Scientia Horticulturae xxx (xxxx) xxx–xxx

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Genotypic and phenotypic identification of olive cultivars from north-

western Spain and characterization of their extra virgin olive oils in terms of
fatty acid composition and minor compounds
⁎
Patricia Reboredo-Rodrígueza, , Carmen González-Barreiroa, Beatriz Cancho-Grandea,
⁎
Jesús Simal-Gándaraa, Isabel Trujillob,
a
Nutrition and Bromatology Group, Department of Analytical and Food Chemistry, Faculty of Sciences, University of Vigo, Ourense Campus, E-32004, Ourense, Spain
b
Agronomy Department, University of Córdoba – International Campus of Excellence on Agrofood (ceiA3), Rabanales Campus, C4 Building, E-14014, Córdoba, Spain

A R T I C L E I N F O A B S T R A C T

Keywords: Galicia (NW Spain) is emerging as a new olive-growing region. Galician oil producers are currently striving to
Olea europaea L. recover old autochthonous cultivars with a view to obtaining high quality extra virgin olive oil (EVOO). In this
SSR markers work, a total of 32 trees were studied in order to established their identity and genetic relationships to the main
Germplasm characterization cultivated material in the Iberian Peninsula. The analysis of 11 morphological features of the endocarp and 14
Genetic diversity
microsatellite markers allowed three different cultivars to be identified among the sampled trees. Comparison
Extra virgin olive oil
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.

1. Introduction
Virgin olive oil (VOO) is the principal source of fat in the
Mediterranean diet and highly appreciated by consumers for its healthy
effects and sensory properties (Aparicio and Harwood, 2003). Spain
ranks first in the world in olive grove area: 2 584 564 ha, which ac-
counts for about 45% and 60% of all olive production in the world and
the European Union, respectively. The average Spanish production
from 2007/08 to 2012/13 was 1 215 798t, with a record of 1 615
million in the 2011/12 year (Magrama, 2017). The main olive-growing
areas in Spain in terms of production are in the south (Andalusia,
60.4%), centre (Extremadura, 10.2%; Castilla–La Mancha, 15.8%) and
northwest (Catalonia, 4.6%; Valencia, 3.7%; Aragón 2.3%). Per capita
olive oil consumption in Spain is 9.6 L per year (Martín Cerdeño, 2015).
Galicia (NW Spain), where annual per capita olive oil consumption
is 11.4 L (Martín Cerdeño, 2015), is emerging as a new Spanish olive-
growing region and a producer of extra virgin olive oil (EVOO). There
are two different policies currently in effect to boost the olive sector in
Galicia. Thus, some major olive oil producers have focused on pro-
moting Spanish varieties widely used in the world (e.g., Arbequina,
Picual) in new plantations. These varieties feature a high pedoclimatic
adaptability and productivity. Thus, the Arbequina cultivar adapts ea-
sily to extremely dense olive groves, and provides early entry into
production, increased productivity and the ability to use modified
mechanical vine harvesters (Proietti et al., 2015). Other producers,
however, have focused on recovering old autochthonous cultivars from
Galicia to obtain high-quality EVOO with specificity of origin, and
special, distinctive sensory, nutritional and health-promoting properties
(Reboredo-Rodríguez et al., 2016a,b). These two policies represent two
different ways of competing on the EVOO market, namely: competi-
tiveness on cost (the former approach) and competitiveness on sensory style
(the latter) (Ilarioni and Proietti, 2014). In the former approach, pro-
duction is geared towards super-intensive cultivation and highly me-
chanized methods; in the latter, traditional methods are favoured
(Ilarioni and Proietti, 2014). In theory, autochthonous cultivars should
be able to adapt to super high-density olive groves for improved pro-
duction while maintaining sensory differences among oils (Proietti
et al., 2012).
Initially, olive cultivars were named for their outstanding morpho-
logical traits or utility of production. Denominations are also frequently
based on the place of origin of the propagating material (Rallo, 2005).
As a result, synonyms (i.e., different names for the same cultivar) and

⁎
Corresponding author.
E-mail addresses: preboredo@uvigo.es (P. Reboredo-Rodríguez), cargb@uvigo.es (C. González-Barreiro), bcancho@uvigo.es (B. Cancho-Grande), jsimal@uvigo.es (J. Simal-Gándara),
ag2trnai@uco.es (I. Trujillo).

https://doi.org/10.1016/j.scienta.2018.01.015
Received 4 July 2017; Received in revised form 7 November 2017; Accepted 9 January 2018
0304-4238/ © 2018 Elsevier B.V. All rights reserved.

Please cite this article as: Reboredo-Rodríguez, P., Scientia Horticulturae (2018), https://doi.org/10.1016/j.scienta.2018.01.015
P. Reboredo-Rodríguez et al.
homonyms (i.e., the same name for different cultivars) are extremely
frequent among and within olive-growing countries (Barranco et al.,
2000). Accurate varietal identification is therefore in order before
specific olives are preserved and used by growers and producers. Sev-
eral studies conducted in recent decades have allowed the main culti-
vated materials within and among countries to be identified and cata-
logued (Barranco et al., 2000; Barranco and Trujillo, 2000; Barranco
et al., 2005; Belaj et al., 2003a, 2003b, 2007, 2012). The diversity of
synonyms and homonyms for olives is illustrated by the facts that Lavee
(1994) have identified more than 2000 varieties and Bartolini et al.
(1998) have documented more than 1200 autochthonous varieties with
more than 3000 names. In Galicia, the olive cultivated material is
known widely for its homonyms (generic names): Brava and Mansa. No
systematic characterization of this material in the Galician region ap-
pears to have been undertaken so far, however.
Recently, a useful protocol for characterizing, identifying and au-
thenticating an olive germplasm bank was established on the basis of
morphological and molecular (SSR marker) traits. The protocol was
used to set up the World Olive Germplasm Bank of Cordoba (WOGBC),
Spain (Trujillo et al., 2014). This is one of the world’s largest collections
and currently includes more than 500 olive accessions from 21 coun-
tries (Trujillo et al., 2014). The protocol also allowed a database in-
cluding 500 morphological profiles and 332 SSR profiles of potential
use for new studies on olives to be created.
Olive cultivars are known to influence the composition of the re-
sulting olive oils, especially with regard to minor compounds such as
phytosterols, which have nutritional and health effects; volatile com-
pounds, which are responsible for oil aroma; and phenolic compounds,
which have been associated to taste and healthy properties (Angerosa
et al., 2004; Gómez-Rico et al., 2008; Inarejos-García et al., 2011).
Kyçyk et al. (2015) recently showed the high genetic significance of
sterol composition and total sterol, which may be especially useful for
new olive breeding projects intended to obtain new cultivars with an
improved VOO sterol fraction. Also, Zarrouk et al. (2009) established a
classification of eighteen Mediterranean olive varieties based on the
abundance of major compounds, fatty acid composition and, more
specifically, the monounsaturated/polyunsaturated fatty acid (MUFA/
PUFA) and oleic/linoleic acid (C18:1/C18:2) ratios.
The primary aim of this work was to promote the use of auto-
chthonous olive cultivars from Galicia and their monovarietal oils.
Available knowledge about the characteristics of Galician olive oils is
scant. In previous work, we characterized VOOs obtained by mixing
Brava and Mansa fruits in different proportions similar to those used by
producers (Reboredo-Rodríguez et al., 2016a, 2016b) and also in mix-
tures with Picual and Arbequina fruits (Reboredo-Rodríguez et al.,
2015). In this work, monovarietal EVOOs from ‘Brava’ and ‘Mansa’
fruits were studied. This required identifying and classifying the ma-
terial of both varieties cultivated in Galicia in terms of morphological
endocarp traits and microsatellite markers. Also, in order to better
understand the peculiarities of the resulting autochthonous mono-
varietal olive oils, these were characterized for quality, stability and
chemical composition, which are associated to the nutritional, func-
tional and sensory properties of the oil. To our knowledge, this is the
first time these characteristics of monovarietal autochthonous olive oils
have been determined, so they may serve as a basis for identifying si-
milarities and differences between cultivars in the future.
2. Materials and methods
2.1. Characterization of cultivars
2.1.1. Plant material
Varieties were identified during the crop year 2014/15 in 11 olive
trees of the Brava, 20 of the Mansa and 1 of the Picuda cultivar. The
studied material was found at different locations in the valley of River
Sil (Lugo, Galicia). Although Galician climate is essentially oceanic and
2
Scientia Horticulturae xxx (xxxx) xxx–xxx
Table 1
Climatological conditions of the studied area over the period 2014–2016 (Source:
MeteoGalicia, 2017).
Climatological conditions
Year R(L/m2) T (°C) TCT7 (days) RH (%) TGR (kJ/m2)
2014 991.1 12.1 8 80.9 15307
2015 670.8 12.6 19 77.0 16577
2016 1097 12.2 15 81.3 17525
R, total rainfall; T, mean air temperature; TCT7, total cold time (T < 7 °C); RH, mean air
relative humidity; TGR, total global radiation.
hence highly rainy (Carballeira et al., 1983), the study area has the
typical climate of the Mediterranean oceanic domain. Table 1 sum-
marizes the climatic conditions for the area over a period of three years
including the crop year (i.e., 2014/16). The rocks in the area are si-
liceous (granites, schists and slates) and under deep soils (particularly
cambisols) (Díaz-Maroto and Vila-Lameiro, 2006). Each tree was given
a unique identifier including the name “UVIGO” and two numbers from
01 to 32 corresponding to the entry order in the material collection
(Table 2b). Putative local olive varieties from Galicia were related to
other olive varieties by using molecular data for the main cultivated
material in the Iberian Peninsula, including 23 Spanish cultivars and 6
Portuguese cultivars reported by Trujillo et al. (2014) (Table 3).
2.1.2. DNA extraction and SSR analysis
Total genomic DNA was extracted from fresh leaves by using the
cetyltrimethylammonium bromide (CTAB) method of Murray and
Thompson (1980) as modified by de la Rosa et al. (2002). Fourteen
olive microsatellite markers including ssrOeUA-DCA3, ssrOeUA-DCA9,
ssrOeUA-DCA11, ssrOeUA-DCA15, ssrOeUA-DCA16 and ssrOeUA-
DCA18 (Sefc et al., 2000); UDO99-011, UDO99-019, UDO99-024 and
UDO99-043 (Cipriani et al., 2002); and GAPU59, GAPU71B, GAPU101
and GAPU103A (Carriero et al., 2002; Cipriani et al., 2002; Sefc et al.,
2000) were examined (Table 2a). All were previously found to be
highly efficient for olive cultivar identification (Baldoni et al., 2009;
Trujillo et al., 2014).
SSR amplification was done in a total volume of 20 μL containing
2 ng genomic DNA, 1 x supplied PCR buffer (Biotools, Spain), 1.5 mM
MgCl2, 200 μM dNTPs (Roche), 0.025 U/μL Taq polymerase (Biotools,
Spain) and 0.2 μM forward primer (fluorescently labelled) and reverse
primer.
Polymerase chain reactions (PCRs) were conducted in a thermal
cycler (GeneAmp PCR system 9600, Applied Biosystems, Foster City,
CA, USA) using the following sequence: initial denaturation at 95 °C for
5 min and 35 cycles with three steps (95 °C for 20 s for denaturation,
50–52 °C depending on the primer combination for 30 s for annealing,
and 72 °C for 30 s for extension). A final extension step at 72 °C for
8 min was also applied. PCR products were separated in an automatic
capillary sequencer (an ABI Prism 3100-Avant Genetic Analyser from
Applied Biosystems), using appropriate fluorescent dyes. Fragment
sizes were determined by using the internal standard GeneScan 400 HD-
Rox. The Frantoio and Picual cultivars were used as controls in all runs.
Amplified fragments were analysed and scored using the software
GeneMapper 3.0 and GenoTyper 3.7 from Applied Biosystems. A nu-
meric code (1–3) was assigned to each SSR profile (Table 2b).
2.1.3. Morphological traits
The putative cultivars defined by the molecular markers were sup-
plemented with 11 characters of the endocarp, namely: weight, length/
width ratio, symmetry at position A, symmetry at position B, position of
the maximum transversal diameter B, shape of apex at position A,
shape of base at position A, surface roughness, number of grooves,
distribution of grooves on the basal end and presence of mucron
(Table 4). These traits are the most discriminating and stable —other
 Table 2
(a) Size range (base pairs), number of alleles (Na) and number of allele patterns (Np) defined at each of the 14 SSRs assessed. (b) Tree code, tree name, molecular characterization and identification of the 32 trees with the 14 SSR used.
P. Reboredo-Rodríguez et al.

(a) LOCUS ssrOeUA- ssrOeUA- ssrOeUA- ssrOeUA- ssrOeUA- ssrOeUA- UDO99-011 UDO99-019 UDO99-024 UDO99-O43 GAPU59 GAPU71B GAPU101 GAPU103A Total
DCA3 DCA9 DCA11 DCA15 DCA16 DCA18

Size range 237–251 160–204 140–178 243–254 124–171 166–176 119–134 129 164–185 172–216 210–220 127–141 191–217 133–184
Na. 4 4 3 3 3 3 3 1 2 3 2 2 2 2 37
Np 2 3 2 3 2 2 2 1 2 2 1 1 1 2 26

(b) Tree Code Tree ssrOeUA- ssrOeUA- ssrOeUA- ssrOeUA- ssrOeUA- ssrOeUA- UDO99-011 UDO99-019 UDO99-024 UDO99-O43 GAPU59 GAPU71B GAPU101 GAPU103A 14 SSR
Name* DCA3 DCA9 DCA11 DCA15 DCA16 DCA18 CODE

UVIGO01 Brava 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO02 Brava 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO03 Brava 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO04 Brava 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO05 Brava 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO06 Brava 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO07 Brava 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO08 Brava 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO09 Brava 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO10 Brava 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1

3
UVIGO11 Brava 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO12 Mansa 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO13 Mansa 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO14 Mansa 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO15 Mansa 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO16 Mansa 243/247 160/160 160/178 263/263 124/152 168/176 127/134 129/129 185/185 172/216 210/220 127/141 191/217 184/184 2
UVIGO17 Mansa 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO18 Mansa 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO19 Mansa 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO20 Mansa 243/247 160/160 160/178 263/263 124/152 168/176 127/134 129/129 185/185 172/216 210/220 127/141 191/217 184/184 2
UVIGO21 Mansa 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO22 Mansa 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO23 Mansa 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO24 Mansa 243/247 160/160 160/178 263/263 124/152 168/176 127/134 129/129 185/185 172/216 210/220 127/141 191/217 184/184 2
UVIGO25 Mansa 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO26 Mansa 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO27 Mansa 243/247 160/160 160/178 263/263 124/152 168/176 127/134 129/129 185/185 172/216 210/220 127/141 191/217 184/184 2
UVIGO28 Mansa 237/251 182/192 140/178 243/254 124/152 166/176 119/134 129/129 164/185 172/204 210/220 127/141 191/217 133/133 1
UVIGO29 Mansa 243/247 160/160 160/178 263/263 124/152 168/176 127/134 129/129 185/185 172/216 210/220 127/141 191/217 184/184 2
UVIGO30 Mansa 243/247 160/160 160/178 263/263 124/152 168/176 127/134 129/129 185/185 172/216 210/220 127/141 191/217 184/184 2
UVIGO31 Mansa 243/247 160/160 160/178 263/263 124/152 168/176 127/134 129/129 185/185 172/216 210/220 127/141 191/217 184/184 2
UVIGO32 Picuda 237/253 160/204 160/178 243/263 152/171 168/176 127/134 129/129 185/185 172/216 210/220 127/141 191/217 184/184 3

*tree name used by the local producers.

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 P. Reboredo-Rodríguez et al.

Table 3
Molecular profile of each variety previously examined by Trujillo et al. (2014).

Cultivar Name Cultivation Area SSR ssrOeUA- ssrOeUA- ssrOeUA- ssrOeUA- ssrOeUA- ssrOeUA- GAPU59 GAPU 71B GAPU 101 GAPU 103 UDO99-011 UDO99-019 UDO99-024 UDO99-043
1
Code DCA3 DCA9 DCA11 DCA15 DCA16 DCA18

Alfafara SP 299 243/247 192/204 160/178 243/243 152/173 168/176 210/220 118/127 191/217 133/133 119/134 129/129 164/185 172/216
Arbequina. SP 33 229/241 182/204 140/178 243/243 122/144 164/174 206/220 121/141 183/205 147/157 116/129 129/153 201/201 175/175
Blanqueta SP 3 227/237 178/204 126/178 243/243 144/148 174/176 220/220 121/141 183/197 157/171 103/125 129/129 185/187 175/216
Carrasquenho de PRT 204 237/251 192/204 140/160 263/263 152/173 168/172 210/210 118/121 191/217 184/184 116/119 129/129 164/185 210/216
Elvas
Castellana SP 316 243/251 160/182 160/178 254/254 124/152 166/176 210/210 127/141 191/199 184/184 119/134 129/129 164/185 208/212
Changlot Real SP 313 243/253 184/204 160/178 254/254 122/154 166/176 210/220 121/121 191/197 147/147 127/134 129/129 164/187 216/216
Çobrancosa PRT 177 237/251 160/204 140/178 243/254 122/124 166/176 210/220 121/141 191/219 133/133 119/134 129/129 185/185 208/216
Cordovil de Castelo PRT 317 243/251 160/182 160/178 263/263 152/173 168/176 210/220 118/121 191/217 133/133 116/119 129/129 185/185 172/212
Branço
Cordovil de Serpa PRT 336 243/251 160/206 140/160 254/254 152/173 168/172 210/220 127/141 191/199 184/184 127/134 129/129 164/185 208/212
Cornicabra SP 382 237/247 182/192 160/178 243/263 122/124 168/176 210/210 121/141 191/199 184/184 127/134 129/129 185/185 172/212
Empeltre SP 220 241/243 184/204 140/178 243/243 144/152 166/176 210/216 121/124 191/197 147/171 114/125 129/129 183/185 187/216
Farga SP 112 237/243 170/184 126/140 243/243 122/148 162/166 206/220 121/124 189/191 157/176 114/125 129/153 183/201 214/216
Galega Vulgar PRT 212 237/251 190/192 126/178 243/243 163/179 168/176 210/222 118/124 183/199 157/184 119/131 129/143 179/185 170/172

4
Gordal Sevillana SP 289 247/251 160/192 160/178 243/243 124/173 172/176 210/210 118/141 199/217 133/184 119/127 129/129 185/185 172/212
Hojiblanca SP 161 237/247 192/204 140/178 243/254 124/152 168/176 210/220 121/141 197/199 147/186 119/134 129/129 185/185 208/216
Lechín de Granada SP 114 237/243 182/204 140/178 254/254 122/152 166/168 210/220 121/127 191/197 147/147 116/134 129/129 164/164 212/216
Lechín de Sevilla SP 261 243/247 160/204 140/178 243/254 124/144 164/172 210/210 121/141 191/217 133/155 119/131 129/129 185/185 172/177
Manzanilla SP 169 237/251 160/182 140/160 254/254 122/124 168/172 210/210 118/127 197/217 133/133 116/119 129/129 185/185 172/216
Cacereña
Manzanilla de SP 322 243/251 160/204 140/160 254/254 152/173 168/176 210/210 121/141 197/217 133/147 119/134 129/129 164/185 210/214
Sevilla
Morisca SP 282 243/247 182/192 140/160 254/254 152/173 166/172 210/220 118/127 191/199 147/184 127/134 129/129 185/185 206/212
Morrut SP 409 241/243 184/206 146/146 243/243 148/175 168/180 210/220 124/127 183/205 147/159 103/119 129/129 164/185 172/175
Picual SP 146 237/247 182/190 140/176 243/254 124/152 166/172 210/220 118/127 191/217 133/133 116/119 129/129 185/185 208/212
Picudo SP 352 243/251 182/192 140/182 263/263 152/173 168/172 210/220 118/121 197/217 133/147 119/134 129/129 164/185 208/216
Sevillenca SP 167 237/251 160/180 130/160 243/243 152/173 170/172 210/227 124/141 183/199 133/157 119/131 129/159 177/185 208/214
Verde Verdelho PRT 5 227/243 182/192 160/178 263/263 144/173 166/172 210/210 118/121 191/199 157/184 116/119 129/129 179/179 208/212
Verdial de Badajoz SP 307 243/253 160/182 160/178 263/263 152/173 168/176 210/220 118/121 191/217 133/133 116/119 129/129 185/185 172/212
Verdial de Huévar SP 144 237/247 182/192 140/160 263/263 152/175 168/176 210/220 118/121 191/199 184/184 116/119 129/129 164/185 172/212
Verdial de Vélez- SP 218 241/243 174/192 146/178 243/243 152/175 168/172 210/220 124/141 191/199 157/190 116/134 97/129 164/164 172/216
Málaga
Villalonga SP 373 251/253 160/180 140/178 243/243 144/173 172/176 206/210 118/121 md* 147/157 119/125 129/129 185/185 172/216

*md Missing datum.

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P. Reboredo-Rodríguez et al. Scientia Horticulturae xxx (xxxx) xxx–xxx

Table 4
Tree code and tree name, morphological characteristics and morphological identification code of endocarps in the 8 trees examined.

Tree Code Tree Name* Morphological characteristics of the endocarps Morph. Code12

Weigth1 Shape.A2 Symm.A3 Symm. B4 T Diam.B5 Apex.A6 Base. A7 Rugosity8 N° grooves9 Dist. Grooves10 Mucro11

UVIGO05 Brava M EP SA S C P P-R R M R P 1

UVIGO07 Brava M EP SA S C P P-R R M R P 1
UVIGO14 Mansa M EP SA S C P P-R R M R P 1
UVIGO16 Mansa M EP SA SA B R P R M R P 2
UVIGO20 Mansa M EP SA SA B R P R M R P 2
UVIGO29 Mansa M EP SA SA B R P R M R P 2
UVIGO31 Mansa M EP SA SA B R P R M R P 2
UVIGO32 Picuda M EP SA SA B P P SC M G P 3

* Tree name used by the local producers.

1
Weight: medium = M (0.30–0.45 g).
2
Shape in position A: elliptic = EP (length/width 1.8–2.2); elongated = EL (length/width > 2.2).
3
Symmetry in position A: slightly asymmetric = SA.
4
Symmetry of position B: symmetric = S; slightly asymmetric = SA.
5
Position of the maximum transversal diameter in position B: towards base = B; central = C.
6
Shape of apex in position A: pointed = P; rounded = R.
7
Shape of base in position A: pointed = P; truncated = T; rounded = R.
8
Rugosity of surface: rough = R; scabrous = SC.
9
Number of grooves on basal end: M (7–10).
10
Distribution of grooves on basal endo: regular = R; grouped around the suture = G.
11
Presence of mucro: present = P.
12
Numeric code assigned to different morphological profiles (1–3).

characteristics such as those of the fruit are more strongly influenced by
the environmental conditions (Trujillo et al., 2014). Moreover, en-
docarps may be conserved for a long time and can be easily exchanged
among collections. For these reasons, endocarp descriptions are fre-
quently used to catalogue olive cultivars (Barranco et al., 2000, 2005;
Fendri et al., 2010; D’Imperio et al., 2011) and large collections of olive
germplasm (Trujillo et al., 2014).
A representative sample of 40–50 endocarps per tree was char-
acterized in the cultivars ‘Brava’ (3 trees), ‘Mansa’ (4 trees) and ‘Picuda’
(1 tree). Only those trees with acceptable production levels over the
studied period were used for this purpose. The morphological profile of
each sample was the combination of its level of expression for each of
the 11 endocarp traits assessed. As before, a numeric code (1–3) was
assigned to each morphological profile (Table 4).
2.1.4. Identification and relationships among cultivars
The previously discriminated cultivars were identified and authen-
ticated by comparing their profiles with the SSRs and morphological
profiles present in the database produced during the varietal catalo-
guing of WOGBC (Spain; Trujillo et al., 2014), as well as from catalo-
guing studies on material recently introduced in the bank (data avail-
able on request from the authors). Collected and identified samples
were assigned a name according to the criteria proposed in several
studies on olive varietal cataloguing (Barranco and Rallo, 1984;
Barranco et al., 2000, 2005; Trujillo et al., 2014), and to the Interna-
tional Code of Nomenclature for Cultivated Plants (Brickell et al.,
2009), as follows: (a) a letter in italics for the initial name of the sample
prior to identification; and (b) a name between quotation marks for
each cultivar after molecular and morphological identification.
Genetic relationships among cultivars were examined by using a
matrix containing SSR profiles only, with amplified alleles scored as
present (1) or absent (0). The matrix was subjected to cluster analysis
based on the UPGMA algorithm by using Dice’s similarity index (Dice,
1945) as implemented in the statistical software NTSYS-pc v. 2.02
(Rohlf, 1998). The goodness of fit of the analysis was assessed through
the correlation coefficient between the similarity matrix and that of
cophenetic values.
2.2. Characterization of olive oils
2.2.1. Oil samples
Olives were harvested in November 2016 in a growing area located
between the municipalities of Ribas do Sil (42° 27′59.8″ N, 7°
17′15.8″W) and Quiroga (42° 29′04.8″ N, 7° 12′33.4″W) in the province
of Lugo (NW Spain). All olive fruits were obtained under organic
agricultural practices. The trees were in dry condition, and their nu-
tritional and sanitary status was controlled by subscriber and sanitary
techniques suitable for cultivation in the studied area.
Two monovarietal olive oils were separately obtained from the
‘Brava’ and ‘Mansa’ cultivars by using an Oliomio 50 processing ma-
chine (Mori-Tem, Tavarnelle Val di Pesa, Italy) equipped with a knife
crusher, a horizontal malaxator and a two-phase decanter. Malaxation
trials were carried out at 18 ± 2 °C for 45 min on one day for ‘Brava’
and the next for ‘Mansa’. Although some authors use data for a three-
year period to avoid the impact of climate on the results for mono-
varietal oil, the climatological conditions in the studied area in 2016
were very similar to those of the previous two years (see Table 1).
Therefore, the slight differences between years are expected to have
had no substantial effect on the physico–chemical properties or che-
mical composition of the oils —rather, cultivar was the determining
factor.
Once in the laboratory, samples were kept at a constant temperature
of 6 °C in amber bottles without headspace until analysis.
2.2.2. Quality-related parameters for classifying olive oils
Free acidity, peroxide value, specific UV extinction coefficients
(K232 and K270) and sensory properties were determined in accordance
with the analytical methods in the Annexes to EU Regulation 2568/91
establishing quality criteria for EVOOs and its subsequent amendments
(European Union Commission, 1991, 2007). The olive oils were as-
sessed sensorily by ten expert tasters according to the official method of
the International Olive Oil Council (IOC) (IOC/T.20/Doc.N°15/Rev.7/
2015) within the framework of EU Regulations 1348/2013. The tasters
evaluated positive gustatory (bitter), olfacto–gustatory (fruity) and
tactile (pungent) attributes, as well as negative attributes. Also, they

5
P. Reboredo-Rodríguez et al.
were invited to assign positive olfactory descriptors among those listed
in IOC/T.20/Doc. N° 22 November 2005.
2.2.3. Genuineness-related parameters
The fatty acid, sterol and triterpene dialcohol composition of the
oils was determined to confirm their authenticity as per EEC/2568/91
and its subsequent amendments (European Union Commission, 1991,
2002).
2.2.4. Phenolic compounds
Lipophilic phenolics (tocopherols) were determined by using IUPAC
2.432 method, and hydrophilic phenolics by UV–VIS spectro-
photometry, using the Folin-Ciocalteau method as described by
Reboredo-Rodríguez et al. (2016a,b); after previous extraction from the
oil with a methanol/water mixture (80:20, v/v) in accordance with the
IOC method (IOC/T.20/Doc N 29).
Bitterness (K225) was evaluated according to Criado et al. (2004).
Briefly, an amount of 1.0 g of oil was dissolved in n-hexane (2 mL) and
passed through a C18 Waters Sep-Pack cartridge previously activated
with methanol (6 mL) and n-hexane (6 mL) to extract bitter compounds.
After the sample was loaded, the cartridge was washed with n-hexane
(10 mL) to remove fat and retained bitter compounds were eluted with
methanol/water (1:1, v/v) to 25 mL. The absorbance at 225 nm of the
extract was measured against a methanol/water mixture (1:1, v/v) in a
1-cm cuvette.
Antioxidant activity was assessed by using the DPPH method
(Gorinstein et al., 2003) with some modifications. A 80 mg/L solution
of DPPH radical in methanol, which exhibited an absorbance at 515 nm
of ca. 1.4 was prepared for this purpose. A hydromethanolic extract
(50 μL) obtained by applying the IOC method to the olive oil was di-
luted with a hydroalcoholic solution (550 μL) of ethanol 70% (v/v) and
added to 400 μL of DPPH solution, the mixture being vigorously stirred
for a few seconds and stored in the dark for 15 min prior to measuring
its absorbance at 515 nm against methanol. Olive oil antioxidants sca-
venge DPPH cation radical and decolorize its purple solution as a result.
Trolox was used as standard and the results were expressed as μmol
Trolox equivalents/kg oil.
3. Results and discussion
3.1. Cataloguing of cultivars
3.1.1. Identification, authentication and naming
The results obtained by using a combined protocol of morphological
and molecular markers to identify the varieties present in olive gene
banks (Trujillo et al., 2014) allowed a total of 37 alleles with its iden-
tified 14 SSR loci to be amplified. The number of alleles per SSR ranged
from 1 (UDO99-019) to 4 (ssrOeUA-DCA3 and ssrOeUA-DCA9), with an
average of 2.6 alleles per locus. Similar values were obtained in other
studies performed on a limited number of olive cultivars (D’Imperio
et al., 2011). Nine of the 14 SSRs (ssrOeUA-DCA3, ssrOeUA-DCA9,
ssrOeUA-DCA11, ssrOeUA-DCA15, ssrOeUA-DCA16, ssrOeUA-DCA18,
UDO99-011, UDO99-024 and UDO99-043) exhibited polymorphism
6
Scientia Horticulturae xxx (xxxx) xxx–xxx
Fig. 1. Endocarps of the cultivars defined as ‘Picuda de Galicia’,
‘Brava’ and ‘Mansa’.
among samples. The remaining SSRs (GAPU-59, UDO99-019, GAPU
71B, GAPU101 and GAPU-103A) were monomorphic (the same profile
was found in all trees) (Table 2a and b). The set of 9 polymorphic SSR
markers amplified 3 different profiles or genotypes (viz., allele se-
quence from all SSR markers used here) among the 32 trees. Each
genotype was coded with an ordinal number (1, 2 and 3) (Table 2b).
Genotypes differed considerably in allele composition (e.g., differ-
ences between genotypes 1 and 3 were observed in all loci of poly-
morphic SSRs, which amounted to 14 alleles in all). This was also the
case with the differences between genotypes 1 and 2 (12 alleles). On the
other hand, genotypes 2 and 3 exhibited small allele differences, with 4
alleles in 3 SSR loci (ssrOeUA-DCA9, ssrOeUA-DCA15 and ssrOeUA-
DCA16) (Table 2b).
The distribution of trees among genotypes was as follows: 24 trees
(75%) shared SSR profile 1; 7 trees (22%) shared profile 2 and only the
Picuda tree represented the third profile (Table 2b).
As regards morphological variability, 6 of the 11 endocarp traits
evaluated were polymorphic and afforded discrimination among three
different morphological profiles. Each profile was coded with an ordinal
number (Table 4, Fig. 1). The morphological traits established were
consistent with those derived from the SSR markers. Thus, all trees
falling in the same group according to SSR profile had identical mor-
phological profiles.
The term cultivar identification has been used in connection with
olives to describe the process of classifying accessions as different cul-
tivars according to the results of their characterization. We used the
nomenclature proposed by Trujillo et al. (2014) here to classify culti-
vars whose trees differed in morphological and/or molecular terms. A
combination of molecular data and endocarp traits allowed the pre-
sence of three different cultivars among the 32 trees to be confirmed
(Table 2b).
The concept cultivar authentication is used in the context of modern
food technology to guarantee that commercial, edible products match
the cultivars stated on their labels (Melchiade et al., 2007). Based on
previous studies with olives, an authentic cultivar is one that can be
matched through DNA and morphological markers to authentic control
samples from its natural area of origin and/or cultivation (Trujillo et al.,
2014). Our three varieties were discriminated by comparing their
profiles with those on the WOGBC database. Although the ‘Brava’ and
‘Mansa’ varieties are not on the list of the Cordoba Olive Collection
examined by Trujillo et al. (2014), their molecular and morphological
profiles are included on the database compiled by the Agronomy De-
partment of the University of Cordoba, Spain. This database is con-
tinually expanded with new material incorporated into the germplasm
bank. The results obtained showed that the combinations of morpho-
logical and molecular profiles (1,1) and (2,2) matched the profiles
corresponding to the true cultivars ‘Brava’ and ‘Mansa’, respectively.
However, the tree named Picuda matched none of the cultivars.
Olive cultivar names are based on generic designations such as out-
standing morphological traits (particularly fruit features), utility of
production (oil or table) or the place of origin of the propagating ma-
terial (Rallo, 2005). For this reason, synonyms (i.e., different names for
the same cultivar) and homonyms (the same name for different
P. Reboredo-Rodríguez et al. Scientia Horticulturae xxx (xxxx) xxx–xxx

Fig. 2. UPGMA dendrogram showing the genetic

Mansa (Ga) relationships of the three local olive varieties from
Picuda (Ga)
Verdial de Badajoz Galicia to the main cultivated material from the
Cordovil C. Branço Iberian Peninsula.
Cornicabra
Verdial de Huévar
Gordal Sevillana
Cordovil de Serpa
Castellana
Morisca
Manzanilla de Sevilla
Picudo
Carrasquenho de Elvas
Hojiblanca
Cobrançosa
Manzanilla Cacereña
Picual
Brava (Ga)
Lechín de Sevilla
Verde Verdelho
Empeltre
Lechín de Granada
Changlot Real
Galega Vulgar
Sevillenca
Villalonga
Morrut
Verdial de Vélez-
Farga
Arbequina
Blanqueta

0.40 0.70 1.00

cultivars) abound among and within olive-growing countries (Barranco
et al., 2000; Trujillo et al., 2014). In Galicia, the names Brava and
Mansa have traditionally been used by the local growers to distinguish
olive trees that are supposed to be autochthonous. We have detected
some confusion among the trees initially named Mansa; thus, 13 trees
(65%) were authenticated as cv Brava, but only 7 (35%) as true cv
Mansa (Table 2b). For this reason, we considered Mansa to be a
homonym of the cultivars ‘Brava’ and ‘Mansa’. Consequently, Mansa is
also a synonym for the cultivar ‘Brava’ in Galicia.
The name Picuda is a synonym for the well-known cultivar ‘Picudo’
(Barranco et al., 2005), which is a major olive cultivar in Andalusia
(southern Spain). However, their profiles did not match those for the
tree with the same name studied in Galicia. We have added the to-
ponym Galicia to the initial name, which has thus become Picuda de
Galicia.
3.1.2. Relationships among cultivars
A UPGMA dendrogram based on Dice’s similarity index (Dice, 1945)
was constructed to examine genetic relationships among the three pu-
tative local olive varieties from Galicia and the main material cultivated
in the Iberian Peninsula (Fig. 2). A wide range of similarity values was
found between all possible pairs of cultivars (results not shown):
0.10–0.96. The cophenetic correlation coefficient between the den-
drogram and the original similarity matrix was high and significant
(r = 0.83; P < 0.01), which indicates good fit of the original data to
the clustering pattern.
The dendrogram clearly separated ‘Brava’ from the cultivars
‘Mansa’-‘Picuda de Galicia’, with similarity indexes (SI) around 0.55. By
contrast, ‘Mansa’ and ‘Picuda de Galicia’shared a high SI (0.92). SI
values among olive cultivars as obtained by SSR markers are usually
less than 0.9 (Bracci et al., 2009; Díez et al., 2011; D’Imperio et al.,
2011; Erre et al., 2010; Fendri et al., 2010; Hannachi et al., 2008;
Haouane et al., 2011; Khadari et al., 2003; Koehmstedt et al., 2010; La
Mantia et al., 2005; Montemurro et al., 2005; Muzzalupo et al., 2014;
Noormohammadi et al., 2007; Sakar et al., 2016; Trujillo et al., 2014).
However, SI may occasionally exceed 0.9. Thus, during the cataloguing
of the WOGBC, Trujillo et al. (2014) found four pairs of cultivars in-
cluding ‘Verdial de Badajoz’-‘Cordovil Castello-Branço’ having identical
or nearly identical SSR profiles but different morphological features.
Identical results were obtained for the pair ‘Mansa’-‘Picuda de Galicia’
(Table 2b; Fig. 1). The two cultivars might thus have originated from
the same cultivar through specific somatic mutations potentially
causing major morphological changes. However, this is only a hy-
pothesis and further research is required to disentangle the genetic
mechanisms behind the change. As regards the remaining cultivars of
the Iberian Peninsula, the Galician varieties grouped with other vari-
eties in Spain and Portugal. For example, ‘Mansa’ and ‘Picuda de Ga-
licia’ grouped with ‘Alfafara’ and ‘Verdial de Badajoz’ from Spain and
‘Cordovil Castelo Branço’ from Portugal. On the other hand, the ’Brava’
cultivar grouped with the Spanish varieties ‘Picual’, ‘Manzanilla Ca-
cereña’ and ‘Hojiblanca’, and also with the Portuguese variety ‘Co-
brançosa’. It should be noted that the two Portuguese varieties come
from northern Portugal and, especially, from Castelo Branço (‘Cordovil
Castelo Branço’ variety) and Trás-os Montes (‘Cobrançosa’ variety).
Both areas are very close to Galicia and share a powerful historic past.
3.2. Characterization of autochthonous VOOs
3.2.1. Quality-related parameters for classifying olive oils
Both monovarietal ‘Brava’ and ‘Mansa’ olive oils were made from
carefully picked olives and the oils extracted under optimal conditions.
Table 5 summarizes the physico-chemical and sensory properties of
these Galician VOOs, which can be classified as Extra Virgin in ac-
cordance with Regulation EU 1348/2013 since its quality indices fall
within the legally established ranges (Table 5) (European Union
Commission, 1991, 2013).
3.2.1.1. Sensory analysis. The target olive oils were analysed sensorily in
terms of positive (fruity, bitter, pungent) and negative attributes (fusty/
muddy sediment, musty–humid–earthy, winey–vinegary–acid–sour, metallic,
rancid). Based on the results, both olive oils were classified as “extra virgin”
since the median of the defects was 0 and that of the fruity attribute exceeded
0 (Table 5). Fruity notes ranged from 3.8–3.9; and bitter and pungent
attributes from 3.3 to 3.6 and 3.6–3.7, respectively. Both olive oils are also
well-balanced because its bitter and pungent scores were 2 units lower than
the median of their fruitiness.
Interestingly, the ‘Brava’ and ‘Mansa’ oils differed in descriptive
sensory profiles. Thus, “tomato” notes were highlighted in ‘Brava’ oil,
and “olive leaf” notes in ‘Mansa’ oil.
3.2.2. Genuineness-related parameters
3.2.2.1. Fatty acid composition. The nutritional properties of olive oil

7
P. Reboredo-Rodríguez et al. Scientia Horticulturae xxx (xxxx) xxx–xxx

Table 5
Physico–chemical and sensory quality properties of the studied VOOs.

‘Brava’ ‘Mansa’ EVOO Reference

Quality-related indices
Free acidity (% oleic acid). 0.17 ± 0.01 0.14 ± 0.01 ≤0.80
Peroxides (meq O2/kg oil) 5± 2 2± 0.2 ≤20
K232 1.95 ± 0.02 1.60 ± 0.05 ≤2.50
K270 0.19 ± 0.02 0.14 ± 0.03 ≤0.22

Sensory analisis
Positive attributes
Fruity 3.8 3.9 >0
Bitter 3.6 3.3
Pungent 3.6 3.7
Negative attributes 0 0 =0

Panel test classification EXTRA VIRGIN EXTRA VIRGIN EXTRA VIRGIN

Genuineness-related indices
Fatty acid composition (% m/m methyl
esters)
Myristic (C14:0) 0.02 ± 0.00 0.01 ± 0.01 ≤0.03
Palmitic (C16:0) 14.70 ± 0.50 9.80 ± 0.40 7.50–20.00
Palmitoleic (C16:1) 1.50 ± 0.08 0.60 ± 0.02 0.30–3.50
Margaric (C17:0) 0.20 ± 0.02 < 0.10 ± 0.01 ≤0.30
Margaroleic C17:1 0.40 ± 0.03 < 0.10 ± 0.01 ≤0.30
Stearic (C18:0) 1.80 ± 0.10 3.40 ± 0.10 0.50–5.00
Oleic (C18:1) 67.80 ± 1.00 77.30 ± 1.40 55.00–83.00
Linoleic (C18:2) 12.00 ± 0.50 7.50 ± 0.20 2.50–21.00
Linolenic (C18:3) 1.00 ± 0.07 0.60 ± 0.05 ≤1.00
Arachidic (C20:0) 0.30 ± 0.01 0.40 ± 0.01 ≤0.60
Eicosenoic (C20:1) 0.20 ± 0.01 0.30 ± 0.01 ≤0.40
Behenic (C22:0) < 0.10 ± 0.01 0.10 ± 0.01 ≤0.20
Lignoceric (C24:0) < 0.10 ± 0.01 < 0.10 ± 0.01 ≤0.20
trans-Oleic isomers C18:1 T < 0.01 ± 0.00 < 0.01 ± 0.00 ≤0.05
trans-Linoleic + trans-Linolenic < 0.01 ± 0.00 < 0.01 ± 0.00 ≤0.05
∑ SFA 17.2 13.9
∑ MUFA 69.9 78.8
∑ PUFA 13.0 8.1
C18:1/C18:2 5.7 10.4
∑ MUFA/∑ PUFA 5.4 9.7

Sterol relative amounts (%)

Cholesterol 0.2 ± 0.00 0.3 ± 0.01 ≤0.5
Brassicasterol < 0.1 ± 0.00 < 0.1 ± 0.00 ≤0.1
Campesterol 2.2 ± 0.02 2.4 ± 0.02 ≤4.0
Stigmasterol 0.7 ± 0.01 0.6 ± 0.01 < Campesterol
Apparent β-sitosterol 95.7 ± 0.2 94.3 ± 0.2 ≥93.0
Δ7-Stigmastenol 0.2 ± 0.01 0.4 ± 0.01 ≤0.5
Total sterols (μg/g) 1752 ± 104 1062 ± 104 ≥1000

Triterpenic dialcohols
Erythrodiol + uvaol 1.3 ± 0.1 2.4 ± 0.2 ≤4.5

Phenolic compounds
Tocopherols (mg/kg)
α-tocopherol 123 ± 7 280 ± 9
β-tocopherol < 0.1 ± 0.0 < 0.1 ± 0.0
γ-tocopherol < 0.1 ± 0.0 < 0.1 ± 0.0
δ −tocopherol 4.6 ± 0.1 15.9 ± 0.1
Total tocopherols 128 ± 6 296 ± 7

Phenolic compounds
Total phenolic content (mg/Kg as g.a.) 489 ± 7 371 ± 6
Antioxidant capacity (μM Trolox/Kg) 1972 ± 19 949 ± 45
K225 7.0 ± 0.2 3.9 ± 0.1

Values are mean ± standard deviation (n = 3).

are related to its balanced fatty acid composition (Salvador et al.,
2001). The fatty acid concentrations of the two oils fall within the
recommended ranges for EVOOs set by EU Regulation 1348/2013
(European Union Commission, 2013). The average fatty acid
composition, expressed as the proportion of total fatty acids, is shown
in Table 5. The fatty acid composition of olive oil is known to depend
strongly on the particular cultivar (Tsimidou and Karakostas, 1993;
Zarrouket al., 2008). Oleic acid (C18:1), palmitic acid (C16:0) and
linoleic acid (C18:2) were the most abundant fatty acids in the
monovarietal olive oils studied. The oleic acid content of ‘Mansa’ oil
(77.3%) was higher than that of ‘Brava’ oil (67.8%), and the opposite
was true of linoleic acid (12.0% in ‘Brava’ oil and 7.5% in ‘Mansa’ oil).
Compared to the most common olive varieties from Spain (viz.,
‘Arbequina’, ‘Picual’ and ‘Cornicabra’), the oleic acid content of
‘Mansa’ oil is similar to that of ‘Cornicabra’ oil (80.6%) and ‘Picual’
oil (80.8%) (Alvarruiz et al., 2015). On the other hand, the linoleic acid
content of ‘Brava’ oil surpasses those of ‘Arbequina’ (10.2%), ‘Mansa’
(7.5%), ‘Cornicabra’ (3.6%) and ‘Picual’ oil (3.3%); also the linolenic

8
P. Reboredo-Rodríguez et al.
acid content of ‘Brava’ oil (1.0%) exceeds those of ‘Arbequina’, ‘Mansa’,
‘Cornicabra’ and ‘Picual’ oils (0.6%). Some authors have suggested that
increased linoleic acid contents may result from enzymatic conversion
of oleic acid into linoleic acid by oleate desaturase during
triacylglycerol biosynthesis (Benito et al., 2013).
Total saturated fatty acids (SFA), monounsaturated fatty acids
(MUFA), polyunsaturated fatty acids (PUFA), and various ratios be-
tween them were estimated. A high content of SFA in oil increases
viscosity and persistence on the oral cavity mucosa, thereby eliciting
“fatty sensation” (Youssef et al., 2011). The main contribution to SFA,
which ranged from 13.9 to 17.2%, was essentially due to palmitic acid
(14.7% in ‘Brava’ oil and 9.8% in ‘Mansa’ oil). The high proportion of
MUFA in ‘Mansa’ oil (78.8%) was due to its high content in oleic acid.
Likewise, the high proportion of PUFA in ‘Brava’ oil (13.0%) was
mainly due to its content in linoleic acid, which is important for human
nutrition but negatively correlated with stability in olive oil. Therefore,
the increased C18:1/C18:2 ratio of ‘Mansa’ oil relative to ‘Brava’ oil
results in increased oxidative stability. The ∑MUFA/∑PUFA ratio is one
other important parameter towards characterizing cultivars. In fact,
Zarrouk et al. (2008) determined the fatty acid composition of oil from
18 Mediterranean olive varieties cultivated in Tunisia and used cluster
analysis to classify VOOs according to the ∑MUFA/∑PUFA and C18:1/
C18:2 ratios. ‘Brava’ oil, with ∑MUFA/∑PUFA = 5.4 and C18:1/
C18:2 = 5.7, fell in the group with low ratios (1.73–5.11 for ∑MUFA/
∑PUFA and 1.72–5.11 for C18:1/C18:2), which includes the varieties
‘Agouromanakolia’,‘Sigoise’,‘Picholine’ and ‘Lechín de Sevilla’. ‘Mansa’
oil, with ∑MUFA/∑PUFA = 9.7 and C18:1/C18:2 = 10.4, fell in the
group with high ∑MUFA/∑PUFA (5.89–17.52) and C18:1/C18:2 ratios
(6.29–21.53), which includes the following nine varieties: ‘Changlot
Real’, ‘Olivière’, ‘Koroneiki’, ‘Verdial de Vélez-Málaga’, ‘Cayon’, ‘Cor-
atina’, ‘Lechín de Granada’, ‘Cornezuelo’ and ‘Leccino’.
3.2.2.2. Sterol and triterpenic dialcohol composition. The sterol
composition of the studied oils was compliant with the limits set by
European Union regulations on EVOOs (European Union Commission,
2013).
The authenticity of olive oil is also related to its total sterol content.
Thus, Kyçyk et al. (2015) studied the sterol composition of VOOs from
43 olive cultivars in WOGBC. They used Principal Component Analysis
(PCA) and Cluster Analysis to establish five VOO categories based on
sterol content (Kyçyket al., 2015). The sterol content of ‘Mansa’ oil
(1062 mg/kg) fell in category V (very low, < 1300 mg/kg), which in-
cludes other Spanish cultivars such as ‘Blanqueta’, whereas that of
‘Brava’ oil (1752 mg/kg) belonged to category III (medium,
1650–1999 mg/kg), which includes ‘Callosina’, ‘Galega Vulgar’, ‘Lechín
de Granada’, ‘Pajarero’ and ‘Racimal’, among other cultivars.
3.2.3. Phenolic compounds
Phenolic compounds including lipophilic and hydrophilic phenols
contribute to the antioxidant properties of VOO. In fact, the phenolic
profile and composition of VOOs are often used to assess their au-
thenticity and potential healthy effects (Angerosa et al., 2004; Gómez-
Rico et al., 2008; Inarejos-García et al., 2011; Rigacci and Stefani,
2016).
3.2.3.1. Lipophilic phenols. Four isomers of tocopherol present in
amounts of 150–250 mg/kg (Uluata et al., 2016) are the main lipid-
soluble antioxidants in olive oil, but can also be found in other
vegetable oils (Servili and Montedoro, 2002). Table 5 shows the
isomer fractions and total tocopherol contents of our oils. As can be
seen, the most abundant isomer (94.6–96.4%) was α-tocopherol
(vitamin E), followed by δ-tocopherol (3.8–5.4%). Also, the total
tocopherol content of ‘Mansa’ oil was higher than that of ‘Brava’ oil
(296 vs 128 mg/kg).
3.2.3.2. Hydrophilic phenols. The hydrophilic phenol profile is specific
9
Scientia Horticulturae xxx (xxxx) xxx–xxx
to olive oils and dependent on the particular olive cultivar (Bendini
et al., 2007). Phenolic compounds in olive oil are usually quantified
with the Folin–Ciocalteau colorimetric method, which, however,
cannot discriminate their nature. The total phenolic content in our
monovarietal olive oils ranged from 371 mg/kg for ‘Mansa’ oil to
489 mg/kg for ‘Brava’ oil. The content of the latter oil is similar to
those of oils from the Spanish cultivars ‘Cornicabra’ (556 mg/kg) and
‘Picual’ (605 mg/kg), which, according to Alvarruiz et al. (2015), are
quite high.
The antioxidant capacity of the studied oils was assessed by de-
termining the ability of their hydrophilic phenolic compounds to sca-
venge free DPPH radicals. Because this property is directly related to
the content in phenolic compounds, ‘Brava’ oil was a more effective
antioxidant capacity (Samaniego Sánchez et al., 2007).
Hydroxytyrosol, a member of the hydrophilic o-diphenol family that
is largely present as a secoiridoid derivative in addition to minor
amounts of free forms and the acetyl derivative, is one of the most re-
presentative phenolic compounds in VOOs. The large fraction of phe-
nolic compounds present in VOO also includes other phenols such as
tyrosol (a hydrophilic mono-phenol) and its secoiridoid derivatives, in
addition to other minor compound classes such as phenolic acids, fla-
vones, lignans and isochromans. Secoiridoids possess several healthy
properties. In fact, EU Commission Regulation 432/2012 (European
Union Commission, 2012) has established a list of permitted health
claims made on foods other than those referring to the reduction of
disease risk and to children’s development and health. For olive oil
polyphenols, the claim “olive oil polyphenols contribute to the protection of
blood lipids from oxidative stress” has been approved and may only be
used in connection with olive oil containing at least 5 mg of hydro-
xytyrosol and its derivatives (e.g., oleuropein complex and tyrosol) per
20 g of oil. Based on the total phenolic content as assessed with the
Folin–Ciocalteu method, and on the fact that hydroxytyrosol secoir-
idoids are the major phenolic constituents of olive oil, the studied oils
can bear the previous health claim since they contain 7.4 mg (‘Mansa’)
and 9.8 mg (‘Brava’) of secoiridoids per 20 g of oil. Therefore, their
beneficial effect on health can be obtained with a daily intake of 20 g of
either oil.
A high phenolic content plays a major role in protecting olive oil
from oxidation; however, it also influences oil taste, which phenols
make bitter and more pungent. As can be seen in Table 5, ‘Brava’ oil had
a higher bitterness index, K225, than ‘Mansa’ oil (7.0 vs 3.9). These
values are consistent with the bitter and pungent attributes noted in
their sensory analysis.
4. Conclusions
This paper is the first to report research on prospecting, collecting
and characterizing olive plant material from the main olive-growing
area of Galicia (NW Spain), where centenary cultivars have survived
since the arrival of Romans to the Iberian Peninsula. The lack of mor-
phological and genetic characterization studies on these autochthonous
olive cultivars, and the high quality of their oils, make the results of this
work especially valuable with a view to improving the scant available
knowledge on them.
Using an effective protocol for characterizing, identifying and authen-
ticating olive plant material on the basis of morphological and molecular
(SSR marker) traits allowed three cultivars to be identified among ancient
olive accessions. This suggests a high genetic potential that could be used by
olive producers to substantially improve their conservation and propagation
strategies for the new identified plant material.
In addition, this work sheds some light on ‘Mansa’ and ‘Brava’ virgin
olive oils by contributing detailed quality and genuineness-related in-
formation, a sensory description and their phenolic profile. As shown
here, both cultivars can be viable alternatives to traditional olive cul-
tivars and expand the current range of commercial high-quality, distinct
virgin olive oils marketable locally and internationally.
P. Reboredo-Rodríguez et al.
The olive crop comprises several clonally propagated traditional
cultivars that emerged through empirical, local selection of exceptional
trees. We thus believe that the Galicia region might have more still
uncatalogued local olive varieties. These encouraging preliminary re-
sults could promote the cataloguing of old olive cultivars with specific
agronomic features affording the production of also specific olive oils in
this emerging olive-growing Spanish region.
Conflict of interests
The authors declare no conflict of interest.
Acknowledgements
This work was financially supported by the Interreg V-A España –
Portugal (POCTEP) 2014–2020 program (project 0181_NANOEATERS_01_E).
The authors are grateful to Miguel Rodríguez González (Aceites Figueiredo)
for providing the oil samples. Also, Patricia Reboredo-Rodríguez acknowl-
edges award of a post-doctoral contract from Xunta de Galicia.
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