The cultivation of olive trees (Olea europaea subsp. europaea var. sativa) is concentrated between the latitudes of 30° and 45° in both the northern and southern hemispheres, primarily in regions with a Mediterranean climate, which is typified by hot, dry summers. In recent decades, the cultivation of olives has expanded beyond the Mediterranean climate zone. In fact, olive groves are currently found in tropical areas, where the climate is influenced by altitude, such as Brazil or the Canary Islands.

The cultivated olive is composed of a vast array of clonally propagated varieties. These cultivated varieties frequently coexist with their wild ancestor, the oleaster (Olea europaea subsp. europaea var. sylvestris), which is native to many humid and sub-humid regions of the thermo-Mediterranean zone where frost is rare. Despite their geographical proximity, the genetic relationship between cultivated and wild olives remains somewhat puzzling and not fully elucidated. Archaeological evidence suggests that olive cultivation originated in the eastern Mediterranean basin approximately 6,000 years ago. Further analysis of chloroplast DNA provides additional support for the hypothesis that the primary domestication site was along the Syrian-Turkish border. 

The introduction of clonal propagation techniques after domestication is likely to have accelerated the spread of olive cultivation across the Mediterranean. This method was also applied to other long-lived perennial crops, such as grapes and figs, thereby facilitating the consistent reproduction of desirable traits in new olive trees. 

Incompatibility is the phenomenon by which pollen deposited on the stigma surface of a flower is usually unable to germinate, grow and fertilise the ovules, thus inhibiting fruit setting. In olive, incompatibility reaction occurs shortly after the pollen grains land on an incompatible stigma and pollen tube growth stops in the first layers of stigmatic cells. (Figure 1).

Most olive varieties were known as self-incompatible (Seifi et al., 2011; Sánchez-Estrada and Cuevas, 2018), but, recently, it has been discovered that, in addition to this, they may also be inter-incompatible.

Recent advances on the genetic and molecular mechanisms underlying self- and inter-incompatibility in olive have highlighted a peculiar system, observed for the first time in numerous genera of the Oleaceae family, including Fraxinus (Saumitou-Laprade et al., 2018), Phillyrea (Carré et al., 2021), Ligustrum (De Cauwer et al., 2021) and Olea (Saumitou-Laprade et al., 2017), known as diallelic self-incompatibility (DSI).

DSI is controlled by a single locus with two alleles, a dominant allele S and a recessive allele s. These alleles lead to only two possible genotypic combinations (Ss and ss), corresponding to the incompatibility groups G1 and G2, respectively, where all varieties of G1 group are self- and inter-incompatible with each other and compatible with all G2 varieties and vice versa.

In many olive-growing areas, despite the richness of olive varietal patrimony, most olive cultivars remain locally important and are progressively displaced by a handful of both traditional and new bred cultivars that meet the requirements of new olive-growing systems. In this sense, the present catalogue bears witness to the standardisation and the globalisation of olive oil, table olives and the plant market, within and beyond Mediterranean olive-growing areas. This trend entails a serious risk of genetic erosion of the crop through progressive abandonment, substitution and, finally, loss of traditional local cultivars. 

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North Hemisphere:

In the late 20th century, when the Iranian government decided to expand the country’s olive cultivation area, researchers prioritised the study of the olive tree’s genetic resources. Surprisingly, they found high diversity levels among Iranian olive ecotypes which were clearly distinct from the Mediterranean olive population. Such noticeable diversity, in addition to the identification of promising ecotypes, paved the way for the implementation of programmes for the improvement and breeding of olive trees. Furthermore, through enhanded access to new DNA sequencing facilities thanks to, an Iranian-Chinese collaboration, all Iranian ecotypes have now been fully sequenced. This has led to adoption of a new approach for selecting suitable parents and shortening the screening and breeding process by combining molecular markers (SSR and SNPs) with morphological and pomological data.

Olea cuspidata, a wild olive tree species located in southeast Iran and expanding towards Pakistan, India and China, is now a major focus of a joint olive breeding programme between Iran and China. The aim is to cross wild and local olives to introduce promising new varieties that are better adapted to climate change. Identifying and producing cultivars suitable for growing olives in steep, drought-prone areas as well as in colder regions could significantly expand the area under olive cultivation in both Iran and China.

The age of genomics:

Four olive genomes have been assembled so far, a development that is allowing advances in the genomics-assisted breeding. However, for an efficient use of the genomics knowledge in breeding we need to improve the quality of the assembled genomes. This goal will be probably achieved in the near future, giving way to the post-genomic era.

Furthermore, considering the high heterozygosity of the olive genome, a necessary step is also to have the two haplotypes of the genome available. In a recent Italian Project (OLGENOME) the sequencing of the Leccino genome was completed where the quality of the assembly and annotation is very high, the anchoring is complete and the haploid versions will soon be available.

QTLs for breeding: 

More and more olive varieties are currently being sequenced. This is allowing the use of GWAS analysis to find genetic markers (GM) associated to olive tree qualities. Those GM have to be confirmed by independent experiments and once this is done, we have good GM for assisted breeding. This process is underway by a number of research groups and it is reasonable to expect that in the next years we will have a number of GM to be use as QTLs in breeding.

Once superior olive genotypes are obtained through genomics-assisted breeding, traditional breeding strategies are expected to become increasingly less efficient. At that point, genome editing will emerge as the most promising option for the genetic improvement of the olive tree. It is reasonable to expect that genome editing will become the leading strategy within a few years.

With the advent of assisted evolution techniques (AET), biotechnological research in the olive sector has gained renewed impetus, particularly towards the development and optimisation of regeneration protocols, both through somatic embryogenesis from adult tissues and from protoplasts. Recent studies have opened new perspectives for the application of AET in olives.

In order to maintain the genetic diversity of the species and to enhance native cultivars, targeted improvements through genome editing could allow major challenges related to climate change to be overcome, while also improving olive oil quality. Genome editing is likely to become the winning strategy for the future of olive cultivation, although it will be necessary to intensify research efforts towards a deeper understanding of gene functions, the identification of editable genes, and, ideally, the selection of functional allelic variants within key genes.
 

Currently the most widely used molecular markers for varietal identification in olive are microsatellites, owing to their high polymorphism and the extensive availability of published data, which enables comparison of molecular profiles across collections. In fact, most international germplasm collections have been characterised with these markers. However, more recently, witht the development of high-throughput genotyping platforms and techniques (e.g. GBS, SPET), panels of single nucleotide polymorphisms (SNPs) are being produced. Thanks to which, their high reproducibility and abundance, SNPs, are particularly well suited for varietal for and intra-varietal identification purposes. The availability of resequenced genomes is also facilitating to the identification of a range of genomic mutations  — including (SNPs, copy number variations (CNVs), insertions and deletions (indels), and genic deletions and duplications  —) which will not only consolidate the varietal identification process but also clarify the evolutionary history of the species, improve the understanding of the genetic structure of olive germplasm, and help identify selection markers and/or markers with functional significance. 

Currently, some markers have already been identified that are significantly associated with phenotypic traits of major agronomic importance, such as drupe shape and oil quality. The identification of the molecular genetic determinants responsible for phenotypic variation will open new perspectives for the assisted selection breeding in olive, as well as for the application of gene editing technologies.
 

Olive oil and table olives are key agricultural commodities, and the ability to accurately distinguish between different olive varieties is crucial for plant nurseries, growers, researchers and inspection authorities. Image analysis offers a non-invasive and efficient way to achieve this goal.

One of the primary applications of image analysis in olive variety identification is the analysis of fruit and endocarp characteristics. In fact, endocarp morphological characteristics are more informative than any other part of the olive tree. Each olive variety exhibits distinct features, such as fruit shape, size and colour, as well as differences in the shape, size and texture of their endocarps. Using high-resolution images, specialised software can be used to extract and analyse these features, allowing for the identification of specific olive varieties.

The characterisation of geometric (e.g., height, projected and transversal areas, volume) and structural (e.g., vigour, vegetative growth habits) canopy parameters of individual olive trees in an orchard is highly valuable for many agronomic decision-making processes. For example, such data support the design and supervision of digitised operations such as pruning or fungicide application; facilitate the study of tree health in response to threats such as Xylella fastidiosa or Verticillium dahliae; and enable the monitoring and phenotyping of varietal performance in field breeding programmes. The latter is particularly focused on the development of new olive cultivars with targeted traits, such as resistance or tolerance to biotic and abiotic stresses, improved suitability for mechanisation (e.g., pruning, harvesting), and adaptability to high- and super-high-density orchards.

However, the measurement of early olive traits for phenotyping presents major challenges, mainly due to the complexity of field sampling and the logistical demands of recording high tree variability across large plots. The advancement of olive breeding programmes has increased the need for more efficient (rapid, low-cost and accurate) tools to alleviate the labour-intensive manual mapping of irregular olive canopies, a crucial step in identifying desirable genotypes as early as possible. This, in turn, contributes to reducing the high costs associated with maintaining large numbers of trees throughout the lengthy evaluation periods required by breeding programmes.

The production of healthy and authentic olive plant material is a vitally important issue, not only for the exchange between research centres and germplasm banks, but also for the commercial transactions undertaken by the nursery industry.

To guarantee the conservation of existing olive varietal biodiversity and promote the international  certification of olive nursery plants, the International Olive Council (IOC) launched the True Healthy Olive Cultivars (THOC) project. This initiative, carried out by the University of Córdoba with the participation of the 23 germplasm banks forming the IOC Network, represents an important milestone in consolidating the IOC Germplasm Bank Network. It has provided an effective tool for the authentication of the most internationally important olive varieties, ensuring the availability of healthy and authentic plant material to facilitate certified plant production across member countries.

At present, authenticity and representativeness are not guaranteed in most olive germplasm banks. Significant confusion persists due to homonyms (the same name for different cultivars), synonyms (different names for the same cultivar) and errors in denomination (in correct names assigned to cultivars). Overcoming this confusion is a fundamental prerequisite for in any germplasm bank that precedes the evaluation of the agronomic and oleotechnical characteristics of its accessions.

The technical guidelines used for the morphological characterization of the olive varieties has been described by the International Union for the Protection of New Varieties of Plants (UPOV) in the TG/99/4, (UPOV, 2011). 

Document TG/99/4 establishes the guidelines for conducting DUS examinations. This document includes expression levels for each character and guidelines for their evaluation (season, shoot types and sample size), as well as reference cultivars and pictures to facilitate character assessment. 

The molecular characterization has been performed by DNA extraction, PCR and fragment analysis using microsatellite markers (Single Sequence Repeats - SSRs), applying an array of 6 SSRs (UDO-43, DCA3, DCA9, DCA16, GAPU-101). 

This set of SSRs was selected given the following characteristics: a) high discriminant capacity; b) low confusion probability; and c) high reproducibility and easy result analysis. The conditions to analyze each SSRs are described in Trujillo et. al., (2014).