Elucidation of the Origin of the Monumental Olive Tree of Vouves in Crete, Greece

Abstract

The olive tree of Vouves in Crete, is considered the oldest producing olive tree in the world with an estimated age exceeding 4000 years. In the present study, we sequenced two samples (from the bottom and the top of the tree) to elucidate the genetic relation of this ancient tree with other olive cvs as well as to gain some insights about its origin. Our results showed that both samples have different genetic origins, proving that this ancient tree has been grafted at least one time. On the basis of whole genome sequences the sample from the top of the Vouves tree showed relation of the same order than half-siblings to one accession corresponding to the present-day Greek cv ‘Mastoidis’. Nevertheless, in the framework of a microsatellite analysis it was found to cluster with the ‘Mastoidis’ samples. The Vouves rootstock (bottom sample) showed a clear grouping with the oleaster samples in a similar way to that of ‘Megaritiki’ Greek cv although it does not show any signal of introgression from them. The genomic analyses did not show a strong relation of this sample with the present-day Greek cvs analyzed in this study so it cannot be proved that it has been used as a source for cultivated olive tree populations represented by available genome sequences. Nevertheless, on the basis of microsatellite analyses, the Vouves rootstock showed affinity with two present-day Greek cvs, one “ancient” rootstock from continental Greece as well as monumental trees from Cyprus. The analysis of the impact of the variants in the gene space revealed an enrichment of genes associated to pathways related with carbohydrate and amino acid metabolism. This is in agreement with what has been found before in the sweep regions related with the process of domestication. The absence of oleaster gene flow, its old age and its variant profile, similar to other cultivated populations, makes it an excellent reference point for domestication studies.

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Article
Elucidation of the Origin of the Monumental Olive Tree of
Vouves in Crete, Greece
Aureliano Bombarely 1,2 , Andreas G. Doulis 3, Katerina K. Lambrou 3, Christos Zioutis 3, Evi Margaritis 4
and Georgios Koubouris 3, *


Citation: Bombarely, A.; Doulis,
A.G.; Lambrou, K.K.; Zioutis, C.;
Margaritis, E.; Koubouris, G.
Elucidation of the Origin of the
Monumental Olive Tree of Vouves in
Crete, Greece. Plants 2021,10, 2374.
https://doi.org/10.3390/
plants10112374
Academic Editors: Luis Rallo,
Fernando Pliego Alfaro, Pilar Rallo,
Concepción Muñoz Díez and
Carlos Trapero
Received: 5 August 2021
Accepted: 26 October 2021
Published: 4 November 2021
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Department of Bioscience, Universita degli Studi di Milano, 20133 Milan, Italy; abombarely@ibmcp.upv.es
2Instituto de Biologıa Molecular y Celular de Plantas (IBMCP), UPV-CSIC, 46022 Valencia, Spain
3Hellenic Agricultural Organization (ELGO) DIMITRA, Institute of Olive Tree, Subtropical Crops and
Viticulture, 73134 Chania, Greece; doulis@elgo.iosv.gr (A.G.D.); lamprou@elgo.iosv.gr (K.K.L.);
zioutisch@gmail.com (C.Z.)
4Science and Technology in Archaeology and Culture Research Center (STARC), The Cyprus Institute,
Nicosia 2121, Cyprus; e.margaritis@cyi.ac.cy
*Correspondence: koubouris@elgo.iosv.gr; Tel.: +30-28210-83434
Abstract:
The olive tree of Vouves in Crete, is considered the oldest producing olive tree in the world
with an estimated age exceeding 4000 years. In the present study, we sequenced two samples (from
the bottom and the top of the tree) to elucidate the genetic relation of this ancient tree with other
olive cvs as well as to gain some insights about its origin. Our results showed that both samples
have different genetic origins, proving that this ancient tree has been grafted at least one time. On
the basis of whole genome sequences the sample from the top of the Vouves tree showed relation
of the same order than half-siblings to one accession corresponding to the present-day Greek cv
‘Mastoidis’. Nevertheless, in the framework of a microsatellite analysis it was found to cluster with
the ‘Mastoidis’ samples. The Vouves rootstock (bottom sample) showed a clear grouping with the
oleaster samples in a similar way to that of ‘Megaritiki’ Greek cv although it does not show any
signal of introgression from them. The genomic analyses did not show a strong relation of this
sample with the present-day Greek cvs analyzed in this study so it cannot be proved that it has been
used as a source for cultivated olive tree populations represented by available genome sequences.
Nevertheless, on the basis of microsatellite analyses, the Vouves rootstock showed affinity with two
present-day Greek cvs, one “ancient” rootstock from continental Greece as well as monumental trees
from Cyprus. The analysis of the impact of the variants in the gene space revealed an enrichment
of genes associated to pathways related with carbohydrate and amino acid metabolism. This is
in agreement with what has been found before in the sweep regions related with the process of
domestication. The absence of oleaster gene flow, its old age and its variant profile, similar to other
cultivated populations, makes it an excellent reference point for domestication studies.
Keywords:
Olea europaea; whole genome DNA resequencing; Illumina; RNA-Seq; gene space
comparison
1. Introduction
Olive (Olea europaea subsp. europaea) is the dominant tree crop in the Mediterranean
countries. In fact, over the 90% of the global olive production is realized in this region [
1
].
The products of this emblematic crop, namely olive oil and table olives are popular in the
framework of a healthy lifestyle [
2
,
3
]. Even in parts of the world far from its traditional
cultivation area, such as Eastern Asia, Australia and America, there is a strong interest in
growing olive trees and consuming its nutritional and rich in biological value products. The
world annual gross production value exceeds 18.3 billion dollars for 2014, 2015 and 2016 [
4
]
depicting the significant contribution in the economic life of producer countries. Even
though that the olive tree has gained a lot of attention for the health benefits of its products
Plants 2021,10, 2374. https://doi.org/10.3390/plants10112374 https://www.mdpi.com/journal/plants

Plants 2021,10, 2374 2 of 18
as well as for its central role in history and culture, the roots of its domestication have
not been unambiguously identified [
5
]. Even in our days, there is an open debate about
whether one major domestication event was realized in the Eastern Mediterranean followed
by subsequent dispersion westwards [
6
] or more than one independent domestication
incidents shaped the richness of olive genetic resources available to date [
7
]. In any case,
it is common belief that the wild olive Olea europaea var. sylvestris is the ancestor of the
cultivated olive Olea europaea var. europaea [
8
–
10
]. Interestingly, wild olive trees still survive
in some Mediterranean forests [11].
Archaeological studies in Greece have identified olive pollen dating to 7000 BC [
12
]
while they suggest that human was firstly attracted by the olive wood during the third
millennium B.C. and that fruit use followed the transformation of olive bush to a tree
through pruning [
13
]. Indeed, early historical references on the use of fruit for its oil
content appear much later [14].
The olive tree was considered as sacred in ancient Greece and it was the highest prize
for the winner athletes in the Olympic Games [
15
]. In an attempt to revive and honor
this tradition, during the modern Olympic Games held in Greece in 2004, the winners
were crowned with an olive wreath from the millennial olive tree of Vouves in Crete,
which is considered the oldest producing olive tree in the world (https://en.wikipedia.org/
wiki/Olive_tree_of_Vouves, accessed on 1 August 2021) with an estimated age exceeding
4000 years.
The oldest indication for the incidence of olive oil based on analysis of residue
on pottery is dated at the 4th millennium in the Gerani Cave in Crete [
16
]. In the case of
Prepalatial Chrysokamino, it was found that olive oil was used to cover wine for preventing
its oxidation to vinegar [
17
]. In another instance, olive oil was noticed in the Early Minoan
I site of Aphrodite’s Kephali [
18
]. After a long period of time, Late Bronze Age findings
were reported from the cemetery of Armenoi [
19
] and Pseira [
20
] in Crete. Taking into
consideration all these findings it is concluded that proof for the production and use of
olive oil in Crete surely precedes the Late Bronze Age.
The millennial olive tree of Vouves in Crete is considered to consist of a Greek cv
resembling ‘Mastoidis’ grafted onto an unknown ancient rootstock yielding a present-day
“monumental” tree. A three-dimensional model of its trunk has been elaborated while
the overall physiognomy is discussed [
21
]. Nevertheless, the tree trunk is presented as a
compact object without differentiating between the two parts (top vs. bottom rootstock).
In ancient times, olive domestication was based on selection of wild trees with desirable
properties and grafting on other trees, as reported by Theophrastus more than 2000 years
ago [22].
The study of ancient olive trees is valuable for detecting unknown genetic resources
and for shedding light in the historical processes of olive domestication [
23
]. Previous
studies on genotyping of old olive trees revealed ancient cvs and confirmed the selection
of cvs [24] as well as rootstocks [25].
The similarity in genetic structure of naturally growing populations with the suckers
of old cultivated trees implies that wild trees were used as rootstocks [
26
]. Pollen archae-
ological studies suggest that a cultivation process seems to have occurred in the Aegean
(Crete)—whether as an independent large-scale management event or as a result of knowl-
edge and/or seedling transfer from the southern Levant around the fourth millennium
BC [27].
In this case, 35 species have been described in the Olea genus in which the most
popular one is O. europaea. This species has been divided into six subspecies: Europaea,
cultivated in the Mediterranean basin; Laperrinei, native to Saharan massifs; Cuspidata,
widely distributed from South Africa to Southern Egypt, and from the Arabian Peninsula to
Southwest China; Guanchica, endemic to the Canary Islands; Maroccana from Morocco and
Cerasiformis native to Madeira [
28
]. The cultivated olive tree (O. europaea subsp. europaea) has
a diploid genome with 46 chromosomes (2n = 2x = 46) and a variable genome size ranging
from 1.65 [
28
] to 2.21 Gb [
29
]. At the time of this publication in 2021, five de-novo olive tree
genome assemblies from four different varieties are available: O. europaea subps. europaea,

Plants 2021,10, 2374 3 of 18
cv. “Farga” version Oe6 [
30
] and its improvement, Oe9 [
31
]; O. europaea var. sylvestris
version Oe451 [
32
]; O. europaea subps. europaea, cv. “Picual” version Oleur0.6.1 [
33
] and the
most recent assembly, O. europaea subps. europaea, cv. “Arberquina” version Oe_Rao [
34
].
Also, some genotyping-by-sequencing studies are available such as [35].
In the present study, we sequenced the genome of the Olive Tree of Vouves, which
is considered the oldest producing olive tree in the world. The upper part of the tree, the
scion, producing fruit and the lower part of the tree, the rootstock, providing the roots for
supplying water and nutrients, were sequenced separately (Figure 1). Further, in order to
gain an initial understanding into the relative placement of the two Vouves parts within the
overall present-day Greek cv diversity landscape, a separate microsatellite (SSR) analysis
was conducted. For that, samples were selected so as to span placement across the SSR
similarity dendrogram from [36] and analyzed anew. In total 17 samples were genotyped
including 12 present-day Greek cvs (alphabetically; Adramytini, Amfissis, Chalkidikis,
Gaidourelia, Karydolia, Koroneiki, Mastoidis, Megareitiki, Pierias, Pikrolia, Tragolia and
Vasilikada), the two Vouves samples, one “ancient” rootstock from Peloponnese and one
Olea europaea subsp. cuspidata (Wall. and G. Don) Cif. sample as an outgroup).
Plants 2021, 10, x FOR PEER REVIEW 3 of 18
olive tree genome assemblies from four different varieties are available: O. europaea subps.
europaea, cv. “Farga” version Oe6 [30] and its improvement, Oe9 [31]; O. europaea var. syl-
vestris version Oe451 [32]; O. europaea subps. europaea, cv. “Picual” version Oleur0.6.1 [33]
and the most recent assembly, O. europaea subps. europaea, cv. “Arberquina” version
Oe_Rao [34]. Also, some genotyping-by-sequencing studies are available such as [35].
In the present study, we sequenced the genome of the Olive Tree of Vouves, which
is considered the oldest producing olive tree in the world. The upper part of the tree, the
scion, producing fruit and the lower part of the tree, the rootstock, providing the roots for
supplying water and nutrients, were sequenced separately (Figure 1). Further, in order to
gain an initial understanding into the relative placement of the two Vouves parts within
the overall present-day Greek cv diversity landscape, a separate microsatellite (SSR) anal-
ysis was conducted. For that, samples were selected so as to span placement across the
SSR similarity dendrogram from [36] and analyzed anew. In total 17 samples were geno-
typed including 12 present-day Greek cvs (alphabetically; Adramytini, Amfissis, Chalki-
dikis, Gaidourelia, Karydolia, Koroneiki, Mastoidis, Megareitiki, Pierias, Pikrolia, Trago-
lia and Vasilikada), the two Vouves samples, one “ancient” rootstock from Peloponnese
and one Olea europaea subsp. cuspidata (Wall. and G. Don) Cif. sample as an outgroup).
Figure 1. Vouves monumental olive tree picture. The places from which the samples were taken are marked in yellow.
Our aims were to (i) verify that the tree is consisted of two different genotypes united
through grafting, (ii) reveal the genetic identity of the two genotypes forming this histor-
ical and millennial tree, (iii) elucidate their genetic relation with other olive cvs, especially
of Greek origin, (iv) characterize their genetic differences in relation to the Olea europaea
var. sylvestris reference genome and (v) propose sound hypotheses on the origin of the
Vouves monumental olive tree.
Figure 1. Vouves monumental olive tree picture. The places from which the samples were taken are marked in yellow.
Our aims were to (i) verify that the tree is consisted of two different genotypes united
through grafting, (ii) reveal the genetic identity of the two genotypes forming this historical
and millennial tree, (iii) elucidate their genetic relation with other olive cvs, especially of
Greek origin, (iv) characterize their genetic differences in relation to the Olea europaea var.
sylvestris reference genome and (v) propose sound hypotheses on the origin of the Vouves
monumental olive tree.

Plants 2021,10, 2374 4 of 18
2. Results and Discussion
2.1. Resequencing, Mapping and Variant Calling with the Olea europaea var. sylvestris Reference
Genome
Leaves from two different parts of the tree (bottom and top) were sampled for the DNA
extraction, library preparation and short read whole genome sequencing. Subsequently,
63.04 and 75.97 Gb of pair end reads were obtained from the bottom and top Illumina
libraries. Then, 411.04 and 496.72 million reads accounting for 58.01 and 70.87 Gb, respec-
tively, were mapped to the reference genome (Olea europaea var. sylvestris version Oe451).
Indeed, 95.49% and 96.71% of the reference genome were covered by at least one read from
the bottom and top samples, leaving 51.48 and 37.61 Mb of the reference uncovered by
any read for each of the samples, respectively. The average mapping coverage was 51.39
and 62.65 X, respectively. The variant calling of the two different samples delivered 23.26
(2.09 variants/100 bp) and 19.54 (1.76 variants/100 bp) millions of variants for bottom and
top samples, respectively. The comparison of both accessions with the reference delivered
7.24 and 4.61 million of homozygous variants of which both samples shared 1.66 million
of variants. Both samples were compared with the Greek samples resequenced in [
33
]
and reanalyzed in this work. These samples included ‘Kalamon’, ‘Koroneiki’, ‘Mastoidis’,
‘Mavreya’, ‘Megaritiki’ and ‘Myrtolia’. Results are summarized in Table 1.
Table 1.
Different type of variants determined following comparison of the two Vouves samples and six previously
sequenced Greek cvs with the genome of Olea europaea var. sylvestris version Oe451.
Sample Total Variants (M) Heterozygous
Variants/100 bp
Total
Variants/100 bp SNP (M) InDels (M) MNP (M)
Vouves bottom 123.26 1.31 2.09 18.79 1.27 0.12
Vouves top 119.54 1.42 1.76 15.73 1.09 0.09
Kalamon 229.47 1.80 2.62 19.26 1.09 0.12
Koroneiki 234.38 2.07 3.07 22.38 1.26 0.19
Mastoidis 229.70 1.76 2.65 19.40 1.09 0.17
Mavreya 231.80 1.99 2.84 21.32 1.18 0.12
Megaritiki 235.70 2.16 3.20 23.24 1.30 0.18
Myrtolia 229.10 1.69 2.60 18.75 1.08 0.16
Note: 1—Samples resequenced in this publication; 2—Samples downloaded from the NCBI SRA public repository from the cite 33.
The levels of heterozygosity were similar between all the samples, ranging from
1.31 heterozygous variants/100 bp of bottom of the Vouves tree to 2.16 of heterozygous
variants/100 bp ‘Megaritiki’. The high levels of heterozygosity are concordant with other
projects in which an olive genome was sequenced such as the ‘Farga’ (5.4%) [
30
] and ‘Picual’
genomes (2.02%) [
33
] whereupon similar values were determined. In a different work,
genotyping of an olive panel using Genotyping-By-Sequencing (GBS) delivered values
ranging from 1.28% of a cv called ‘Zhonglan’ to 6.36% of the Italian cv ‘Nociara’ [
35
], in-
cluding some Greek samples such as ‘Koroneiki’ with 2.19%. Olive genome heterozygosity
is much higher than in other tree crops. For example, the apple (Malus domestica) variety
‘Golden Delicious’ is considered highly heterozygous reaching values of 0.32 heterozygous
variants each 100 bp [
37
]. Peach (Prunus persica) is another example of a tree crop where the
average heterozygosity for cvs and wild relatives are 0.07% and 0.25%, respectively [
38
].
Avocado trees (Persea americana) have heterozygosity levels of the same order with olive
trees. Indeed, estimated heterozygosity of the ‘Hass’ variety is 1.05% [39].
2.2. Origin of the Vouves Monumental Olive Tree in the Context of Olive Domestication
All RNASeq data from NCBI SRA project PRJNA525000 [
6
] as well as the Whole
Genome DNA Resequencing (WGR) data from the SRA project PRJNA556567 [
33
] were
used in an effort to propose sound hypotheses regarding the origins and phylogenomic/

Plants 2021,10, 2374 5 of 18
phylogenetic relations of the Vouves’ olive tree. The first dataset contains 56 samples
of wild and cultivated olive trees from 14 different countries across the Mediterranean
basin. The second dataset, eventually used to produce Figure 2a, contains 41 different
cultivated varieties (Olea europaea subsp. europaea) as well as 10 wild accessions (i.e., a
total of 51 taxons), including different subspecies such as laperrinei and guanchica and wild
Olea europaea subsp. europaea varieties (Olea europaea subsp. europaea var. sylvestris), syn
Olea europaea var. sylvestris (also called oleasters). After the read mapping, variant calling
and filtering, 299, 435 biallelic SNPs were obtained for 117 individuals. Subsequently,
samples coming from RNASeq and WGR were compared so as to assess if it is feasible to
combine data sets produced from two different methodologies (i.e., RNASeq and WGR).
It was found that samples clustered by methodology and not by origin or cv (Figure S1).
Consequently, and based on this result, data derived from RNASeq analyses were filtered
out, retaining only the WGR data for subsequent analyses. An additional filtering was
applied to remove linked variants obtaining a total of 71,040 biallelic SNPs.
The distance tree produced using these variants was employed to construct the phy-
logenomic NJ tree depicted in Figure 2a. Accession (Olea europaea subsp. laperrinei) termed
‘Adjelella10’ was employed as an outgroup. In Figure 2a it can be observed that accession
‘Gran Canaria’ is sister to the outgroup accession as is expected for a different subspecies
(Olea europaea subsp. guanchica) although the other guanchica accession, ‘Tenerife’, is nested
with the oleaster accessions (Olea europaea var. sylvestris). Accession ‘Dokkar ’ is also nested
with the oleaster accessions. The rest of the accessions are part of the same clade. There
are three oleaster accessions nested with the cultivated accessions, ‘Croatia’ acting as an
outgroup of the cultivated accessions and ‘Extremadura’ and ‘Morocco’ that are nested
with three accessions which originate from southern Spain (‘Temprano’, ‘Zarza’ and ‘Lechin
de Sevilla’) and the Algerian accession (‘Chemlal De Kabylie’).
In the phylogenomic tree it can be seen that the Vouves tree bottom sample (more
than 4000-year-old) is external to all the cultivated samples except for ‘Megaritiki’. The
Vouves top tree sample is sister to the ‘Mastoidis’ accession and it clusters with other
present Greek samples. The Italian samples (‘Frantoio’, ‘Leccino’ and ‘Grappolo’) are
a monophyletic group as well as all the Syrian and Iranian accessions. The Spanish
samples are divided into four groups. The first one is nested with two oleaster accessions
(‘Extremadura’ and ‘Morocco’). The 1 (‘Pinonera’ and ‘Menya’) is sister to the Greek
accession ‘Mavreya’. The third one (‘Farga’, ‘Llumeta’ and ‘Forastera de Tortosa’) are sister
to the Israel accession ‘Barnea’. The fourth group contains accessions from southern Spain
and is sister to the Syrian/Iranian clade. The ‘Kalamon’ accession is sister to the Turkish
accession ‘Uslu’. In the UPGMA similarity tree produced with 11 SSR loci (Figure 2b) it can
be seen that ‘Vouves bottom’ exhibits high similarity with another “ancient” rootstock from
the Greek province of Peloponnese. This province is isolated from Crete by sea. Further, the
Peloponnese rootstock shares high similarity with present-day Greek cvs “Pikrolia” and
“Vasilikada’. This is in full agreement with a subsequent—yet unpublished—populational
study involving few hundred olive tree samples from all over Greece genotyped with
SSR markers. In this study each Greek cv is represented by a series of newly analyzed
independent genotypes (data not shown).
A PCA analysis on the samples show similar results (Figure 3). No cultivated sub-
species or wild varieties such as O. europaea subsp. laperrinei (‘Adjelella10’),
O. europaea subsp.
guanchica (‘Tenerife’ and ‘Gran Canaria’) and O. europaea var. sylvestris (‘Minorca’, ‘Pal-
maRio’, ‘Jaen’, ‘Albania’, ‘Croatia’, ‘Extremadura’ and ‘Morocco’) appear to be separated
from the main cluster of cultivated olives (O. europaea subsp. europaea). ‘Dokkar’ is close
to the oleaster accessions, indicating a possible gene flow with the wild populations. The
bottom of the Vouves tree is also close to the oleaster accessions, while the sample from the
top of the tree clusters with the Greek accession ‘Mastoidis’ (Figure 3).

Plants 2021,10, 2374 6 of 18
Plants 2021, 10, x FOR PEER REVIEW 5 of 18
in an effort to propose sound hypotheses regarding the origins and phylogenomic/phylo-
genetic relations of the Vouves’ olive tree. The first dataset contains 56 samples of wild
and cultivated olive trees from 14 different countries across the Mediterranean basin. The
second dataset, eventually used to produce Figure 2a, contains 41 different cultivated va-
rieties (Olea europaea subsp. europaea) as well as 10 wild accessions (i.e., a total of 51 taxons),
including different subspecies such as laperrinei and guanchica and wild Olea europaea
subsp. europaea varieties (Olea europaea subsp. europaea var. sylvestris), syn Olea europaea
var. sylvestris (also called oleasters). After the read mapping, variant calling and filtering,
299, 435 biallelic SNPs were obtained for 117 individuals. Subsequently, samples coming
from RNASeq and WGR were compared so as to assess if it is feasible to combine data
sets produced from two different methodologies (i.e., RNASeq and WGR). It was found
that samples clustered by methodology and not by origin or cv (Figure S1). Consequently,
and based on this result, data derived from RNASeq analyses were filtered out, retaining
only the WGR data for subsequent analyses. An additional filtering was applied to remove
linked variants obtaining a total of 71,040 biallelic SNPs.
(a)
Plants 2021, 10, x FOR PEER REVIEW 6 of 18
(b)
Figure 2. (a) Phylogenomic NJ tree made with the 71,040 filtered biallelic SNPs produced with whole genome resequencing
data. Taxa names encode the subspecies (OEL, for Olea europaea subsp. laperrinei; OEG, Olea europaea subsp. guanchica; OES,
Olea europaea var. sylvestris and OEE, Olea europaea subsp. europaea), country of origin, also with different colors [ALB (red),
Albania; ALG (orange), Algeria; CRO (yellow), Croatia; GRE (light green), Greece; IRA (green), Iran; ISR (green-blue),
Israel; ITA (light blue), Italy; MOR (blue), Morocco; SPA (purple), Spain; SYR (light purple), Syria and TUR (pink), Turkey]
and variety name (n = 51). Bootstrap values are in cursive over their respective nodes. Target samples (Vouves Bottom and
Top) have been marked with an asterisk (b) UPGMA similarity dendrogram of 12 present-day Greek cvs, the two Vouves
samples and one “ancient” olive-tree genotype based on Jaccard’s index (n = 16). Distances based on Jaccard's index are
indicated on each branch of the tree. Olea europaea subsp. cuspidata (Wall. and G. Don) Cif. (denoted as OEC) was employed
as an outgroup. Other taxa names correspond to the Olea europaea subsp. europaea (OEE) cvs (data from [36] reprocessed
within the framework of present study). Distances are indicated above each tree branch.
The distance tree produced using these variants was employed to construct the phy-
logenomic NJ tree depicted in Figure 2a. Accession (Olea europaea subsp. laperrinei) termed
‘Adjelella10’ was employed as an outgroup. In Figure 2a it can be observed that accession
‘Gran Canaria’ is sister to the outgroup accession as is expected for a different subspecies
(Olea europaea subsp. guanchica) although the other guanchica accession, ‘Tenerife’, is
nested with the oleaster accessions (Olea europaea var. sylvestris). Accession ‘Dokkar’ is also
nested with the oleaster accessions. The rest of the accessions are part of the same clade.
There are three oleaster accessions nested with the cultivated accessions, ‘Croatia’ acting
as an outgroup of the cultivated accessions and ‘Extremadura’ and ‘Morocco’ that are
nested with three accessions which originate from southern Spain (‘Temprano’, ‘Zarza’
and ‘Lechin de Sevilla’) and the Algerian accession (‘Chemlal De Kabylie’).
In the phylogenomic tree it can be seen that the Vouves tree bottom sample (more
than 4000-year-old) is external to all the cultivated samples except for ‘Megaritiki’. The
Vouves top tree sample is sister to the ‘Mastoidis’ accession and it clusters with other
present Greek samples. The Italian samples (‘Frantoio’, ‘Leccino’ and ‘Grappolo’) are a
monophyletic group as well as all the Syrian and Iranian accessions. The Spanish samples
are divided into four groups. The first one is nested with two oleaster accessions (‘Extre-
madura’ and ‘Morocco’). The 1 (‘Pinonera’ and ‘Menya’) is sister to the Greek accession
‘Mavreya’. The third one (‘Farga’, ‘Llumeta’ and ‘Forastera de Tortosa’) are sister to the
Israel accession ‘Barnea’. The fourth group contains accessions from southern Spain and
is sister to the Syrian/Iranian clade. The ‘Kalamon’ accession is sister to the Turkish acces-
sion ‘Uslu’. In the UPGMA similarity tree produced with 11 SSR loci (Figure 2b) it can be
seen that ‘Vouves bottom’ exhibits high similarity with another “ancient” rootstock from
the Greek province of Peloponnese. This province is isolated from Crete by sea. Further,
the Peloponnese rootstock shares high similarity with present-day Greek cvs “Pikrolia”
Figure 2.
(
a
) Phylogenomic NJ tree made with the 71,040 filtered biallelic SNPs produced with whole genome resequencing
data. Taxa names encode the subspecies (OEL, for Olea europaea subsp. laperrinei; OEG, Olea europaea subsp. guanchica; OES,
Olea europaea var. sylvestris and OEE, Olea europaea subsp. europaea), country of origin, also with different colors [ALB (red),
Albania; ALG (orange), Algeria; CRO (yellow), Croatia; GRE (light green), Greece; IRA (green), Iran; ISR (green-blue), Israel;
ITA (light blue), Italy; MOR (blue), Morocco; SPA (purple), Spain; SYR (light purple), Syria and TUR (pink), Turkey] and
variety name (n= 51). Bootstrap values are in cursive over their respective nodes. Target samples (Vouves Bottom and
Top) have been marked with an asterisk (
b
) UPGMA similarity dendrogram of 12 present-day Greek cvs, the two Vouves
samples and one “ancient” olive-tree genotype based on Jaccard’s index (n= 16). Distances based on Jaccard’s index are
indicated on each branch of the tree. Olea europaea subsp. cuspidata (Wall. and G. Don) Cif. (denoted as OEC) was employed
as an outgroup. Other taxa names correspond to the Olea europaea subsp. europaea (OEE) cvs (data from [
36
] reprocessed
within the framework of present study). Distances are indicated above each tree branch.

Plants 2021,10, 2374 7 of 18
Plants 2021, 10, x FOR PEER REVIEW 7 of 18
and “Vasilikada’. This is in full agreement with a subsequent—yet unpublished—popu-
lational study involving few hundred olive tree samples from all over Greece genotyped
with SSR markers. In this study each Greek cv is represented by a series of newly analyzed
independent genotypes (data not shown).
A PCA analysis on the samples show similar results (Figure 3). No cultivated sub-
species or wild varieties such as O. europaea subsp. laperrinei (‘Adjelella10’), O. europaea
subsp. guanchica (‘Tenerife’ and ‘Gran Canaria’) and O. europaea var. sylvestris (‘Minorca’,
‘PalmaRio’, ‘Jaen’, ‘Albania’, ‘Croatia’, ‘Extremadura’ and ‘Morocco’) appear to be sepa-
rated from the main cluster of cultivated olives (O. europaea subsp. europaea). ‘Dokkar’ is
close to the oleaster accessions, indicating a possible gene flow with the wild populations.
The bottom of the Vouves tree is also close to the oleaster accessions, while the sample
from the top of the tree clusters with the Greek accession ‘Mastoidis’ (Figure 3).
Figure 3. Principal Component Analysis (PCA) of the distance matrix of the different varieties used
in this study. PC1 represents 18.77% of the variance and PC2 5.46%. Subspecies is encoded with the
shape of the point (OEL, a diamond, for Olea europaea subsp. laperrinei; OEG, a square, Olea europaea
subsp. guanchica; OES, a triangle, Olea europaea var. sylvestris and OEE, a circle, Olea europaea subsp.
europaea) while country of origin is encoded with different colors (ALB (red), Albania; ALG (orange),
Algeria; CRO (yellow), Croatia; GRE (light green), Greece; IRA (green), Iran; ISR (green-blue), Israel;
ITA (light blue), Italy; MOR (blue), Morocco; SPA (purple), Spain; SYR (light purple), Syria and TUR
(pink), Turkey). Samples of interest (Vouves bottom and top) have been marked with a star.
The topology of the phylogenomic tree is similar to previously published phylo-
genomic trees [31,33], with Italian and Syrian/Iranian samples as monophyletic groups,
Spanish samples grouped in two branches with one of them being a sister group to the
Syrian/Iranian groups. Greek samples were also distributed in a similar fashion with
‘Kalamon’ grouped with the Syrian samples and ‘Myrtolia’, ‘Mastoidis’ and ‘Koroneiki’
comprising a monophyletic group sister to the Italian one. ‘Megaritiki’ appeared as one of
Figure 3.
Principal Component Analysis (PCA) of the distance matrix of the different varieties
used in this study. PC1 represents 18.77% of the variance and PC2 5.46%. Subspecies is encoded
with the shape of the point (OEL, a diamond, for Olea europaea subsp. laperrinei; OEG, a square,
Olea europaea subsp.
guanchica; OES, a triangle, Olea europaea var. sylvestris and OEE, a circle, Olea
europaea subsp. europaea) while country of origin is encoded with different colors (ALB (red), Albania;
ALG (orange), Algeria; CRO (yellow), Croatia; GRE (light green), Greece; IRA (green), Iran; ISR
(green-blue), Israel; ITA (light blue), Italy; MOR (blue), Morocco; SPA (purple), Spain; SYR (light
purple), Syria and TUR (pink), Turkey). Samples of interest (Vouves bottom and top) have been
marked with a star.
The topology of the phylogenomic tree is similar to previously published phyloge-
nomic trees [
31
,
33
], with Italian and Syrian/Iranian samples as monophyletic groups,
Spanish samples grouped in two branches with one of them being a sister group to the
Syrian/Iranian groups. Greek samples were also distributed in a similar fashion with
‘Kalamon’ grouped with the Syrian samples and ‘Myrtolia’, ‘Mastoidis’ and ‘Koroneiki’
comprising a monophyletic group sister to the Italian one. ‘Megaritiki’ appeared as one of
the most outer taxa of the cultivated olives similarly to ‘Dokkar’, so it is possible that this
cv has some contribution from the wild olive populations [31,33].
The clustering analysis using STRUCTURE software and DAPC evidenced four clus-
ters as the most probable number (Figure S2), with two, three and six being the alternative
scenarios. The grouping of the different samples using four groups with Structure showed
a first group (G1) composed by non-cultivated olives such as O. europaea subsp. laperrinei,
O. europaea subsp. guanchica and most of the O. europaea var. sylvestris with the exception of
the ‘Extremadura’, ‘Morocco’ and the ‘Croatia’ accessions that cluster in the groups “G2”,
“G2” and “G4”, respectively. ‘Dokkar’ and the sample of the bottom of the Vouves tree also
cluster in the group “G1”. Both samples show some component of the group “G4”. The
second group, “G2”, is composed by accessions from southern Spain such as ‘Lechin de
Sevilla’, ‘Zarza’ and ‘Temprano’. In this group, there can also be found accessions with

Plants 2021,10, 2374 8 of 18
some components of the groups “G1 + G4” such as ‘Chemlal Kabile’ and “G4 + G3” such
as ‘Forastera T.’. A third group, “G3”, is composed by most of the cvs from Syria such as
‘Abou Kanami’, ‘Mari’ and ‘Barri’. In this group, there can also be found the Greek sample
‘Kalamon’ as well as some southern Spain samples such as ‘Verdial’, ‘Ocal’ or ‘Picudo’ as
admixture between this “G3” group and the “G2”. The fourth group, “G4” is composed
mostly by Greek, Italian and Northwest Spanish accessions including the top of Vouves
tree sample (Figure 4). The results for the DAPC analysis are similar (Figure S3). The
clustering analysis is also similar to the previously published analysis if the groups G2 and
G3 are considered as a single group [
33
] or G1 and G2 are considered as one group and then
G3 and G4 as another one [
31
]. A recent work with a wider sampling of wild accessions
evidenced two separate groups; one comprising O. europaea var. sylvestris and another
comprising O. europaea subsp. guanchica [
40
] in contrast to the present work where only
one group is detected. This could evidence one of the limitations of the sampling of the
present study associated with non-representative number of accessions for some groups,
such as guanchica. Nevertheless, the sylvestris accessions appears as a group separate from
the group of cvs giving enough resolution to distinguish both groups even if this study did
not sample hundreds of accessions.
Plants 2021, 10, x FOR PEER REVIEW 8 of 18
the most outer taxa of the cultivated olives similarly to ‘Dokkar’, so it is possible that this
cv has some contribution from the wild olive populations [31,33].
The clustering analysis using STRUCTURE software and DAPC evidenced four clus-
ters as the most probable number (Figure S2), with two, three and six being the alternative
scenarios. The grouping of the different samples using four groups with Structure showed
a first group (G1) composed by non-cultivated olives such as O. europaea subsp. laperrinei,
O. europaea subsp. guanchica and most of the O. europaea var. sylvestris with the exception
of the ‘Extremadura’, ‘Morocco’ and the ‘Croatia’ accessions that cluster in the groups
“G2”, “G2” and “G4”, respectively. ‘Dokkar’ and the sample of the bottom of the Vouves
tree also cluster in the group “G1”. Both samples show some component of the group
“G4”. The second group, “G2”, is composed by accessions from southern Spain such as
‘Lechin de Sevilla’, ‘Zarza’ and ‘Temprano’. In this group, there can also be found acces-
sions with some components of the groups “G1 + G4” such as ‘Chemlal Kabile’ and “G4 +
G3” such as ‘Forastera T.’. A third group, “G3”, is composed by most of the cvs from Syria
such as ‘Abou Kanami’, ‘Mari’ and ‘Barri’. In this group, there can also be found the Greek
sample ‘Kalamon’ as well as some southern Spain samples such as ‘Verdial’, ‘Ocal’ or
‘Picudo’ as admixture between this “G3” group and the “G2”. The fourth group, “G4” is
composed mostly by Greek, Italian and Northwest Spanish accessions including the top
of Vouves tree sample (Figure 4). The results for the DAPC analysis are similar (Figure
S3). The clustering analysis is also similar to the previously published analysis if the
groups G2 and G3 are considered as a single group [33] or G1 and G2 are considered as
one group and then G3 and G4 as another one [31]. A recent work with a wider sampling
of wild accessions evidenced two separate groups; one comprising O. europaea var. syl-
vestris and another comprising O. europaea subsp. guanchica [40] in contrast to the present
work where only one group is detected. This could evidence one of the limitations of the
sampling of the present study associated with non-representative number of accessions
for some groups, such as guanchica. Nevertheless, the sylvestris accessions appears as a
group separate from the group of cvs giving enough resolution to distinguish both groups
even if this study did not sample hundreds of accessions.
Figure 4. Admixture analysis for K = 4. Four populations are represented with the following colors: Red (noted in the text
as G1) composed mostly by wild accessions (Olea europaea subsp. laperrinei; Olea europaea subsp. guanchica; and Olea euro-
paea var. sylvestris); Purple (noted in the text as G2) has southern Spain accessions and some Olea europaea var. sylvestris;
Figure 4.
Admixture analysis for K = 4. Four populations are represented with the following colors:
Red (noted in the text as G1) composed mostly by wild accessions (Olea europaea subsp. laperrinei;
Olea europaea subsp. guanchica; and Olea europaea var. sylvestris); Purple (noted in the text as G2) has
southern Spain accessions and some Olea europaea var. sylvestris; Blue (noted in the main text as G3) is
composed by individuals from Syria and Iran; Green (noted in the text as G4) present-day Greek and
Italian accessions and some accessions from north-western Spain. Vouves tree samples have been
marked with a star.
Examining the composition of the individuals in admixture analysis it appears pos-
sible that some introgression and gene flow has taken place between the wild and some
cultivated accessions such as “Dokkar”. Consequently, and in order to test this possibility,
an ABBA-BABA analysis was performed using the Italian varieties as sister group, the wild
accessions “Minorca”, “Palma del Rio” and “Jaen. The Olea europaea subsp. laperrinei was
used as outgroup. The results evidenced that three cvs (“Chemlal Kabil”, “Megaritiki” and
“Dokkar”) experienced introgression from wild olive trees: (Table S1). This is in agreement
with the position of these three cvs in the phylogenetic tree (Figure 2a). The Vouves tree

Plants 2021,10, 2374 9 of 18
bottom sample did not present any Olea europaea var. sylvestris introgression according to
the ABBA-BABA analysis.
The relation between the top of the Vouves tree sample and the Greek accession
‘Mastoidis’ was tested calculating the relatedness Ajk statistic between all the samples
(Figure S4). Individuals with themselves will have values of 1 or higher, individuals in the
same population will have values close to 0 and unrelated individuals will have negative
values. The top of the Vouves tree sample has an Ajk statistics value of 0.53 with the
‘Mastoidis’ accession indicating a relation of the same order than half-siblings, and verifying
that the Vouves tree was grafted with a present-day cv. This is in full agreement with present
SSR analysis whereupon Vouves top sample fell within the ‘Mastoidis’ cluster
(Figure 2b)
.
The Vouves rootstock (bottom sample) showed a clear grouping with the oleaster samples
in a similar way that the ‘Megaritiki’ Greek cv. A previous work hypothesized that
‘Megaritiki’ has an introgression of oleaster populations [
31
]. This result agrees with the
Ajk statistics with values of 0.13, 0.10, 0.10, 0.09 and 0.09 with the ‘Croatia’, ‘Palma del
Rio’, ‘Minorca’, ‘Jaen’ and ‘Albania’ oleaster accessions, respectively. The Vouves rootstock
(bottom sample) shows higher values with the oleaster samples, especially with ‘Minorca’
(0.16), ‘Albania’ (0.15) and ‘Palma del Rio’ (0.14). These values are similar to the values that
the oleaster samples show between them. Even though this agrees with the hypothesis
that this sample could have oleaster introgressions, the estimated age of the tree could
indicate an early stage of domestication and diversification from the oleaster accessions. A
more extensive sampling of other Greek accessions as well as other oleaster samples of the
West Mediterranean area could help to clarify this result. Greek cvs whose genomes were
available for inclusion in the present study (Table 1; ‘Kalamon’, ‘Koroneiki’, ‘Mastoidis’,
‘Mavreya’, ‘Megaritiki’, ‘Myrtolia’) didn’t show any high Ajk values with the Vouves
bottom sample so it cannot be documented that the original monumental tree has a special
role in the development of these present-day Greek varieties. Nevertheless, previous SSR
studies showed that the Vouves tree is genetically related with other “monumental” trees
from the ‘Sotira’ area in Cyprus [
24
] in addition to an “ancient” tree from Peloponnese)—a
region in continental Greece (present study). Both localities are geographically isolated
from Crete, by sea, the first by appr. 700 Km and the second by appr. 300 Km. Similar
to the Vouves bottom sample, the Sotira area “monumental” samples appear genetically
remote by comparison to present-day local, Cypriot cvs. Specifically, [
25
] had performed
Maximum Likelihood (ML) clustering analysis, employing 17 SSR loci data, of 51 old
rootstocks (also dubbed ‘living fossils’ or ‘centennial olive germplasm’) and 12 present-day
cvs from Cyprus. They showed that the ‘Vouves tree’ (‘Vouves bottom’ of present study)
is genetically related to other monumental trees of the Sotira area in Cyprus (the two
islands of Cyprus and Crete are 700 Km apart). The same authors subsequently performed
coalescent modelling employing the same data as for ML. Similar to what is found for
‘Vouves bottom’ in the present study, [
25
] concluded that “most of the rootstocks were
positioned externally to the core of the olive entries, thus underlining their lack of genetic
affinity, but without ruling out the possible contribution to the establishment of the current
cultivars”.
Overall, we believe that we have shown that the bottom of the Vouves tree is a well-
supported separate branch with no gene flow from the sylvestries trees indicating that
probably its cultivation was a separate event and that, at another level, wild cultivars from
the eastern cluster together with those from the western Mediterranean basin.
The analysis of the Vouves tree can bring forth some interesting points about the date
of the early diversification of the East/West cultivated populations. The ‘Farga’ taxon
represents an ancient branch of domesticated olive trees dated between 300 and
1000 years
old [
41
]. The phylogenetic tree shows a clear divergency of some lineages (e.g., Italian
or some Greek popular accessions) from the ‘Farga’ lineage indicating that the ancestor
of these cvs could have existed more than a thousand years ago. The estimated age
of diversification between Eastern and Western cultivated populations is dated around
6000 years
ago [
5
]. The position of the bottom of the Vouves tree sample, dated more

Plants 2021,10, 2374 10 of 18
than 4000 years ago could indicate a later diversification (appears as an outgroup for the
cultivated olives without any apparent gene flow with the oleaster populations) although
more ancient monumental trees should be studied before any solid conclusion is drown.
Taking together the conclusions of [
25
,
41
] and of the present study it could be proposed
that, in the Mediterranean Basin, there existed an ancient olive tree common genetic pool
which is only partly represented in few present-day cvs.
2.3. Gene Space Variation in the Vouves Monumental Olive Tree
The genome resequencing allows for analysis of possible changes occurred in the gene
space of a genome. Most of the variants between the reference genome (Oe451) and the
Vouves tree samples were intergenic variants (55.30% and 46.53% of the annotated variants
for bottom and top samples, respectively), followed by 5 Kb downstream (14.90% and
12.98%) and upstream gene variants (20.63% and 16.81%). Intron variants accounted for
5.12% and 4.15% of the total variants in both datasets. The impact of the variants in the
Vouves samples was compared with other Greek genotypes (Table 2). Between 36.73%
(Vouves Top) and 56.36% (‘Megaritiki’) of the genes presented at least one variant with
high impact. Most of these types of variants are associated with frameshift or gaining of a
stop codon (Table 3).
Table 2. Percentage of the different variant categories produced in the variant annotation for each of the samples.
Sample % Intergenic
Variants
% 5 Kb
Upstream
Variants
% 5 Kb
Downstream
Variants
%Intron
Variants
% Genic Variants
Genes 4with
HI Variants
High Impact 1Moderate
Impact 2Low Impact 3
Vouves
bottom 55.30 20.63 14.90 5.12 0.23 2.26 1.56 21,559
Vouves top 46.53 16.81 12.98 4.15 0.19 1.87 1.31 18,618
Kalamon 59.45 18.32 13.72 4.46 0.27 2.32 1.45 25,917
Koroneiki 59.93 18.25 13.64 4.32 0.26 2.20 1.40 26,334
Mastoidis 60.12 18.01 13.56 4.34 0.26 2.27 1.43 25,041
Mavreya 59.77 18.11 13.69 4.43 0.27 2.28 1.44 26,558
Megaritiki 59.45 18.43 13.76 4.45 0.26 2.23 1.40 28,567
Myrtolia 59.79 18.28 13.67 4.32 0.26 2.26 1.42 26,164
Notes:
1
—High impact (HI) variants are defined as those variants with exon lost, frameshift, splice acceptor or donor variant, loss of the
start or gain of a stop of the translation.
2
—Moderate impact (MI) variants are those with a conservative or disruptive in-frame insertions or
deletions, missense variants and splice region variants.
3
—Low impact (LI) variants are those as codon initiation variants and synonymous
variants and variants in the stop codon. 4—The total number of gene models for the Oe451 genome is 50,684.
Table 3. Summary of the different sources of high impact variants affecting each of the Greek accessions.
Sample
Genes with High Impact Variants
Exon Lost Frameshift Splice
Acceptor
Splice
Donor Start Lost Stop Gain Stop Lost
Vouves
Bottom 2 12,651 4697 4632 2112 11,624 3126
Vouves Top 1 10,588 3781 3723 1792 9748 2598
Kalamon 1 14,752 5954 5790 2860 17,086 3603
Koroneiki 2 16,355 6503 6204 3275 17,037 4003
Mastoidis 3 14,896 5798 5570 2757 16,170 3593
Mavreya 5 15,829 6318 6071 3111 17,307 3736
Megaritiki 3 16,924 6956 6629 3386 19,322 4199
Myrtolia 1 14,565 5789 5451 2747 16,332 3427

Plants 2021,10, 2374 11 of 18
In the present study, 11,553 genes with high impact variants were shared between
all the Greek accessions and the Vouves tree samples. Indeed, 13,269 and 12,909 genes
with high impact variants were shared between the Greek accessions and the bottom and
the top Vouves tree sample, respectively, meanwhile 14,984 genes were shared between
both Vouves samples (Figure 5). To further understand the processes in which these genes
may be involved, a Gene Set Enrichment Analysis (GSEA) was performed in the different
groups. The first group, formed by 4859 genes with high impact variants in the Vouves
bottom tree sample, presented an enrichment in eight terms: “IMP Salvage” (GO: 0032264),
“Heme Oxidation” (GO: 0006788), “rRNA processing” (GO: 0006364), “cellulose biosyn-
thetic process” (GO: 0030244), “Glycolytic process” (GO: 0006096), “Telomere maintenance”
(GO: 0000723), “DNA repair” (GO: 0006281) and “Carbohydrate metabolic process” (GO:
0005975) (Figure 6A). The second group had 2,278 genes with high impact variants for the
Vouves top tree sample. It presented enrichment in the following terms “Regulation of pro-
ton transport” (GO: 0010155), “Mitochondrial electron transport ubiquinol to cytochrome
c” (GO: 0006122), “Terpenoid biosynthetic process” (GO: 0016114), “Response to metal
ion” (GO: 0010038), “Biosynthetic process” (GO: 0009058), “L-phenylalanine catabolic
process” (GO: 0006559), “tyrosine metabolic process” (GO: 0006570), “tyrosine biosynthetic
process” (GO: 0006571), “Metabolic process” (GO: 0008152), “Double-strand break repair
via homologous recombination” (GO: 0000724), “Anaphase-promoting complex dependent
catabolic process” (GO: 0031145), “Dolichol-linked oligosaccharide biosynthetic process”
(GO: 0006488), “Phytochelatin biosynthetic process” (GO:0046938) and “Photosynthesis
light harvesting” (GO: 0009765) (Figure 6B).
Plants 2021, 10, x FOR PEER REVIEW 11 of 18
Kalamon 1 14,752 5954 5790 2860 17,086 3603
Koroneiki 2 16,355 6503 6204 3275 17,037 4003
Mastoidis 3 14,896 5798 5570 2757 16,170 3593
Mavreya 5 15,829 6318 6071 3111 17,307 3736
Megaritiki 3 16,924 6956 6629 3386 19,322 4199
Myrtolia 1 14,565 5789 5451 2747 16,332 3427
In the present study, 11,553 genes with high impact variants were shared between all
the Greek accessions and the Vouves tree samples. Indeed, 13,269 and 12,909 genes with
high impact variants were shared between the Greek accessions and the bottom and the
top Vouves tree sample, respectively, meanwhile 14,984 genes were shared between both
Vouves samples (Figure 5). To further understand the processes in which these genes may
be involved, a Gene Set Enrichment Analysis (GSEA) was performed in the different
groups. The first group, formed by 4,859 genes with high impact variants in the Vouves
bottom tree sample, presented an enrichment in eight terms: “IMP Salvage” (GO:
0032264), “Heme Oxidation” (GO: 0006788), “rRNA processing” (GO: 0006364), “cellulose
biosynthetic process” (GO: 0030244), “Glycolytic process” (GO: 0006096), “Telomere
maintenance” (GO: 0000723), “DNA repair” (GO: 0006281) and “Carbohydrate metabolic
process” (GO: 0005975) (Figure 6A). The second group had 2,278 genes with high impact
variants for the Vouves top tree sample. It presented enrichment in the following terms
“Regulation of proton transport” (GO: 0010155), “Mitochondrial electron transport ubiq-
uinol to cytochrome c” (GO: 0006122), “Terpenoid biosynthetic process” (GO: 0016114),
“Response to metal ion” (GO: 0010038), “Biosynthetic process” (GO: 0009058), “L-phenyl-
alanine catabolic process” (GO: 0006559), “tyrosine metabolic process” (GO: 0006570), “ty-
rosine biosynthetic process” (GO: 0006571), “Metabolic process” (GO: 0008152), “Double-
strand break repair via homologous recombination” (GO: 0000724), “Anaphase-promot-
ing complex dependent catabolic process” (GO: 0031145), “Dolichol-linked oligosaccha-
ride biosynthetic process” (GO: 0006488), “Phytochelatin biosynthetic process”
(GO:0046938) and “Photosynthesis light harvesting” (GO: 0009765) (Figure 6B).
Figure 5. Venn diagram showing the number of High Impact (HI) variants shared between the
Vouves tree bottom (green), the Vouves tree top sample (blue) and the other Greek accessions (yel-
low).
Figure 5.
Venn diagram showing the number of High Impact (HI) variants shared between the Vouves
tree bottom (green), the Vouves tree top sample (blue) and the other Greek accessions (yellow).
The third group had 970 genes associated with high impact changes shared by all the
Greek accessions (‘Kalamon’, ‘Koroneiki’, ‘Mastoidis’, ‘Mavreya’, ‘Megaritiki’ and ‘Myrto-
lia’) but absent in the Vouves tree. This cluster presented an enrichment in the following
Gene Ontology Terms: “Mitotic cell cycle” (GO: 0000278), “DNA repair” (GO: 0006281),
“Golgi to plasma membrane transport” (GO: 0006893), “ATP metabolic process” (GO:
0046034), “Chromosome segregation” (GO: 0007059), “Sucrose biosynthetic process” (GO:
0005986), “DNA integration” (GO: 0015074), “Regulation of cytokinesis” (GO: 0032465),
“Peptidyl-lysine modification to peptidyl-hypusine” (GO: 0008612) and “Telomere mainte-
nance” (GO: 0000723) (Figure 6C). The list of the genes as well as the variants and their
impacts have been summarized in the Table S2. The fourth group contained 11,553 genes
with high impact variants common for all the datasets (Vouves tree and Greek accessions).

Plants 2021,10, 2374 12 of 18
This group presented a GO enrichment for the terms “valyl-tRNA aminoacylation” (GO:
0006438), “ribosomal large subunit biogenesis” (GO: 0042273), “ribosomal large subunit
export from nucleus” (GO: 0000055), “dimethylallyl diphosphate biosynthetic process”
(GO: 0050992), “DNA integration” (GO: 0015074), “proteolysis” (GO: 0006508), “DNA
topological change” (GO: 0006265), “DNA repair” (GO: 0006281), “photosynthetic elec-
tron transport chain” (GO: 0009767), “photosynthetic electron transport in photosystem
II” (GO: 0009772), “methylation” (GO: 0032259), “transcription DNA-templated” (GO:
0006351), “isopentenyl diphosphate biosynthetic process methylerythritol 4-phosphate
pathway” (GO: 0019288), “transcription initiation from RNA polymerase III promoter” (GO:
0006384), “recognition of pollen” (GO: 0048544), “retrograde transport endosome to Golgi”
(GO: 0042147), “protein N-linked glycosylation via asparagine” (GO: 0018279), “protein
phosphorylation” (GO: 0006468) and “telomere maintenance” (GO: 0000723) (Figure 6D).
Plants 2021, 10, x FOR PEER REVIEW 12 of 18
Figure 6. Gene Ontology analysis of the genes affected by high impact variants. The x-axis represents the value of the over
or under-representation of these categories. The size of the dot represents the number of genes of the category and the
color intensity its statistical significance as p-value. (A) Vouves bottom tree sample; (B) Vouves top tree sample; (C) High
impact changes shared by all the Greek accessions (‘Kalamon’, ‘Koroneiki’, ‘Mastoidis’, ‘Mavreya’, ‘Megaritiki’ and ‘Myr-
tolia’) but absent in the Vouves tree; (D) Genes affected by high impact variants common for all the datasets (Vouves tree
and Greek accessions).
The third group had 970 genes associated with high impact changes shared by all the
Greek accessions (‘Kalamon’, ‘Koroneiki’, ‘Mastoidis’, ‘Mavreya’, ‘Megaritiki’ and ‘Myr-
tolia’) but absent in the Vouves tree. This cluster presented an enrichment in the following
Gene Ontology Terms: “Mitotic cell cycle” (GO: 0000278), “DNA repair” (GO: 0006281),
“Golgi to plasma membrane transport” (GO: 0006893), “ATP metabolic process” (GO:
0046034), “Chromosome segregation” (GO: 0007059), “Sucrose biosynthetic process” (GO:
0005986), “DNA integration” (GO: 0015074), “Regulation of cytokinesis” (GO: 0032465),
“Peptidyl-lysine modification to peptidyl-hypusine” (GO: 0008612) and “Telomere
maintenance” (GO: 0000723) (Figure 6C). The list of the genes as well as the variants and
their impacts have been summarized in the Table S2. The fourth group contained 11,553
genes with high impact variants common for all the datasets (Vouves tree and Greek ac-
cessions). This group presented a GO enrichment for the terms “valyl-tRNA aminoacyla-
tion” (GO: 0006438), “ribosomal large subunit biogenesis” (GO: 0042273), “ribosomal
carbohydrate metabolic process
DNA repair
telomere maintenance
glycolytic process
cellulose biosynthetic process
rRNA processing
heme oxidation
IMP salvage
02468
R a tio H I V a r ia n t G e n e s / E x p e c te d G e n e s
G e n e O n t lo g y T e r m
HI_VariantGenes
25
50
75
0.01
0.02
0.03
0.04
pValue
A
biosynthetic process
metabolic process
photosynthesis
light harvesting
terpenoid biosynthetic process
double−strand break repair
via homologous recombination
mitochondrial electron tr ansport
ubiquinol to cytochrome c
regulation of proton transpor t
response to metal ion
tyrosine biosynthetic process
phytochelatin biosynthetic process
tyrosine metabolic process
L−phenylalanine catabolic process
anaphase−promoting complex
dependent catabolic process
dolichol−linked oligosacchar ide
biosynthetic process
0 5 10 15 20 25
R a tio H I V a r ia n t G e n e s / E x p e c te d G e n e s
G e n e O n t lo g y T e r m
0.01
0.02
0.03
0.04
pValue
HI_VariantGenes
100
200
300
B
DNA integration
DNA repair
mitotic cell cycle
chromosome segregation
telomere maintenance
peptidyl−lysine modification
to peptidyl−hypusine
Golgi
to plasma membrane transpor t
sucrose biosynthetic process
regulation of cytokinesis
0 255075100
R atio H I V arian t G enes / E xp ected G en es
G e n e O n t lo g y T e r m
0.01
0.02
pValue
HI_VariantGenes
3
6
9
12
C
protein phosphorylation
methylation
DNA repair
proteolysis
telomere maintenance
recognition of pollen
photosynthetic electron tr ansport
in photosystem II
DNA topological change
DNA
integration
photosynthetic electron transpor t chain
dimethylallyl diphosphate biosynthetic process
isopentenyl diphosphate biosynthetic process
methylerythritol 4−phosphate pathw ay
valyl−tRNA aminoacylation
retrograde transport
endosome to Golgi
protein N−linked glycosylation
via asparagine
ribosomal large subunit biogenesis
ribosomal large subunit expor t from nucleus
transcription
initiation from RNA polymerase III promoter
02468
R atio H I Varian t G enes / E xpected G en es
G e n e O n t lo g y T e r m
0.01
0.02
0.03
pValue
HI_VariantGenes
100
200
300
D
Figure 6.
Gene Ontology analysis of the genes affected by high impact variants. The x-axis represents the value of the over
or under-representation of these categories. The size of the dot represents the number of genes of the category and the color
intensity its statistical significance as p-value. (
A
) Vouves bottom tree sample; (
B
) Vouves top tree sample; (
C
) High impact
changes shared by all the Greek accessions (‘Kalamon’, ‘Koroneiki’, ‘Mastoidis’, ‘Mavreya’, ‘Megaritiki’ and ‘Myrtolia’) but
absent in the Vouves tree; (
D
) Genes affected by high impact variants common for all the datasets (Vouves tree and Greek
accessions).

Plants 2021,10, 2374 13 of 18
The comparison of the Gene Ontology Terms of the genes with high impact variants
between the Greek samples and the Vouves bottom and top tree didn’t show any term
related with fatty acid metabolism or accumulation. This comparison didn’t reveal other
terms related with adaptation to biotic or abiotic stresses. Nevertheless, it is interesting that
pathways related with carbohydrate and amino acid metabolism were found, in the same
way that has been found in the sweep regions related with the process of domestication [
31
].
An important proportion of these genes are related with the cell wall biosynthesis (e.g.,
“cellulose synthase 1” or “heteroglycan glucosidase 1”). The impact of these variations in
the phenotype of the different varieties is uncertain. These genes are part of extensive gene
families (e.g., 42 genes were annotated as “cellulose synthase” in the reference genome used)
while mutations in some of the copies can be a part of the process of non-functionalization
of multiple gene copies that may be risen by duplications. The study of the evolution of
the cellulose synthase revealed that some of them are indeed pseudogenes [
42
] so it is
expected to find high impact variants on those. It is also interesting to find a high number
of genes with HI variants associated with DNA repair. Most of these genes have the
descriptor “PIF1 helicase”. PIF1 DNA helicases are important players in the stability of
the genome [
43
], but their multiple copies and their redundancy has limited the number
of functional studies associated to this gene family. The olive genome has annotated
103 genes under the descriptor of “PIF1 helicase”. Indeed, 9, 2, 7 PIF1 Helicase genes
presented high impact variations in the Vouves bottom and top samples and the other
Greek accessions, respectively. One possibility of this elevated number of PIF1 genes as
well as the high impact variations on those may be related with the fact that Helitron
HelRel proteins have an S1 helicase domain similar to the PIF1 helicase [
44
]. This may lead
to the misidentification of these helitrons as PIF1 helicases.
3. Conclusions
The olive tree of Vouves, which is considered to be one of the oldest olive trees in the
world, was studied to reveal its genetic relations with present-day cultivated and wild
genotypes and to shed light into potential origin or routes of domestication of olive. The top
of the Vouves tree sample has an Ajk statistics value of 0.53 with the ‘Mastoidis’ accession
indicating a relation of the same order than half-siblings, and verifying that the Vouves
tree was grafted with a present-day cv. The Vouves rootstock (bottom sample) showed a
clear grouping with the oleaster samples in a similar way that the ‘Megaritiki’ Greek cv.
An ABBA-BABA analysis showed that it doesn’t have any introgression from the oleaster
population. The Greek cvs didn’t show any high Ajk values with the Vouves bottom
sample so it cannot be documented that the original monumental tree has a special role in
the development of some important present-day Greek varieties. Nevertheless, previous
results showed that the Vouves tree is genetically related with other monumental trees of
the Sotira area in Cyprus. An ongoing survey on centennial olive trees allover Greece and
the comparative analysis of resequencing data with other projects will shed more light on
the history of olive growing and potentially reveal ancient varieties to be reserved as natural
heritage and genotypes with desirable traits to be exploited as invaluable pre-breeding
material.
4. Materials and Methods
4.1. Plant Samples and DNA Extraction
Fresh healthy leaves were collected during July 2018 separately (i) from the highest
part of the Vouves monumental olive tree (referred as ‘top’) and (ii) from the lowest part of
the tree at the base of the trunk (referred as ‘bottom’) and were immediately transferred in a
cool container to the laboratory for DNA extraction. For all analysis described herein, total
genomic DNA was isolated from leaf material using the DNeasy Plant Mini kit (Qiagen cat.
No 69104, Düsseldorf, Germany) according to manufacturer’s instructions. The hypothesis
that the tree is consisted of two genotypes united through grafting was tested.

Plants 2021,10, 2374 14 of 18
4.2. DNA Sequencing and SSR Analysis
1 ug of DNA for each of the Vouves monumental olive tree (top and bottom) were
sent to Novogene Co. Ltd. (Sacramento, CA, USA, https://en.novogene.com/, accessed
on 25 June 2018) for whole genome sequencing. The libraries were prepared following the
standard Illumina Nextera
®
protocol for insert sizes ~350 bp. Then, they were sequenced
with an Illumina Novaseq6000 platform, paired-end 150 bp.
SSR analysis followed [
45
] with the inclusion, in the SSR set, of two new loci while
omitting one, previously used. Total SSR loci presently employed were 11 (DCA3, DCA5,
DCA9, DCA14, DCA16, DCA18, EMO90, GAPU71B, GAPU101, GAPU103A, UDO99).
Allelic data were used to construct a UPGMA similarity dendrogram based on Jaccard
distances, similarly to [36].
4.3. Read Processing, Reference Mapping and Variant Calling
Reads were processed with Fastq-mcf v1.04.676 from the Ea-utils package [
46
], then
they were mapped with BWA v0.7.17-r1188 [
47
] to the Olea europaea var. sylvestris genome
reference v1.0 [
32
] downloaded from Phytozome (https://phytozome.jgi.doe.gov/, ac-
cessed on 20 May 2019) [
48
]. Mapping coverage was evaluated with BEDtools genome-
cov v2.29.0 [
49
] with the option -bga. Variants were called using Freebayes v1.3.1-16-
g85d7bfc [
50
] with a minimum read coverage of 5, a minimum mapping quality of 20 and
discarding complex variants. Non-biallelic variants were filtered with Vcftools v0.1.15 [
51
].
The impact of the variants was evaluated with Snpeff v4.3 [52].
4.4. Origin Analysis
RNA-Seq reads representing 55 olive accessions from 14 different countries were
downloaded from GenBank SRA (ProjectID PRJNA525000 [
6
] as well as 50 olive accessions
Whole Genome DNA Resequencing (WGR) data from the SRA project PRJNA556567 [33].
Reads were processed with Fastq-mcf v1.04.676 from the Ea-utils package [
46
] and them
mapped to the reference genome Olea europaea var. sylvestris genome reference v1.0 [
32
]
using Hisat2 v2.1.0 [
53
]. Variants were called using Freebayes v1.3.1-16-g85d7bfc [
50
] with
a minimum read coverage of 5, a minimum mapping quality of 20 and discarding complex
variants. The VCF file was filtered using Vcftools v0.1.15 [
51
] removing the variants
that were not present in all the samples and keeping only biallelic Single Nucleotide
Polymorphisms (SNPs). The VCF file was upload in RStudio v1.1.463 running R v3.5.1
using Adegenet v2.1.1 [
54
] and Poppr v2.8.3 [
55
] packages. A distance matrix between all
the samples’ SNP was calculated with the function dist() with the default parameters. A
distance tree was calculated with the function aboot() function from the Poppr package
using Nei distance, NJ tree and 1000 samples. A Principal Components Analysis (PCA)
was performed using the prcomp() function from the Stats R core package with the default
parameters and it was plotted with the Ggplot2 v3.2.0 package. The DAPC analysis was
performed with the function dapc() from the Adegenet package.
Population structure was inferred using two alternative procedures: (1) a Bayesian,
model-based algorithm employed through STRUCTURE software (release: V2.3.4, July
2012) [
56
] and (2) Discriminant Analysis of Principal Components [
57
] which produces
genetic clusters using a few “synthetic” variables constructed as linear combinations of
the original variables (alleles). These alleles are in turn selected as having the largest
between-group variance and the smallest within-group variance.
The ABBA-BABA analysis was performed using the same VCF file that was de-
scribed before. The VCF files was converted to the EIGENSTRAT format with the script
convertVCFtoEigenstrat.sh from Joanam at Github (https://github.com/joanam/scripts/
blob/master/convertVCFtoEigenstrat.sh/, accessed on 8 September 2021). The “admixr”
R package v0.9.1 was used to perform the ABBA-BABA analysis. In summary, the EIGEN-
STRAT files were uploaded in R with the eigenstrat() function. The sister group to the
targets was the Italian cvs (“Frantoio”, “Grappolo”, “Leccino) being a monophyletic branch
in the Figure 2. The wild accessions were the O. europaea var. sylvestris accessions: “Mi-

Plants 2021,10, 2374 15 of 18
norca”, “Jaen”, “PalmaRio” being also a monophyletic clade for sylvestris. Finally, as
outgroup the O. europaea subsp. laperrinei was used.
4.5. Gene Space Comparison
Gene space comparison was performed using three different approaches by comparing
the two Vouves olive samples, the cv ‘Farga’ (SRA accession ERR1346608) [
30
] and the wild
type reference Olea europaea var. sylvestris genome v1.0 [
32
]. For the first approach, the
presence or absence of the genes annotated in the reference genome was analyzed using
BEDtools v2.29.0 [
49
]. First the BAM files containing the mapped reads was converted to
BED with the function bamtobed. Then, the bed rows were merged using the function
merge. Finally, the function complement was applied to the previous BED file and the
reference gene annotation GFF file, producing a filtered GFF with the genes that were not
covered by the mapped reads. For the second approach the impact of the variants (see
Section 3) on the gene space was evaluated with Snpeff v4.3 [52].
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/
10.3390/plants10112374/s1, Figure S1: PCA analysis of the genetic distances between the different
samples. Samples with SNPs produced from RNASeq data have the prefix “t”. Samples with SNPs
produced by DNA WGR data have the prefix “g”. Figure S2: Most probable number of clusters
based in the lowest entropy value for the DAPC analysis. Number of ancestral populations are
compared with the entropy values; Figure S3: Assignment of the different individuals to the ancestral
populations for K = 2, K = 3, K = 4 and K = 5 for the DAPC analysis; Figure S4: Analysis of the
relatedness between the different samples based in the AJK index. Positive relationships are colored
in red and negative relationship are in blue; Table S1: ABBA-BABA analysis performed to compare
individuals with possible admixture with the groups Italian cvs (X), O. europaea var. sylvestris (Y) and
Olea europaea subsp. perrinei accession used as outgroup (Z). Significant values for the introgressions
from O. europaea var. sylvestris have been colored in green. Significant values for the introgressions
from the Italian cvs have been colored in blue including the name of the accession; Table S2: List of
genes associated with the GSEA for genes containing High Impact (HI) variants.
Author Contributions:
Conceptualization, A.G.D. and G.K.; data curation, A.B. and C.Z.; formal
analysis, A.B. and C.Z.; funding acquisition, G.K,; investigation, K.K.L. methodology, G.K., A.G.D.
and A.B.; project administration, G.K.; supervision, A.G.D. and G.K.; Visualization, A.B. and C.Z.,
resources, A.B. and G.K.; writing—original draft preparation, A.B.; writing—review and editing G.K.,
A.G.D., E.M. and A.B. All authors have read and agreed to the published version of the manuscript.
Funding:
This research has been partially financed by (i) Greek national funds through the Action
“Establishment of a National Research Network in the Olive Value Chain”, code 2018
Σ
E01300000 of
GSRT, (ii) the General Secretariat for Research and Innovation of the Ministry of Development and
Investments under the PRIMA Programme for the project Freeclimb (PRIMA is an Art. 185 initiative
supported and co-funded under Horizon 2020, the European Union’s Programme for Research and
Innovation) and (iii) the European Union’s Horizon 2020 research and innovation programme under
grant agreement No. 101000427 for the project Gen4Olive.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The two Vouves trees re-sequencing data was submitted to the NCBI
SRA database with the BioProject accession number PRJNA721943.
Acknowledgments:
The authors would like to thank the Municipality of Platanias in Chania, Crete,
Greece for generously providing full access to the Monumental Olive Tree of Vouves for conducting
the present study. We also thank Georgios Kostelenos for providing plant material of a centennial
olive tree in Peloponnese employed in the SSR analysis. We would like to thank also the reviewers
for their comments and suggestions.
Conflicts of Interest: The authors declare no conflict of interest.

Plants 2021,10, 2374 16 of 18
Abbreviations
AJK Averaged pairwise relatedness index
BED Browser Extensible Data
DAPC Discriminant Analysis of Principal Components
GBS Genotyping-By-Sequencing
GFF Generic Feature Format
GO Gene Ontology
GSEA Gene Set Enrichment Analysis
HI High Impact variant
LI Low Impact variant
MI Moderate Impact variant
PCA Principal Component Analysis
VCF Variant Call Format
cv(s) cultivar(s)
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