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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.
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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
Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, O.A, M.P. (INIA)
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.
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:
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
Pilar Gago, José L. Santiago, Susana Boso and María C. Martínez
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e0702
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
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
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
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
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
C for
5 min, 35 cycles of 94oC for 20 s, 50-59oC for 30 s,
C for 30 s, and a nal extension at 72
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.
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
Pilar Gago, José L. Santiago, Susana Boso and María C. Martínez
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e0702
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-
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
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
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
227-253 6 1.000 0.800 0.070 0.770
160-206 7 1.000 0.810 0.061 0.785
130-178 4 0.900 0.745 0.113 0.697
243-263 3 0.500 0.585 0.262 0.495
122-171 6 1.000 0.795 0.072 0.765
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
dierence between mean leaves 5, 12 and 18 in Fig.
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
morphology to be dierent too, with the leaves
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
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
Pilar Gago, José L. Santiago, Susana Boso and María C. Martínez
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e0702
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
Ancient olive genotypes of NW Spain
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e0702
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
in peripheral growing areas; where only a small pro-
portion of those examined represented genotypes
a commercial use (Díez et al., 2011; Salimonti et
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-
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
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
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-
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.
6.23cm 6.06cm 4.36cm
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
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.
Over colour
at full
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
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
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
position Ac
position Bc
Position of the maxi-
mum transverse diamd
Shape of
the apexe
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
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.
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.
Shape of
the basee
of the
Number of
Distribution of
Presence of
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
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
full range of Galicia's olive tree diversity, followed by
agricultural studies that might indicate the potential of
the region's rediscovered genotypes.
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.
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... Éstos, si bien presentan un fenotipo similar al de variedades tradicionales bien definidas, poseen un comportamiento productivo diferente y estable a lo largo del tiempo (Prataviera, 1998;Diez et al., 2012). Este complejo panorama varietal, dificulta la caracterización e identificación indubitable de los cultivares y materiales nativos e impone para su clasificación un abordaje que considere aspectos tanto morfológicos como moleculares (Trujillo et al., 2014;Gago et al., 2019) para dilucidar además, los frecuentes casos de homonimia (mismo nombre varietal en genotipos diferentes) (Barranco et al., 2005) o sinonimia (diferentes nombres para genotipos idénticos) . El olivo es un ejemplo único dentro de la fruticultura debido a que su caracterización, identificación y discriminación entre variedades aún no ha sido totalmente esclarecida (Torkzaban et al., 2015). ...
... El análisis de marcadores moleculares junto al de caracteres morfológicos estructurados a partir de los descriptores cuantitativos y cualitativos del árbol, hoja, inflorescencia, fruto y endocarpo, han sido de gran utilidad para catalogar los cultivares de olivo depositados en el Banco Mundial de Germoplasma de Olivo (BMGO), sito en Córdoba, España y para la descripción y caracterización de genotipos nativos con denominación incierta o desconocida y de larga persistencia en el tiempo en las principales regiones olivícolas (Barranco et al., 2005;Gago et al., 2019 La Estación Experimental Agropecuaria del Instituto Nacional de Tecnología Agropecuaria (EEA-INTA) de la localidad de Sumalao, provincia de Catamarca (28° 33' 07,7" S, 65° 43' 40,3" O), en la región noroeste de Argentina (NOA), posee una colección de olivos que preserva entre otros materiales, clones derivados de plantas de identidad varietal reconocida pertenecientes al Banco de Germoplasma de la EEA-INTA Junín y genotipos autóctonos con aptitud agroecológica superior respecto a algunos caracteres tales como precocidad, vecería y tamaño de frutos, pero de origen genético incierto. ...
... Si bien, la ausencia de efecto ambiental sobre los marcadores microsatélites les confiere el doble de capacidad discriminante que la proporcionada por la variabilidad de los descriptores del endocarpo, su uso resultaría especialmente informativo en casos como éste donde la diferencia molecular resulta pequeña (Trujillo et al., 2014) por cuanto, dentro de los caracteres morfológicos, es el más discriminante y estable (Barranco et al., 2005). De igual manera, este carácter morfológico, así como otras variables botánicas cualitativas como las propuestas por Gago et al. (2019) aportaría información para el esclarecimiento de la posible sinonimia entre las accesiones Israelí de Catamarca y Mendoza con el cv. Sevilla de Mendoza, todos los cuales presentaron idéntico perfil de microsatélites. ...
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El olivo (Olea europaea L.) presenta un complejo panorama varietal aún no caracterizado e identificado totalmente. En este trabajo se estudió material con incertidumbre identitaria: se analizaron seis loci microsatélites de 13 accesiones relacionadas al cultivar Manzanilla de la colección del INTA Catamarca y de dos genotipos de huertos comerciales del departamento Cruz del Eje, Córdoba. Para confirmar la identidad de cultivares y la predominante base germoplásmica en genotipos de origen incierto, se compararon sus perfiles alélicos con los nueve cultivares referentes depositados en la colección del EEA INTA Junín (Mendoza), en el Banco Mundial de Germoplasma de Olivo (Córdoba, España) y con información disponible en OLEA databases. Con la composición alélica obtenida se generó una matriz de distancia genética que permitió estimar parámetros de diversidad y realizar análisis multivariados de ordenación y agrupamiento. Los resultados permitieron conocer la base genética de genotipos nativos, constatándose un elevado nivel de heterocigocidad. Se confirmó la identidad de la accesión Carmona y de dos variantes alélicas (Imperial y 4So.CE). Se detectó una posible sinonimia entre las accesiones Israelí y Fina y una homonimia para el cv. Española. Estos resultados contribuyen a la valorización y uso eficiente de recursos genéticos del olivo.
... In this sense, an emerging interest in the Spanish scientific community has been growing in the last few years. Several reports have been published, for example, (i) reporting the localisation of ancient olive trees in Galicia, their characterisation using botanical and molecular markers, and examining whether or not these trees represent unknown native genotypes [1]; (ii) implementing the scientific basis for the creation of a Protected Designation of Origin (PDO) [2]; (iii) evaluating the consumer acceptance of commercial EVOOs elaborated with autochthonous Galician cultivars [3]; and (iv) evaluating simple sequence repeats (SSR) and single-nucleotide polymorphism (SNP)-based methods in two autochthonous varieties, and their potential for integration in a microfluidic device [4]. ...
... At this point, it is important to note that the variety reported herein is genetically different from 'Mansa Gallega', which is one of the two Galician varieties currently recognized by the Spanish Department of Agriculture [42]. As stated before, extensive efforts are being put into the identification of other varieties [1,2]. Up to now, 20 varieties have been discovered and the process of protection following the UPOV (International Union for the Protection of New Varieties of Plants) system has already been initiated for some of them-i.e., 'Brava Gallega', 'Mansa Gallega', 'Brétema', 'Carapucho', 'Carmeliña', 'Folgueira', 'Hedreira', 'Maruxiña', 'Santiagueira', 'Susiña', and 'Xoana' [43]. ...
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In this work, the quality and physicochemical parameters, phenolic composition, and antidiabetic potential of olive oils obtained from olives belonging to centenarian olive trees of the so-called ‘Mansa de Figueiredo’ cultivar were evaluated during three consecutive crop seasons (2017–2019). The oils produced during the three crop years were classified as extra virgin based on the quality-related indices, sensory analysis, and the genuineness-related parameters. In addition, LC-ESI-TOF MS was used to get a comprehensive characterisation of the phenolic fraction while LC-ESI-IT MS was applied for quantitation purposes. The content of phenolic compounds (ranging from 1837 to 2434 mg/kg) was significantly affected by the harvest year due to the environmental conditions and ripening index. Furthermore, although significant differences in the inhibitory effects against the α-glucosidase enzyme for the EVOOs extracted throughout the three successive years were detected, all the studied EVOOs exhibited a stronger inhibitor effect than that found for acarbose.
... This varieties are mainly cultivated in Galicia, in the NW of Spain, where the area under olive cultivation increased from 10 ha in 2007 to 275 ha in 2019 (MAPA, 2019). Those particular varieties, although just a part of this total area, are highly appreciated and efforts towards the identification of other varieties are currently going on (Gago, Santiago, Boso, & Martínez, 2019). ...
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Miniaturization of DNA-based techniques can bring interesting advantages for food analysis, such as portability of complex analytical procedures. In the olive oil industry, miniaturization can be particularly interesting for authenticity and traceability applications, through in situ control of raw materials before production and/or the final products. However, variety identification is challenging, and implementation on miniaturized settings must be carefully evaluated, starting from the selected analytical approach. In this work, SSR- and SNP-based genotyping strategies were investigated for the identification and differentiation of two olive varieties from the Northwest of Spain. For the selected SNPs two genotyping methods were tested: real-time allele-specific PCR and high resolution melting analysis. These methods were compared and evaluated regarding their potential for integration in a microfluidic device. Both SNP-based methods proved to be successful for identification of the selected varieties, however real-time allele-specific PCR was the one that achieved the best results when analyzing mixtures, allowing the identification of both monovarietal samples and mixtures of the varieties tested with up to 25%.
... The high regenerative potential of the olive tree and its extensive longevity have determined the presence of centenary and millennial olive trees growing in various Countries. The presence of ancient olive trees, with monumental characters, is documented across the Mediterranean Basin where they represent an important naturalistic and historical heritage (Petruccelli et al. 2014;Laaribi et al. 2017;Adakalic and Lazović 2018;Ninot et al. 2018;Gago et al. 2019). In Italy, historical and/or monumental olive trees are present in all regions with an olive-growing vocation, such as Apulia, Sicily, Umbria, Tuscany and Emilia-Romagna (Cipriani et al. 2002;Ganino et al. 2007;Pannelli et al. 2010;Salimonti et al. 2013;Perrino et al. 2014;Rotondi et al. 2018). ...
The olive is a long-lived evergreen tree, capable of regenerating itself from any part of the plant (pollard tree). This characteristic has allowed the olive trees to survive across the centuries until they become real natural monuments. The aim of this work has been to study the monumental “Olivo della Strega” (“Witch’s Olive tree”) olive tree, located in the olive orchard of Santissima Annunziata church, in Magliano (Grosseto, Italy), a tree with a special place in local history and folklore and with estimated age over 700 years. The morphometric data of leaf, fruit, stone and pollen were used to characterize the phenotype, while genetic diversity was assessed on the basis of microsatellite markers. The morphological and genetic analysis pointed to two clearly distinguishable parts in the “Olivo della Strega” tree, called sample left (SL) and sample right (SR). This ancient plant consists of two different genotypes: one part (SL) was identified as cv Frantoio and the other (SR) as an unknown genotype. Moreover, traditional (in vivo conservation) and advanced techniques (cryopreservation) have made conservation achievable to preserve its genetic heritage. In particular “Olivo della Strega” pollen was stored for 1 year in liquid nitrogen. The morphology of pollen grains, viability and germinability of fresh and long term cryopreserved pollen were observed at different times during preservation, without revealing significant differences.
... However, a recent study carried out between 2012 and 2018 by the Superior Council of Scientific Investigations (CSIC) discovered the existence of 13 autochthonous Galician varieties, as the olive cultivation was highly extended in this region during the sixteenth century. The main autochthonous varieties are the Galician ''Brava'' and the Galician ''Mansa'' (Gago et al. 2019). ...
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An increase in the cultivation of olive trees was prompted in the Northwestern Spain during recent years favored by the agricultural politics in the region. The aim of this study is to know the phenological and aerobiological behavior of the olive trees at the northern limit of the Mediterranean bioclimatic area in order to determine their thermal requirements for the development of predictive agro-phenological models for two varieties (Arbequina and Frantoio) of the new olive cultivated areas. The BBCH scale was used for the phenological observations. Chill accumulation during dormancy and heat requirements to overcome the successive phenological stages were calculated following different methods. To complete the phenology predictive models, the Mitscherlich’s monomolecular equation was applied. The monitoring of the atmospheric pollen content was conducted by means a volumetric pollen Hirst trap placed in the middle of the plot. The period of chilling accumulation was completed during the first fortnight of January with an average of 654 chilling hours and a base temperature of 7.5 °C. Considering the duration of the vegetative olive cycle around 2390°GDD (growing degree days) was necessary to overcome the ripening of berries. The proposed model for the prediction of the successive ecological events showed a deviation between 1.5 and 3.5 days on the phenological scale. The presence of Olea pollen grains in the atmosphere of the olive grove was registered during stage 5 and stage 6 (flowering). The Hybrid Single Particle Lagrangian Integrated Trajectory Models confirmed that pollen peaks during the previous days to the flowering stage came from the extensive olive groves of the Northern Portugal which flowers some days in advance.
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Olive (Olea europaea) is an ancient and important crop in both olive oil production and table use. It is important to identify the genetic diversity of olive genetic resources for cultivar development and evaluation of olive germplasm. In the study, 14 microsatellite markers (UDO4, UDO8, UDO9, UDO11, UDO12, UDO22, UDO24, UDO26, UDO28, DCA9, DCA11, DCA13, DCA15, and DCA18) were used to assess the genetic variation on 76 olive (Olea europaea L.) genotypes from Mardin province together with 6 well-known Turkish and 4 well-known foreign reference cultivars. All microsatellite markers showed polymorphism and the number of alleles varied between 9 and 22, with an average of 14.57. The most informative loci were DCA 11 (22 alleles) and DCA 9 (21 alleles). Dendrogram based on genetic distances was constructed for the 86 olive genotypes/cultivars, which revealed the existence of different clusters. The high genetic similarity was evident between Bakırkire2 and Zinnar5 (0.74) genotypes, while the most genetically divergent genotypes were Gürmeşe5 and Yedikardeşler2 (0.19). It was concluded that there was abundant SSR polymorphism in olive germplasm in southern Anatolia in Turkey and could be important for future breeding activities.
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The conservation of cultivated plants in ex-situ collections is essential for the optimal management and use of their genetic resources. For the olive tree, two world germplasm banks (OWGB) are presently established, in Córdoba (Spain) and Marrakech (Morocco). This latter was recently founded and includes 561 accessions from 14 Mediterranean countries. Using 12 nuclear microsatellites (SSRs) and three chloroplast DNA markers, this collection was characterised to examine the structure of the genetic diversity and propose a set of olive accessions encompassing the whole Mediterranean allelic diversity range. We identified 505 SSR profiles based on a total of 210 alleles. Based on these markers, the genetic diversity was similar to that of cultivars and wild olives which were previously characterised in another study indicating that OWGB Marrakech is representative of Mediterranean olive germplasm. Using a model-based Bayesian clustering method and principal components analysis, this OWGB was structured into three main gene pools corresponding to eastern, central and western parts of the Mediterranean Basin. We proposed 10 cores of 67 accessions capturing all detected alleles and 10 cores of 58 accessions capturing the 186 alleles observed more than once. In each of the 10 cores, a set of 40 accessions was identical, whereas the remaining accessions were different, indicating the need to include complementary criteria such as phenotypic adaptive and agronomic traits. Our study generated a molecular database for the entire OWGB Marrakech that may be used to optimise a strategy for the management of olive genetic resources and their use for subsequent genetic and genomic olive breeding. Electronic supplementary material The online version of this article (doi:10.1007/s10709-011-9608-7) contains supplementary material, which is available to authorized users.
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Vineyards in the Asturias Princedom (in the north of Spain) are on the verge of extinction. Their existence has been recorded since the 9th century, and they are as old as other prominent vineyards in Spain and Europe. Some autochthonous grapevine cultivars of this region have never been described. Well-known winegrape cultivars are also cultivated together with these autochthonous cultivars. The former were introduced after the phylloxera crisis in the late 19th century. For the past 10 years, vineyards in Cangas del Narcea (heart of the Asturian vine-growing area) have been studied. Four white-berried cultivars (Albarin blanco, Albillo, Blanca extra, and Moscatel blanco), eight black-berried cultivars (Albarin francés, Albarin negro, Cabernet, Carrasco, Garnacha tintorera, Mencía, Negrona, and Verdejo negro), one rose-berried cultivar (Jaen), and one red-berried cultivar (Moscatel rojo) were found. The average leaf of each of these cultivars was graphically reconstructed. An ampelographic description of the young shoot, the adult leaf, the cluster, the berry, and the seeds was also carried out using the descriptor list for grapevine cultivars and Vitis species of the OIV. Other parameters considered appropriate for a full description of these cultivars were also measured.
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Most traditional olive-producing countries possess a diversified genetic patrimony in Olea europaea L. Since the emergence ofmodern olive growing system, the identification, classification, and conservation of autochthonous olive cultivars is a priority for these countries. In this work, a total of 84 accessions belonging to the "Boughrara"-Sfax olive germplasm collection located in Tunisia have been screened using a powerful set of eight simple sequence repeat markers (SSRs). The study revealed a high genetic variability among the collection and detected a total of 64 alleles. For better management of the mentioned germplasm bank, an improved classification of the entries, including new denominations, has been proposed. In addition, several cases of mislabeling, synonymy, and homonymy have been clarified. Genetic relationships among cultivars have been analyzed showing four major clusters. Finally, a correspondence factor analysis demonstrated that cultivars tend to cluster depending on their main use as oil or table olives. No clear clustering tendencies were observed when the geographical origin of cultivars was used as the criteria for the analysis. All results obtained by SSR screening and classification were in accordance with classification based on morphological traits of fruit endocarps.
Background and Aims Numerous descriptions have been made of the wines produced in the past, however, the grapevine genotypes have been poorly recorded. Naming errors are also common, a problem that could negatively impact scientific work and cause economic damage to wine markets. The aim of the present work was to compare the accessions from Galicia and Asturias held in the Misión Biológica de Galicia‐Consejo Superior de Investigaciones Científicas and El Encín‐Instituto Madrileño de Investigación y Desarrollo Rural, Agrario y Alimentario collections in Spain in order to resolve synonyms, homonyms and naming errors in both. Materials and Methods The accessions of these collections were compared in terms of their leaf and bunch ampelographic characteristics, via simple sequence repeat analysis and via the examination of old photographs and other historic documentation. Results The two collections were found to hold 59 distinct genotypes from the two regions. Ten were unique to the Misión Biológica de Galicia‐Consejo Superior de Investigaciones Científicas collection and four to the El Encín‐Instituto Madrileño de Investigación y Desarrollo Rural, Agrario y Alimentario collection – rather fewer than expected. Traces of intra‐genotype variation were seen for some genotypes. Conclusions The comparison of the accessions allowed some problems of genotype identity and origin to be identified and resolved. The possible intra‐genotype variation detected should be examined in greater depth. The results also highlight the value of well documented live collections, gathered at different times and curated in different places, for this type of work, as well as in the conservation of world viticultural heritage. Significance of this Study These findings open up paths for future research on the material conserved, and its commercial exploitation.
Galicia (NW Spain) is emerging as a new olive-growing region. Galician oil producers are currently striving to recover old autochthonous cultivars with a view to obtaining high quality extra virgin olive oil (EVOO). In this work, a total of 32 trees were studied in order to established their identity and genetic relationships to the main cultivated material in the Iberian Peninsula. The analysis of 11 morphological features of the endocarp and 14 microsatellite markers allowed three different cultivars to be identified among the sampled trees. Comparison with the morphological and molecular profiles available in the World Olive Germplasm Bank of Cordoba (WOGBC) revealed that 24 trees (75%) were of the 'Brava' cultivar and 7 (22%) of the 'Mansa' cultivar. The other tree, labelled as Picuda, matched no specific cultivar in WOGBC. Characterizing the oils obtained from the studied cultivars revealed a high potential for producing high-quality EVOOs of specific origin.