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Evaluation of genetic
diversity among olive trees
(Olea europaea L.) from Jordan
Mazen A. Al-Kilani
1
, Francesca Taranto
2
, Nunzio D’Agostino
3
,
Cinzia Montemurro
4
, Angjelina Belaj
5
, Salam Ayoub
1
,
Randa Albdaiwi
6
, Shireen Hasan
7
and Ayed M. Al-Abdallat
8
*
1
National Center for Agriculture Research (NARC), Amman, Jordan,
2
Institute of Biosciences and
Bioresources, National Research Council (CNR-IBBR), Bari, Italy,
3
Department of Agricultural Sciences,
University of Naples Federico II, Portici, Italy,
4
Department of Soil: Plant and Food Sciences (DiSSPA),
University of Bari Aldo Moro, Bari, Italy,
5
Centro “Alameda del Obispo”, Instituto Andaluz de
Investigacio
´n y Formacio
´n Agraria, Pesquera, Alimentaria y de la Produccio
´n Ecolo
´gica (IFAPA),
Co
´rdoba, Spain,
6
Department of Allied Medical Sciences, Zarqa University College, Al-Balqa Applied
University, Al-Salt, Jordan,
7
Hamdi Mango Center for Scientific Research, The University of Jordan,
Amman, Jordan,
8
Department of Horticulture and Crop Science, School of Agriculture, The University
of Jordan, Amman, Jordan
This study aimed to identify and evaluate the genetic diversity of olive trees in
Jordan, a country located in the eastern Mediterranean, where olive domestication
originated. For this purpose, a total of 386 olive trees were analyzed, including 338
collected from two surveys (JOCC-1 and JOCC-2) across seven regions, and 48
selected accessions from the Olive Germplasm Bank of Jordan (JGBOC). These
trees underwent comprehensive phenotypic and molecular characterization using
different tools. Significant differences in morphological traits were detected among
tested regions using the Chi-square test. Principal components analysis revealed
that fruit color change and growth habit as the most discriminating traits,
segregating the trees into two groups, with the first group including the KANABISI
cultivar and the second group including the KFARI BALADI cultivar. Utilizing
Kompetitive Allele Specific PCR assay, two sets of informative SNPs were used
for the genetic diversity analysis. Cladograms were constructed using the
maximum likelihood method, revealing a consistent pattern where two clades
containing identical genotypes were observed to cluster with the KFARI BALADI or
KANABISI. In addition, the SNP data was used to perform a comparative analysis with
the Worldwide Olive Germplasm Bank of Co
rdoba, which revealed 73 unreported
olive genotypes from Jordan. Genetic structure analyses using Discriminant
Analysis of Principal Components (DAPC) identified four clusters with distinctive
patterns of relatedness among 149 unique accessions, including 52 olive
accessions from various Mediterranean countries (IOCC-3). ADMIXTURE analysis
revealed four genetic clusters, consistent with the clustering observed in DAPC and
cladogram analysis, indicating a high level of genetic admixture among Jordanian
olive germplasm. In conclusion, the results show that olive trees in Jordan are
highly diverse, providing valuable information for future conservation and
management plans.
KEYWORDS
genetic variation, center of domestication, molecular markers, olive, phylogenetic
analysis, population structure, single nucleotide polymorphisms
Frontiers in Plant Science frontiersin.org01
OPEN ACCESS
EDITED BY
Isabel Mafra,
University of Porto, Portugal
REVIEWED BY
Antonio Giovino,
Council for Agricultural Research and
Agricultural Economy Analysis | CREA, Italy
Jalal Kassout,
National Institute for Agricultural Research,
Morocco
*CORRESPONDENCE
Ayed M. Al-Abdallat
a.alabdallat@ju.edu.jo
RECEIVED 23 May 2024
ACCEPTED 15 July 2024
PUBLISHED 06 August 2024
CITATION
Al-Kilani MA, Taranto F, D’Agostino N,
Montemurro C, Belaj A, Ayoub S, Albdaiwi R,
Hasan S and Al-Abdallat AM (2024)
Evaluation of genetic diversity among olive
trees (Olea europaea L.) from Jordan.
Front. Plant Sci. 15:1437055.
doi: 10.3389/fpls.2024.1437055
COPYRIGHT
© 2024 Al-Kilani, Taranto, D’Agostino,
Montemurro,Belaj,Ayoub,Albdaiwi,Hasanand
Al-Abdallat. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). The
use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.
TYPE Original Research
PUBLISHED 06 August 2024
DOI 10.3389/fpls.2024.1437055
1 Introduction
The olive tree (Olea europaea L.) is a fruit tree native to the
Mediterranean region that belongs to the Oleaceae family (Besnard,
2016). It has become an economically important crop, with
approximately 10.95 million hectares cultivated globally with an
estimated production of 18.40 million tons of olives (FAOSTAT,
2022). The Mediterranean region hosts 95% of the world’s
cultivated olive growing area, with Spain (21.41%), Turkey
(16.19%) and Italy (11.74%), as the main producers (FAOSTAT,
2022). It also has cultural and historical significance and is
considered a sacred tree in various civilizations and one of the
oldest domesticated plants in the world (Green, 2002).
The cultivated olive tree (O. europaea subsp. europaea)is
believed to have originated from its wild ancestor, oleaster (O.
europaea subsp. sylvestris), in the eastern parts of the Mediterranean
basin (Barazani et al., 2023). Fossil pollen datasets obtained across
the Mediterranean basin showed that the southern Levant served as
the primary site for olive cultivation as early as ~6500 years BP,
followed by a subsequent domestication process in Crete/Greece
(Langgut et al., 2019). This indicates that the eastern Mediterranean
basin is the primary center of olive domestication and is considered
a natural habitat for wild and cultivated olive species (Julca et al.,
2020). However, the history of olive domestication remains
complex and somewhat mysterious, with archaeological and
genetic studies suggesting several possible origins (Besnard et al.,
2013;Lavee and Zohary, 2011). It remains unclear whether olive
cultivars originated from a single initial domestication event in the
Levant, followed by secondary diversification (Gros-Balthazard
et al., 2019), or if the cultivated lineages resulted from multiple,
independent primary domestication events (Jimenez-Ruiz
et al., 2020).
In Jordan, a country situated in the eastern parts of the
Mediterranean basin, olives are highly believed to have been
domesticated as early as 6200 BCE, with evidence of oil pressing
dating back to at least 5200 BCE in Pella, located in the northern
Jordan valley (Dighton et al., 2017). During this period, the size and
shape of olive endocarps changed, indicating a selection process for
larger, more uniform olives fruits. An archeological study
conducted by Jordanian and French scientists suggests that
Hadeib Al-Reeh, a village in Wadi Rum, may be the oldest site in
the world where olive tree cultivation dates back more than 7,500
years ago (Al-Shdiefat et al., 2012). Analysis of ashes from fireplaces
in the village ruins revealed that olive cultivation dates back to the
Chalcolithic period (around 5400 BC). This evidence strongly
suggests that Jordan lies within the primary center of olive
domestication in the Mediterranean region (Barazani et al., 2023).
To identify olive cultivars, scientists rely on morphological,
physiological, and biochemical traits, as well as more recently on
DNA molecular markers technologies (Boucheffa et al., 2019).
Molecular tools have been used for olive cultivar identification,
fingerprinting, and determining genetic relatedness patterns among
them (Sebastiani and Busconi, 2017;Kaya et al., 2019;Marchese
et al, 2023). Recently, new techniques based on next-generation
sequencing technologies such as genotyping-by sequencing (GBS),
double digest restriction-site associated DNA (ddRADseq), and
diversity arrays technology (DArTseq) have enabled the
generation of thousands of single nucleotide polymorphisms
(SNPs). These SNPs have been used for cultivar characterization,
analysis of genetic diversity, and have significantly enhanced our
understanding of the genetic makeup of olive trees (D’Agostino
et al., 2018;Kaya et al., 2019;Zhu et al., 2019;Islam et al., 2021;
Slobodova et al., 2023). SNP markers have also proven to be an
excellent tool for verifying the authenticity of table and oil olives
(Ben Ayed and Rebai, 2019;Piarulli et al., 2019). Such new
technologies have empowered genetic studies on the olive tree,
enabling gene discovery, mapping of quantitative trait loci (QTL)
and a deeper understanding of its domestication process
(Koubouris et al., 2019;Mariotti et al., 2020).
In Jordan, the predominant olive cultivars include NABALI
BALADI,NABALI MUHASSAN,andRASIE (Brake et al., 2014).
However, numerous clones of these cultivars are dispersed
throughout the country, resulting in the assignment of various
common names. These names often differ based on the specific
region where the olives are grown. The NABALI BALADI cultivar is
regarded as one of the oldest olive cultivars in the Levant, believed
to have originated on the banks of the Jordan River. It is favored by
many farmers for its robust resilience and adaptation to dry
environments, as well as its resistance against various pathogens
(Brake et al., 2014). Furthermore, in Jordan, farmers continue to
utilize centuries-old olive trees known as ROMI, a term referring to
the era of the Romans. These trees grow naturally in various regions
across Jordan and are preferred over introduced cultivars from
foreign origins (Brake et al., 2014). The centenary age of the ROMI
trees, their adaptation to harsh environment, resilience to various
stresses, and high quality of their extracted oil indicate a rich genetic
diversity. This highlights their significance and emphasizes the
urgency of conserving and integrating them into the local olive
production system.
One of the primary challenges facing the cultivation of olive
trees in Jordan is the limited understanding of their origins, degree
of genetic purity and diversity, particularly whether they are
considered clones or not. Therefore, scientists are actively
investigating the genetic relationships between wild and cultivated
olive trees that naturally thrive in various parts of the country. The
second major problem is the loss of such valuable genetic material
from its natural habitat, exacerbated by climate change,
urbanization, and insufficient awareness of their historical
importance (Belaj et al., 2016). Furthermore, such material,
growing naturally in remote and marginal areas, is often
neglected, lacking proper care and good agricultural practices that
are crucial for optimal growth and production. Therefore, it is
important to shed the light on the genetic diversity of olive
germplasm in Jordan, a country where olive cultivation is
believed to have originated. This will aid in better understanding
their importance and underscore the necessity of developing action
plans to preserve them using both in situ and ex situ approaches.
This study assesses the extent of genetic diversity among
Jordanian olive trees using molecular markers and morphological
traits, with the aim of guiding olive conservation and management
strategies, especially in the context of climate change and genetic
Al-Kilani et al. 10.3389/fpls.2024.1437055
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erosion. Understanding the genetic variability of Jordan olive trees in
arid conditions may facilitate the identification of genes/alleles to be
introduced in olive breeding programs across the Mediterranean
region. To this end, two targeted prospecting surveys were conducted
across Jordan to identify olive trees growing across different habitats.
The identified trees were characterized by different morphological
traits, which were used to categorize them into distinct sets. The
genetic identity and level of variation among olive genotypes were
assessed with a selected set of SNP markers through the KASP
(kompetitive allele-specific PCR) genotyping assay. The obtained
results provide valuable insights into the distribution and
characteristics of olive trees across different regions of Jordan,
enhancing our understanding of the genetic diversity within the
country’s olive tree heritage and their ecological significance. The data
highlights the importance of Jordanian olive genetic resources and
emphasizes the pressing need for future conservation plans,
innovative propagation strategies, and integration into the national
olive production system.
2 Materials and methods
2.1 Plant material, survey procedure and
description of the collection sites
In this study, two field surveys were conducted in 2020 and 2022
across various regions of Jordan to identify and characterize olive
trees (Table 1;Supplementary Figure S1). Both surveys spanned from
April to December and targeted olive trees older than 100 years,
estimated by measuring the trunk diameter at 130 cm from the
ground following the procedure outlined by Arnan et al. (2012).
Comprehensive information, including site description (regions
representing governorates and areas representing municipalities
within each governorate) with respective geographic coordinates
(latitude, longitude, elevation), physiography, habitat, micro-climate
conditions, human management practices, density, and distribution,
was collected for each Jordanian locality (Supplementary Table S1).
Additionally, meteorological data such as average annual rainfall,
temperature, and humidity were obtained from the nearest
meteorological station for each site, sourced from the Jordanian
Meteorological Department (data not shown).
In the first prospecting survey (Jordan olive core collection-1;
JOCC-1), a total of 200 olive trees were sampled from seven
different regions: Irbid (IR) (n. = 62), Jarash (JA) (n. = 39),
Ajloun (AJ) (n. = 32), Balaqa (BA) (n. = 24), Al-Karak (KR)
(n. = 16), At-Tafilah (TF) (n. = 13), and Ma’an (MN) (n. = 14).
In the second prospecting survey (Jordan olive core collection-2;
JOCC-2), two regions from the first survey, namely BA and JA, were
extensively studied where both regions were re-targeted based on
preliminary observations of significant morphological variation and
abundant genetic diversity observed during the molecular analysis
of olive trees sampled in the initial prospecting survey. A total of
138 olive trees were sampled in the second prospecting survey,
including 94 trees from BA and 44 from JA. Additionally, this study
included 48 cultivated varieties from the Olive Germplasm Bank of
Jordan (Jordan Gene Bank Olive Collection (JGBOC), located in Al-
Mushagar (NARC, Jordan), bringing the total number of Jordanian
olive trees under study to 386 (Supplementary Table S1). Finally, 52
trees representing olive accessions from various Mediterranean
countries were selected from the open-field collection (Italy Olive
Core Collection (IOCC-3)) conserved in Palagiano, Taranto, Italy
(Supplementary Table S1) and were used for the genetic structure
and relationships analysis.
In this study, each recorded Jordanian olive tree received a
unique identification code to specify its origin and collection site
(Supplementary Table S1). For this purpose, the 386 trees of Jordan
were grouped based on their geographical region: IR (Irbid), AJ
(Ajloun), JA (Jarash), BA (Balaqa), and KR (Karak), TF (Tafilah),
MN (Ma’an), while the last group included accessions retrieved
form JGBOC (Supplementary Table S1).
2.2 Morphological characterization
The morphological characterization involved the examination of
28 traits following the methodology outlined in Barranco et al. (2000).
The analysis included both the olive trees sampled during the field
surveys (334 trees, with data missing for four trees) and the 48
JGBOC accessions. A complete list of the studied morphological
traits, along with descriptions, measurement methods, and recorded
data are given in Supplementary Table S2. In addition to the traits
outlined in Barranco et al. (2000), measurements of trunk diameter
(Arnan et al., 2012) and fruit color change location (starting point of
fruit coloration: apex; uniform; base) were included. Morphological
data were subjected to statistical analysis using the Chi-square test
based on the region of origin of the trees, as described above
(Supplementary Table S1).
To explore patterns of variation in morphological traits among
individuals, Principal Component Analysis (PCA) was performed
using ‘FactoMineR’version 2.4 (Leet al., 2008;https://cran.r-
project.org/web/packages/FactoMineR/index.html) and ‘factoextra’
version 1.0.7 (Kassambara, 2017;https://cran.r-project.org/web/
packages/factoextra/index.html). To visualize clustering patterns
within the dataset, a hierarchical cluster was built using the
‘pheatmap’package v1.0.8 in R (Kolde and Kolde, 2015;https://
cran.r-project.org/web/packages/pheatmap/index), using Euclidean
distances and the Ward.D2 method.
Additionally, a heatmap was generated, that visually represents
the morphological data of the 382 olive trees. Furthermore,
Pearson’s correlation coefficients were calculated using the
‘corrplot’package in R (Wei et al., 2017;https://cran.r-
project.org/web/packages/corrplot/vignettes/corrplot-intro.html)
to quantify the relationships and correlations between various
morphological traits.
2.3 Molecular characterization
Total genomic DNA (gDNA) was extracted from fresh young
leaves from the canopy of each olive tree using the Wizard®
Genomic Purification Kit (Promega, Madison, WI, USA)
following the manufacturer’s instructions. The quantity and
Al-Kilani et al. 10.3389/fpls.2024.1437055
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TABLE 1 Collection, locations, regions, area names, climatic zones, and the number of sampled trees in the study.
Collection Location Region Area Number of
Sampled Trees Climatic Zone
JOCC-1
North of Jordan
Ajloun Ain Janna 6 Mediterranean sub-humid
Ajloun Al Hashimiyya 3 Mediterranean semi-arid
Ajloun Anjara 3 Mediterranean sub-humid
Ajloun Khirbat al Wahadinah 13 Mediterranean semi-arid
Ajloun Kufranjah 7 Mediterranean semi-arid
Irbid Al Taybeh 12 Mediterranean semi-arid
Irbid Al-Korah 12 Mediterranean semi-arid
Irbid Al-Mazar-N 12 Mediterranean sub-humid
Irbid Bani Kananeh 14 Mediterranean semi-arid
Irbid Kufr-Asad 12 Mediterranean semi-arid
Jarash Al-Kittah 18 Mediterranean semi-arid
Jarash Borma 15 Mediterranean semi-arid
Jarash Kausheba 6 Mediterranean semi-arid
Central of Jordan
Balaqa ArRumaymin 2 Mediterranean sub-humid
Balaqa Buyoida 2 Mediterranean semi-arid
Balaqa El-Baque 4 Mediterranean sub-humid
Balaqa Maysarah 9 Mediterranean semi-arid
Balaqa Shafa-Badran 5 Mediterranean semi-arid
Balaqa Umm-Joza 2 Mediterranean sub-humid
South of Jordan
Karak Aiy 7 Mediterranean semi-arid
Karak Al Taybeh-S 7 Mediterranean arid cool
Karak Iraq 2 Mediterranean arid cool
Ma’an Megaraeh 4 Mediterranean arid cool
Ma’an Wadi Musa 8 Mediterranean arid cool
Ma’an Wadi Rum 2 Saharan Mediterranean
Tafilah Aema 7 Mediterranean semi-arid
Tafilah EL-Balad 3 Mediterranean semi-arid
Tafilah El-Meshref 3 Mediterranean semi-arid
JOCC-2
North of Jordan Jarash Borma 44 Mediterranean semi-arid
Central of Jordan
Balaqa ArRummain 16 Mediterranean semi-arid
Balaqa Buyoida 13 Mediterranean semi-arid
Balaqa Maysarah 49 Mediterranean semi-arid
Balaqa Shafa-Badran 4 Mediterranean semi-arid
Balaqa Umm Jozeh 12 Mediterranean sub-humid
JGBOC Central of Jordan Madaba Gene Bank 48 Mediterranean semi-arid
IOCC-3 Italy Taranto Palagiano 52 Mediterranean humid
Al-Kilani et al. 10.3389/fpls.2024.1437055
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quality of the extracted gDNA were assessed using a 1% agarose gel
stained with Red Safe (Intron, Bio-tek, Seoul, Korea) and a
spectrophotometer (BIO-RAD, SmartSpecTM Plus, Hercules, CA,
USA). Subsequently, a gDNA stock solution (30 ng/µL) was
prepared for each sample using sterile distilled water and stored
at -20°C until further analysis.
Two sets of SNP markers were used for KASP analysis
(Supplementary Table S3). KASP technology was selected for its
ability to specifically target individual SNP loci with high accuracy.
It offers cost-effectiveness for large-scale studies, scalability across
hundreds to thousands of SNP markers, and consistent
reproducibility across different laboratories. Additionally, its
compatibility with automation enhances workflow efficiency and
minimizes potential human errors. The SNP markers in both sets
were selected based on their highest degree of authenticity score.
The first set of markers included 24 SNPs sourced from two
previous studies: Biton et al. (2015) (N. = 12) and Belaj et al.
(2018) (N. = 12). This set was successfully tested on 173 trees from
JOCC-1, along with 42 trees from JGBOC. The second set included
48 SNPs from Belaj et al. (2022) and was tested on 203 trees,
encompassing 132 trees from JOCC-2, 37 selected trees from JOCC-
1, and 34 accessions from JGBOC.
The SNP profile from the second set was utilized for two
primary objectives. Firstly, it was compared with the World Olive
Genebank Collection (WOGBC) database to evaluate the reliability
of the EST-SNP markers across various laboratories and techniques,
and to determine how the genetic profile or SNP markers of olive
trees from Jordan compare or match with those already recorded in
the WOGBC database (Belaj et al., 2022). Secondly, the same set of
markers was employed for conducting the Discriminant Analysis of
Principal Components (DAPC) and population structure analysis.
This analysis involved 52 trees from the IOCC-3 and 203 olive trees
from Jordan, as described previously.
The SNP marker sequence data was used to design KASP assays,
which were performed and analyzed by LGC-Genomics (Hoddesdon,
UK). For the first and second sets of SNP markers, 22 and 45 assays
were successfully designed and tested, respectively. The genotypic data
obtained from these assays were analyzed using LGC’s KlusterCaller
software (https://www.biosearchtech.com/products/pcr-reagents-kits-
and-instruments/pcr-instruments-and-software/genotyping-and-lims-
software/klustercaller-genotyping-software) and visualized using
LGC’sSNP-viewersoftware(https://www.biosearchtech.com/
products/pcr-reagents-kits-and-instruments/pcr-instruments-and-
software/genotyping-and-lims-software/snpviewer). Each marker
was scored based on its allele call in each individual, and the
results were transformed into a binary matrix. In this matrix, the
presence of homozygous alleles was scored as 00 or 11, while
heterozygous alleles were represented as 01.
2.4 Genetic diversity and population
structure analysis
The heterozygosity index (H), polymorphism information
content (PIC), and discriminating power (D) were calculated
using iMEC (https://irscope.shinyapps.io/iMEC/). SNP data were
used to construct cladograms using the maximum likelihood
method (with default parameters under the BINGAMMA
substitution model), with 1,000 bootstrap replicates using RAxML
(Randomized Axelerated Maximum Likelihood) (Edler et al., 2021).
The constructed tree was refined and visualized using the
Interactive Tree of Life online tool (Letunic and Bork, 2021).
PLINK v.1.90 (Purcell et al., 2007) was used to compute the IBS
(Identical-By-State) distance matrix between pairs of accessions
using the second set of markers. Identical genotypes within different
regions (JA: Borma and BA: Maysarah, ArRummain, Byouida and
Umm Jozeh) were found by setting an IBS value ≥0.95 (to capture
as much intra-region variability as possible).
After removing duplicates within regions, a more stringent IBS
analysis was conducted with a threshold of ≥0.98 to search for
duplicates in the JOCC-1, JOCC-2 and JGBOC collections and
reduce putative redundancy. Only one tree per unique genotype was
retained for downstream analyses. Population structure was
investigated using two different approaches. Allele frequency and
ancestry estimation were performed using ADMIXTURE v. 1.3.0
(Alexander and Lange, 2011), with 10-fold cross validation (CV) for
subpopulations (K) between 1 and 15, and 1,000 bootstrap
replicates. CV scores were used to estimate the optimal K value.
A membership coefficient (q
i
) > 0.55 was used to assign individuals
to each cluster. A DAPC analysis was performed using the
‘adegenet’package in R (Jombartetal.,2010;Jombart and
Ahmed, 2011). The optimal number of principal components
(PCs) was determined using a value ≥1:200. The optimal number
of subpopulations was assessed using the Bayesian information
criterion (BIC). Pairwise genetic distance between subpopulations
identified by DAPC was estimated using the Weir and Cockerham’s
average F
ST
, implemented in SVS v.8.9.1.
3 Results
3.1 Occurrence of olive trees in Jordan
In this study, two surveys were conducted to prospect and
identify olive trees in their native range and on farmers’fields
across seven distinct regions in Jordan, encompassing a total of 28
selected areas (Table 1;Supplementary Figure S1;Supplementary
Table S1). Thus, 388 olive trees were sampled. These trees were
located at altitudes ranging from 166 m in Khirbat Wahadinah to
1220 m in Wadi Musa. The regions targeted in this study had an
average annual rainfall between 50 mmand 586 mm, with the highest
recorded in AJ and the lowest in Wadi Rum. The number of trees
varied depending on the region examined, with the highest
concentration of olive trees found in northern Jordan, where the
density of olive trees is considerably high (Supplementary Figure S1;
Supplementary Table S1). The collected trees were found in 28 areas
(municipalities) of the seven targeted regions (governorates), with the
numbers of trees per area ranging from two (Wadi Rum (MN)) to 59
(Borma (JA)). The olive trees were found growing in nature (hillsides,
hilltops, rock face, forests, rangelands, forests and mountains) or in
cultivated fields and were in various topographies, with the
majority in the plains (Figure 1;Supplementary Table S1).
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The predominant habitat of the sampled trees was arable lands, with
two trees identified in the Wadi Rum desert. Most olive trees were
found in managed farmers’fields, while 41 trees grew naturally in
remote areas. In southern areas of the country olive trees thrived and
remained productive thanks to supplementary irrigation practices by
local farmers, despite the dry conditions. In contrast, northern areas
relied predominantly on rainfed production systems. Despite drought
stress from precipitation fluctuations, trees in these regions
continued to grow and produce satisfactorily.
3.2 Morphological data
Data analysis indicated significant differences in several
morphological traits, as confirmed by the Chi-square test using
phenotypic data for 382 trees across eight identified regions
(Supplementary Table S4). Out of the examined traits, 24 showed
statistically significant differences (p≤0.05). Traits related to the
presence and size of lenticels and color at full maturity did not
exhibit significant differences and were excluded from downstream
analyses. Significant differences were observed in fruit weight
among olive trees, with notable frequencies across different
regions. Specifically, trees from the JA region showed the highest
frequency (51.61%) of “low weight”fruits, while JGBOC accessions
exhibited the highest frequency (87.50%) of “the very high weight”
fruits (Supplementary Table S4). These results indicate distinctive
variations in morphological characteristics among the olive trees,
providing valuable insights into their diversity and potential
genetic differentiation.
To analyze the most discriminating traits among the defined
groups, principal components analysis (PCA) was performed. The
Eigenvalues of the top 10 principal components (PCs) explained
78.01% of the total variation, with PC1 explaining 24.10% and PC2
contributing 15.20% (Figure 2;Supplementary Figure S2). Among
the variables tested, the largest contribution in the PCA was
observed for traits related to the location of the color change and
growth habit. These traits clustered closely together in in quadrant
IV of the PCA plot, distinctly separated from the other traits
(Supplementary Figure S2). Moreover, “stone termination”was
loaded separately in quadrant II of the PCA plot, indicating its
potential role in discriminating among the identified olive trees.
Finally, several traits were found to be highly correlated in the PCA,
such as fruit and stone diameter, fruit apex with stone base, tree
vigor, fruit weight, fruit symmetry, and stone symmetry
(Supplementary Figure S2). Overall, the PCA identified key
morphological traits that significantly contribute to differentiation
and genetic variation among the olive tree populations under study.
The PCA results depicted a clear separation of the sampled trees
into two distinctive groups (Figure 2). The first group was
positioned in quadrant IV and included a JGBOC accession
known as KANABISI, along with several olive trees collected from
different sites across the country that were also recognized by local
farmers as KANABISI. The second group was positioned in quadrant
III of the PCA plot and included a JGBOC accession known as KFARI
BALADI, along with several other accessions from JGBOC (such as
B
CD
A
FIGURE 1
Olive trees from selected areas of Jordan (A) Irbid, (B) Ajloun, (C) Wadi Rum and (D) Karak.
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NABALI BALADI,KFARI BALADI,ARABI TAFILAH,KETAT, etc.) and various
olive trees collected from different sites across the country, which
were recognized by local farmers as ROMI or BALADI. Out of the two
defined groups, most of the remaining trees were either positioned
in quadrant III near to the KFARI BALADI group or randomly
scattered, indicating a higher degreeofphenotypicdiversity
(Figure 2). This set included several trees identified in survey 2
(JA and BA regions) as well as several accessions retrieved
from JGBOC.
As mentioned above, several morphological traits can act as
discriminators between KFARI BALADI and KANABISI olive genotypes.
These traits include growth habit, stone weight, fruit and stone
diameter, site of initial fruit color change, longitudinal curvature of
the leaf blade, stone termination, and stone base (Supplementary
Figure S2). In terms of growth habit, trees of KFARI BALADI genotype
exhibited a higher prevalence of the drooping growth habit, whereas
the erect growth habit was more frequent in KANABISI and trees that
showed the same genotype with it. KFARI BALADI trees tended to
show a drooping phenotype, characterized by slender shoots and
branches that curve downward from the outset (Supplementary
Figure S3). Conversely, the growth habit of KANABISI trees was
characterized by strong apical dominance, with branches tending to
grow vertically, resulting in a crown that takes on a distinct conical
shape, transitioning to cylindrical upon reaching maturity.
Regarding the location of the onset of color change, the KFARI
BALADI fruits showed the onset of color change from the base of the
fruit, while the KANABISI genotype showed the onset of color change
from the apex of the fruit. For “the longitudinal curvature of the leaf
blade”,K
FARI BALADI trees predominantly exhibited an epinastic
curvature type, whereas the flat curvature type was more prevalent
in the KANABISI trees. As for stone weight, the highest frequency of a
“medium weight”score was noted in KFARI BALADI trees, whereas the
frequency of “high weight”was more pronounced in the KANABISI
trees. These distinct morphological traits offer a basis for
discriminating between the olive trees of KFARI BALADI and
KANABISI (Supplementary Figure S3).
Significant positive and negative pairwise correlations (p≤0.05)
were observed between different traits (Supplementary Figure S4).
Interestingly, a strong positive correlation was observed between
growth habit and fruit diameter, stone diameter, and the location of
coloration change, while stone termination showed a strong
negative correlation with these traits. This is in general agreement
with the PCA results (Supplementary Figure S2). The heatmap in
Figure 3 allows three distinct clusters to be distinguished based on
genetic background that overlaid with the PCA clustering of
KANABISI,KFARI BALADI, and diverse (i.e., the set of individuals not
included in any of the previous clusters). Overall, the analysis of
morphological traits provided a complete overview of the
relationships and existing variation among the olive trees under
study. However, these results alone may not resolve the issue of
redundancy among Jordan’s olive trees.
3.3 Molecular data analysis
Two sets of informative SNPs, originally derived from previous
studies (Biton et al., 2015;Belaj et al., 2018,2022), were converted into
KASP assays to assess genetic diversity among the olive trees under
investigation (Supplementary Table S3). The first set of markers was
used to genotype 215 olive trees (comprising 173 trees from JOCC-1
FIGURE 2
Principal component analysis (PCA) plot based on morphological traits measured for 382 olive trees showing the distribution of the of 382 olive
trees (cos2: quality of the representation of individuals based on the principal components).
Al-Kilani et al. 10.3389/fpls.2024.1437055
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and 42 trees from JGBOC). Unfortunately, 39 individuals were
excluded from downstream analyses due to failures of the KASP
assay. The second set of markers was used to genotype 203 olive trees
(consisting of 132 trees from JOCC-2, 34 accessions from JGBOC,
and 37 selected trees from JOCC-1). Only three olive trees failed to
produce informative genotypic data and were consequently excluded
from downstream analysis. The analysis of the KASP data from the
first set revealed that the SNP marker OLIVESNP191 exhibited the
highest heterozygosity index (0.59) and polymorphic information
content (PIC) (0.50) (Supplementary Table S5). Conversely, the
lowest heterozygosity index (0.10) and polymorphic information
content (PIC) (0.09) were observed for OLIVESNP600. For the
second set, SNP marker EContig5183 exhibited the highest
heterozygosity index (0.55) and polymorphic information content
(PIC) (0.45), while the lowest heterozygosity index (0.12) and
polymorphic information content (PIC) (0.12) were observed for
EContig2937_4 (Supplementary Table S5).
Molecular marker data from both sets of SNPs were used to
construct cladograms. The cladogram in Figure 4 was generated
using KASP data from 173 olive trees from JOCC-1 and 42 trees
from JGBOC, which organized olive trees into four distinct clades.
Clade 1 (yellow colored) comprised 12 genotypes, including five
accessions from JGBOC (BARNEA (K18), CORATINA,KALAMATA,
ENABY and SORANI), and seven Jordanian trees (BA-4, BA-6, BA-
20, IR-62, JA-30, JA-33, and TF-4). The second clade (green
colored) accounted for 62.09% of the total olive genotypes (131
out of 211) and consisted mainly of KFARI BALADI trees. Thus, within
this cluster, 89 trees were found to be very similar (as inferred by
zero length branches) suggesting that KFARI BALADI is a master
cultivar widely cultivated in various regions of Jordan. In addition,
several accessions from JGBOC (SOURANI,KFARI BALADI,ARABI
TAFILEH,KETAT, and NABALI BALADI-2) also shared the same SNP
genotypes with KFARI BALADI. Two smaller groups were also
identified within this cluster, the first including five olive trees
(BA-18, IR-20, TF-10, and two similar trees, JA-35 and JA-36)
grouped with four accessions from JGBOC (NABALI MUHASSAN,
NABALI BALADI-1, BAROUNI, and PICHOLINE)(Figure 4). The second
group included five accessions from JGBOC (CHEMLALI SFAX,OFF
THE BIGON,MANZANILLA DE SEVILLA,LECCINO and TUFAHI) and eight
olive trees from Jordan. The third clade (blue colored) included 34
trees that shared the same genotype with KANABISI indicating thus
the presence of another master cultivar grown predominantly
in various regions of Jordan. Interestingly, the genotypes within
clade-3 overlap with the ones identified as “KANABISI”in the
morphological analysis. Additionally, olive trees from the IR
region (IR-1, IR-3, IR-24, IR-35, IR-55) grouped together,
indicating close kinship between them (Figure 4). Within this
clade, several accessions from JGBOC were found to be identical,
indicating redundancy even in the gene bank material. The fourth
clade (red colored) included six JGBOC accessions and seven trees,
of which two (JA38 and JA39) were found to be identical (Figure 4).
The cladogram in Figure 5 was generated using KASP data from
45 SNP loci, allowing the distinction of four clades. Despite the
increased number of markers, a significant portion of the collected
material displayed 100% similarity, once again indicating the
presence of redundant genotypes within the collection (Figure 5).
The first clade (yellow colored) comprised 10 genotypes, including
six JGBOC accessions, three trees from JOCC-1 (IR-1, IR-24, and
IR-55), and a single tree from JOCC-2, which is identical to RASIE,a
Jordanian cultivar. Notably, the three trees from JOCC-1 also
grouped together in the cladogram of Figure 4. The second clade
(red colored) comprised six genotypes, five of which were from
FIGURE 3
Heatmap and two-dimensional hierarchical clustering using data from 24 phenotypic traits measured in the 382 olive trees under study.
Al-Kilani et al. 10.3389/fpls.2024.1437055
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JGBOC (BARNEA (K18), CANINO,SORANI,KHODEIRY, and EVOLEK).
This clade also included BA-40 collected in JOCC-2. The third clade
(blue colored), the second largest clade (comprising 87 genotypes,
i.e., 42.86% of the total), showed the highest diversity (Figure 5).
This clade incorporated 10 accessions from JGBOC, all classified as
Eastern Mediterranean cultivars with the exception of SIGOISE .
Among these, was the cultivar ‘KANABISI’, which showed complete
identity with 23 genotypes within the clade. The fourth clade (green
colored) consisted mainly of KFARI BALADI trees, indicating the
dominance of this genotype in different regions of Jordan
(Figure 5). Several genotypes closely related to KFARI BALADI were
observed within the same clade, including 11 trees and three
JGBOC accessions (BAROUNI,NABALI BALADI-1, and NABALI
MUHASSAN). Furthermore, the clade included a smaller subgroup
of 16 genotypes, consisting of three JGBOC accessions (MANZANILLA
DE SEVILLA and LECCINO, oddly identified as identical being thus a
possible JBOC management error, and CORATINA) and 13 genotypes,
with 10 trees collected from the BA region, of which nine were from
JOCC-2 (Figure 5). Interestingly, six trees (BA-63, BA-66, BA-67,
BA-68, BA-127 and BA-130) were found to be identical, indicating
the presence of a new and previously unknown cultivar uniquely
adapted to the BA region.
3.4 Genetic structure and relationships of
olive trees from Jordan and
other countries
The KASP data obtained from the second survey (45 SNP
markers) were used to confirm the identity of the Jordanian olive
germplasm. They revealed a high reliability when compared with
the EST-SNP profiles from the WOGBC database (Belaj et al.,
2022). In addition, as expected and considering that the Jordanian
plant material was provided from the JGBOC to the international
olive collection of Cordoba, the data comparison between the two
collections confirmed the redundant germplasm described above
within the Jordanian collection. Besides, including a large WOGBC
database of 668 different genotypes the comparison made possible
to confirm previously known synonyms of Jordanian olive
germplasm with cultivars from neighboring countries. Thus, as
expected, the 62 olive trees that were identified as KFARI BALADI as
well six JGBOC accessions (KFARI ROMI,ARABI TAFILAH,KETAT,KFARI
ROMI,NABALI BALADI-2 and SOURANI) shared the same genotype with
the Lebanon cultivar ‘BALADI’and its synonyms (Supplementary
Table S6). On the other hand, it was confirmed that 23 prospected
olive trees shared the same genotype with KANABISI, as shown
FIGURE 4
Cladogram constructed using the maximum likelihood method (bootstrapping value of 1,000) based on the genotyping data from 22 SNP loci. The
analysis included 173 olive trees from JOCC-1 and 42 accessions from JGBOC.
Al-Kilani et al. 10.3389/fpls.2024.1437055
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previously (Belaj et al., 2022), that belongs to the synonymy group
of the Syrian cultivar ‘SAFRAWI’. Interestingly, two olive trees
collected from BA were genetically identical to two accessions
from JGBOC. Specifically, BA-38 was identical to LECCINO (ITA),
which might be a collection mistake or sampling error, while BA-42
was identical to RASIE (synonymous with NABALI MUHASSAN (JOR),
GORDAL DEGRANADA (ESP) etc.). The six unique trees that were
found to be identical did not match with any cultivar from the
WOGBC database, indicating the presence of unknown cultivated
germplasm among the prospected trees in Jordan.
The analysis also revealed 14 possible errors of donor collections
and/or mislabeling or propagation errors in the JGBOC, especially in
the case of introduction of foreign plant material. For instance, the
JBBOC cultivars ‘MANZANILLA DE SEVILLA’and ‘CORATINA’,two
important cultivars from Spain and Italy, respectively, showed
different SNP profile with the same cultivars from WOGBC that
were previously authenticated (Trujillo et al., 2014). Similarly, SIGOISE,
a Western Mediterranean cultivar from Algeria (synonymous of
PICHOLINE MAROCAINE;Belaj et al., 2022), was found to share the
same SNP genotype with a totally different (by both morphological
and molecular description at WOGBC) Greek cultivar named
‘GAYDOYRELIA’(Supplementary Table S6). This finding could explain
its initial grouping with eastern Mediterranean varieties (Figure 5).
Interestingly, the comparative analysis confirmed the presence of 73
new and unique genotypes among the prospected olive trees
(Supplementary Table S6).
To eliminate the presence of duplicate and redundant olive
genotypes in JOCC-2 and IOCC-3 (comprising 252 samples), an
initial IBS analysis was performed within each sampling area of
Jordanian trees. Using a threshold of IBS ≥0.95, 26, 34, 2, 1, and 2
individuals were retained for the Borma (43 samples), Maysarah (57
samples), Umm Jozeh (12 samples), Byouida (10 samples), and
ArRummain (16 samples) groups, respectively. After removal of
these duplicated or redundant genotypes, a subsequent IBS analysis
(IBS ≥0.98) was carried out on the remaining 183 olive trees
(Supplementary Table S7). A total of 34 duplicate individuals were
identified and subsequently excluded from the dataset, resulting in a
final collection of 149 unique olive genotypes that included 28 trees
from JOCC-1, 48 trees from JOCC-2, 21 cultivars from JOGBC and
52 cultivars from IOCC-3. These 149 different genotypes were then
used for the subsequent genetic structure analysis. To gain insights
into the genetic structure, two separate analyses were performed
(Figure 6). DAPC analysis identified four clusters of genetically
related individuals (Supplementary Figure S5). Cluster 1 (C1)
encompassed 70 individuals, predominantly from the Borma
(n. = 21) and Maysarah (n. = 22) sites including few
FIGURE 5
Cladogram constructed using the maximum likelihood method (bootstrapping value of 1,000) based on the genotyping data of 45 SNP loci. The
analysis included 132 olive trees from JOCC-2, 34 olive trees from JOCC-1 and 34 accessions from JGBOC.
Al-Kilani et al. 10.3389/fpls.2024.1437055
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Mediterranean cultivars (Figure 6A;Supplementary Table S8).
Cluster 2 (C2) consisted of genotypes (n. = 16) with different
Jordanian origins and the two cultivars, KFARI BALADI and
KANABISI. Most olive cultivars from Italy and other Mediterranean
countries were grouped into clusters 3 (C3) and 4 (C4), with a few
Jordanian trees included in cluster 4 (IR-1, IR-24, IR-55, and BA-3).
ADMIXTURE analysis identified three and four genetic pools (with
cross-validation error values of 0.6627 and 0.6621, respectively)
(Figure 6B,Supplementary Figure S6). At K = 3, a group of 26
Mediterranean cultivars was separated into the q1K3 gene pool,
corresponding to cluster C3 in the DAPC analysis. While 16
genotypes belonging to the q2K3 gene pool largely overlapped
with the DAPC C2 cluster. The q3K3 gene pool included
genotypes with different origins, similar to the C1 cluster in
DAPC, resulting in 44 trees that showed high admixture
(Supplementary Table S8). At K = 4, it was observed that the
gene pools q1K4 and q4K4 corresponded to Mediterranean
cultivars included in cluster C3 (DAPC) and q1K3, while q2K4
and q3K4 corresponded to cluster C2 and gene pool q2K3,
respectively. The gene pools q2K4 and q3K4 separated genotypes
B
C
A
FIGURE 6
Population genetic structure assessed on the 149 olive accessions by (A) DAPC scatter plot. The axes represent the first two Linear Discriminants
(LD). Each circle represents a cluster and each dot represents an individual. Numbers and colors refer to four different groups identified by BIC
values. (B) Pairwise F
ST
distance values between the four clusters identified by DAPC. (C) Population structure with 3 and 4 ancestries.
Al-Kilani et al. 10.3389/fpls.2024.1437055
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belonging to q3K3, and 89 genotypes were identified as admixed
(Supplementary Table S8). Genetic relationship analysis supported
the clustering with minor differences (Supplementary Figure S7). To
investigate the genetic differentiation among the four clusters
defined by DAPC, pairwise F
ST
values were computed. The
analysis showed that genetic differentiation was strong between
C3 and C4 versus C1 and C2 (F
ST
> 0.21), high between C1 versus
C2 and C3 (F
ST
= 0.14 and 0.13), and moderate between C1 versus
C2 (F
ST
= 0.11) and C3 versus C4 (F
ST
= 0.09) (Figure 6C).
4 Discussion
Nowadays, climate change and its associated conditions have
posed significant challenges to olive tree cultivation (Kaniewski
et al., 2023). As a result, there is a pressing need for novel
approaches to effectively address these challenges and develop
adaptation strategies, both in the short and long term, for the
effective use of olive genetic resources (D’Agostino et al., 2018). In
the present study, a great effort was made to explore the genetic
diversity of olive trees germplasm overspread in Jordan, revealing a
remarkable richness of genetic diversity, and the potential of this
germplasm for various purposes.
Field surveys revealed the extensive distribution of olive trees in
a wide range of conditions and habitats. Noteworthy, these olive
trees have shown impressive resilience and have thrived in
environments ranging from unmanaged farmer’sfields to natural
desert habitats. Furthermore, many of these olive trees were in
marginal areas, characterized by a broad spectrum of micro-
environments and spanning a wide altitude range from 166 to
1220 meters, fluctuating rainfall patterns, and received little or no
agricultural interventions (Supplementary Table S1). Despite these
harsh conditions, these trees have continued to grow and produce
high quality yields. The remarkable resilience of the prospected and
identified germplasm highlights its value as native genetic material
suitable for adaptation to various stresses in harsh environments
and future adoption to cope with climate change worse scenarios
(Sales et al., 2021). The inclusion and evaluation of such germplasm
into ex situ germplasm collections and its further use for
comparative field trials and in olive breeding programs will
potentially expand the narrow genetic base of the current elite
olive cultivars in modern olive orchards and improve resilience to
climate change (Dıez et al., 2015;Arenas-Castro et al., 2020).
In this study, the morphological traits proved to be effective and
informative for olive tree discrimination. The morphological
characterization of olive trees identified a considerable level of
phenotypic variations within the identified genotypes, as shown
in the PCA and heatmap analysis (Figures 2,3;Supplementary
Figure S2). The analysis allowed the identification of three main
groups that formed independently of their geographical origin
including the cultivars KFARI BALADI and KANABISI as well as a
diverse set of Jordanian germplasm bank accessions besides
Jordanian trees not associated with the KFARI BALADI and KANABISI
cultivars. Similarly, Chalak et al. (2015) identified three main
groups of olive trees from Lebanon, which are in general
agreement with the results of this study. The PCA analysis of
morphological traits indicated that some traits have higher
discriminative power between the identified groups. These traits
included growth habit, location of fruit color change, fruit and stone
diameter, stone termination, and leaf blade curvature. Previous
studies confirmed that fruit and stone traits were the most
informative traits for olive cultivars identification with a high
discriminating power (Trujillo et al., 2014;Titouh et al., 2021;
Khadivi et al., 2022). In general agreement with this study,
El Bakkali et al. (2019), found that fruit-associated traits were
very effective in identifying a total of 251 different morphological
profiles in a large olive collection. Stone shape was found to have
high discriminating power compared to other traits and can be used
to effectively differentiate between major identified groups as
previously reported (Veloso et al., 2018;Koubouris et al., 2019).
In this study, KFARI BALADI trees had an elliptic stone shape, while
KANABISI had an elongated shape, irrespective to their collection site
(Supplementary Table S2;Supplementary Figure S3). These results
agreed with the previous studies, which found that stone traits are
less affected by environmental conditions than leaf and fruit traits,
due to their high heritability (Terral et al., 2004;Trujillo et al., 2014;
D’Agostino et al., 2018). Furthermore, the positive correlation
between endocarp and fruit traits are in general agreement with
Bazakos et al. (2023). On the other hand, growth habit also proved
to be a useful trait for discriminating between olive tree genotypes
and particularly between KFARI BALADI and KANABISI trees, which
have an upright growth habit. This may highlight the potential use
of KANABISI trees in intensive farming system due to its upright
growth habit as well as its “easy to harvest”features, which make it
suitable for mechanized production systems (Tous et al., 2010).
Furthermore, KFARI BALADI trees were characterized by medium
fruit weight, while KANABISI and JOGBC accessions had high fruit
weight values. In summary, the results of this study highlighted the
potential of olive trees in Jordan as a useful genetic resource for key
horticultural traits such as fruit weight and growth habit.
Here, informative SNP markers described in previous studies
(Biton et al., 2015;Belaj et al., 2018,2022) were used to develop an
effective genotyping assay using the KASP technology (Jatayev et al.,
2017). SNPs have proven to be efficient, reliable, and robust
molecular markers in the study of the genetic diversity of
different plant species, including olive trees (Belaj et al., 2018;
D’Agostino et al., 2018;Sion et al., 2021). Recently, 96 EST-SNP
markers were used to genotype 1,273 accessions collected from 29
countries in the WOGBC-Cordoba-Spain (Belaj et al., 2022).
Despite the high overall genetic diversity, a notable percentage of
redundant accessions was observed within and between collected
materials, regardless of their countries or regions of origin. In
addition, the results of this study highlight that previously tested
SNP markers data are reliable and reproducible in discriminating
between genotypes, especially when it comes to olives, which have
been the subject of numerous studies using various molecular
techniques (Marchese et al., 2023;Gomez-Galvez et al., 2024).
Furthermore, this study describes the first use of KASP
technology in olive trees, which had proven successful and
beneficial in genotyping and discriminating olive accessions.
Recently, KASP markers have been used in genetic studies for
several fruit trees such as: almond (Goonetilleke et al., 2018), apple
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(Winfield et al., 2020), and peach (Fleming et al., 2022). The
conversion of selected SNPs into KASP markers for olive tree
genotyping also proved to be reliable, practical, and cost-effective
and their use allowed characterizing the collected olive tree material
and discriminating olive accessions. The use of this technology was
not only powerful in distinguishing different cultivars, but it also
provided a cheaper and automated markers genotyping for used in
future olive studies (Winfield et al., 2020).
Using SNP data, the genetic relationship analysis grouped olive
trees of Jordan into three main clusters regardless of their
geographical origin. This clustering was in general agreement
with the results obtained from the analysis of morphological
traits. Molecular analysis was able to distinguish between the
cultivars KFARI BALADI and KANABISI and most of the olive trees as
well as accessions from the JOGBC. As expected, the JOGBC
accessions and most of the sampled trees, with a few regional
exceptions shared the same genotype with KFARI BALADI, thus
confirming it is a widespread cultivated genotype across the
country where it has been named differently. The second cluster
included the cultivar KANABISI and all the trees that displayed the
same SNP genotype with this cultivar, indicating that this cultivar is
widespread throughout the country and particularly in arid
environments of Jordan. The third group of olive trees showed
high genetic variation and included accessions from JOGBC and
several olive trees from Jordan. The identification of unique
genotypes among the prospected trees from regions such as TF,
BA, IR, JA and AJ suggests the existence of new and previously
uncharacterized genotypes. These findings are in general agreement
with previous studies in which it was possible to identify genetically
diverse olive trees using molecular markers that were represented
only as a small percentage of the dominant olive cultivars (Baldoni
et al., 2006;Erre et al., 2010;Gomez-Galvez et al., 2024). Such
autochthonous or native olive tree material represents a genetic
reservoir of important alleles, and it is considerably important to
increase the percentage of unique genotypes to be collected in
hotspot regions (Dıez et al., 2011). Furthermore, some of these trees
were found to have special agronomic characteristics, especially
fruit-related traits, including fruit weight that ranged from medium
to very high. These agronomic characteristics are considerably
important because they yield larger quantities of olives, which can
translate to higher oil production or greater fruit yield for table
olives. Furthermore, trees with varying fruit weights may indicate
adaptation to different environmental conditions, such as varying
levels of rainfall or soil types. Understanding these traits helps in
selecting cultivars that are resilient and productive under diverse
agronomic settings.
The SNP marker-based cladogram used in this study revealed the
presence of redundant genotypes in the collection, that were reported
in previous studies (D’Imperio et al., 2011;Mariotti et al., 2020;Belaj
et al, 2022). Previous studies using EST-SNP markers identified the
largest group of redundant accessions that included the Lebanese
cultivar BALADI and 39 identical accessions from six countries
including the SOURANI cultivar from Syria and several accessions
from Jordan (KETAT,KFARI BALADI,KFARI ROMI,ARABI AL-TAFILAH,
and NABALI BALADI). Those results are in total agreement with the
molecular data obtained in this study. In the Eastern Mediterranean
region, local terminology recognizesfourcultivarsintraditionalolive
cultivation: SOURI,NABALI BALADI,NAB ALI MUHASSAN, and MALLISI,
among which SOURI is considered the oldest and most predominant
variety in the region (Ben-Ari et al., 2014). Genetic diversity of some
olive cultivars from the southern Levant using SSR confirmed that the
SOURI cultivar was highly related to the ROMI,NABALI BALADI,NABALI
MUHASSAN, and MAILSI cultivars (Barazani et al., 2023). The second
most prevalent cultivar in Jordan, KANABISI, included trees from desert
areas and was identified as synonymous with the Syrian cultivar
SAFRAWI, as along with other cultivars from eight different countries
(Belaj et al., 2022). It is interesting to note that SAFRAWI cultivar
alongside GORDAL SEVILLANA cultivar have been considered two main
founders of more than 60% of the olive cultivars in the Mediterranean
Basin (Mariotti et al., 2023). In another study, the Greek cultivar
THROUBOLIA, which is synonymous with the cultivar SAFRAWI (Belaj
et al., 2022)andK
ANABISI, showed IBS value of 0.967 with the
monumental olive tree THROUBA NAXOS, estimated to be about
3000 years, linking this cultivar to the early domestication of olive
trees in Greece (Bazakos et al., 2023). These results highlight the
central role played by KANABIS I in olive domestication in the
Mediterranean basin and support the concept of a primary
domestication event in the eastern Mediterranean basin followed by
a subsequent dispersion towards the West accompanied by secondary
domestication events with wild olive populations (Gros-Balthazard
et al., 2019;Bazakos et al., 2023).
In this study, the identity of several Jordanian olive
germplasms was confirmed by comparing SNP data with
WOGBC EST-SNP markers (Belaj et al., 2022). This analysis
facilitated the identification and correction of errors within the
JGBOC, which is crucial for ensuring the accuracy and reliability of
genetic resources for future research. Incorrect labeling or
identification of genotypes due to human error can lead to
confusion and hinder accurate research and breeding efforts. The
implementation of molecular markers for verification can improve
record-keeping practices and help in including missing cultivars
and wild olive genotypes in gene banks to enhance diversity and
prioritize proper conservation methods (Belaj et al., 2022). This
will enhance collaboration and information sharing among
institutions, thereby contributing to the overall enhancement and
sustainability of olive germplasm collections, both within Jordan
and globally. The analysis also identified 73 unique and novel
genotypes from Jordan that had not been previously reported,
highlighting the presence of rich genetic diversity within the
country. One of these unique genotypes is represented by as
many as six individuals in our collection, suggesting evidence of
clonal vegetative propagation, and indicating the cultivated nature
of this unknown genotype. The remaining genotypes are
considered unique, and this suggests the presence of previously
unknown genotypes and potentially novel traits within Jordan’s
olive germplasm, underscoring the importance of further
exploration and conservation efforts. For instance, a unique olive
tree TF-1 produced high fruit weight mean value and flourished in
a region where annual rainfall is less than 200 millimetres. In the
same region, TF-9 produced very high fruit weight mean value and
was closely related to TOFFEHI, a well-known cultivar with high fruit
weight mean value. Besides TF region, high fruit weight has been
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observed in olive trees from AJ, JA and BA, making them a suitable
choice for table olive production. Interestingly, unique olive trees
from Borma area (JA) were found in feral status and had small fruit
weight mean value when compared with other unique trees from
other regions. At the same area, occurrence of wild oleaster is
common indicating a hotspot for future collection missions.
Furthermore, these unique genotypes exhibited a wide range of
phenotypic and genetic variations that can provide a valuable
resource for breeding programs aiming to develop olive cultivars
adapted to local conditions. It is worth to mention that such unique
genotypes could represent previously unrecognized introductions
from neighboring regions or as germinating seeds from existing
cultivars or even ancient lineages that have persisted in Jordan for
millennia (Mousavi et al., 2017). Therefore, future research should
focus on understanding the ancestry and genetic relationships of
these unique genotypes with genetic material within the
Mediterranean basin.
When Jordanian samples were analyzed together with 52
Mediterranean accessions (IOCC-3), they formed distinct clusters
separate from Central and Western Mediterranean cultivars,
indicating a high degree of genetic diversity unique to Jordan.
Similar separation of Eastern Mediterranean cultivars from those in
Central and Western regions has been reported previously
(El Bakkali et al., 2019;Haddad et al., 2020). In the DAPC
analysis, Jordanian olive trees grouped into two clusters distinct
from other Mediterranean genotypes. One cluster included 70 trees
predominantly from Borma and Maysarah, indicating these
locations harbor a hotspot of genetic diversity within Jordan.
Furthermore, ancestry-specific allele frequency estimation
revealed a high degree of genetic admixture within the Jordanian
germplasm. Using a model with four ancestries (K =4), 89
accessions were tagged as admixed, with 60 of these trees
originating from Jordan, indicating a complex evolutionary
history within the region. Previous studies by Belaj et al. (2010)
have observed distinct genetic patterns in both wild olive
populations and cultivars in Spain, suggesting regional divergence
influenced by local and introduced varieties. This parallels the
findings here, suggesting similar dynamics in Jordanian olive
populations shaped by historical and modern gene flow.
Additionally, Julca et al. (2020) and Zunino et al. (2023) provided
lines of evidence of recurrent genetic admixture during
domestication, particularly in the Mediterranean Basin. Bazakos
et al. (2023) emphasized the prevalence of admixed olive
populations in the Western Mediterranean basin and identified
distinct genetic clusters within olive populations across the
Mediterranean basin, corresponding to different domestication
events. These findings are generally consistent with the results of
this study, which highlight the complex nature of olive diversity.
This diversity is shaped by regional differentiation, domestication
processes, and ongoing genetic mixing and evolution, all of which
are evident in Jordan, a region considered to be one of the centers of
olive domestication. Collectively, these studies highlight the
intricate genetic history and emphasize the importance of
understanding and conserving olive genetic resources across their
native range.
5 Conclusions
The study of olive trees in Jordan has revealed their widespread
distribution across diverse environments, highlighting their
adaptability to different growing conditions. Morphological analysis
demonstrated a high degree of diversity in the studied traits among
olive tree populations, providing evidence of rich genetic variation
within Jordanian olive germplasm. Genetic analysis using informative
SNP markers grouped Jordanian olive trees into distinct clades and
uncovered potential redundancy among genotypes, particularly within
KFARI BALADI and KANABISI cultivars. Comparative analysis with the
Worldwide Olive Germplasm Bank confirmed the reliability of EST-
SNP markers and the presence of synonyms, propagation errors and/
or mislabeling within the Jordanian olive germplasm collection. These
results underscore the critical importance of accurate documentation
and in-depth characterization for the effective management and
conservation of olive tree genetic resources in germplasm collections
as well as in situ/on farm. The analysis of population structure resulted
into distinct genetic groups, along with the presence of admixed
individuals, thus suggesting historical or modern gene flow. Finally,
the identification of 73 new olive genotypes in Jordanian
environments highlights their potential as genetic resources for
future improvement and utilization. These findings remark the
urgent need for comprehensive conservation efforts to preserve the
rich genetic diversity of Jordanian olive germplasm.
Data availability statement
The original contributions presented in the study are included
in the article/Supplementary Material. Further inquiries can be
directed to the corresponding author.
Author contributions
MA-K: Conceptualization, Data curation, Investigation,
Methodology, Writing –original draft. FT: Conceptualization,
Data curation, Formal analysis, Funding acquisition,
Investigation, Methodology, Supervision, Writing –review &
editing. ND’A: Data curation, Formal analysis, Methodology,
Resources, Software, Writing –review & editing. CM: Data
curation, Formal analysis, Methodology, Writing –review &
editing. AB: Data curation, Formal analysis, Investigation,
Methodology, Validation, Writing –review & editing. SA: Data
curation, Methodology, Writing –review & editing. RA: Data
curation, Formal analysis, Visualization, Writing –review &
editing. SH: Data curation, Methodology, Software, Writing –
review & editing. AA-A: Conceptualization, Data curation,
Formal analysis, Funding acquisition, Investigation, Methodology,
Project administration, Supervision, Writing –original draft.
Funding
The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. This work
Al-Kilani et al. 10.3389/fpls.2024.1437055
Frontiers in Plant Science frontiersin.org14
was supported by a grant from Come ho indicato in precedenza, io
metterei questo “bilateral agreement-HCST/NCRD. National
Centre for Research and Development/The Higher Council for
Science and Technology (Jordan), Project: Assessment of Genetic
Diversity among Indigenous Populations of Ancient Olive (Olea
europaea L.) Trees from Jordan”and in part by a grant from the
Deanship of Scientific Research, The University of Jordan.
Acknowledgments
We express our sincere gratitude to Jordanian farmers for
granting permission to utilize their fields and olive material in
our experimental work. Special thanks are extended to Emeritus
Professor Mostafa Qrunfleh of the University of Jordan for
providing invaluable information about Romi olive trees. We
appreciate the assistance of Eng. Abdul Rahman Al-Trawneh
from Ministry of Agriculture-Jordan during the field surveys in
southern Jordan. We are grateful for finical support by the National
Center for Research and Development (The Higher Council for
Science and Technology-Jordan) and the Deanship of Scientific
Research at the University of Jordan.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
The author(s) declared that they were an editorial board
member of Frontiers, at the time of submission. This had no
impact on the peer review process and the final decision.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fpls.2024.1437055/
full#supplementary-material
SUPPLEMENTARY FIGURE 1
Map of Jordan showing the targeted regions for olive trees collection; Capital
letters refer to sampling area: (A) northern parts (Irbid, Ajloun and Jarash); (B)
central parts (Balaqa and Gene Bank); (C) southern parts (Karak and Tafilah;
(D) northern parts (Ma’an).
SUPPLEMENTARY FIGURE 2
(A) Biplot showing the distribution of the 24 morphological traits (contrib: the
contributions in percentage). (B) Biplot showing the distribution of the 24
morphological traits for the 382 olive tress.
SUPPLEMENTARY FIGURE 3
Morphological characteristics of Baladi and Kanabisi olive trees, fruits, leaves,
stones and fruit coloration pattern.
SUPPLEMENTARY FIGURE 4
Pairwise correlation (Pearson’s coefficients) using 24 phenotypic traits of 382
olive trees identified in this study.
SUPPLEMENTARY FIGURE 5
Discriminant analysis of principal components (DAPC) of 149 olive
accessions. (A) The Bayesian information criteria (BIC) supported four
distinct genetic groups; (B) Variance explained by PCA; barplot of
eigenvalues for the discriminant analysis.
SUPPLEMENTARY FIGURE 6
Plot of ADMIXTURE cross validation error from K=1 through K=15. In red were
indicated the two best K (3 and 4), as the value that minimizes the error.
SUPPLEMENTARY FIGURE 7
Phylogenetic tree constructed using the maximum likelihood method
(bootstrapping value of 1000) based on the genotyping data of 45 SNP loci.
The analysis included 149 olive trees from JOCC-2, 32 olive trees from
JOCC-1 and 34 accessions from JGBOC.
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