this crop has largely been forgotten, even though olive oil was once an important product of the area. Sadly, apart from a few scraps of information handed down orally, little information exists regarding the genotypes grown, or from where they may have originally come. Many centuries-old olive trees, however, can still be found in the area, some even forming groves now part of open woodland but which may harbour an important genetic reservoir. The present work describes a botanical and molecular analysis of these ancient trees, following a survey of allegedly native genotypes surviving in different locations in Galicia. Comparison of their molecular profiles with those in the World Olive Germplasm Bank of Cordoba, and those in the database compiled by the Agronomy Department of the University of Cordoba, revealed two known Galician genotypes, ´Brava Gallega´ and ´Mansa Gallega´, and the Portuguese genotype ´Cobrancoça´. Six genotypes present in neither database were also detected. In addition, some misidentifications of the ´Mansa´ genotype in recent studies were clarified. Botanical analysis confirmed the molecular results in all cases. The findings suggest a larger survey should be performed so that the full olive genetic diversity of this region can be recorded and preserved.

Full text

RESEARCH ARTICLE OPEN ACCESS
The forgotten, ancient olive trees of the Spanish northwest: A rst
molecular and botanical analysis
Pilar Gago, José L. Santiago, Susana Boso and María C. Martínez
Misión Biológica de Galicia (MBG-CSIC), Consejo Superior de Investigaciones Cientícas, Carballeira 8, Salcedo, 36143 Pontevedra, Spain.
Spanish Journal of Agricultural Research
17 (2), e0702, 15 pages (2019)
eISSN: 2171-9292
https://doi.org/10.5424/sjar/2019172-13572
Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, O.A, M.P. (INIA)
Abstract
No country has a larger area under olive (Olea europaea subs. europaea var. europaea) cultivation than Spain. In the Spanish
northwest, however, this crop has largely been forgotten, even though olive oil was once an important product of the area. Sadly, apart
from a few scraps of information handed down orally, little information exists regarding the genotypes grown, or from where they may
have originally come. Many centuries-old olive trees, however, can still be found in the area, some even forming groves now part of
open woodland but which may harbour an important genetic reservoir. The present work describes a botanical and molecular analysis
of these ancient trees, following a survey of allegedly native genotypes surviving in dierent locations in Galicia. Comparison of their
molecular proles with those in the World Olive Germplasm Bank of Cordoba, and those in the database compiled by the Agronomy
Department of the University of Cordoba, revealed two known Galician genotypes, ´Brava Gallega´ and ´Mansa Gallega´, and the
Portuguese genotype ´Cobrancoça´. Six genotypes present in neither database were also detected. In addition, some misidentications
of the ´Mansa´ genotype in recent studies were claried. Botanical analysis conrmed the molecular results in all cases. The ndings
suggest a larger survey should be performed so that the full olive genetic diversity of this region can be recorded and preserved.
Additional keywords: Olea europaea L; ´Brava Gallega´; ´Mansa Gallega´; unknown genotypes; Galicia; morphological
descriptors; SSRs.
Authors’ contributions: Conception and design of the experiments: MCM, JLS. Surveying for plant material and data analysis:
MCM, JLS, SB, PG. Botanical analysis and drafting of the manuscript: MCM, PG. Microsatellite analysis: PG. Fund raising and
overall supervision: MCM.
Citation: Gago, P.; Santiago, J. L.; Boso, S.; Martínez, M. C. (2019). The forgotten, ancient olive trees of the Spanish northwest:
A rst molecular and botanical analysis. Spanish Journal of Agricultural Research, Volume 17, Issue 2, e0702. https://doi.org/10.5424/
sjar/2019172-13572
Supplementary material (Tables S1 and S2) accompanies the paper on SJAR’s website.
Received: 06 Jun 2018. Accepted: 20 May 2019.
Copyright © 2019 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0
International (CC-by 4.0) License.
Funding: Invatia Research, the Centre for the Development of Industrial Technology (CDTI) (project INNGAL-AGROMAR-
SALUD 2013 – EXP 00064360 / ITC-20133014); Spanish Research Council (CSIC).
Competing interests: The authors have declared that no competing interests exist.
Correspondence should be addressed to María C. Martínez: carmenmartinez@mbg.csic.es
Introduction
Olives (Olea europaea subs. europaea var. euro-
paea), wheat and grapes are some of the oldest of all
crops (Zohary & Hopf, 1994). Olives are normally cul-
tivated between 30º and 45º N and S, and in other areas
where the climate is Mediterranean (Barranco et al.,
2000). Spain has 2,554,829 ha under olive cultivation,
and is the world's foremost producer of olive oil
(Ministerio de Agricultura, Pesca y Alimentación-
MA PA, 2018); its output accounts for 60% of all the
EU's olive oil and 45% of that produced worldwide
(International Olive Oil Council, 20151). These data
provide an idea of the economic and environmen tal
importance of olives in Spain.
Many olive genotypes are grown around the world,
and many of those growing in the most important o li ve
oil-producing countries have been described (Barranco
et al., 2000; Belaj et al., 2002; Bartolini et al., 2005;
Rallo et al., 2005; Fendri et al., 2010 and 2014;
Haouane et al., 2011; Lazovic et al., 2016; Sakar et al.,
2016). In Spain, over 250 are reported in use (Barranco
et al., 2005; Vargas-Gómez & Ta lavera-Lozano, 2012),
but the current number used in the main commercial
plantations is small (Rallo et al., 2005). The variation
in Spanish olive germplasm has been studied in certain
areas (Viñuales, 2007; Díez et al., 2011; Gómez et
al., 2012; Trujillo et al., 2014; Martí et al., 2015).
In marginal areas, however, much less work of this
kind has been done, and in some pla ces no surveys or
1 http://www.internationaloliveoil.org/estaticos/view/131-world-olive-oil-gures?lang=en_US

Pilar Gago, José L. Santiago, Susana Boso and María C. Martínez
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e0702
2
characterisations have been underta ken at all. Such is
the case of Galicia in the NW Peninsu lar.
The Atlantic-inuenced climate of this region is
not currently associated with olive cultivation, yet
old references cite olives trees being grown here
(Alonso de Herrera, 1513; Contreras, 1798; Hidalgo-
Tablada, 1870). The importance of olive production
in the past is evident in archaeological nds such as
primitive oil mills dating from the 1st-2nd centuries
BCE (Fernández de la Cigoña & Martínez, 2003), and
numerous references to olive trees and olive oil in the
region's toponymy. A strong oral tradition also exists
among the region's inhabitants that testify to families
having produced their own olive oil for generations.
This residual cultivation of olive trees has persisted in
the area until the present day, but in the last 10 years
there have been several initiatives that have attempted
to recover olive production as part of the regional
economy. Indeed, between 2008 and 2017, the area
under olive cultivation increased from just 10 ha to
272 ha
(MAPA, 2018).
While a number of recent studies have examined
the olive oils produced in Galicia (Espinosa-Sánchez,
2010; Reboredo-Rodríguez et al., 2014a, 2014b and
2015), most of these oils were not produced by native
trees (Reboredo-Rodríguez et al., 2015) but by recent ly
planted and commonly cultivated genotypes from An-
dalusia, such as ´Picual´ and ´Arbequina´. Indeed, while
the agricultural biodiversity of Galicia's woody-plant
crops-grapes (Gago et al., 2009; Martínez et al., 2018),
apples (Pereira-Lorenzo et al., 2007), pears (dos San tos
et al., 2011; Pereira-Lorenzo et al., 2012) and chest nuts
(Pereira-Lorenzo et al., 1997) has been studied, that of
the region's olive trees is almost unk nown. Localising,
characterising and conserving the genotypes that may
still be found in this geographi cal area is vital to avoid
the genetic erosion of the species and to save their traits
for use in olive improvement programmes. A recent ar
ti-
cle by Reboredo-Rodríguez et al. (2018), and the doc-
toral thesis of Reboredo-Rodríguez (2015), identied a
number of olive genoty pes from this region. However,
these contributions co vered only a very small part of
the territory and some of the molecular results were
contradictory. Wider and more rigorous and systematic
surveying is required to catalogue the area's olive tree
biodiversity and to allow their inclusion in the Spa nish
list of olive varieties of commercial interest.
The present work reports the localisation of anci ent
olive trees in Galicia, their characterisation using bo-
tanical and molecular markers, and examines whe ther
or not these trees represent unknown native genoty-
pes. Back in the 19th century, Hidalgo-Tablada (1870)
suggested that olive genotypes might be characteri-
sed via certain leaf, fruit and endocarp variables, the
shape of the tree, and other features. Nowadays the
International Olive Council (IOC) uses the genotype
classication system of Barranco et al. (2005), which
employs botanical and agronomic markers. The pre-
sent work, however, introduces a further morphome-
tric inspection of the leaf. Martínez & Grenan (1999)
developed a graphic method for visualizing the dif-
ferences that appeared in biometric studies of the gra-
pevine leaf. This method provides a highly realistic
representation of the foliar morphology and has been
used to compare genotypes (Martínez & Pérez, 2000;
Santiago et al., 2005; Martínez, 2007; Gago et al.,
2009; Martínez et al., 2018) and clones (Martínez et
al., 2005). Martínez & Grenan´s (1999) method has
been adapted in the present work, in order to be used
in
the study of olive average leaves. Finally, simple se-
quence repeats (SSRs) markers were also used in geno-
type identications. Many genetic characterisation stu-
dies have used dierent sets of SSRs, and the results
have greatly increased our knowledge of olive genetic
heritage in dierent areas (Cipriani et al., 2002; Belaj
et al., 2004 and 2011; Gil et al., 2006; Sarri et al., 2006;
Baldoni et al., 2009; Muzzalupo et al., 2010; Fendri et
al.,
2010; Diez et al., 2011; Martí et al., 2015; Lazo-
vic et al., 2016; Sakar et al., 2016). Together, all these
techniques provide a glimpse of the possibly notable
olive diversity of the Spanish Northwest.
Material and methods
Plant material
A literature review was performed on olive culti-
vation in Galicia in order to determine the priority
areas to be surveyed. Orally transmitted information
was then collected from growers in the chosen areas
to record people's recollections of olive trees, and to
make note of any locally used genotype names. An
initial survey was then undertaken to nd old trees.
Some of these were clearly centuries old, as manifested
by the size of their trunks and the references made to
them by dierent generations of the owning families.
Some were no longer used in an agricultural sense,
although a number of these retired trees had taken on
an ornamental role. A total of 18 trees were sampled for
the present work. Each tree was given a code number
(Table 1 and Fig. 1).
Molecular characterisation
Genomic DNA was extracted from fresh young
lea ves of all 18 trees located, using the cetyltrimethy-
lammonium bromide (CTAB) protocol method origi-

Ancient olive genotypes of NW Spain
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e0702
3
nally developed by Murray & Thompson (1980) and
modied by De la Rosa et al. (2002).
A set of 13 SSRs were analysed: ssrOeUA- DC A 03,
ssrOeUA-DCA09, ssrOeUA-DCA11, ssrOeUA-
DC A15, ssrOeUA-DCA16, ssrOeUA-DCA18 (Sefc
et al., 2000) GAPU59, GAPU71B, GAPU101,
GA PU 103 (Carriero et al., 2002); UDO99–019,
UDO99–024 and UDO99-043 (Cipriani et al.,
2002). These markers were selected for their high
eciency and resol ving power in previous olive
genotype characterisa tion studies (Baldoni et al.,
2009; Trujillo et al., 2014).
Polymerase chain reactions (PCR), performed in
20 μL volumes, involved 2 ng of genomic DNA, 1X
supplied PCR buer (Biotools, Spain), 200 μM of
each dNTP (Roche), 1.5 mM MgCl, 0.25 units of Taq
DNA polymerase (Biotools, Spain) and 0.2 μΜ of
forward (uorescently labelled) and reverse primers.
All reactions were performed in a Perkin-Elmer 9600
thermocycler as follows: denaturation at 94
o
C for
5 min, 35 cycles of 94oC for 20 s, 50-59oC for 30 s,
72
o
C for 30 s, and a nal extension at 72
o
C for 8 min.
Amplicons were detected using an ABI 3130 Genetic
Analyzer (Applied 181 Biosystems/HITACHI) using
the GeneScan 400 HD-Rox internal standard. The
genotypes ´Frantoio´ and ´Arbequina´ were used as
controls in all runs.
The allele proles were sized in base pairs (bp) and
characterized using Genescan 3.7 software (Applied
Biosystems). A code number was assigned to the
dierent SSR proles dened.
Additionally, for each SSR marker, the total number
of alleles at each locus (Na), and the observed (Ho)
and expected (He) heterozygosity, were determined
using GenAlex v.6.503 software (Peakall & Smouse,
2006 and 2012). The probability of identity index (PI)
and the polymorphism information content (PIC) were
calculated using Power Marker v.3.25 software (Liu &
Muse, 2005). Genotypes showing only one fragment
am plied by a pair of primers at a particular locus were
deemed homozygous at that locus.
Botanical characterisation
The qualitative botanical characteristics examined
were those described by Barranco et al. (2005) and
adopted by the International Union for the Protection of
New Varieties of Plants (UPOV Code: OLEAA_EUR)
for the description and identication of olive cultiva ted
genotypes. These characteristics include:
⸻ Leaf: shape, width, and longitudinal curvature of
the leaf blade (40 leaves were taken from the mid area
of 8-10 of the year's shoots, chosen from among the
most representative of each tree, and always from the
south-facing side).
⸻ Drupe: weight, shape, symmetry, maximum
transverse diameter, apex and base shape, and presence/
absence of a tip (40 drupes were examined).
⸻ Endocarp: weight, shape, symmetry position
A, symmetry position B, position of the maximum
transverse diameter, shape of the apex, shape of the
base, roughness of the surface, number of vascular
bundles, distribution of vascular bundles, and presence
of mucron (40 endocarps were examined).
Table 1. List of the olive samples included in the study.
Sample
code
Collection site
(Province) Cultivation status
1 Ourense Abandoned cultivation
2 Ourense Fruit production
3 Ourense Fruit production
4 Ourense Ornamental
5 Ourense Ornamental
6 Lugo Fruit production
7 Lugo Abandoned cultivation
8 Lugo Abandoned cultivation
9 Lugo Abandoned cultivation
10 Ourense Fruit production
11 Pontevedra Ornamental
12 A Coruña Ornamental
13 A Coruña Abandoned cultivation
14 Pontevedra Ornamental
15 Pontevedra Ornamental
16 A Coruña Ornamental
17 A Coruña Ornamental
18 Pontevedra Ornamental
Figure 1. Map of Galicia, a region in northwestern Spain,
showing the location of the 18 trees examined (see Table
1).

Pilar Gago, José L. Santiago, Susana Boso and María C. Martínez
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e0702
4
The characterisation of the leaf was complemented
using an adapted version of the method of Martínez
& Grenan (1999) used to construct 'mean leaves' of
grapevine genotypes. Forty young leaves were taken
from shoots of the present year in the crown of each
tree. These were then herborized, photographed, and the
images used to determine the lengths and angles shown
in Fig. 2 (performed using AnaliSIS FIVE® software).
The mean values were then used to construct a mean
leaf for each tree. This method provides a recognisable
image that can be compared against others.
Principal components analysis (PCA) was also
performed to group the trees depending upon their
morphology using the measured leaf variables, and
upon certain quantitative variables recorded for the
drupes and endocarps (drupe length, drupe width,
drupe weight, endocarp length, endocarp width, endo-
carp weight, and pulp weight). Since the dierent
trees were found growing under dierent soil, climatic
and cultivation conditions, the raw values for these
variables were not used in this analysis, but rather the
relationships between them (Table 2), which reect the
resulting morphology. All statistical calculations were
performed using SAS software v.9.3 (SAS Inst. Inc.,
Cary, NC, USA).
Genotype identication
The criteria used in genotype identication were
those described by Trujillo et al. (2014), i.e., the pair-
wise comparison of SSR and morphological proles
with those in databases (the World Olive Germ plasm
Bank of Cordoba [WOGBC] and the Agronomy De -
partment of the University of Cordoba [UCO] data-
bases).
Results
Molecular characterisation
SSR variability
A total of 57 alleles were detected for the 13 SSR loci
examined. The number of alleles per locus ranged from
two (UDO99-19 and GAPU59) to seven (ssrOeUA-
DCA09 and UDO99-43) with an average of 4.38 alleles
per locus (Table 3).
The He value ranged from 0.180 (UDO99-019) to
0.810 (ssrOeUA-DCA09 and UDO99-43), with a mean
value of 0.654. The PIC values were always over 0.5
(Table 3), except for UDO99-019 (0.164), UDO99-
024 (0.442), GAPU59 (0.375) and ssrOeUA-DCA15
(0.495).
Ten dierent molecular proles or genotypes were
recorded among the 18 trees examined (Table 4 ) which
were grouped as follow: 7 trees gave rise to unique SSR
proles (not duplicated in any other tree) and 1 trees
had SSR proles in common with other trees resulting
in the identication of three SSR proles among them.
Genotype identication
When the molecular proles were compared (in
2015) with those in the WOGBC and UCO databases
(performed by the person responsible for molecular
identications), three matches were returned. Tree 11
was identied as belonging to the genotype ´Mansa
Gallega´, trees 6 and 7 as belonging to ´Brava Gallega´,
and trees 1, 2, 4, 5 and 10 to the Portuguese genotype
´Cobrancoça´ (Table 4).
The literature search and conversations with growers
returned only two cultivated genotypes names, ´Brava´
and ´Mansa´, which were used generically to describe
ostensibly native Galician olive trees. Interestingly,
both names are recorded by the WOGBC as referring to
material introduced elsewhere from Galicia.
Figure 2. Lengths and angles measured for the preparation
of the mean leaf of each tree. Lengths: L, A2, A1, A3 and
P; Angles: α1 and α 2.
Table 2. Relationships between dierent leaf, drupe and
endocarp variables.
Leaf relationshipsa
Rel 1 = A2/L
Rel 2 = A1/L
Rel 3 = A3/L
Rel 4 = A1/A2
Rel 5 = A3/A2
Drupe & endocarp relationships
Rel A = length/drupe width at position Ab
Rel B = length/width of endocarp at position Ab
Rel C = pulp weight/drupe weight
Rel D = endocarp weight/drupe weight
Rel E = pulp weight/endocarp weight
Rel F = drupe width/endocarp width
Rel G = drupe length/endocarp length
aSee Fig. 2. bPosition A, according to the UPOV code.

Ancient olive genotypes of NW Spain
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e0702
5
Table 3. Size range (base pairs), number of alleles
(Na), observed (Ho) and expected (He) heterozygosity,
probability of identity (PI) and polymorphism information
content (PIC) for each SSR locus.
SSR locus Size
range Na HoHePI PIC
ssrOeUA-
DCA03
227-253 6 1.000 0.800 0.070 0.770
ssrOeUA-
DCA09
160-206 7 1.000 0.810 0.061 0.785
ssrOeUA-
DCA11
130-178 4 0.900 0.745 0.113 0.697
ssrOeUA-
DCA15
243-263 3 0.500 0.585 0.262 0.495
ssrOeUA-
DCA16
122-171 6 1.000 0.795 0.072 0.765
ssrOeUA-
DCA18
166-183 5 1.000 0.720 0.126 0.672
UDO99-019 97-129 2 0.200 0.180 0.689 0.164
UDO99-024 164-185 3 0.600 0.505 0.308 0.442
UDO99-043 170-216 7 1.000 0.810 0.063 0.784
GAPU59 210-220 2 0.800 0.500 0.375 0.375
GAPU71B 121-141 4 0.900 0.655 0.171 0.603
GAPU101 189-217 4 1.000 0.685 0.161 0.623
GAPU103 133-184 4 0.100 0.715 0.135 0.661
All loci 57
Mean 4.38 0.769 0.654 0.200 0.603
diered from the rest in shape (ovoid), position of
maximum diameter (toward the base) and shape of
the base (round).
Quantitative drupe and endocarp variables mea-
sured, and the relationships between them were
calculated (Table S2 [Suppl.]).
The results of the PCA on the leaf variables (Table
1 and Fig. 4) show the two rst axes account for
85.68% of the variance (Prin 1 accounted for 51.37%
of the variance, and Prin 2 for 34.31%).With respect
to axis 1 (Prin 1), the variables with the greatest
weight were Rel 1 (A2/L) and Rel 3 (A3/L). Both
relation ships provide information regarding leaf
shape (elliptical, elliptic-lanceolate, or lanceolate).
With respect to axis 1 (Prin 2), the variables with the
greatest weight we re Rel 4 (A1/A2) and Rel 5 (A3/A2),
which provide information on the longitudinal prole
of the leaf, i.e., the proportional distance over which
the two sides of the leaf remain parallel (e.g., note
the
dierence between mean leaves 5, 12 and 18 in Fig.
3).
With respect to Prin 1 (Fig. 4), the trees with
elliptical leaves (12, 13 and 17) are distributed more
to the right, and those with more lanceolate leaves
(5, 9 and 18) towards the left. The majority, i.e.,
trees with elliptic-lanceolate leaves (as shown in
Table 5), are situated between these other positions.
With respect to Prin 2 (Fig. 4), the leaves of trees 9
and 5
were clearly se parated from the rest, indicating
their
morphology to be dierent too, with the leaves
of
tree 9 wider and tho se of tree 5 narrower than all
others. In addition, the reduction in width at the apex
and peduncle was less in the leaves of tree 5 than in
all others. Finally, trees 9 and 5 also diered from all
others in terms of the pattern of change in leaf width
along the length of the blade.
The results of PCA (Fig. 5) on the calculated drupe
and endocarp variables from Table 1, show the two
rst axes to account for 95.51% of the variance (Prin
1 accounted for 73.67% of the variance, and Prin 2 for
20.84%).
For Prin 1, the variable with the most positive
weight was Rel C (pulp weight/drupe weight), and
that with most negative weight was Rel D (endo-
carp weight/drupe weight). For Prin 2, the variable
with the most positive weight was Rel B (endocarp
length/endocarp width), followed by Rel A (drupe
length/drupe width); these provide information on
the sha pe of the endocarp and drupe respectively.
With respect to Prin 1, those trees with drupes
with a more meaty pulp (i.e., less endocarp) fall to
the right of the diagram (Fig. 5); these correspond
to the ge notypes ´Cobrancoça´, ´Brava Gallega´
and Unk nown Genotype 5. Those trees with drupes
Botanical characterisation
Leaf qualitative botanical variables (Table 5) were
noted. The three types of leaf blade shape cited by
Barranco et al. (2005) were found among the trees
studied although only tree 9 (Unknown Genotype 5)
showed the lanceolate shape. Most of the trees have a
medium width and at leaf blade.
Leaf lengths and angles were measured (Table S1
[suppl.]), and the relationships between them were
calculated and used for drawing mean leaves (Fig. 3).
Drupe and endocarp qualitative botanical variables
were recorded following the method of Barranco et al.
(2005) (Tables 6 and 7). Only fruits from trees identi-
ed as belonging to genotype ‘Cobrancoça’ showed
a high weight (Table 6). Tree number 3 (Unknown
Genotype 3) presented fruits with spherical shape and
with the maximum transverse diameter toward the base,
the rest of the studied fruits were ovoid or elongated
with the maximum diameter centred (Table 6). Finally,
none of the fruits studied presented an evident nipple
(Table 6). Regarding the endocarp qualitative botanical
variables (Table 7), only genotype ´Mansa Gallega´
(tree 11) presented endocarps with a low weight and,
again, endocarps from tree 3 (Unknown Genotype
3)

Pilar Gago, José L. Santiago, Susana Boso and María C. Martínez
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e0702
6
Table 4. Continued.
Sample UDO99-019 UDO99-024 UDO99-043 GAPU59 GAPU71B GAPU101 GAPU103 Identicationa
6 129 129 164 185 172 204 210 220 127 141 191 217 133 133 Brava Gallega
7 129 129 164 185 172 204 210 220 127 141 191 217 133 133 Brava Gallega
1 129 129 185 185 208 216 210 220 121 141 191 217 133 133 Cobrancoça
2 129 129 185 185 208 216 210 220 121 141 191 217 133 133 Cobrancoça
4 129 129 185 185 208 216 210 220 121 141 191 217 133 133 Cobrancoça
5 129 129 185 185 208 216 210 220 121 141 191 217 133 133 Cobrancoça
10 129 129 185 185 208 216 210 220 121 141 191 217 133 133 Cobrancoça
11 97 129 164 177 170 216 220 220 124 127 189 191 159 159 Mansa Gallega
13 129 129 177 185 170 212 210 220 124 141 189 191 159 159 Unknown 1
15 129 129 177 185 170 212 210 220 124 141 189 191 159 159 Unknown 1
16 129 129 177 185 170 212 210 220 124 141 189 191 159 159 Unknown 1
17 129 129 177 185 170 212 210 220 124 141 189 191 159 159 Unknown 1
18 97 129 177 185 170 216 210 220 124 141 189 217 133 159 Unknown 2
3 129 129 185 185 172 216 210 210 121 141 197 217 184 184 Unknown 3
8 129 129 185 185 172 204 210 220 141 141 191 217 184 184 Unknown 4
9 129 129 185 185 172 216 210 220 127 141 191 217 184 184 Unknown 5
14 129 129 177 185 170 212 210 220 124 141 189 191 161 161 Unknown 6
12 129 129 177 185 170 214 210 220 124 141 189 191 159 159 Unknown 7
aIdentied by comparison with molecular proles held in the WOGBC (World Olive Germplasm Bank of Cordoba) and UCO
(University of Cordoba) databases.
Table 4. Allelic proles (bp) of the 18 olive trees with respect to the 13 microsatellite loci examined.
Sample ssrOeUA-DCA03 ssrOeUA-DCA09 ssrOeUA-DCA11 ssrOeUA-DCA15 ssrOeUA-DCA16 ssrOeUA-DCA18
6 237 251 182 192 140 178 243 254 124 152 166 176
7 237 251 182 192 140 178 243 254 124 152 166 176
1 237 251 160 204 140 178 243 254 122 124 166 176
2 237 251 160 204 140 178 243 254 122 124 166 176
4 237 251 160 204 140 178 243 254 122 124 166 176
5 237 251 160 204 140 178 243 254 122 124 166 176
10 237 251 160 204 140 178 243 254 122 124 166 176
11 227 243 180 182 130 140 254 254 144 152 166 183
13 227 251 170 182 130 160 243 254 144 159 166 176
15 227 251 170 182 130 160 243 254 144 159 166 176
16 227 251 170 182 130 160 243 254 144 159 166 176
17 227 251 170 182 130 160 243 254 144 159 166 176
18 237 243 180 204 140 140 254 254 122 152 166 183
3 237 251 160 206 160 178 243 243 124 152 168 172
8 237 247 182 204 140 178 243 243 124 152 168 176
9 243 247 160 204 160 178 263 263 152 171 168 176
14 227 251 170 182 130 160 243 254 144 159 166 176
12 227 253 170 182 130 160 243 254 144 159 166 176
possessing heavier endo carps and less pulp (´Mansa
Gallega´ and Unknown Genotype 1) fall towards
the left (Fig. 5). With respect to Prin 2, Unknown
Genotype 3 remains clearly separated from the rest.
This was represented by the only tree with spherical-
to-oval drupes and oval endocarps (see Tables 6 and
7). All the other trees had fruits with an elliptical
endocarp
.

Ancient olive genotypes of NW Spain
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e0702
7
Discussion
This study on the almost forgotten olive trees of
northwestern Spain aims to provide their rst botanical
and molecular characterisation, and to compare this
local germplasm with that conserved in databases. The
results provide a glimpse of the olive diversity that the
region may still hold.
The molecular proles of the 18 examined trees
grouped them into nine genotypes, of which three
could be identied: ´Brava Gallega´, ´Mansa Gallega´
and ´Cobrancoça´. For now, the identity of the other
six genotypes remains unknown. These results agree
with those of other studies that have genetically or
morphologically characterised centuries-old olive
trees
in peripheral growing areas; where only a small pro-
portion of those examined represented genotypes
with
a commercial use (Díez et al., 2011; Salimonti et
al.,
2013; Martí et al., 2015; Lazovic et al., 2016; Sakar
et al., 2016). Similar results have been repor ted also
for centuries-old grapevines (Martínez & Pérez, 2000;
Santiago et al., 2005; Gago et al., 2009).
SSRs are widely used as markers in the identi ca tion
of olive genotypes (Cipriani et al., 2002; Bal doni et al.,
2009; Díez et al., 2012; Jakše et al., 2013; Reboredo
et al., 2018). In the present work, the loci GAPU059
and UDO99-019 showed low-le vel polymorphism,
and were therefore little informative in identifying the
genotypes of the examined trees. Reboredo et al. (2018)
reported the same for these two loci. Loci UDO043 and
ssrOeUA-DCA9 showed the greatest discriminatory
power, in agreement with the results of other authors
who examined olive material from dierent areas
(Baldoni et al., 2009; Salimonti et al., 2013; Trujillo et
al., 2014).
The morphological characteristics of the endocarp,
which are considered very stable, are also widely used
in olive genotype identication (Barranco et al., 2000;
Fendri et al., 2010). It is also usual to make use of the
characteristics of the leaves or drupes. Certainly, the size
of the leaves and drupes may dier depending upon the
edaphoclimatic conditions, but it should be remembered
that in grapevine the eect of 'genotype' dominates that
of 'edaphoclimatic conditions' (Martínez & Grenan,
1999). In other words, although the size of the leaves
and drupes may be dierent, their shape is constant.
Further, the use of relationships between measure-
ments
of dierent variables eliminates the eect of
Table 5. Qualitative leaf characteristics of the analysed trees, showing the mode values for 40 leaves.
Sample code Genotype name
Leaf blade: shapeaLeaf blade: widthbLeaf blade: curvature
of longitudinal axisc
CPVO 6 CPVO 5 CPVO 7
UPOV 7 UPOV 6 UPOV 9
6 Brava Gallega EP M FL
7 Brava Gallega EP-LA N FL
1 Cobrancoça EP-LA M FL
2 Cobrancoça EP-LA M FL
4 Cobrancoça EP-LA M FL
5 Cobrancoça EP-LA M FL
10 Cobrancoça EP-LA M FL
11 Mansa Gallega EP-LA M FL
13 Unknown 1 EP M FL
15 Unknown 1 EP-LA M FL
16 Unknown 1 EP-LA M/W EP
17 Unknown 1 EP WFL
18 Unknown 2 EP-LA M FL
3 Unknown 3 EP-LA M FL
8 Unknown 4 EP-LA M FL
9 Unknown 5 LA N FL
14 Unknown 6 EP-LA WFL
12 Unknown 7 EP WEP
CPVO: Community Plant Variety Oce code characteristic number; UPOV: International Union for the Protection
of New Varieties of Plants code characteristic number. aelliptic = EP; elliptic-lanceolate = EP-LA; lanceolate = LA.
bnarrow = N; medium = M; wide = W. cFlat = FL; Epinasty = EP.

Pilar Gago, José L. Santiago, Susana Boso and María C. Martínez
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e0702
8
growing conditions, and the mean leaves constructed
from them provide an excellent identication tool.
In the present work, the trees with the same
molecular proles fell into the same PCA-determined
groups based on their endocarp characteristics. This did
not always happen, however, with respect to the leaves;
indeed, large qualitative and quantitative dierences
were seen between trees with identical molecular
proles. Such was the case for the ´Cobrancoça´ trees;
these grouped together in terms of their leaf qualitative
variables (leaf blade shape, leaf blade width and
longitudinal curvature of the leaf), but not in terms of
their quantitative variables (leaf lengths and angles).
In contrast, the Unknown Genotype 1 trees (13, 15, 16
and 17) grouped together in terms of their qualitative
but not their quantitative variables. The same was true
for the ´Brava Gallega´ trees (trees 6 and 7). With
respect to drupe qualitative characteristics, Unknown
Genotype 1 was also heterogeneous, especially in terms
of fruit colour (Table 6). This might be explained in that
although all fruits were collected on the same day (by
dierent teams), the trees grew in dierent areas and
their fruit may not have been of equal ripeness. Sali mon ti
et al. (2013) suggests that many of the dierences seen
within genotypes could be the result of the existence of
dierent clones, as reported for grapevine (Boso et al.,
2004; Martínez et al., 2005).
It is possible that a larger number of SSR markers
might have led to dierent genotype identications,
though this is unlikely given that 13 were examined.
This has been reported in grapevine, although a redu-
ced number (just six) of highly discriminatory SSRs are
now recognised that can identify nearly all genotypes
(OIV, 2009).
Recently, Reboredo et al. (2018) published an arti-
cle
in which cultivated olive material from the same
region was examined, and three dierent genotypes
were found among a 32-olive-tree sample; also, using a
set of 14 SSRs loci, a total of 37 alleles were reported
in the cited work. In the present work, nine dierent
SSRs proles were found in an 18-olive-tree sample,
and a total of 55 alleles detected with a set of 13 SSRs
loci. This might be explained in that the present survey
covered a much wider sampling area where locations
with dierent numbers of olive trees are present.
Historical records for these locations conrmed
their past association with active olive cultivation. In
addition, the present work selected centuries-old olive
trees; these were documented as such in some cases,
and at least referred to as such by oral tradition in others.
Figure 3. Mean leaf for each of the 18 examined trees, produced according to the
adapted method of Martínez & Grenan (1999). Leaves 1, 2, 4, 5 and 10 = Cobrancoça;
leaf 3 = Unknown Genotype 3; leaves 6 and 7 = Brava Gallega; leaf 8 = Unknown
Genotype 4; leaf 9 = Unknown Genotype 5; leaf 11 = Mansa Gallega; leaf 12 =
Unknown Genotype 7; leaves 13, 15, 16 and 17 = Unknown Genotype 1; leaf 14 =
Unknown Genotype 6; and leaf 18 = Unknown Genotype 2.
4.52cm
6.23cm 6.06cm 4.36cm
6.05cm
4.65cm
5.03cm
4.77cm
5.76cm
5.76cm 6.07cm 4.98cm
6.37cm 5.89cm
6.19cm 5.99cm
5.68cm 5.52cm

Ancient olive genotypes of NW Spain
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e0702
9
The molecular prole assigned to the genotype
´Brava´ by Reboredo-Rodríguez et al. (2018) matches
that of ´Brava Gallega´ in the present work (in both cases
the SSR proles were compared to those held in the
WOGBC and UCO databases). However, the molecu-
lar and morphological (which included only endocarp
information) proles assigned by Reboredo-Rodríguez
et al. (2018) to the genotype ´Mansa´ (reported as
´Unknown´ by Reboredo-Rodríguez, 2015) did not
match those of ´Mansa Gallega´ as determined in the
present work and in the consulted WOGBC and UCO
databases. It is important to note that the molecular
prole and botanical characterisation reported here as
identifying the genotype ´Mansa Gallega´ correspond
exactly to those recognized by the Spanish Department
of Agriculture (MAPAMA, 2017).
The correct molecular characterization of genotypes
is important to prevent confusion with other genoty-
pes with similar morphological characteristics and also
to use this plant material in breeding programs and in
commercial propagation. SSR analysis is a powerful
tool for genotype characterization. In olive, intra-
genotype genetic diversity has been reported using SSR
markers (Muzzalupo et al., 2010; Caruso et al., 2014;
Trujillo et al., 2014), for these authors, SSR proles that
are dierentiated by one or several dissimilar alleles are
classied into the same genotype. These are classied as
´molecular variants´ and are treated as ´clones´ within
the main variety due to somaclonal mutations. But in
other woody species SSR markers are not considered
as an eective approach to detect genetic dierences
among clones (Imazio et al., 2002; Bouhadida et al.,
2007; Pereira-Lorenzo et al., 2007).
The ´Mansa Gallega´ identied in the present work
was located in the south of the Province of Ponteve-
dra − a long way from the sampling area studied by
Reboredo-Rodríguez et al. (2018). However, the trees
studied that were identied as belonging to ´Brava
Gallega´ were located in the same area studied by the
latter authors. Finally, the molecular prole assigned
to the genotype ´Picuda´ by Reboredo-Rodríguez et
al. (2018) was not found among those detected in the
Table 6. Drupe qualitative characteristics (as set out in the method of Barranco et al., 2005) for the studied trees. Results
represent the mode for 40 examined drupes; trees 8, 10 and 18 were not included since they produced no fruit.
Sample
code
Genotype
name
WeightaShapebSymmetryc
Maximum
transverse
diameterd
ApexeBasefNippleg
Over colour
at full
maturityh
CPVO 8 CPVO 9 CPVO 11 CPVO 12 CPVO 14 CPVO 13 CPVO 10
UPOV 16 UPOV 18 UPOV 23 UPOV 24 UPOV 26 UPOV 25 UPOV 22
6 Brava Gallega M O S C R T A B
7 Brava Gallega M O S C R R A B/RW
1 Cobrancoça M EL S C P T S V
2 Cobrancoça H O AS C R T S B
4 Cobrancoça H O SA C R T S B
5 Cobrancoça H O SA C R T S B
10 Cobrancoça - - - - - - - -
11 Mansa Gallega L O SA C R T A B
13 Unknown 1 L EL AS C P T S V
15 Unknown 1 M O AS C P T S B
16 Unknown 1 L EL AS C R R S V
17 Unknown 1 L EL AS C P T S RW
18 Unknown 2 - - - - - - - -
3 Unknown 3 M S S B R T S RW
8 Unknown 4 - - - - - - - -
9 Unknown 5 M O S C R T A B/RW
14 Unknown 6 M EL AS C R T S V
12 Unknown 7 M O SA C P T A B
CPVO: Community Plant Variety Oce code characteristic number; UPOV: International Union for the Protection of New Varieties of
Plants code characteristic number. alow = L; medium = M; high = H. bspherical = S; ovoid = O; elongated = EL. cSymmetry of position
A: symmetric = S; slightly asymmetric = SA; asymmetric = AS. dtoward the base = B; centred = C. eForm of the apex in position A:
pointed = P; rounded = R. fForm of the base in position A: truncated = T; rounded = R. gTip or Nipple: absent = A; slight= S; present
= P. hviolet = V; red wine = RW; black = B.

Pilar Gago, José L. Santiago, Susana Boso and María C. Martínez
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e0702
10
present work. Indeed, neither ´Picual´ nor ´Arbequina´,
nor indeed any other genotype cultivated in Spain's most
important olive-producing regions, was represented
by the examined trees. The olive-growing area closest
to Galicia is in northern Portugal; the detection of the
Portuguese genotype ´Cobrancoça´ (trees 1, 2, 4, 5 and
10) is therefore not very surprising. Fig. 1 shows all
these ´Cobrancoça´ trees to be located within a few
kilometres of the Portuguese border. It is rather more
surprising that no other specimen of this genotype was
found away from this area. It is also of note that no
specimens of a genotype extensively grown in Portugal,
known as ´Galega´ (Cordeiro et al., 2008) - a name that
suggests it originated in Galicia - were found in the
present study.
Trees 1-10, all known locally under the name of
´Brava´, were found in areas where olive growing has
more of a tradition. However, only trees 6 and 7 had a
molecular prole that matched with the prole recorded
for the genotype ´Brava Gallega´ in the WOGBC and
UCO databases. Trees 1, 2, 4, 5 and 10 were found to
belong to the genotype ´Cobrancoça´ (Cordeiro et al.,
2008), and others belonged to unknown genotypes
(both in terms of their molecular prole and botanical
characteristics). The name ´Brava´ appears to be used
locally to refer to many dierent genotypes; only one
of them, of course, is the ´Brava Gallega´ genotype.
The term ´brava´ in fruticulture is used to refer to plant
grown from a seed and normally used as a seedling
rootstock, but in this particular case the olive growers in
this area use this term to refer to a number of genotypes
with a high agronomic quality and clearly distinct from
a wild olive or a rootstock and that they propagate using
cuttings.
The second most locally used genotype name was
´Mansa´, but only one tree (tree 11) actually had a
molecular prole that matched that deposited in the
WOGBC and UCO databases.
The problems of homonyms and synonyms aec-
ting Galicia's olive trees is not the same as that which
aects grapevine genotypes (Martínez et al., 2018).
While grapevine genotypes may have synonyms, they
always identify the same genotype. For example, the
genotype that goes by the name ´Tempranillo´ in the
Rioja winemaking region, is called Tinta Fina in the
Ribera del Duero region, and has dierent names in
other areas. However, even though viticulturists may
use these dierent names, they all identify the same
genotype through association with the same leaf
and cluster characteristics. ´Brava´ and ´Mansa´, in
Table 7. Endocarp qualitative characteristics (as set out in the method of Barranco et al., 2005) of olives from the
studied trees. Results represent the mode for 40 examined endocarps; trees 8, 10 and 18 were not included since
they produced no fruit.
Sample code Genotype
name
WeightaShapebSymmetry
position Ac
Symmetry
position Bc
Position of the maxi-
mum transverse diamd
Shape of
the apexe
CPVO16 CPVO15 CPVO17 CPVO18 CPVO CPVO21
UPOV32 UPOV31 UPOV33 UPOV34 UPOV UPOV37
6 Brava Gallega H EP SA S C P
7 Brava Gallega H EP SA S C P
1 Cobrancoça H EL A S C P
2 Cobrancoça VH EP SA S C P
4 Cobrancoça H EL SA S C P
5 Cobrancoça VH EP SA S C P
10 Cobrancoça - - - - - -
11 Mansa Gallega L EP SA S C P
13 Unknown 1 M EP SA S C P
15 Unknown 1 M EP SA S C P
16 Unknown 1 M EP A/SA S C P
17 Unknown 1 M EP A S A P
18 Unknown 2 - - - - - -
3 Unknown 3 M O S S B R
8 Unknown 4 - - - - - -
9 Unknown 5 M EP A/SA S A R
14 Unknown 6 M EP SA S C P
12 Unknown 7 H EP SA S C P

Ancient olive genotypes of NW Spain
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e0702
11
contrast, are not terms that identify respective olive
genotypes in Galicia. In conversations with growers in
the present work, it was noted that they used the terms
with entirely dierent genotypes. The armation by
Reboredo-Rodríguez et al. (2018) that Galicia ´Mansa´
is a homonym of the genotypes ´Brava´ and ´Mansa´,
and that ´Mansa´ is a synonym of the genotype ´Brava´,
seems not to hold up.
Trees 3, 8 and 9, which were located very close to
one another, each represented an unknown genotype
(Unknown Genotypes 3, 4 and 5 respectively), with each
showing dierent molecular and botanical dierences.
The presence of dierent unknown genotypes in such a
small area hints at the diversity yet to be discovered.
Also,
tree 18, which was located close to tree 11, was of
another unknown genotype (Unknown Genotype 2).
Trees 13, 15, 16 and17 all belonged to Unknown
Genotype 1. The age of these trees, plus their being
found over a wide area, suggests that the vegetative
propagation of olive trees has long been performed in
the region. Tree 14 (Unknown Genotype 6) was found
in the same cultivation area that trees 13, 16 and 17
(Unknown Genotype 1) but it has a molecular prole
that diers in one SSR locus from this genotype (trees
13, 16 and 17). Tree 12 was also found in the same area
but its molecular prole diers in one allele for two loci
from the Unknown Genotype 1; in addition, this tree
also diers from trees of Unknown Genotype 1 in some
morphological characteristics, as the absence of nipple
in the fruit or the high weight in the endocarp.
The results suggest that Galicia may be a reservoir
of olive diversity. This agrees with the thinking of other
authors (Trujillo et al., 1990; Zohary & Hopf, 1994;
Claros et al., 2000; Cordeiro et al., 2008) who suggest
the majority of the region's olive genotypes to be native
and to have spread little to other areas. Apart from
providing new genetic material, such native genotypes
could provide information of use in other scientic
studies. For example, studies on the domestication and
parentage of olive trees (Trujillo et al., 2014; Diez et
al., 2015) have normally examined genotypes native to
more Mediterranean areas. Galicia's native genotypes
could add new variability and molecular heterogeneity
to be considered in such studies.
Table 7. Continued.
Sample
code
Genotype
name
Shape of
the basee
Roughness
of the
surfacef
Number of
vascular
bundlesg
Distribution of
vascular
bundlesh
Presence of
mucroni
CPVO23 CPVO24 CPVO20 CPVO22
UPOV39 UPOV40 UPOV36 UPOV38
6 Brava Gallega P S M R P
7 Brava Gallega P R M R P
1 Cobrancoça P S L R P
2 Cobrancoça P R M R P
4 Cobrancoça P R L/M R P
5 Cobrancoça P R M R P
10 Cobrancoça - - - - -
11 Mansa Gallega P S L R A
13 Unknown 1 P S L R P
15 Unknown 1 P S L/M R P
16 Unknown 1 P S L R P
17 Unknown 1 P S L R P
18 Unknown 2 - - - - -
3 Unknown 3 R R M R A
8 Unknown 4 - - - - -
9 Unknown 5 P R M R P
14 Unknown 6 P S L R P
12 Unknown 7 P S L R P
CPVO: Community Plant Variety Oce code characteristic number; UPOV: International Union for the Protection of
New Varieties of Plants. code characteristic number. alow = L; medium = M; high = H; very high = VH. bovoid = O;
elliptic = EP; elongated = EL. csymmetric = S; slightly asymmetric = SA; asymmetric = A. dtoward the base = B; centred
= C; toward the apex = A. epointed = P; rounded = R. fsmooth = S; rough = R. glow = L (less than 7); medium = M (7
to 10). hregular = R. ipresent = P; absent = A.

Pilar Gago, José L. Santiago, Susana Boso and María C. Martínez
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e0702
12
Figure 5. Results of PCA analysis for the drupe and endocarp relationships Rel A,
Rel B, Rel C, Rel D, Rel E, Rel F and Rel G. No values were available for trees 8,
10 or 18. The dierent colours identify the trees shown to be identical in the SSR
analysis (name of genotypes explained in Fig. 3).
Prin 1
Prin 2
Figure 4. Results of PCA analysis for the leaf relationships Rel 1, Rel 2, Rel 3,
Rel 4 and Rel 5, and leaf angle measurements α1 and α 2. The dierent colours
identify the trees shown to be identical in the SSR analysis (name of genotypes
explained in Fig. 3).
The present work provides the molecular proles and
complete botanical descriptions of some unreported,
local olive genotypes surviving in Galicia. The results
identied two potentially native genotypes ´Brava
Gallega´ and ´Mansa Gallega´, and claried certain
misidentications of the latter by other authors. Six
unknown genotypes were also detected, as well as
the Portuguese genotype ´Cobrancoça´. The evidence
suggests that olive trees have been cultivated in the
region for centuries, and that the diversity of native
genotypes is high. This diversity should be preserved as
part of Europe's agricultural heritage, but also because
it may oer scientic and commercial opportunities.
A larger survey should be performed to determine the
Prin 1
Prin 2

Ancient olive genotypes of NW Spain
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e0702
13
full range of Galicia's olive tree diversity, followed by
agricultural studies that might indicate the potential of
the region's rediscovered genotypes.
Acknowledgements
Dr. I. Trujillo provided assistance in SSR analysis
during a period at the Laboratorio de Elaiografía y
Marcadores Moleculares at the Dept. of Agronomy,
University of Córdoba. Dr. Trujillo also compared the
proles obtained with those in the WOBG and UCO
databases (performed in 2015). Iván González and
Elena Zubiaurre are thanked for technical assistance, as
is Adrian Burton for the English translation of the text.
References
Alonso de Herrera G, 1513. Agricultura general. Imprenta
Real, Madrid, Spain.
Baldoni L, Cultrera NG, Mariotti R, Ricciolini C, Arcioni S,
Vendramin GG, Buonamici A, Porceddu A, Sarri V, Ojeda
MA, et al., 2009. A consensus list of microsatellite markers
for olive genotyping. Mol Breeding 24 (3): 213-231. https://
doi.org/10.1007/s11032-009-9285-8
Barranco D, Cimato A, Fiorino P, Rallo L, Touzani A, Castañeda
C, Serani F, Trujillo I, 2000. World olive catalogue of olive
varieties. International Olive Oil Council, Madrid, Spain.
Barranco D, Trujillo I, Rallo L, 2005. Variedades de olivo en
España. Mundi-Prensa, Madrid, Spain.
Bartolini G, Prevost G, Messeri C, Carignani C, 2005. Olive
germplasm: genotypes and world-wide collections. FAO
SaPGRSo (ed), FAO, Rome.
Belaj A, Satovic Z, Rallo L, Trujillo I, 2002. Genetic diversity
and relationships in olive (Olea europaea L.) germplasm
collections as determined by randomly amplied
polymorphic DNA. Theor Appl Genet 105: 638-644.
https://doi.org/10.1007/s00122-002-0981-6
Belaj A, Cipriani G, Testolin R, Rallo L, Trujillo I, 2004.
Characterization and identication of the main Spanish
and Italian accessions by simple sequence repeat markers.
Hort-Science 39: 1557-1561. https://doi.org/10.21273/
HORTSCI.39.7.1557
Belaj A, Leon L, Satovic Z, de la Rosa R, 2011. Variability of
wild olives (Olea europaea subsp europaea var. sylvestris)
analyzed by agro-morphological traits and SSR markers.
Sci Hortic 129 (4): 561-569. https://doi.org/10.1016/j.
scienta.2011.04.025
Boso S, Santiago JL, Martínez MC, 2004. Intravarietal
agronomic variability in the cv. Albariño (Vitis vinifera L.).
Am J Enol Vitic 55: 279-282.
Bouhadida M, Casas AM, Moreno MA, Gogorcena Y, 2007.
Molecular characterization of Miraores peach variety
and relatives using SSRs. Sci Hort 111: 140-145. https://
doi.org/10.1016/j.scienta.2006.10.018
Carriero F, Fontanazza G, Cellini F, Giorio G, 2002.
Identication of simple sequence repeats (SSRs) in olive
(Olea europaea L.). Theor Appl Genet 104: 301-307.
https://doi.org/10.1007/s001220100691
Caruso T, Marra FP, Costa F, Campisi G, Macaluso L, Marchese
A, 2014. Genetic diversity and clonal variation within
the main Sicilian olive cultivars based on morphological
traits and microsatellite markers. Sci Hortic 180: 130-138.
https://doi.org/10.1016/j.scienta.2014.10.019
Cipriani G, Marrazzo MT, Marconi R, Cimato A, Testolin
R, 2002. Microsatellite markers isolated in olive (Olea
europaea L) are suitable for individual ngerprinting
and reveal polymorphism within ancient genotypes.
Theor Appl Genet 104: 223-228. https://doi.org/10.1007/
s001220100685
Claros MG, Crespillos R, Aguilar ML, Canovas FM,
2000. DNA ngerprinting and classication of
geographically related genotypes of olive-tree (Olea
europaea L.) Euphytica 116: 131-142. https://doi.
org/10.1023/A:1004011829274
Contreras JM, 1798. Historia del célebre santuario de Nuestra
Señora de Las Hermitas, situada en las montañas que
baña el rio Bibey en tierra del Bollo, Reyno de Galicia, y
Obispado de Astorga. Ed. Francisco de Toxar, Salamanca,
Spain.
Cordeiro AI, Sanchez-Sevilla JF, Alvarez-Tinaut MC,
Gomez-Jimenez MC, 2008. Genetic diversity assessment
in Portugal accessions of Olea europaea by RAPD
markers. Biol Plantarum 52 (4): 642-647. https://doi.
org/10.1007/s10535-008-0125-1
De La Rosa R, James CM, Tobutt KR, 2002. Isolation and
characterization of polymorphic microsatellites in olive
(Olea europaea L.) and their transferability to other
genera in the Oleaceae. Mol Ecol Notes 2 (3): 265-267.
https://doi.org/10.1046/j.1471-8286.2002.00217.x
Diez CM, Trujillo I, Barrio E, Belaj A, Barranco D, Rallo
L, 2011. Centennial olive trees as a reservoir of genetic
diversity. Ann Bot 108: 797-807. https://doi.org/10.1093/
aob/mcr194
Diez CM, Imperato A, Rallo L, Barranco D, Trujillo I,
2012. Worldwide core collection of olive genotypes
based on simple sequence repeat and morphological
markers. Crop Sci 52: 211-221. https://doi.org/10.2135/
cropsci2011.02.0110
Diez CM, Trujillo I, Martínez-Urdiroz N, Barranco D, Rallo
L, Marl P, Gaut BS, 2015. Olive domestication and
diversication in the Mediterranean Basin. New Phytol
206: 436-447. https://doi.org/10.1111/nph.13181
Dos Santos ARF, Ramos-Cabrer AM, Diaz-Hernandez
MB, Pereira-Lorenzo S, 2011. Genetic variability and
diversication process in local pear genotypes from
northwestern Spain using microsatellites. Tree Genet

Pilar Gago, José L. Santiago, Susana Boso and María C. Martínez
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e0702
14
Genomes 7 (5): 1041-1056. https://doi.org/10.1007/
s11295-011-0393-3
Espinosa-Sánchez J, 2010. Mito y realidad del aceite de
Quiroga. GDR Ribeira Sacra, Lugo, Spain.
Fendri M, Trujillo I, Trigui A, Rodriguez-Garcia MI, Ramirez
JDA, 2010. Simple sequence repeat identication and
endocarp characterization of olive tree accessions in a
Tunisian germplasm collection. Hortscience 45: 1429-
1436. https://doi.org/10.21273/HORTSCI.45.10.1429
Fendri M, Ferreira E, Calado L, Abreu I, Rodríguez-
García MI, Amorim MI, Alche JD, 2014. Discrimination
of Portuguese and Spanish olive genotypes using
microsatellite markers. Acad J Agric Res 2: 58-61.
Fernández de la Cigoña E, Martínez X, 2003. O aceite en
Galicia guía das lagaretas castrexo-romanas, medievais
e modernas. Ed. Asociación Galega para a Cultura e a
Ecoloxía, Pontevedra, Spain.
Gago P, Santiago JL, Boso S, Alonso-Villaverde V, Grando S,
Martínez MC, 2009. Biodiversity and characterization of
twenty-two Vitis vinifera L. genotypes in the Northwestern
Iberian Peninsula. Am J Enol Vit 60 (3): 293-301.
Gil FS, Busconi M, Machado AD, Fogher C, 2006.
Development and characterization of microsatellite loci
from Olea europaea. Mol Ecol Notes 6 (4): 1275-1277.
https://doi.org/10.1111/j.1471-8286.2006.01513.x
Gómez MC, Delgado FJ, Parra MC, 2012. Identicación de
variedades de olivo cultivadas en Extremadura mediante
marcadores morfológicos y moleculares. Universidad de
Extremadura Servicio de Publicaciones, Badajoz, Spain.
Haouane H, El Bakkali A, Moukhli A, Tollon C, Santoni
S, Oukabli A, El Modafar C, Khadari B, 2011. Genetic
structure and core collection of the World Olive
Germplasm Bank of Marrakech: towards the optimised
management and use of Mediterranean olive genetic
resources. Genetica 139 (9): 1083-1094. https://doi.
org/10.1007/s10709-011-9608-7
Hidalgo-Tablada J, 1870. Tratado del cultivo de la vid en
España y modo de mejorarlo. Librería de la Señora Viuda
e Hijos de Don José Cuesta, Madrid.
Imazio S, Labra M, Grassi F, Wineld M, Bardini M,
Scienza A, 2002. Molecular tools for clone identication:
The case of the grapevine cultivar 'Traminer'. Plant
Breed 121 (6): 531-535. https://doi.org/10.1046/j.1439-
0523.2002.00762.x
Jake J, tajner N, Tomi L, Javornik B, 2013. Application
of microsatellite markers in grapevine and olives. In:
The Mediterranean genetic code - Grapevine and olive;
Sladonja B & Poljuha D (eds.). pp: 25-50. IntechOpen.
https://doi.org/10.5772/53411
Lazovic B, Adakalic M, Pucci C, Perovic T, Bandelj D,
Belaj A, Mariotti R, Baldoni L, 2016. Characterizing
ancient and local olive germplasm from Montenegro.
Sci Hortic 209: 117-123. https://doi.org/10.1016/j.
scienta.2016.06.022
Liu K, Muse SV, 2005. Power marker: Integrated analysis
environment for genetic marker data. Bioinformatics 21
(9): 2128-2129. https://doi.org/10.1093/bioinformatics/
bti282
MAPA, 2018. Anuario de estadística agraria 2017. Ministerio
de Agricultura, Pesca y Alimentación. https://www.mapa.
gob.es/es/estadistica/temas/publicaciones/anuario-de-
estadistica [11/04/2019].
MAPAMA, 2017. Resolución de la Dirección General de
Producciones y Mercados Agrarios de 23 de Octubre
de 2017, por la que se reconocen ocialmente una serie
de variedades de especies frutales, propuestas por la
Dirección General de Ganadería, Agricultura e Industrias
Alimentarias de la Xunta de Galicia. Dirección de
Validación: https://sede.administracion.gob.es/pagSede-
Front / servicios/consultaCSV.htm (Código Seguro de
Vericación: CSV: GEN-68e0-ec35-2596-15ab-d855-
e52f-2900-2339). [11/04/2017].
Martí AFI, Forcada CFI, Company RSI, Rubio-Cabetas,
MJ, 2015. Genetic relationships and population structure
of local olive tree accessions from Northeastern Spain
revealed by SSR markers. Acta Physiol Plant 37: 1726.
https://doi.org/10.1007/s11738-014-1726-2
Martínez MC, 2007. La colección de variedades de vid del
Consejo Superior de Investigaciones Cientícas (Misión
Biológica de Galicia). La Semana Vitivinícola 3198:
3926-3929.
Martínez MC, Genan S, 1999. A graphic reconstruction
method of an average leaf of vine. Agronomie 19: 491-
507. https://doi.org/10.1051/agro:19990607
Martínez MC, Pérez JE, 2000. The forgotten vineyard of the
Asturias Princedom (north of Spain) and ampelographic
description of its genotypes (Vitis vinifera L). Am J Enol
Vit 51 (4): 370-378.
Martínez MC, Boso S, Santiago JL, 2005. Los clones de
Albariño (Vitis vinifera L) seleccionados en el Consejo
Superior de Investigaciones cientícas. Editorial CSIC,
Madrid, Spain.
Martínez MC, Boso S, Gago P, Muñoz-Organero G, De
Andrés M, Gaforio L, Cabello F, Santiago JL, 2018. Value
of two Spanish live grapevine collections in the resolution
of synonyms, homonyms and naming errors. Aus J
Grape Wine Res 24 (4): 430-438. https://doi.org/10.1111/
ajgw.12348
Murray MG, Thompson WF, 1980. Rapid isolation of high
molecular weight plant DNA. Nucl Acid Res 8: 4321-
4325. https://doi.org/10.1093/nar/8.19.4321
Muzzalupo I, Chiappetta A, Benincasa C, Perri E, 2010. Intra-
cultivar variability of three major olive cultivars grown in
dierent areas of central-southern Italy and studied using
microsatellite markers. Sci Hortic 126: 324-329. https://
doi.org/10.1016/j.scienta.2010.07.014
OIV, 2009. OIV descriptor list for grape varieties and Vitis
species (2nd ed). International Organisation of Vine and

Ancient olive genotypes of NW Spain
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e0702
15
Wine, http://www.oiv.int/oiv/info/enplubicationoiv#grape.
[28/04/2017].
Peakall R, Smouse PE, 2006. GENALEX 6: Genetic
analysis in Excel Population genetic software for teaching
and research. Mol Ecol Notes 6: 288-295. https://doi.
org/10.1111/j.1471-8286.2005.01155.x
Peakall R, Smouse PE, 2012. GenAlEx 65: Genetic analysis
in Excel Population genetic software for teaching and
research-an update. Bioinformatics 28: 2537-2539. https://
doi.org/10.1093/bioinformatics/bts460
Pereira-Lorenzo S, Fernandez-Lopez J, 1997. Description
of 80 genotypes and 36 clonal selections of chestnut
(Castanea sativa Mill) from Northwestern Spain. Fruit
Var J 51 (1): 13-27.
Pereira-Lorenzo S, Ramos-Cabrer AM, Díaz-Hernández,
MB, 2007. Evaluation of genetic identity and variation
of local apple cultivars (Malus x domestica Borkh.) from
Spain using microsatellite markers. Genet Resour Crop
Evol 54: 405-420. https://doi.org/10.1007/s10722-006-
0003-7
Pereira-Lorenzo S, dos Santos ARF, Ramos-Cabrer AM, Sau
F, Diaz-Hernandez MB, 2012. Morphological variation in
local pears from north-western Spain. Sci Hortic 138: 176-
182. https://doi.org/10.1016/j.scienta.2012.02.007
Rallo L, Barranco D, Caballero JM, Del Rio C, Martin A,
Tous J, Trujillo I, 2005. Variedades de olivo en España.
Coed. Junta de Andalucía, MAPA & Ed Mundi-Prensa,
Madrid.
Reboredo-Rodríguez P, 2015. Caracterización aromática
y fenólica de aceitunas y aceites de oliva producidos en
Galicia. Doctoral thesis. Univ. Vigo, Ourense, Spain.
Reboredo-Rodríguez P, González-Barreiro C, Cancho-
Grande B, Simal-Gándara J, 2014a. Improvements in the
malaxation process to enhance the aroma quality of extra
virgin olive oils. Food Chem 158: 534-545. https://doi.
org/10.1016/j.foodchem.2014.02.140
Reboredo-Rodríguez P, González-Barreiro C, Cancho-
Grande B, Simal- Gándara J, 2014b. Quality of extra
virgin olive oils produced in an emerging olive growing
area in north-western Spain. Food Chem 164 (1): 418-426.
https://doi.org/10.1016/j.foodchem.2014.05.043
Reboredo-Rodríguez P, González-Barreiro C, Cancho-
Grande B, Fregapane G, Salvador MD, Simal-Gándara
J, 2015. Characterisation of extra virgin olive oils from
Galician autochthonous varieties and their co-crushings
with Arbequina and Picual cv. Food Chem 176: 493-503.
https://doi.org/10.1016/j.foodchem.2014.12.078
Reboredo-Rodríguez P, González-Barreiro C, Cancho-
Grande B, Simal-Gándara J, Trujillo I, 2018. Genotypic
and phenotypic identication of olive genotypes from
north-western Spain and characterization of their extra
virgin olive oils in terms of fatty acid composition and
minor compounds. Sci Hort 232: 269-279. https://doi.
org/10.1016/j.scienta.2018.01.015
Sakar E, Unver H, Ercisli S, 2016. Genetic diversity among
historical olive (Olea europaea L) genotypes from
southern Anatolia based on SSR markers. Biochem
Genet 54 (6): 842-853. https://doi.org/10.1007/s10528-
016-9761-x
Salimonti A, Simeone V, Cesari G, Lamaj F, Cattivelli L,
Perri E, Desiderio F, Fanizzi FP, Del Coco L, Zelasco
S, 2013. A rst molecular investigation of monumental
olive trees in Apulia region. Sci Hortic 162: 204-212.
https://doi.org/10.1016/j.scienta.2013.08.005
Santiago JL, Boso S, Martín JP, Ortiz, JM, Martínez, MC,
2005. Characterization and identication of grapevine
genotypes (Vitis vinifera L) from northwestern Spain
using microsatellite markers and ampelometric
methods. Vitis 44 (2): 67-72.
Sarri V, Baldoni L, Porceddu A, Cultrera NGM, Contento
A, Frediani M, Belaj A, Trujillo I, Cionini PG,
2006. Microsatellite markers are powerful tools for
discriminating among olive genotypes and assigning
them to geographically dened populations. Genome
49 (12): 1606-1615. https://doi.org/10.1139/g06-126
Sefc KM, Lopes S, Mendonca D, Dos Santos MR,
Machado MLD, Machado AD, 2000. Identication of
microsatellite loci in olive (Olea europaea L) and their
characterization in Italian and Iberian olive trees. Mol
Ecol 9: 1171-1173. https://doi.org/10.1046/j.1365-
294x.2000.00954.x
Trujillo I, Ojeda MA, Urdiroz NM, Potter D, Barranco
D, Rallo L, Diez CM, 2014. Identication of the
Worldwide Olive Germplasm Bank of Córdoba
(Spain) using SSR and morphological markers. Tree
Genet Genomes 10: 141-155. https://doi.org/10.1007/
s11295-013-0671-3
Vargas-Gómez P, Talavera-Lozano S, 2012. Olea. In:
Flora Ibérica Plantas vasculares de la Península Ibérica
e Islas Baleares; Andrés C, Talavera S, Quintanar A
(eds.). Vol 11, pp: 136-139. Real Jardín Botánico, CSIC,
Madrid. http://www.oraiberica.org/ [29/04/2017].
Viñuales J, 2007. Variedades de olivo del Somontano.
Área de Desarrollo de la Diputación de Huesca,
Instituto de Estudios Altoaragoneses, Huesca, Spain.
Zohary D, Hopf M, Weiss E, 1994. Olive: Olea europaea.
In: Domestication of plants in the Old World, pp: 137-
143. Clarendon Press, Oxford, England.