An olive parentage atlas: founder cultivars, regional diversification, and implications for breeding programs

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Gómez-Gálvez, R. Rosa-Navarro, G. Besnard, I. J. Lorite, A. Belaj This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8524821/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Mar, 2026 Read the published version in BMC Plant Biology → Version 1 posted 11 You are reading this latest preprint version Abstract Background Olive pedigree has been scarcely explored beyond domestication and diversification studies, even though it can be valuable for breeding programs and germplasm management. This study presents a new and comprehensive exploration of olive parentage relationships by combining a large dataset of 840 cultivars with a cost-effective and highly informative panel of 96 EST-SNP markers routinely applied at the World Olive Germplasm Bank of Córdoba. Parentage assignments were performed combining two approaches, SambaR and CERVUS, and using 13 seedlings of known crosses as validation controls. Results This strategy revealed 1,218 parent-offspring duos and 280 robust parents pair-offspring trios, involving more than 85% of the genotypes analysed. Four founder cultivars, ‘Gordal Sevillana’, ‘Lechín de Granada’, ‘Toffehi Tataouine’, and ‘Safrawi’, emerged as central nodes in the pedigree network, highlighting their crucial role in the diversification of olive cultivars across the Mediterranean Basin. ‘Gordal Sevillana’, in combination with ‘Lechín de Granada’ and ‘Toffehi Tataouine’, contributed substantially to the origin of Western and, to a lesser extent, Central Mediterranean cultivars, while ‘Safrawi’ acted as a key connector across the entire basin. Conclusions This study provides the first olive parentage atlas, providing new insights into the diversification processes, but also a practical tool for the management of genetic resources. In particular, these results demonstrate that a small but informative SNP set can generate reliable pedigree information to identify compatible parents, resolve uncertain genealogies of cultivars of agronomic interest, reconstruct unknown pedigrees in open-pollination, and guide the selection of balanced parental sets for developing new cultivars. pedigree atlas genealogy breeding cultivar diversification inter-compatibility founder cultivars Figures Figure 1 Figure 2 Figure 3 Figure 4 Background The olive tree ( Olea europaea L. subsp. europaea var. europaea ) is one of the earliest woody crops [ 1 , 2 ]. With an annual worldwide production of 23 million tons of olives produced across 11 million hectares, it represents an important source of local livelihood, mainly in the Mediterranean Basin [ 3 ]. Despite its economic and cultural relevance, the genetic background of olive cultivars remains only partially understood. Exploitation of its wild relative [ O. e. subsp. europaea var. sylvestris (Mill.) Lehr] is estimated to have occurred since the Neolithic in different parts of the Mediterranean Basin [ 1 , 2 ]. The domestication of this crop took place ~ 6000–7000 years ago in the Fertile Crescent region of the Middle East, according to evidence from archaeological, palaeobotanical, and genetic studies [ 1 , 2 , 4 , 5 ]. As with other cultivated woody species, farmers should have first learned to propagate the most promising individuals by transplanting hard-cuttings and later by grafting [ 1 , 6 ]. Thus, early olive farmers might have selected the best genotypes from wild olive populations according to their fruit size, oil content, productivity, and adaptation to the local environment, giving rise to the first cultivars [ 1 , 5 , 7 ]. Reproduction from seeds may also have occurred during the early stages of domestication, either unintentionally through seed dispersal by migrating people or intentionally promoted by farmers as a means to generate new genetic diversity, as it has been described in other clonally propagated crops [ 8 ]. The expansion of the olive crop over centuries, combining both vegetative and sexual propagation, has resulted in several thousand of cultivars. Several multilocus genetic analyses have revealed a clear geographical structuring of olive diversity in Eastern, Central and Western Mediterranean gene pools [ 5 , 9 , 10 ]. However, the modern oliviculture motivates the extensive use of few elite cultivars, which poses a risk of genetic erosion and a decrease in the variability of the crop [ 11 ]. This loss of diversity could affect the ability to respond to various challenges of the future, such as climate change, so a comprehensive effort has been put over decades to safeguard, characterize and valorise olive genetic resources in ex situ germplasm collections in Mediterranean countries and beyond [ 12 ]. Accurately identified and agronomically evaluated ex situ germplasm collections can serve as a library that feeds breeding programs in search of new cultivars adapted to the evolving challenges of olive cultivation. In this sense, documenting the history of selected genotypes, including their parentage relationships, is a prerequisite for managing germplasm collections and material used in breeding or research. In recent times, molecular markers such as microsatellite and Single Nucleotide Polymorphism (SNP) markers have been used to explore this purpose [ 13 , 14 ]. Thus, pedigrees have been reconstructed in a wide range of clonally propagated crops, including almond, apple, grape, and peach [ 15 – 20 ]. In the case of olive, few studies have incorporated parentage relationships in their analysis [ 21 – 24 ]. However, these studies were conducted with a contrasting and limited number of cultivars, heterogeneous marker sets, and/or a primary focus on uncovering aspects of domestication history and diversification. As a result, the genealogical links among cultivars of agronomic relevance and the contribution of key founders remain insufficiently assessed. Beyond reconstructing domestication and diversification events, pedigree analysis can be useful for other purposes in the framework of breeding and genetic management of material in research. First, it may assist for deducing mating behaviours and variations in reproductive success [ 25 ]. In this sense, the study of parent-offspring relationships can give important information about the cross compatibility of olive cultivars as olive possesses a self-incompatibility (SI) system characterized by two groups G1/G2: genotypes belonging to one group are only compatible with genotypes of the other group and vice versa [ 26 – 29 ]. Second, it can help to elucidate an unknown parent in open-pollination crosses. Knowing the parents allows to identify successful genetic combinations, facilitates the selection of genotypes with desirable traits, and helps to understand how variability is transmitted to offspring [ 30 ]. Third, the selection of appropriate starting material in breeding programs should be facilitated by pedigree analyses, for example by considering the parentage of progenitors to limit inbreeding. This is particularly relevant in the context of contemporary olive growing which increasingly demands the development of low-vigour genotypes adapted to high-density hedgerow systems [ 31 , 32 ]. Many of the emerging cultivars developed through both public and private breeding initiatives are primarily or secondarily derived from the same progenitor, the ‘Arbequina’ cultivar, raising concerns about inbreeding and the resulting lack of diversification in olive orchards [ 31 – 33 ]. Finally, pedigree information also enhances the management and use of germplasm banks, for example by guiding the selection of maximally diverse cultivar sets for building core collections, comparative trials, or large-scale phenotyping experiments. In this context, the present study aims to enlarge the knowledge in parentage relationships in olive and complementing it from a more practical point of view for breeding and management of olive germplasm collections. To achieve this, it takes advantage of a large dataset of cultivars accurately identified by a set of 96 EST-SNP markers routinely applied at the World Olive Germplasm Bank of Córdoba (WOGBC-ESP046) and its associated prospecting trials [ 10 , 11 , 34 ]. It is worth mentioning that the use of a relatively small panel of SNPs genotyped via real-time PCR (Fluidigim) has proven efficacy for parentage analysis in other species [ 14 ]. Our study represents the first attempt to do so in olive, demonstrating that informative set of 96 EST-SNP markers is enough to routinely perform Germplasm-Bank-scale pedigree analysis within a reasonable timeframe and without the need for high-performance hardware i.e., in a cost-efficient way. Using this marker set, we conducted paternity assignments for 853 olive genotypes, representing, to our knowledge, the largest dataset used for parentage analysis in olive to date. The resulting pedigree relationships provide novel insights into the diversification processes of the crop across the Mediterranean, shed light on mating behaviours among cultivars, and offer valuable guidance for the selection of genitors in future breeding crosses. Material and methods Material All the distinct cultivars identified in previous studies were subjected to parentage analysis in the present work. Thus, 668 different international cultivars identified by Belaj et al . together with 172 different Spanish cultivars identified by Gómez-Gálvez et al ., were used [ 10 , 11 ]. In addition, 13 seedlings were included as validation controls, 12 of them from both known genitors and one from a known mother fertilized in open pollination. To sum up, a total of 853 distinct EST-SNP profiles were analysed (Table S1 ). All genotypic data were generated using the same standardized genotyping pipeline based on the 96 EST-SNP panel routinely applied at the WOGBC-ESP046. DNA extraction, genotyping method and allele calling procedures were identical across studies, ensuring full data consistency and comparability. Detailed information about the 96 EST-SNP has been previously published [ 10 ]. Information of chloroplast DNA data, and stigmatic groups of andro-sterility (G1/G2) was added for each cultivar in Table S1 according to information available in literature (see references in Table S1 ). Some andro-sterility information obtained from the periodic phenology records routinely carried out in the WOGBC-ESP046 was also added (at the beginning of flowering, andro-sterile cultivars can be detected by observing dehiscence of the anthers and/or absence of pollen) [ 27 ]. Finally, information of population structure was also presented in Table S1 . Since this information was separately explored in previous works for the different sets of cultivars [ 10 , 11 ], a new exploration was conducted here combining all the cultivars under study. Thus, this new information of population structure was obtained using ‘LEA’ admixture analysis within the pipeline of SambaR package [ 35 ] ( https://github.com/mennodejong1986/SambaR ) performed in R 4.2.2 [ 36 ]. The snmf function was run, and the optimal number of populations ( K ) was assessed based on the cross-entropy criterion through examining a range of K from 1 to 10 with 10 replicates for each scenario [ 37 ]. The proportions of membership (Q) of each individual in each cluster were exported. A cultivar was considered belonging to a cluster when its Q > 75%; otherwise, it was considered as admixed. Parent-offspring analysis (duos) Parentage analysis was conducted following two steps, similar to the procedure published by Khadari et al . [ 22 ]. First, a single-parent search was performed in the whole set of 853 distinct genotypes to identify putative parent-offspring duos. Then, a parental pair search was conducted on the genotypes inferred in some putative parent-offspring duo to identify putative parents-offspring trios. For parent-offspring duos, two different approaches were used: i) First, the kinship analysis available in the R package SambaR was used. This analysis does not require allele frequency information and is based on the approach of Manichaikul et al . [ 38 ]. Thus, a parent-offspring duo was retained if all SNP loci shared at least one allele (i.e., the “zero identity-by-state”, R0, coefficient is zero) and if the KING-robust coefficient (φ) was within the inference criteria range (0.177 < φ < 0.354) [ 38 ]; ii) Second, the parentage analysis available in CERVUS v.3.0.7 was computed [ 39 ]. This analysis relies on allele frequencies and is based on the difference in the log-likelihood ratio (LOD) between related and unrelated relationships to assign parentage combined with simulation of parentage analysis to determine the confidence of assignments [ 40 ]. The default settings for the simulation, recommended by the software [ 39 ], were the following: number of offspring = 10000; number of candidate parents = 200; proportion of candidate parents sampled = 0.3; proportion of loci typed = 0.8; and proportion of loci mistyped = 0.01. Two criteria were finally considered to establish strict parentage relationships: a confidence level of the LOD score higher than 95% and mismatches on a maximum of two loci. Such parameters have also been used in previous works on woody crops [ 22 , 24 , 41 , 42 ]. Parents pair-offspring analysis (trios) For parents-offspring trios, all those cultivars inferred in any duo were included in the likelihood-based method of CERVUS v.3.0.7 using the same parameters and criteria as above [ 39 ]. From the putative trios obtained, a selection of the most robust ones for each offspring was done and represented in a pedigree network. The selection was done according to the higher trio LOD score. Those cases in which several parent pairs with close trio LOD scores were inferred for the same offspring were carefully studied and selected according to the number of trio-mismatches, number of parents significantly inferred in the duo analysis, and logical geographic distribution. Similarly, conflictive cases in which several cultivars appeared together in several trios, sometimes acting as offspring and sometimes as parents, were solved by selecting the trio with the highest trio LOD score value. Use of additional information in robust trios Information of chloroplast data, stigmatic groups of compatibility and andro-sterility (see reference sources in Table S1 ) was incorporated to all the robust trios selected. This information was first used to corroborate the selection of robust trios, i.e., checking the plausibility of the matching when the information of the trio was complete. Chlorotype and andro-sterility information were also used to deduce the sex of candidate parents when possible. Finally, the stigmatic information was used to deduce the compatibility groups in those robust trios with partial information, i.e., if a candidate parent with an unknown group was inferred as a pair of a candidate parent with stigmatic group G1, it was deduced as G2, and vice versa . Results Genetic structure The ‘LEA’ admixture analysis detected K = 3 as the most likely number of genetic groups, so three clusters were considered, namely A, B and C. The proportions of membership (Q) of each individual in each cluster are presented in Table S1 . For Q > 75%, 145 cultivars were considered belonging to cluster A, 51 to B, and 163 to C. The rest of cultivars (494; 58%) were considered to be admixed (Q < 75% in any cluster). As expected [ 10 ], most of cultivars assigned to clusters A, B and C belong to cultivars mainly grown in Eastern, Central and Western Mediterranean countries, respectively (Table 1 ). Table 1 Averaged proportion of membership (Q) observed for cultivars grown in Eastern (EM), Central (CM) or Western (WM) Mediterranean countries and number of cultivars (N) assigned to each cluster (Q > 75%) or admixed (Q < 75%). Region of cultivation Q mean for A Q mean for B Q mean for C N assigned to A N assigned to B N assigned to C N assigned as admixed EM 0.764 0.089 0.147 97 0 1 56 CM 0.345 0.406 0.249 19 39 2 153 WM 0.283 0.190 0.527 29 12 160 285 Parent-offspring duo analysis A total of 1,696 different putative parent-offspring duos were identified in SambaR. With this method, around 89% (759 out of 853) of the genotypes under study conformed a duo (Table S2). The CERVUS approach identified 3,221 different putative parent-offspring with 95% confidence level (LOD duo > 4.65) and less than two mismatching loci (Table S2). In this case, 815 cultivars (95.6%) were inferred in at least one duo. The number of putative duos validated by the two approaches was 1,218, and more than 85% of the genotypes conformed pairwise relationship(s) (Table S2). Of these, 255 genotypes were inferred only once, while 27 were inferred in 10 or more parent-offspring duos. Among the latter, ‘Gordal Sevillana’, ‘Safrawi’, ‘Toffehi Tataouine’, ‘Lechín de Granada’ and ‘Frantoio’ show more than 25 parent-offspring relationships (Fig. 1 ), pointing out their founding importance in the current olive germplasm. It is worth noting the wide geographical range of relationships for two of them: firstly, ‘Gordal Sevillana’ shows direct relationship with well-known cultivars found in the East (e.g. ‘Izmir Sofralik, ‘Gemlik’, or ‘Sebhawy’), the Centre (e.g. ‘Amygdalolia Nana’, ‘Ascolana Tenera’, or ‘Buga’), and the West (e.g. ‘Azapa’, ‘Bouteillan’, or ‘Manzanilla de Sevilla’); and secondly, ‘Safrawi’ is also related to eastern (e.g. ‘Rowghani’, ‘Tebabs’ and ‘Ayvalik’), central (e.g. ‘Karydolia’, ‘Gerboui’ and ‘Dolce Agogia’), and western cultivars (e.g. ‘Sauzen Vert-2218’, ‘Panseñera’ and ‘Empeltre’; Table S2). In contrast, the three other founder cultivars (i.e. ‘Toffehi Tataouine’, ‘Lechín de Granada’ and ‘Frantoio’) were related to cultivars mainly found in the Central and Western olive growing areas. Most duos were identified among cultivars belonging to the same cluster or among cultivars belonging to a cluster and admixed ones (Fig. 2 ). Thus, cultivars belonging to cluster A (Eastern Mediterranean) were inferred in 46% of the cases with other cultivars of the same cluster and 49% with admixed cultivars. As for cluster B (Central Mediterranean area), 21% of the duos were identified within the same cluster, and 69% were found among admixed cultivars. Finally, those assigned to cluster C (Western Mediterranean) appeared in duos with cultivars from the same cluster (43%) or with admixed ones (56%). Only 13 duos included a cultivar belonging to cluster A (either ‘Toffehi Tataouine’ or ‘Ayvalik’) and a cultivar belonging to cluster B; only two duos (‘Toffehi Tataouine’ – ‘Verdale de l’Hérault’, and ‘Bassiols’ – ‘Cornicabra’) were conformed among a cultivar belonging to cluster A and cluster C; and none of the duos was inferred among cultivars belonging to B and C clusters (Fig. 2 ). Parental pair-offspring trio analysis The parentage relationships of the genotypes inferring duos were further examined by searching parental pairs trios with the likelihood approach. A total of 1,000 putative trios were identified under the criteria considered, i.e. LOD pp > 14.88 for 95% success rate, maximum two mismatching loci, and both parents inferred as significant in at least one of the approaches conducted in previous duo analysis (data not shown). From these putative trios, a selection was done to conserve the most likely parent pair for each offspring (according to the higher trio LOD score and other considerations; see criteria above). Thus, a total of 142 genotypes were involved in 131 different parent pairs leading to 280 different offspring genotypes, with LOD pp scores ranging from 15.69 to 58.38 (Table S3). All the validation controls were successfully inferred within these robust trios, confirming the reliability of the approach. All the seedlings with both parents known were correctly assigned, with LODpp scores ranging from 19.65 to 37.30. For example, ‘Martina’, ‘Chiquitita’ and ‘Sikitita-2’ were correctly assigned to the expected pair of parents ‘Arbequina’ × ‘Picual’ with a LODpp scores of 19.65, 28.37, and 27.27, respectively (Table S3). Among the 280 robustly identified trios, the distribution of offspring cultivars by main region of cultivation was as follows: 51 from the Eastern Mediterranean (EM), 61 from the Central Mediterranean (CM), and 168 from the Western Mediterranean (WM) (Table S3). If combining with the main region of cultivation of parents-pairs, footprints of local selection were revealed, with a clear predominance of within-region crosses. Thus, crosses among WM cultivars led to 119 offspring cultivars in the same region; crosses among CM cultivars gave 31 CM cultivars; and crosses among EM cultivars produced 29 EM cultivars. Nevertheless, a notable number of inter-regional combinations were also observed, including EM–CM (25 offspring cultivars), CM–WM (22) and EM-WM (9). Offspring from the WM exhibited the highest number and diversity of parental combinations, which may be attributed, partially, to their greater representation in the dataset (≈ 57%). In contrast, EM offspring showed more limited parental origins, mostly involving EM or CM cultivars (Table S3). When the selected robust trios were represented in a pedigree network, it was observed a main, continuous network connecting most of the genotypes, with eight subnetworks conformed by few cultivars (Fig. 3 ). In the continuous network, several highly connected nodes could be differentiated, representing founder cultivars connections. As expected according to the duos analysis, the cultivars ‘Gordal Sevillana’, ‘Lechín de Granada’, ‘Toffehi Tataouine’ and ‘Safrawi’ were identified as the most likely candidate parents of 47.5% of the offspring (132 out of 280 cultivars; Table S3, Figs. 3 and 4 ). Contribution of major founders to the Mediterranean olive germplasm The cultivar ‘Gordal Sevillana’ was inferred as candidate parent in a total of 91 robust trios (Table S3; Figs. 3 and 4 ). It was inferred in 12 different parent pairs, three of them accounting for most of the offspring: i) ‘Gordal Sevillana’ × ‘Lechín de Granada’, that was identified as the most likely parental pair for 53 cultivars, mainly cultivated in Southwestern Mediterranean and including important commercial cultivars such as ‘Cornicabra’, ‘Manzanilla de Sevilla’, ‘Cordovil de Serpa’, ‘Picholine Marocaine’ or the South American cultivar ‘Azapa’; ii) the parent pair ‘Toffehi Tataouine’ × ‘Gordal Sevillana’, reported here for the first time, was directly linked to 19 cultivars mainly cultivated in northern Spain but also in the southern part of the country (‘Amargoso’), in France (‘Grossanne’), and in the Italian island of Sicily (‘Abunara’); and iii) the pair ‘Gordal Sevillana’ × ‘Morchiaio’, with 10 offspring cultivars, mainly from the Balkan area (7), such as ‘Buga’ and ‘Oblica’, but also including the well-known Italian cultivar ‘Ascolana Tenera’ (Fig. 4 ). In addition, ‘Gordal Sevillana’ showed close genetic relationships with cultivars from both the Eastern Mediterranean (‘Izmir Sofralik’, ‘Samanli’, ‘Samsun Salamuralik’, and ‘Samsun Tuzlamalik’) and Central Mediterranean (‘Derdi’, ‘Semidana’, and ‘Rotondella di Melfi’). This frequent connection across regions is consistent with its hypothesized eastern origin [ 24 ] and later diffusion to the West under different local names, such as ‘Ters Yaprak’ or ‘Tavsan Yuregui’ in Turkey, ‘Seviljska Maslina’ in Croatia, ‘Bella di Spagna’, ‘Giarrafa’, ‘Pizzo di Corvo’ or ‘Santa Caterina’ in Italy, ‘Grosse du Hamma’ in Algeria, or ‘Boube’ in France (Fig. 4 ). In line with this, chloroplast data further indicate that ‘Gordal Sevillana’ (E1.2 chlorotype) acted as a pollen donor in most of these crosses (offspring generally harbouring the E1.1 chlorotype), reinforcing its role as a male founder (Table S3). The cultivar ‘Lechín de Granada’ was identified as putative parent of a large group of offspring cultivars (57) in the Southwestern Mediterranean Basin. Most of these cultivars (51) were originated from a cross with ‘Gordal Sevillana’. However, additional parentage combinations were detected in this region, including crosses with ‘Safrawi’ and the local Eastern Spanish cultivar ‘Genovesa’. With the latter, it gave rise to the commercial cultivar ‘Changlot Real’. Beyond this regional influence in the Southwestern Mediterranean Basin, ‘Lechín de Granada’, which is known to be synonymous of the Greek cultivar ‘Dafnelia’, was also identified as a putative parent of the Egyptian cultivar ‘Sebhawy’ and the Italian cultivar ‘Rotondella di Melfi’ (Table S3, Fig. 4 ). The founder cultivar ‘Toffehi Tataouine’, introduced in the WOGBC-ESP046 collection from southern Tunisia, and recently found in an ancient tree in northern Spain [ 43 ], was inferred in four different parent pairs. Apart from the above-mentioned parent pair with ‘Gordal Sevillana’, its most prolific pairing was with ‘Safrawi’, producing 11 offspring cultivars. These descendants were mainly found across the central and western Mediterranean, including Greek (‘Agouromanakolia’, ‘Kothreiki’), Albanian (‘Kokerramdh Elbasani’, ‘Kotruvsi’), and northern Spanish cultivars (‘Claramunt’, ‘Domengues’). The well-known Turkish cultivar ‘Ayvalik’, synonymy of ‘Edremit Yaglik’ and ‘Adramitini’, was also found to be originated from this cross. Additionally, part of the local Tunisian germplasm may have resulted from crosses between ‘Toffehi Tataouine’ and other national cultivars, such as ‘Chemlali Sig’ and ‘Dokkar’ (Fig. 4 ). The cultivar ‘Safrawi’ was inferred as candidate parent in 14 different parent pairs, generating 34 offspring cultivars. It was the most frequently inferred founder cultivar in parent pairs (7) that generated more than one descendant. Thus, besides the above-mentioned pairings with ‘Toffehi Tataouine’ and ‘Lechín de Granada’, it was involved in five additional crosses generating more than one offspring: with Eastern Mediterranean cultivars from Syria (‘Saifi’), or Turkey (‘Ayvalik), as well as with Central Mediterranean ones from Greece (‘Amygdaloia Nana’), or Italy (‘Nocellara del Bellice’ and ‘Ogliarola del Bradano’). It is noteworthy to mention that ‘Safrawi’ was the founder cultivar with the most widely distributed progeny across the Mediterranean area. In addition to local offspring produced in Syria (‘Khoukhe’, ‘Sayfi’, etc), it generated other cultivars all along the Mediterranean Basin, such as the cultivars ‘Rowghani’ in Iran, ‘Memeli’ in Turkey, ‘Karydolia, in Greece, ‘Ulliri shekullor Berat’ in Albania, ‘Aitana’ in Italy, ‘Gerboui’ in Tunisia, and ‘Farga’ in Spain (Fig. 3 ). This broad distribution and high number of ‘Safrawi’ descendants may probably be due to the extensive dissemination of this variety along the Mediterranean basin where it “was baptised” with different local names such as ‘Antawi’ or ‘Dan’ in Syria, ‘Dal’ or ‘Baladi Remadieh’ in Lebanon, ‘Karabisi’ in Jordan, ‘Celebi (Silifke)’, ‘Erkence’, ‘Hurma Kaba’, ‘Sari Hebsi (Hatay)’ or ‘Yag Zeytini’ in Turkey, ‘Throubolia’ in Greece, ‘Marski’ in Albania, ‘Grossolana’ or ‘Santagatesse’ in Italy, ‘Filayre Noir’ in France, and ‘Cirujal’, or ‘’Grossal de Pallars’ in Spain (Fig. 4 ). Apart from the founder cultivars mentioned above, the network reflected a high number of further connections. They included cultivars that generated offspring in their nearby areas of cultivation, therefore representing local diversification foci. Thus, in the Eastern Mediterranean, the parent pair ‘Zaity’ × ‘Saifi’ originated several cultivars in Syria, while ‘Zaity’ × ‘Baladi’, from neighbouring Lebanon, generated at least seven local descendants. Similarly, six cultivars grown in Egypt were inferred as descendant from the parent pair ‘Chemlali Sfax’ × ‘Balady’, from Tunisia and Egypt, respectively (Table S3). In Central Mediterranean, it was observed local diversity derived from second generation of crosses, i.e. some inferred offspring cultivars were also detected as putative parents of other cultivars. Thus, the Sicilian cultivar ‘Abunara’ (inferred as offspring of the parent pair ‘Toffehi Tataouine‘ × ‘Gordal Sevillana’) was found to be candidate parent of the well-known Tunisian cultivar ‘Meski’. Similarly, four local cultivars in this country seem to have been derived from crosses of the cultivar ‘Jemri Bouchouka’ (a local offspring of the parent pair ‘Toffehi Tataouine’ × ‘Chemlali Sig’) with the Sicilian cultivar ‘Nocellara del Belice’ that was also found in Tunisia with different names (‘Beldi’, ‘Fakhari’, ‘Meski Zarzis’ or ‘Zarrazi’) [ 10 ]. This was also found in Western Mediterranean, where, as an example, the cultivar ‘Claramunt’, derived from the parent pair ‘Toffehi Tataouine’ × ‘Safrawi’, gave rise to new local germplasm by pairing with the local cultivar ‘Verdal de Pallars’. Similarly, second-generation crosses seem to have contributed to the local diversity of olive cultivars in the New World, as some cultivars identified in South America, such as ‘Carrasqueña Huasco’ and ‘Ascolana Huasco’, appear to be descendants of ‘Azapa’, which, as mentioned above, is derived from the parent pair ‘Lechín de Granada’ × ‘Gordal Sevillana’. Valuable information deduced from robust trios Information added about chloroplast DNA, stigmatic groups and andro-sterility matched perfectly in the robust trios inferred, corroborating therefore the plausibility of these parentage assignments. In cases where such information was incomplete or lacked, logical deductions allowed to predict it. Notably, the deduced information aligned well with the parentage assignments, even for trios without bibliographic support (Table S3). The logical deductions allowed to predict the stigmatic group of 74 cultivars, including cultivars of major agronomic relevance such as ‘Chemlali Sfax’ (G1), ‘Galega Vulgar’ (G1), ‘Azapa’ (G2), ‘Zaity’ (G1), or the founder cultivar ‘Toffehi Tataouine’. (G2). Likewise, the analysis revealed new insights into the reproductive compatibilities of cultivars with observed potential for hedgerow systems, such as ‘Arróniz’, ‘Rotondella di Melfi’, or ‘Zeitoun Boubezzoula’. Also, the robust trios obtained enabled to deduce the chlorotype information in 52 cultivars and the sex of candidate parents in 60 trios. Most of the chlorotypes deduced (46) were E1.1, as a result of obtaining an offspring with lacking information but originated from a parent pair with both candidate parents classified as E1.1. The combination with andro-sterility information enabled to deduce the chlorotype on a group of cultivars from eastern Spain, for which both types of information were consistent and mutually reinforcing. In particular, ‘ Carrasco’, ‘Cuquello de la Jana’, ‘Picuda de Luis’, and ‘Rufina’ were found to share the same chlorotype (E3.1) and andro-sterility profile, both likely inherited from ‘Farga’, their inferred maternal parent. Finally, the information provided by the robust trios made possible to identify the male parent in progenies derived from open-pollination in some breeding programs. As an example, the cultivar ‘Favolosa’, obtained in a breeding program from ‘Frantoio’ in open pollination, was identified as offspring of ‘Frantoio’ and ‘Ascolana Tenera’ with a high LOD pp score (43.32). In addition, the approach allowed for the reconstruction of complete pedigrees for cultivars whose origin was only partially known. Notable examples include ‘Dwarf D’, ‘Cairo-7’, or ‘S:27_4’. Similarly, ‘Arbosana’, a commercial cultivar adapted to hedgerow system, was correctly inferred to its known female parent, ‘Arbequina’, and the initially unknown male parent was newly postulated as ‘Vaneta’, a local cultivar that is present in the same area of cultivation. Discussion This present study provides the most comprehensive analysis to date of parentage relationships in olive, revealing both parent-offspring duos and parent pairs-offspring trios within a set of 853 genotypes. This large-scale approach, based on a cost-effective set of 96 EST-SNPs, uncovered four founder cultivars, namely ‘Gordal Sevillana’, ‘Lechín de Granada’, ‘Toffehi Tataouine’ and ‘Safrawi’, that played central roles in the diversification of the crop. Moreover, the resulting pedigree atlas sheds new light on the genealogical structure of Mediterranean olive germplasm and provided practical tools for breeding and germplasm management, moving beyond previous studies that focused mainly on domestication history and diversification. Founder cultivars shaping Mediterranean olive diversification As in other woody plants, analysing parentage is a useful approach to trace the origins of cultivated germplasm. The few generations since domestication and the vegetative propagation of key cultivars may help to reconstruct genealogies and identify founder genotypes [ 19 , 44 ]. Here, various cultivars have been identified as founder cultivars, as evidenced by the large number of the offspring generated. Three of them, ‘Gordal Sevillana’, ‘Lechín de Granada’ and ‘Safrawi’ have been already distinguished as founder cultivars in previous works [ 21 , 22 , 24 ], but here their offspring relationships were expanded and refined thanks to the broader dataset of 853 genotypes analysed. In addition, we provide the first evidence about ‘Toffehi Tataouine’ acting as a founder, with descendants in both Central and Western Mediterranean. This represents a surprising finding, as the impact of this cultivar, primarily discovered in Tunisia, has not been suggested until now. Its previously unrecognized influence highlights the importance of including ancient, forgotten cultivars in pedigree studies. Interestingly, an ancient olive tree recently genotyped in northern Spain matched with this cultivar [ 43 ], reinforcing the results obtained in our study and supporting for its historical diffusion beyond Tunisia. A common feature of the four founder cultivars is their association with very old olive trees [ 9 , 24 ], which reinforces their antiquity and probable early selection. Moreover, ‘Gordal Sevillana’, ‘Lechín de Granada’ and ‘Safrawi’ present well-known synonymies in different parts of the Mediterranean area [ 10 , 22 , 24 ], indicating long-distance dissemination and local renaming that contributed to diversification in different areas. Although ‘Toffehi Tataouine’ currently remains a little-known local cultivar in southern Tunisia [ 45 ], the recent evidence of its genetic match in Spain [ 43 ] and its prominent role in the inferred offspring suggest that it may also have experienced wider historical diffusion, and future prospecting may reveal additional synonymies. Overall, the identification of these four founders, especially their wide geographical spread and contribution to cultivar diversification highlight the long-distance dispersal and hybridization events that shaped the current structure of Mediterranean olive germplasm. The resulting olive pedigree atlas provides, for the first time, an integrated view of these genealogical links, revealing patterns of crossing between local and foreign cultivars and identifying multiple foci of diversification across the Mediterranean Basin. Regional patterns of diversification: Eastern, Central and Western Mediterranean In the Eastern Mediterranean region, considered the primary centre of olive domestication, our parentage analysis revealed a remarkable number of cultivars involved in different duos and trios, confirming the important role of eastern cultivars in the early diversification of the crop. Despite the relatively limited number of cultivars from this area included in our dataset (≈ 18%), the results highlight their contribution to multiple, locally adapted hybridization events. This observation is consistent with the high levels of genetic diversity previously reported for the region and with archaeological and genetic evidences indicating that the Levant is the cradle of olive cultivation [ 1 , 4 , 5 , 24 ]. In the Central Mediterranean, the parentage network reveals a more complex and heterogeneous pattern of cultivar origin and diversification. This area appears as a transitional zone where both autochthonous an allochthonous genotypes contributed to shaping the current olive germplasm. This pattern has been particularly observed in studies conducted in some islands such as Malta, Sicily, or Capri, considered crossroads of the Mediterranean [ 46 – 48 ]. In the Western Mediterranean, the parentage patterns showed a strong founder effect driven by a limited number of recurrent parent pairs. The allochthonous origin of most cultivars of Western Mediterranean has been postulated in previous studies [ 5 , 21 , 22 , 24 ]. In this study, it has been corroborated that the parent pair ‘Lechín de Granada’ × ‘Gordal Sevillana’ had a crucial role in this area, generating a large number of offspring cultivars. Among them, some popular and relevant cultivars such as ‘Cordovil de Serpa’, ‘Manzanilla de Sevilla’, or ‘Picholine Marocaine’, were robustly inferred. Additionally, other commercially important Spanish cultivars, namely ‘Picual’ and ‘Hojiblanca’, were also inferred as descendant of this parent pair, although their trio assignments did not reach the criteria required under our robustness thresholds (data not shown). These genealogical connections should be re-evaluated and confirmed in future analyses using higher-density SNP arrays or whole-genome sequencing. In the New World, as in the Central and Western Mediterranean, second-generation crosses appear to have contributed to the local diversification of olive cultivars. For instance, the South American variety ‘Azapa’, identified as an offspring of the parent pair ‘Lechín de Granada’ × ‘Gordal Sevillana’ and likely introduced from the Iberian Peninsula, was found to act as a progenitor of several local cultivars in that region. Thus, throughout the history of olive cultivation in the New World, both the introduction and preservation of ancient Mediterranean cultivars—some of which may have been lost or remain to be identified in their regions of origin—together with the local selection and further propagation of seedlings, may have shaped the current olive diversity observed in that area [ 10 ]. It is noteworthy that most of the cultivars identified as offspring of this parent pair belong to gene pool C and share the dominant E1.1 chlorotype, corroborating the bottleneck of diversity described for Western Mediterranean in other studies [ 5 , 21 , 22 , 24 ]. The high number of offspring attributed to this pair may reflect both the long-term coexistence of these founders and their high efficiency as crossing pair [ 28 , 49 ]. Since ‘Lechín de Granada’ has the E1.1 chlorotype and ‘Gordal Sevillana’ the E1.2, it implies that the latter usually had to act as male progenitor [ 22 ]. This fact could be explained by a high pollinic viability of ‘Gordal Sevillana’, and an optimal phenological overlapping between these two founder cultivars. Another possible explanation could be the easier dispersal of seeds from ‘Lechín de Granada’ than form ‘Gordal Sevillana’, whose smaller fruit size allows better ingestion by bird species known to act as olive seed dispersers. Hybridization between local and introduced germplasm The diverse origin of olive cultivars can also be observed at the scale of individual countries. In the case of Spain, which provided the best-represented germplasm in our study, the cultivars mainly grown in northern and north-eastern regions presented a broader diversity of origins respect to those from the south, which is in accordance with results obtained previously [ 11 ]. Many cultivars of these regions were traced back to diverse foreign ancestors or their first-degree relatives, resulting in a much more diversified network of relationships than in the south of the peninsula. ‘Gordal Sevillana’, ‘Toffehi Tataouine’, and ‘Safrawi’ were found to be a progenitor in a significant number of trios of Northern and North-eastern Spain. Interestingly, ‘Lechín de Granada’ was never inferred as a putative parent in cultivars of these region. This suggests an influx of cultivars linked to different historical periods and different civilizations with dominium in northern or southern coast of the Mediterranean. On the other hand, the autochthonous material also seems to have played an important role in this part of the Western Mediterranean. The high level of admixture observed in this area, together with the genetic similarity to local wild forms, led some authors to suggest an autochthonous influence in some cultivars [ 50 – 53 ]. A cultivar that clearly exemplifies this admixture is ‘Farga’. Although ‘Farga’ harbours the western E3.1 chlorotype, its nuclear genome is more similar to cultivars carrying the eastern E1 lineage, suggesting a cytoplasmic capture through backcrossing with cultivars originating from the Eastern Mediterranean basin [ 50 , 53 ]. This can be better understood if we analyse the putative progenitors inferred for this cultivar in our study: ‘Patrón de Cabús’ and ‘Safrawi’. There are two clues that suggest that ‘Patrón de Cabús’ may represent autochthonous material. The first is that it was assigned to gene pool B, as were other cultivars known to be closely related to wild forms, such as ‘Dokkar’ [ 53 , 54 ]. The second clue comes from its name: since “Patrón” means rootstock in Spanish, it may refer to local wild material that was grafted in the past. In contrast, ‘Safrawi’ seems to be a cultivar introduced from the East by ancient civilizations, as mentioned above and in other studies [ 10 , 24 , 55 ]. Together, these findings support the hypothesis of hybridization between local and introduced germplasm and highlight ‘Farga’ as a likely outcome of such genetic exchange. Furthermore, ‘Farga’ has been observed to be linked in the single parent-offspring duo to other 19 local cultivars of Northeastern Spain, suggesting that it acted as a focus of introgression and local diversification. In this context, it is worth noting that cytoplasmic male sterility (CMS) has been described in olive and shown to be maternally inherited and associated with the E3 chlorotype [ 56 ]. Androsterility within the ‘Farga’ group had already been reported by Rojas-Gómez et al. , who proposed a potential founder effect [ 49 ]. This observation is consistent with our results, since ‘Farga’ carries the E3.1 chlorotype and several of its inferred descendants exhibit andro-sterility, such as ‘Carrasco’, ‘Cuquello de la Jana’, and ‘Rufina’. The co-occurrence of andro-sterility and the E3.1 haplotype therefore suggests that CMS could have been transmitted through the maternal line of ‘Farga’, further reinforcing its role as a cytoplasmic donor and as a centre of diversification in the Northeastern Iberian Peninsula. Practical implications for breeding and germplasm management Apart from shedding light on the diversification history of olive, the pedigree information inferred in this study may provide substantial value for breeding programs and germplasm management. In particular, we combined paternity results with information on stigmatic compatibility groups to deduce mating behaviours, which reinforced the robustness of trios inferred with incomplete information and expanded the knowledge already available on compatibility groups [ 57 ]. This approach can improve the selection of inter-compatible genitors in future breeding programs. In this context, our analysis revealed the compatibility groups of cultivars of potential interest for modern high-density systems, such as ‘Arróniz’, ‘Rotondella di Melfi’ and ‘Zeitoun Boubezzoula’. These cultivars could represent promising alternatives to the widely used ‘Arbequina’, contributing to the diversification of intensive olive cultivation and helping to reduce risks associated with a narrow genetic base. Also, pedigree information has resulted useful for knowing the origin of progenies obtained from open pollination, or progenies received in exchanges of plant material between collections but with incomplete or lost passport information. Thirdly, knowledge of pedigrees is also useful for assembling balanced sets of progenitors in terms of inbreeding. Otherwise, released cultivars may become susceptible to new biotic and abiotic stresses due to their genetic similarity [ 58 ]. Although inbreeding depression has not yet been documented in olive, it has been widely reported in other woody crops, where it leads to reduction in vigour, flower number and fruit set, increase in fruit abortion, lower seed germination and seedling survival, abnormal growth, and a loss of disease resistance [ 59 ]. Finally, the construction of a pedigree atlas may also have additional practical applications, as highlighted by other authors. For example, it can help optimize whole-genome sequencing by using key founders as reference points to estimate missing genetic data in other cultivars [ 17 ]. In addition, pedigree information can be exploited for the description and valorisation of cultivars, serving as a resource for storytelling in cultivar marketing and providing added value, as demonstrated in other woody crops [ 15 , 17 , 19 , 20 ]. Finally, paternity analysis should be seen as a systematic task that should be continuously updated as new cultivars are genotyped and included in the reference databases. The approach used here is based on a set of 96 informative EST-SNPs [ 10 ] (mean H O = 0.501, mean Minimum Allele Frequency, MAF value, of 0.380). A substantial number of studies have demonstrated that SNP markers are as suitable for parentage analysis as classical markers, such as SSRs, and that a relatively small number of SNPs, from 60 to 200, with higher MAF values, are sufficient for most parentage analyses [ 14 ]. Our results confirm that this set of 96 EST-SNPs markers provides robust pedigree inferences across a wide and diverse olive germplasm collection. Many of these inferences agree with those reported in previous studies using diverse sets of SSRs [ 21 – 23 ] or a larger set of SNPs [ 24 ]. Thus, although the resolution could be improved further through the use of whole-genome sequencing or higher-density SNP arrays, the cost-effective and standardisable nature of the current method makes it particularly suitable for systematic pedigree analyses applied in germplasm banks and breeding platforms. Conclusion The comprehensive analysis of paternity conducted here provides the first olive pedigree atlas, with insights into the spread of olive cultivars along the Mediterranean Basin, but also with practical tools for modern breeding programs. The results not only confirm previously suggested genealogical links but also uncover new key founder cultivars and robust parent-offspring relationships, shedding light on the historical processes that have shaped olive cultivation. Additionally, the pedigree atlas offers actionable information for breeding and germplasm management: it enables the identification of inter-compatible parents, the reconstruction of previously unknown pedigrees, and the design of balanced genitor sets to ensure genetic diversity while minimizing the risks of inbreeding. The methodological approach used in this study demonstrates the effectiveness of a practical set of SNP markers for a rapid and a continuous pedigree analysis, which can be widely applied in germplasm management and breeding programs. This study establishes a strong basis for future research and advancements in breeding programs, emphasizing the need for continuous updates to the olive pedigree atlas as new cultivars are discovered and characterized. Declarations Ethics approval and consent to participate Not applicable. This study does not involve human participants, animals, or clinical trials. Consent for publication Not aplicable Availability of data and materials The datasets and material analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This research was financially supported by the regional IFAPA projects PR.CRF.CRF201900.004 and PR.CRF.CRF202200.004, partially funded by European Agricultural Fund for Rural Development (EAFRD). The management, identification and conservation of WOGBC-ESP046 have also been financially supported by the contract CAICEM 23-76. FJ G-G has been supported by PR.CRF.CRF202200.004, partially funded by EAFRD. G.B. is supported by PatrimOlea and is a member of the CRBE laboratory, which is supported by the Laboratory of Excellence (LabEx) CEBA (grant ANR-10-LABX-25-01) and LabEx TULIP (grant ANR-10-LABX-0041), both managed by the French National Research Agency (ANR). Authors’ contributions FG-G: Data curation, Formal analysis, Methodology, Resources, Software, Visualization, Writing– original draft. RR-N: Conceptualization, Resources, Formal analysis, Funding acquisition, Methodology, Validation, Writing review & editing, Writing– original draft. G-B: Formal analysis, Validation, Writing review & editing. IL: Data curation, Formal analysis, Writing review & editing. AB: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing– review & editing. All authors read and approved the final manuscript. Acknowledgements The authors are grateful to all the field and laboratory technicians for their work in the conservation and identification of accessions. The authors are also grateful for the EST-SNP genotyping support of staff at UPV/EHU—Scientific Park Maria Goyri Biotechnology Center (Bizkaia, Spain), especially to Fernando Rendo. 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Supplementary Files SupplementaryTables.xlsx Cite Share Download PDF Status: Published Journal Publication published 11 Mar, 2026 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 22 Jan, 2026 Reviews received at journal 22 Jan, 2026 Reviewers agreed at journal 14 Jan, 2026 Reviewers agreed at journal 14 Jan, 2026 Reviews received at journal 12 Jan, 2026 Reviewers agreed at journal 12 Jan, 2026 Reviewers invited by journal 09 Jan, 2026 Editor invited by journal 09 Jan, 2026 Editor assigned by journal 09 Jan, 2026 Submission checks completed at journal 09 Jan, 2026 First submitted to journal 05 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Gómez-Gálvez","email":"data:image/png;base64,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","orcid":"","institution":"Instituto Andaluz de Investigación y Formación Agraria, Centro Alameda del Obispo","correspondingAuthor":true,"prefix":"","firstName":"F.","middleName":"","lastName":"Gómez-Gálvez","suffix":""},{"id":573310259,"identity":"ee669386-2398-4c84-ae58-ba1fb9b217c3","order_by":1,"name":"R. Rosa-Navarro","email":"","orcid":"","institution":"Institute for Sustainable Agriculture","correspondingAuthor":false,"prefix":"","firstName":"R.","middleName":"","lastName":"Rosa-Navarro","suffix":""},{"id":573310271,"identity":"f6378638-86ce-4b3a-a63f-3dee4f4daf46","order_by":2,"name":"G. Besnard","email":"","orcid":"","institution":"UMR 5300 CNRS-IRD- TINP-UT3, Université Toulouse III – Paul Sabatier","correspondingAuthor":false,"prefix":"","firstName":"G.","middleName":"","lastName":"Besnard","suffix":""},{"id":573310272,"identity":"0db1496b-39bb-4f2a-83c7-770dde1ff73d","order_by":3,"name":"I. J. Lorite","email":"","orcid":"","institution":"Instituto Andaluz de Investigación y Formación Agraria, Centro Alameda del Obispo","correspondingAuthor":false,"prefix":"","firstName":"I.","middleName":"J.","lastName":"Lorite","suffix":""},{"id":573310274,"identity":"f00b517a-caa2-4529-948b-faa1e9f7877a","order_by":4,"name":"A. Belaj","email":"","orcid":"","institution":"Instituto Andaluz de Investigación y Formación Agraria, Centro Alameda del Obispo","correspondingAuthor":false,"prefix":"","firstName":"A.","middleName":"","lastName":"Belaj","suffix":""}],"badges":[],"createdAt":"2026-01-05 21:23:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8524821/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8524821/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-026-08504-y","type":"published","date":"2026-03-11T15:59:49+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":100176453,"identity":"cfea7ffd-0ec2-481a-af14-6d5e5ac56272","added_by":"auto","created_at":"2026-01-13 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18:07:13","extension":"xml","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":159770,"visible":true,"origin":"","legend":"","description":"","filename":"75a6c4e86a244edd94b279c0c4ff38f11structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8524821/v1/ca24cc4dc733297fd08d8340.xml"},{"id":100176462,"identity":"fb58f3ce-9fd0-4dd9-a285-8aea0d985fa3","added_by":"auto","created_at":"2026-01-13 18:07:13","extension":"html","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":173326,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8524821/v1/bda9c483bdde003c6f80902e.html"},{"id":100176449,"identity":"14bfed09-8e7f-442f-8b08-b8f4e4b499b3","added_by":"auto","created_at":"2026-01-13 18:07:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":33926,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of direct parent-offspring duos inferred for each cultivar jointly by SambaR and CERVUS. The name of the five main progenitors (with more than 25 putative descendants) is indicated.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8524821/v1/4e478c1db9a76af1d015809a.png"},{"id":100176451,"identity":"084fdba7-6dac-4d05-bd76-19301436c214","added_by":"auto","created_at":"2026-01-13 18:07:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17891,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage distribution of parent-offspring (PO) duos according to the cluster assignment in the LEA analysis. The x-axis shows the cluster assignment of the parental cultivars, and each column represents the cluster assignment of the offspring. A (red), B (blue) or C (green) represent the three clusters with membership assignations Q \u0026gt; 0.75. The ADMIX (grey) category represents admixed cultivars with Q \u0026lt; 0.75.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8524821/v1/3010449502b043d9823137d2.png"},{"id":100369349,"identity":"a9551bd4-b7b9-4228-9543-666743b5435f","added_by":"auto","created_at":"2026-01-16 07:58:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":536928,"visible":true,"origin":"","legend":"\u003cp\u003ePedigree network of robust selected trios. Blue spheres = offspring cultivars; Red circles = candidate genitors; Green diamond = offspring and genitors cultivars. Created with NodeXL. Cultivars with more connections are numbered: 1= Gordal Sevillana; 2 = Lechín de Granada; 3 = Safrawi; 4 = Toffehi Tataouine; 5 = Zaity; 6 = Chemlali Sfax; 7 = Arbequina; 8 = Morchiaio; 9 = Picual; 10 = Verdal del Pallars.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8524821/v1/65323f741fc7326828b6adbc.png"},{"id":100368667,"identity":"3260c942-90cc-46ad-9cbf-be12c78c8bb0","added_by":"auto","created_at":"2026-01-16 07:58:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":381129,"visible":true,"origin":"","legend":"\u003cp\u003eOffspring distribution from the four main founder cultivars. Each circle represents the offspring number derived from parent pairs. Each parent contribution is represented by a colour. Dashed circles represent areas of cultivation of the founder cultivar. The names next to the dashed circles represent synonymies identified in those areas.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8524821/v1/827886b54a64b7f05a14f369.png"},{"id":104740446,"identity":"4052c3d7-e458-4ae6-8bac-908bc5c05479","added_by":"auto","created_at":"2026-03-16 16:18:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1724589,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8524821/v1/15078f4e-3b0d-4bea-be95-4a6f2b1a11f9.pdf"},{"id":100369235,"identity":"bc16bff3-cb1f-4fc6-872f-b8b27f253f83","added_by":"auto","created_at":"2026-01-16 07:58:50","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":342986,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8524821/v1/b30c82cdf247ff7dc90bd58d.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"An olive parentage atlas: founder cultivars, regional diversification, and implications for breeding programs","fulltext":[{"header":"Background","content":"\u003cp\u003eThe olive tree (\u003cem\u003eOlea europaea\u003c/em\u003e L. subsp. \u003cem\u003eeuropaea\u003c/em\u003e var. \u003cem\u003eeuropaea\u003c/em\u003e) is one of the earliest woody crops [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. With an annual worldwide production of 23\u0026nbsp;million tons of olives produced across 11\u0026nbsp;million hectares, it represents an important source of local livelihood, mainly in the Mediterranean Basin [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Despite its economic and cultural relevance, the genetic background of olive cultivars remains only partially understood.\u003c/p\u003e \u003cp\u003eExploitation of its wild relative [\u003cem\u003eO. e.\u003c/em\u003e subsp. \u003cem\u003eeuropaea\u003c/em\u003e var. \u003cem\u003esylvestris\u003c/em\u003e (Mill.) Lehr] is estimated to have occurred since the Neolithic in different parts of the Mediterranean Basin [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The domestication of this crop took place\u0026thinsp;~\u0026thinsp;6000\u0026ndash;7000 years ago in the Fertile Crescent region of the Middle East, according to evidence from archaeological, palaeobotanical, and genetic studies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. As with other cultivated woody species, farmers should have first learned to propagate the most promising individuals by transplanting hard-cuttings and later by grafting [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Thus, early olive farmers might have selected the best genotypes from wild olive populations according to their fruit size, oil content, productivity, and adaptation to the local environment, giving rise to the first cultivars [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Reproduction from seeds may also have occurred during the early stages of domestication, either unintentionally through seed dispersal by migrating people or intentionally promoted by farmers as a means to generate new genetic diversity, as it has been described in other clonally propagated crops [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe expansion of the olive crop over centuries, combining both vegetative and sexual propagation, has resulted in several thousand of cultivars. Several multilocus genetic analyses have revealed a clear geographical structuring of olive diversity in Eastern, Central and Western Mediterranean gene pools [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the modern oliviculture motivates the extensive use of few elite cultivars, which poses a risk of genetic erosion and a decrease in the variability of the crop [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This loss of diversity could affect the ability to respond to various challenges of the future, such as climate change, so a comprehensive effort has been put over decades to safeguard, characterize and valorise olive genetic resources in \u003cem\u003eex situ\u003c/em\u003e germplasm collections in Mediterranean countries and beyond [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccurately identified and agronomically evaluated \u003cem\u003eex situ\u003c/em\u003e germplasm collections can serve as a library that feeds breeding programs in search of new cultivars adapted to the evolving challenges of olive cultivation. In this sense, documenting the history of selected genotypes, including their parentage relationships, is a prerequisite for managing germplasm collections and material used in breeding or research. In recent times, molecular markers such as microsatellite and Single Nucleotide Polymorphism (SNP) markers have been used to explore this purpose [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Thus, pedigrees have been reconstructed in a wide range of clonally propagated crops, including almond, apple, grape, and peach [\u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In the case of olive, few studies have incorporated parentage relationships in their analysis [\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, these studies were conducted with a contrasting and limited number of cultivars, heterogeneous marker sets, and/or a primary focus on uncovering aspects of domestication history and diversification. As a result, the genealogical links among cultivars of agronomic relevance and the contribution of key founders remain insufficiently assessed.\u003c/p\u003e \u003cp\u003eBeyond reconstructing domestication and diversification events, pedigree analysis can be useful for other purposes in the framework of breeding and genetic management of material in research. First, it may assist for deducing mating behaviours and variations in reproductive success [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In this sense, the study of parent-offspring relationships can give important information about the cross compatibility of olive cultivars as olive possesses a self-incompatibility (SI) system characterized by two groups G1/G2: genotypes belonging to one group are only compatible with genotypes of the other group and \u003cem\u003evice versa\u003c/em\u003e [\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Second, it can help to elucidate an unknown parent in open-pollination crosses. Knowing the parents allows to identify successful genetic combinations, facilitates the selection of genotypes with desirable traits, and helps to understand how variability is transmitted to offspring [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Third, the selection of appropriate starting material in breeding programs should be facilitated by pedigree analyses, for example by considering the parentage of progenitors to limit inbreeding. This is particularly relevant in the context of contemporary olive growing which increasingly demands the development of low-vigour genotypes adapted to high-density hedgerow systems [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Many of the emerging cultivars developed through both public and private breeding initiatives are primarily or secondarily derived from the same progenitor, the \u0026lsquo;Arbequina\u0026rsquo; cultivar, raising concerns about inbreeding and the resulting lack of diversification in olive orchards [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Finally, pedigree information also enhances the management and use of germplasm banks, for example by guiding the selection of maximally diverse cultivar sets for building core collections, comparative trials, or large-scale phenotyping experiments.\u003c/p\u003e \u003cp\u003eIn this context, the present study aims to enlarge the knowledge in parentage relationships in olive and complementing it from a more practical point of view for breeding and management of olive germplasm collections. To achieve this, it takes advantage of a large dataset of cultivars accurately identified by a set of 96 EST-SNP markers routinely applied at the World Olive Germplasm Bank of C\u0026oacute;rdoba (WOGBC-ESP046) and its associated prospecting trials [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. It is worth mentioning that the use of a relatively small panel of SNPs genotyped via real-time PCR (Fluidigim) has proven efficacy for parentage analysis in other species [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our study represents the first attempt to do so in olive, demonstrating that informative set of 96 EST-SNP markers is enough to routinely perform Germplasm-Bank-scale pedigree analysis within a reasonable timeframe and without the need for high-performance hardware i.e., in a cost-efficient way. Using this marker set, we conducted paternity assignments for 853 olive genotypes, representing, to our knowledge, the largest dataset used for parentage analysis in olive to date. The resulting pedigree relationships provide novel insights into the diversification processes of the crop across the Mediterranean, shed light on mating behaviours among cultivars, and offer valuable guidance for the selection of genitors in future breeding crosses.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterial\u003c/h2\u003e \u003cp\u003eAll the distinct cultivars identified in previous studies were subjected to parentage analysis in the present work. Thus, 668 different international cultivars identified by Belaj \u003cem\u003eet al\u003c/em\u003e. together with 172 different Spanish cultivars identified by G\u0026oacute;mez-G\u0026aacute;lvez \u003cem\u003eet al\u003c/em\u003e., were used [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition, 13 seedlings were included as validation controls, 12 of them from both known genitors and one from a known mother fertilized in open pollination. To sum up, a total of 853 distinct EST-SNP profiles were analysed (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). All genotypic data were generated using the same standardized genotyping pipeline based on the 96 EST-SNP panel routinely applied at the WOGBC-ESP046. DNA extraction, genotyping method and allele calling procedures were identical across studies, ensuring full data consistency and comparability. Detailed information about the 96 EST-SNP has been previously published [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInformation of chloroplast DNA data, and stigmatic groups of andro-sterility (G1/G2) was added for each cultivar in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e according to information available in literature (see references in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Some andro-sterility information obtained from the periodic phenology records routinely carried out in the WOGBC-ESP046 was also added (at the beginning of flowering, andro-sterile cultivars can be detected by observing dehiscence of the anthers and/or absence of pollen) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Finally, information of population structure was also presented in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Since this information was separately explored in previous works for the different sets of cultivars [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], a new exploration was conducted here combining all the cultivars under study. Thus, this new information of population structure was obtained using \u0026lsquo;LEA\u0026rsquo; admixture analysis within the pipeline of SambaR package [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/mennodejong1986/SambaR\u003c/span\u003e\u003cspan address=\"https://github.com/mennodejong1986/SambaR\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) performed in R 4.2.2 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The \u003cem\u003esnmf\u003c/em\u003e function was run, and the optimal number of populations (\u003cem\u003eK\u003c/em\u003e) was assessed based on the cross-entropy criterion through examining a range of \u003cem\u003eK\u003c/em\u003e from 1 to 10 with 10 replicates for each scenario [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The proportions of membership (Q) of each individual in each cluster were exported. A cultivar was considered belonging to a cluster when its Q\u0026thinsp;\u0026gt;\u0026thinsp;75%; otherwise, it was considered as admixed.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eParent-offspring analysis (duos)\u003c/h3\u003e\n\u003cp\u003eParentage analysis was conducted following two steps, similar to the procedure published by Khadari \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. First, a single-parent search was performed in the whole set of 853 distinct genotypes to identify putative parent-offspring duos. Then, a parental pair search was conducted on the genotypes inferred in some putative parent-offspring duo to identify putative parents-offspring trios. For parent-offspring duos, two different approaches were used: i) First, the kinship analysis available in the R package SambaR was used. This analysis does not require allele frequency information and is based on the approach of Manichaikul \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Thus, a parent-offspring duo was retained if all SNP loci shared at least one allele (i.e., the \u0026ldquo;zero identity-by-state\u0026rdquo;, R0, coefficient is zero) and if the KING-robust coefficient (φ) was within the inference criteria range (0.177\u0026thinsp;\u0026lt;\u0026thinsp;φ\u0026thinsp;\u0026lt;\u0026thinsp;0.354) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]; ii) Second, the parentage analysis available in CERVUS v.3.0.7 was computed [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This analysis relies on allele frequencies and is based on the difference in the log-likelihood ratio (LOD) between related and unrelated relationships to assign parentage combined with simulation of parentage analysis to determine the confidence of assignments [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The default settings for the simulation, recommended by the software [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], were the following: number of offspring\u0026thinsp;=\u0026thinsp;10000; number of candidate parents\u0026thinsp;=\u0026thinsp;200; proportion of candidate parents sampled\u0026thinsp;=\u0026thinsp;0.3; proportion of loci typed\u0026thinsp;=\u0026thinsp;0.8; and proportion of loci mistyped\u0026thinsp;=\u0026thinsp;0.01. Two criteria were finally considered to establish strict parentage relationships: a confidence level of the LOD score higher than 95% and mismatches on a maximum of two loci. Such parameters have also been used in previous works on woody crops [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eParents pair-offspring analysis (trios)\u003c/h3\u003e\n\u003cp\u003eFor parents-offspring trios, all those cultivars inferred in any duo were included in the likelihood-based method of CERVUS v.3.0.7 using the same parameters and criteria as above [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. From the putative trios obtained, a selection of the most robust ones for each offspring was done and represented in a pedigree network. The selection was done according to the higher trio LOD score. Those cases in which several parent pairs with close trio LOD scores were inferred for the same offspring were carefully studied and selected according to the number of trio-mismatches, number of parents significantly inferred in the duo analysis, and logical geographic distribution. Similarly, conflictive cases in which several cultivars appeared together in several trios, sometimes acting as offspring and sometimes as parents, were solved by selecting the trio with the highest trio LOD score value.\u003c/p\u003e\n\u003ch3\u003eUse of additional information in robust trios\u003c/h3\u003e\n\u003cp\u003eInformation of chloroplast data, stigmatic groups of compatibility and andro-sterility (see reference sources in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) was incorporated to all the robust trios selected. This information was first used to corroborate the selection of robust trios, i.e., checking the plausibility of the matching when the information of the trio was complete. Chlorotype and andro-sterility information were also used to deduce the sex of candidate parents when possible. Finally, the stigmatic information was used to deduce the compatibility groups in those robust trios with partial information, i.e., if a candidate parent with an unknown group was inferred as a pair of a candidate parent with stigmatic group G1, it was deduced as G2, and \u003cem\u003evice versa\u003c/em\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGenetic structure\u003c/h2\u003e \u003cp\u003eThe \u0026lsquo;LEA\u0026rsquo; admixture analysis detected \u003cem\u003eK\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3 as the most likely number of genetic groups, so three clusters were considered, namely A, B and C. The proportions of membership (Q) of each individual in each cluster are presented in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. For Q\u0026thinsp;\u0026gt;\u0026thinsp;75%, 145 cultivars were considered belonging to cluster A, 51 to B, and 163 to C. The rest of cultivars (494; 58%) were considered to be admixed (Q\u0026thinsp;\u0026lt;\u0026thinsp;75% in any cluster). As expected [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], most of cultivars assigned to clusters A, B and C belong to cultivars mainly grown in Eastern, Central and Western Mediterranean countries, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAveraged proportion of membership (Q) observed for cultivars grown in Eastern (EM), Central (CM) or Western (WM) Mediterranean countries and number of cultivars (N) assigned to each cluster (Q\u0026thinsp;\u0026gt;\u0026thinsp;75%) or admixed (Q\u0026thinsp;\u0026lt;\u0026thinsp;75%).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRegion of cultivation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQ mean for A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eQ mean for B\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eQ mean for C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN assigned to A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN assigned to B\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN assigned to C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eN assigned as admixed\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.764\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.345\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.406\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.249\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e153\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.283\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.190\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.527\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e285\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eParent-offspring duo analysis\u003c/h3\u003e\n\u003cp\u003eA total of 1,696 different putative parent-offspring duos were identified in SambaR. With this method, around 89% (759 out of 853) of the genotypes under study conformed a duo (Table S2). The CERVUS approach identified 3,221 different putative parent-offspring with 95% confidence level (LOD duo\u0026thinsp;\u0026gt;\u0026thinsp;4.65) and less than two mismatching loci (Table S2). In this case, 815 cultivars (95.6%) were inferred in at least one duo.\u003c/p\u003e \u003cp\u003eThe number of putative duos validated by the two approaches was 1,218, and more than 85% of the genotypes conformed pairwise relationship(s) (Table S2). Of these, 255 genotypes were inferred only once, while 27 were inferred in 10 or more parent-offspring duos. Among the latter, \u0026lsquo;Gordal Sevillana\u0026rsquo;, \u0026lsquo;Safrawi\u0026rsquo;, \u0026lsquo;Toffehi Tataouine\u0026rsquo;, \u0026lsquo;Lech\u0026iacute;n de Granada\u0026rsquo; and \u0026lsquo;Frantoio\u0026rsquo; show more than 25 parent-offspring relationships (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), pointing out their founding importance in the current olive germplasm. It is worth noting the wide geographical range of relationships for two of them: firstly, \u0026lsquo;Gordal Sevillana\u0026rsquo; shows direct relationship with well-known cultivars found in the East (e.g. \u0026lsquo;Izmir Sofralik, \u0026lsquo;Gemlik\u0026rsquo;, or \u0026lsquo;Sebhawy\u0026rsquo;), the Centre (e.g. \u0026lsquo;Amygdalolia Nana\u0026rsquo;, \u0026lsquo;Ascolana Tenera\u0026rsquo;, or \u0026lsquo;Buga\u0026rsquo;), and the West (e.g. \u0026lsquo;Azapa\u0026rsquo;, \u0026lsquo;Bouteillan\u0026rsquo;, or \u0026lsquo;Manzanilla de Sevilla\u0026rsquo;); and secondly, \u0026lsquo;Safrawi\u0026rsquo; is also related to eastern (e.g. \u0026lsquo;Rowghani\u0026rsquo;, \u0026lsquo;Tebabs\u0026rsquo; and \u0026lsquo;Ayvalik\u0026rsquo;), central (e.g. \u0026lsquo;Karydolia\u0026rsquo;, \u0026lsquo;Gerboui\u0026rsquo; and \u0026lsquo;Dolce Agogia\u0026rsquo;), and western cultivars (e.g. \u0026lsquo;Sauzen Vert-2218\u0026rsquo;, \u0026lsquo;Panse\u0026ntilde;era\u0026rsquo; and \u0026lsquo;Empeltre\u0026rsquo;; Table S2). In contrast, the three other founder cultivars (i.e. \u0026lsquo;Toffehi Tataouine\u0026rsquo;, \u0026lsquo;Lech\u0026iacute;n de Granada\u0026rsquo; and \u0026lsquo;Frantoio\u0026rsquo;) were related to cultivars mainly found in the Central and Western olive growing areas.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMost duos were identified among cultivars belonging to the same cluster or among cultivars belonging to a cluster and admixed ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Thus, cultivars belonging to cluster A (Eastern Mediterranean) were inferred in 46% of the cases with other cultivars of the same cluster and 49% with admixed cultivars. As for cluster B (Central Mediterranean area), 21% of the duos were identified within the same cluster, and 69% were found among admixed cultivars. Finally, those assigned to cluster C (Western Mediterranean) appeared in duos with cultivars from the same cluster (43%) or with admixed ones (56%). Only 13 duos included a cultivar belonging to cluster A (either \u0026lsquo;Toffehi Tataouine\u0026rsquo; or \u0026lsquo;Ayvalik\u0026rsquo;) and a cultivar belonging to cluster B; only two duos (\u0026lsquo;Toffehi Tataouine\u0026rsquo; \u0026ndash; \u0026lsquo;Verdale de l\u0026rsquo;H\u0026eacute;rault\u0026rsquo;, and \u0026lsquo;Bassiols\u0026rsquo; \u0026ndash; \u0026lsquo;Cornicabra\u0026rsquo;) were conformed among a cultivar belonging to cluster A and cluster C; and none of the duos was inferred among cultivars belonging to B and C clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eParental pair-offspring trio analysis\u003c/h3\u003e\n\u003cp\u003eThe parentage relationships of the genotypes inferring duos were further examined by searching parental pairs trios with the likelihood approach. A total of 1,000 putative trios were identified under the criteria considered, i.e. LOD\u003csub\u003epp\u003c/sub\u003e \u0026gt; 14.88 for 95% success rate, maximum two mismatching loci, and both parents inferred as significant in at least one of the approaches conducted in previous duo analysis (data not shown). From these putative trios, a selection was done to conserve the most likely parent pair for each offspring (according to the higher trio LOD score and other considerations; see criteria above). Thus, a total of 142 genotypes were involved in 131 different parent pairs leading to 280 different offspring genotypes, with LOD\u003csub\u003epp\u003c/sub\u003e scores ranging from 15.69 to 58.38 (Table S3). All the validation controls were successfully inferred within these robust trios, confirming the reliability of the approach. All the seedlings with both parents known were correctly assigned, with LODpp scores ranging from 19.65 to 37.30. For example, \u0026lsquo;Martina\u0026rsquo;, \u0026lsquo;Chiquitita\u0026rsquo; and \u0026lsquo;Sikitita-2\u0026rsquo; were correctly assigned to the expected pair of parents \u0026lsquo;Arbequina\u0026rsquo; \u0026times; \u0026lsquo;Picual\u0026rsquo; with a LODpp scores of 19.65, 28.37, and 27.27, respectively (Table S3).\u003c/p\u003e \u003cp\u003eAmong the 280 robustly identified trios, the distribution of offspring cultivars by main region of cultivation was as follows: 51 from the Eastern Mediterranean (EM), 61 from the Central Mediterranean (CM), and 168 from the Western Mediterranean (WM) (Table S3). If combining with the main region of cultivation of parents-pairs, footprints of local selection were revealed, with a clear predominance of within-region crosses. Thus, crosses among WM cultivars led to 119 offspring cultivars in the same region; crosses among CM cultivars gave 31 CM cultivars; and crosses among EM cultivars produced 29 EM cultivars. Nevertheless, a notable number of inter-regional combinations were also observed, including EM\u0026ndash;CM (25 offspring cultivars), CM\u0026ndash;WM (22) and EM-WM (9). Offspring from the WM exhibited the highest number and diversity of parental combinations, which may be attributed, partially, to their greater representation in the dataset (\u0026asymp;\u0026thinsp;57%). In contrast, EM offspring showed more limited parental origins, mostly involving EM or CM cultivars (Table S3).\u003c/p\u003e \u003cp\u003eWhen the selected robust trios were represented in a pedigree network, it was observed a main, continuous network connecting most of the genotypes, with eight subnetworks conformed by few cultivars (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the continuous network, several highly connected nodes could be differentiated, representing founder cultivars connections. As expected according to the duos analysis, the cultivars \u0026lsquo;Gordal Sevillana\u0026rsquo;, \u0026lsquo;Lech\u0026iacute;n de Granada\u0026rsquo;, \u0026lsquo;Toffehi Tataouine\u0026rsquo; and \u0026lsquo;Safrawi\u0026rsquo; were identified as the most likely candidate parents of 47.5% of the offspring (132 out of 280 cultivars; Table S3, Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eContribution of major founders to the Mediterranean olive germplasm\u003c/h2\u003e \u003cp\u003eThe cultivar \u0026lsquo;Gordal Sevillana\u0026rsquo; was inferred as candidate parent in a total of 91 robust trios (Table S3; Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). It was inferred in 12 different parent pairs, three of them accounting for most of the offspring: i) \u0026lsquo;Gordal Sevillana\u0026rsquo; \u0026times; \u0026lsquo;Lech\u0026iacute;n de Granada\u0026rsquo;, that was identified as the most likely parental pair for 53 cultivars, mainly cultivated in Southwestern Mediterranean and including important commercial cultivars such as \u0026lsquo;Cornicabra\u0026rsquo;, \u0026lsquo;Manzanilla de Sevilla\u0026rsquo;, \u0026lsquo;Cordovil de Serpa\u0026rsquo;, \u0026lsquo;Picholine Marocaine\u0026rsquo; or the South American cultivar \u0026lsquo;Azapa\u0026rsquo;; ii) the parent pair \u0026lsquo;Toffehi Tataouine\u0026rsquo; \u0026times; \u0026lsquo;Gordal Sevillana\u0026rsquo;, reported here for the first time, was directly linked to 19 cultivars mainly cultivated in northern Spain but also in the southern part of the country (\u0026lsquo;Amargoso\u0026rsquo;), in France (\u0026lsquo;Grossanne\u0026rsquo;), and in the Italian island of Sicily (\u0026lsquo;Abunara\u0026rsquo;); and iii) the pair \u0026lsquo;Gordal Sevillana\u0026rsquo; \u0026times; \u0026lsquo;Morchiaio\u0026rsquo;, with 10 offspring cultivars, mainly from the Balkan area (7), such as \u0026lsquo;Buga\u0026rsquo; and \u0026lsquo;Oblica\u0026rsquo;, but also including the well-known Italian cultivar \u0026lsquo;Ascolana Tenera\u0026rsquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In addition, \u0026lsquo;Gordal Sevillana\u0026rsquo; showed close genetic relationships with cultivars from both the Eastern Mediterranean (\u0026lsquo;Izmir Sofralik\u0026rsquo;, \u0026lsquo;Samanli\u0026rsquo;, \u0026lsquo;Samsun Salamuralik\u0026rsquo;, and \u0026lsquo;Samsun Tuzlamalik\u0026rsquo;) and Central Mediterranean (\u0026lsquo;Derdi\u0026rsquo;, \u0026lsquo;Semidana\u0026rsquo;, and \u0026lsquo;Rotondella di Melfi\u0026rsquo;). This frequent connection across regions is consistent with its hypothesized eastern origin [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and later diffusion to the West under different local names, such as \u0026lsquo;Ters Yaprak\u0026rsquo; or \u0026lsquo;Tavsan Yuregui\u0026rsquo; in Turkey, \u0026lsquo;Seviljska Maslina\u0026rsquo; in Croatia, \u0026lsquo;Bella di Spagna\u0026rsquo;, \u0026lsquo;Giarrafa\u0026rsquo;, \u0026lsquo;Pizzo di Corvo\u0026rsquo; or \u0026lsquo;Santa Caterina\u0026rsquo; in Italy, \u0026lsquo;Grosse du Hamma\u0026rsquo; in Algeria, or \u0026lsquo;Boube\u0026rsquo; in France (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In line with this, chloroplast data further indicate that \u0026lsquo;Gordal Sevillana\u0026rsquo; (E1.2 chlorotype) acted as a pollen donor in most of these crosses (offspring generally harbouring the E1.1 chlorotype), reinforcing its role as a male founder (Table S3).\u003c/p\u003e \u003cp\u003eThe cultivar \u0026lsquo;Lech\u0026iacute;n de Granada\u0026rsquo; was identified as putative parent of a large group of offspring cultivars (57) in the Southwestern Mediterranean Basin. Most of these cultivars (51) were originated from a cross with \u0026lsquo;Gordal Sevillana\u0026rsquo;. However, additional parentage combinations were detected in this region, including crosses with \u0026lsquo;Safrawi\u0026rsquo; and the local Eastern Spanish cultivar \u0026lsquo;Genovesa\u0026rsquo;. With the latter, it gave rise to the commercial cultivar \u0026lsquo;Changlot Real\u0026rsquo;. Beyond this regional influence in the Southwestern Mediterranean Basin, \u0026lsquo;Lech\u0026iacute;n de Granada\u0026rsquo;, which is known to be synonymous of the Greek cultivar \u0026lsquo;Dafnelia\u0026rsquo;, was also identified as a putative parent of the Egyptian cultivar \u0026lsquo;Sebhawy\u0026rsquo; and the Italian cultivar \u0026lsquo;Rotondella di Melfi\u0026rsquo; (Table S3, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe founder cultivar \u0026lsquo;Toffehi Tataouine\u0026rsquo;, introduced in the WOGBC-ESP046 collection from southern Tunisia, and recently found in an ancient tree in northern Spain [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], was inferred in four different parent pairs. Apart from the above-mentioned parent pair with \u0026lsquo;Gordal Sevillana\u0026rsquo;, its most prolific pairing was with \u0026lsquo;Safrawi\u0026rsquo;, producing 11 offspring cultivars. These descendants were mainly found across the central and western Mediterranean, including Greek (\u0026lsquo;Agouromanakolia\u0026rsquo;, \u0026lsquo;Kothreiki\u0026rsquo;), Albanian (\u0026lsquo;Kokerramdh Elbasani\u0026rsquo;, \u0026lsquo;Kotruvsi\u0026rsquo;), and northern Spanish cultivars (\u0026lsquo;Claramunt\u0026rsquo;, \u0026lsquo;Domengues\u0026rsquo;). The well-known Turkish cultivar \u0026lsquo;Ayvalik\u0026rsquo;, synonymy of \u0026lsquo;Edremit Yaglik\u0026rsquo; and \u0026lsquo;Adramitini\u0026rsquo;, was also found to be originated from this cross. Additionally, part of the local Tunisian germplasm may have resulted from crosses between \u0026lsquo;Toffehi Tataouine\u0026rsquo; and other national cultivars, such as \u0026lsquo;Chemlali Sig\u0026rsquo; and \u0026lsquo;Dokkar\u0026rsquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe cultivar \u0026lsquo;Safrawi\u0026rsquo; was inferred as candidate parent in 14 different parent pairs, generating 34 offspring cultivars. It was the most frequently inferred founder cultivar in parent pairs (7) that generated more than one descendant. Thus, besides the above-mentioned pairings with \u0026lsquo;Toffehi Tataouine\u0026rsquo; and \u0026lsquo;Lech\u0026iacute;n de Granada\u0026rsquo;, it was involved in five additional crosses generating more than one offspring: with Eastern Mediterranean cultivars from Syria (\u0026lsquo;Saifi\u0026rsquo;), or Turkey (\u0026lsquo;Ayvalik), as well as with Central Mediterranean ones from Greece (\u0026lsquo;Amygdaloia Nana\u0026rsquo;), or Italy (\u0026lsquo;Nocellara del Bellice\u0026rsquo; and \u0026lsquo;Ogliarola del Bradano\u0026rsquo;). It is noteworthy to mention that \u0026lsquo;Safrawi\u0026rsquo; was the founder cultivar with the most widely distributed progeny across the Mediterranean area. In addition to local offspring produced in Syria (\u0026lsquo;Khoukhe\u0026rsquo;, \u0026lsquo;Sayfi\u0026rsquo;, etc), it generated other cultivars all along the Mediterranean Basin, such as the cultivars \u0026lsquo;Rowghani\u0026rsquo; in Iran, \u0026lsquo;Memeli\u0026rsquo; in Turkey, \u0026lsquo;Karydolia, in Greece, \u0026lsquo;Ulliri shekullor Berat\u0026rsquo; in Albania, \u0026lsquo;Aitana\u0026rsquo; in Italy, \u0026lsquo;Gerboui\u0026rsquo; in Tunisia, and \u0026lsquo;Farga\u0026rsquo; in Spain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This broad distribution and high number of \u0026lsquo;Safrawi\u0026rsquo; descendants may probably be due to the extensive dissemination of this variety along the Mediterranean basin where it \u0026ldquo;was baptised\u0026rdquo; with different local names such as \u0026lsquo;Antawi\u0026rsquo; or \u0026lsquo;Dan\u0026rsquo; in Syria, \u0026lsquo;Dal\u0026rsquo; or \u0026lsquo;Baladi Remadieh\u0026rsquo; in Lebanon, \u0026lsquo;Karabisi\u0026rsquo; in Jordan, \u0026lsquo;Celebi (Silifke)\u0026rsquo;, \u0026lsquo;Erkence\u0026rsquo;, \u0026lsquo;Hurma Kaba\u0026rsquo;, \u0026lsquo;Sari Hebsi (Hatay)\u0026rsquo; or \u0026lsquo;Yag Zeytini\u0026rsquo; in Turkey, \u0026lsquo;Throubolia\u0026rsquo; in Greece, \u0026lsquo;Marski\u0026rsquo; in Albania, \u0026lsquo;Grossolana\u0026rsquo; or \u0026lsquo;Santagatesse\u0026rsquo; in Italy, \u0026lsquo;Filayre Noir\u0026rsquo; in France, and \u0026lsquo;Cirujal\u0026rsquo;, or \u0026lsquo;\u0026rsquo;Grossal de Pallars\u0026rsquo; in Spain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eApart from the founder cultivars mentioned above, the network reflected a high number of further connections. They included cultivars that generated offspring in their nearby areas of cultivation, therefore representing local diversification foci. Thus, in the Eastern Mediterranean, the parent pair \u0026lsquo;Zaity\u0026rsquo; \u0026times; \u0026lsquo;Saifi\u0026rsquo; originated several cultivars in Syria, while \u0026lsquo;Zaity\u0026rsquo; \u0026times; \u0026lsquo;Baladi\u0026rsquo;, from neighbouring Lebanon, generated at least seven local descendants. Similarly, six cultivars grown in Egypt were inferred as descendant from the parent pair \u0026lsquo;Chemlali Sfax\u0026rsquo; \u0026times; \u0026lsquo;Balady\u0026rsquo;, from Tunisia and Egypt, respectively (Table S3). In Central Mediterranean, it was observed local diversity derived from second generation of crosses, i.e. some inferred offspring cultivars were also detected as putative parents of other cultivars. Thus, the Sicilian cultivar \u0026lsquo;Abunara\u0026rsquo; (inferred as offspring of the parent pair \u0026lsquo;Toffehi Tataouine\u0026lsquo; \u0026times; \u0026lsquo;Gordal Sevillana\u0026rsquo;) was found to be candidate parent of the well-known Tunisian cultivar \u0026lsquo;Meski\u0026rsquo;. Similarly, four local cultivars in this country seem to have been derived from crosses of the cultivar \u0026lsquo;Jemri Bouchouka\u0026rsquo; (a local offspring of the parent pair \u0026lsquo;Toffehi Tataouine\u0026rsquo; \u0026times; \u0026lsquo;Chemlali Sig\u0026rsquo;) with the Sicilian cultivar \u0026lsquo;Nocellara del Belice\u0026rsquo; that was also found in Tunisia with different names (\u0026lsquo;Beldi\u0026rsquo;, \u0026lsquo;Fakhari\u0026rsquo;, \u0026lsquo;Meski Zarzis\u0026rsquo; or \u0026lsquo;Zarrazi\u0026rsquo;) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This was also found in Western Mediterranean, where, as an example, the cultivar \u0026lsquo;Claramunt\u0026rsquo;, derived from the parent pair \u0026lsquo;Toffehi Tataouine\u0026rsquo; \u0026times; \u0026lsquo;Safrawi\u0026rsquo;, gave rise to new local germplasm by pairing with the local cultivar \u0026lsquo;Verdal de Pallars\u0026rsquo;. Similarly, second-generation crosses seem to have contributed to the local diversity of olive cultivars in the New World, as some cultivars identified in South America, such as \u0026lsquo;Carrasque\u0026ntilde;a Huasco\u0026rsquo; and \u0026lsquo;Ascolana Huasco\u0026rsquo;, appear to be descendants of \u0026lsquo;Azapa\u0026rsquo;, which, as mentioned above, is derived from the parent pair \u0026lsquo;Lech\u0026iacute;n de Granada\u0026rsquo; \u0026times; \u0026lsquo;Gordal Sevillana\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eValuable information deduced from robust trios\u003c/h2\u003e \u003cp\u003eInformation added about chloroplast DNA, stigmatic groups and andro-sterility matched perfectly in the robust trios inferred, corroborating therefore the plausibility of these parentage assignments. In cases where such information was incomplete or lacked, logical deductions allowed to predict it. Notably, the deduced information aligned well with the parentage assignments, even for trios without bibliographic support (Table S3).\u003c/p\u003e \u003cp\u003eThe logical deductions allowed to predict the stigmatic group of 74 cultivars, including cultivars of major agronomic relevance such as \u0026lsquo;Chemlali Sfax\u0026rsquo; (G1), \u0026lsquo;Galega Vulgar\u0026rsquo; (G1), \u0026lsquo;Azapa\u0026rsquo; (G2), \u0026lsquo;Zaity\u0026rsquo; (G1), or the founder cultivar \u0026lsquo;Toffehi Tataouine\u0026rsquo;. (G2). Likewise, the analysis revealed new insights into the reproductive compatibilities of cultivars with observed potential for hedgerow systems, such as \u0026lsquo;Arr\u0026oacute;niz\u0026rsquo;, \u0026lsquo;Rotondella di Melfi\u0026rsquo;, or \u0026lsquo;Zeitoun Boubezzoula\u0026rsquo;.\u003c/p\u003e \u003cp\u003eAlso, the robust trios obtained enabled to deduce the chlorotype information in 52 cultivars and the sex of candidate parents in 60 trios. Most of the chlorotypes deduced (46) were E1.1, as a result of obtaining an offspring with lacking information but originated from a parent pair with both candidate parents classified as E1.1. The combination with andro-sterility information enabled to deduce the chlorotype on a group of cultivars from eastern Spain, for which both types of information were consistent and mutually reinforcing. In particular, \u003cem\u003e\u0026lsquo;\u003c/em\u003eCarrasco\u0026rsquo;, \u0026lsquo;Cuquello de la Jana\u0026rsquo;, \u0026lsquo;Picuda de Luis\u0026rsquo;, and \u0026lsquo;Rufina\u0026rsquo; were found to share the same chlorotype (E3.1) and andro-sterility profile, both likely inherited from \u0026lsquo;Farga\u0026rsquo;, their inferred maternal parent.\u003c/p\u003e \u003cp\u003eFinally, the information provided by the robust trios made possible to identify the male parent in progenies derived from open-pollination in some breeding programs. As an example, the cultivar \u0026lsquo;Favolosa\u0026rsquo;, obtained in a breeding program from \u0026lsquo;Frantoio\u0026rsquo; in open pollination, was identified as offspring of \u0026lsquo;Frantoio\u0026rsquo; and \u0026lsquo;Ascolana Tenera\u0026rsquo; with a high LOD\u003csub\u003epp\u003c/sub\u003e score (43.32). In addition, the approach allowed for the reconstruction of complete pedigrees for cultivars whose origin was only partially known. Notable examples include \u0026lsquo;Dwarf D\u0026rsquo;, \u0026lsquo;Cairo-7\u0026rsquo;, or \u0026lsquo;S:27_4\u0026rsquo;. Similarly, \u0026lsquo;Arbosana\u0026rsquo;, a commercial cultivar adapted to hedgerow system, was correctly inferred to its known female parent, \u0026lsquo;Arbequina\u0026rsquo;, and the initially unknown male parent was newly postulated as \u0026lsquo;Vaneta\u0026rsquo;, a local cultivar that is present in the same area of cultivation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis present study provides the most comprehensive analysis to date of parentage relationships in olive, revealing both parent-offspring duos and parent pairs-offspring trios within a set of 853 genotypes. This large-scale approach, based on a cost-effective set of 96 EST-SNPs, uncovered four founder cultivars, namely \u0026lsquo;Gordal Sevillana\u0026rsquo;, \u0026lsquo;Lech\u0026iacute;n de Granada\u0026rsquo;, \u0026lsquo;Toffehi Tataouine\u0026rsquo; and \u0026lsquo;Safrawi\u0026rsquo;, that played central roles in the diversification of the crop. Moreover, the resulting pedigree atlas sheds new light on the genealogical structure of Mediterranean olive germplasm and provided practical tools for breeding and germplasm management, moving beyond previous studies that focused mainly on domestication history and diversification.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFounder cultivars shaping Mediterranean olive diversification\u003c/h2\u003e \u003cp\u003eAs in other woody plants, analysing parentage is a useful approach to trace the origins of cultivated germplasm. The few generations since domestication and the vegetative propagation of key cultivars may help to reconstruct genealogies and identify founder genotypes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Here, various cultivars have been identified as founder cultivars, as evidenced by the large number of the offspring generated. Three of them, \u0026lsquo;Gordal Sevillana\u0026rsquo;, \u0026lsquo;Lech\u0026iacute;n de Granada\u0026rsquo; and \u0026lsquo;Safrawi\u0026rsquo; have been already distinguished as founder cultivars in previous works [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], but here their offspring relationships were expanded and refined thanks to the broader dataset of 853 genotypes analysed. In addition, we provide the first evidence about \u0026lsquo;Toffehi Tataouine\u0026rsquo; acting as a founder, with descendants in both Central and Western Mediterranean. This represents a surprising finding, as the impact of this cultivar, primarily discovered in Tunisia, has not been suggested until now. Its previously unrecognized influence highlights the importance of including ancient, forgotten cultivars in pedigree studies. Interestingly, an ancient olive tree recently genotyped in northern Spain matched with this cultivar [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], reinforcing the results obtained in our study and supporting for its historical diffusion beyond Tunisia.\u003c/p\u003e \u003cp\u003eA common feature of the four founder cultivars is their association with very old olive trees [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], which reinforces their antiquity and probable early selection. Moreover, \u0026lsquo;Gordal Sevillana\u0026rsquo;, \u0026lsquo;Lech\u0026iacute;n de Granada\u0026rsquo; and \u0026lsquo;Safrawi\u0026rsquo; present well-known synonymies in different parts of the Mediterranean area [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], indicating long-distance dissemination and local renaming that contributed to diversification in different areas. Although \u0026lsquo;Toffehi Tataouine\u0026rsquo; currently remains a little-known local cultivar in southern Tunisia [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], the recent evidence of its genetic match in Spain [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] and its prominent role in the inferred offspring suggest that it may also have experienced wider historical diffusion, and future prospecting may reveal additional synonymies. Overall, the identification of these four founders, especially their wide geographical spread and contribution to cultivar diversification highlight the long-distance dispersal and hybridization events that shaped the current structure of Mediterranean olive germplasm. The resulting olive pedigree atlas provides, for the first time, an integrated view of these genealogical links, revealing patterns of crossing between local and foreign cultivars and identifying multiple foci of diversification across the Mediterranean Basin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRegional patterns of diversification: Eastern, Central and Western Mediterranean\u003c/h2\u003e \u003cp\u003eIn the Eastern Mediterranean region, considered the primary centre of olive domestication, our parentage analysis revealed a remarkable number of cultivars involved in different duos and trios, confirming the important role of eastern cultivars in the early diversification of the crop. Despite the relatively limited number of cultivars from this area included in our dataset (\u0026asymp;\u0026thinsp;18%), the results highlight their contribution to multiple, locally adapted hybridization events. This observation is consistent with the high levels of genetic diversity previously reported for the region and with archaeological and genetic evidences indicating that the Levant is the cradle of olive cultivation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the Central Mediterranean, the parentage network reveals a more complex and heterogeneous pattern of cultivar origin and diversification. This area appears as a transitional zone where both autochthonous an allochthonous genotypes contributed to shaping the current olive germplasm. This pattern has been particularly observed in studies conducted in some islands such as Malta, Sicily, or Capri, considered crossroads of the Mediterranean [\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the Western Mediterranean, the parentage patterns showed a strong founder effect driven by a limited number of recurrent parent pairs. The allochthonous origin of most cultivars of Western Mediterranean has been postulated in previous studies [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In this study, it has been corroborated that the parent pair \u0026lsquo;Lech\u0026iacute;n de Granada\u0026rsquo; \u0026times; \u0026lsquo;Gordal Sevillana\u0026rsquo; had a crucial role in this area, generating a large number of offspring cultivars. Among them, some popular and relevant cultivars such as \u0026lsquo;Cordovil de Serpa\u0026rsquo;, \u0026lsquo;Manzanilla de Sevilla\u0026rsquo;, or \u0026lsquo;Picholine Marocaine\u0026rsquo;, were robustly inferred. Additionally, other commercially important Spanish cultivars, namely \u0026lsquo;Picual\u0026rsquo; and \u0026lsquo;Hojiblanca\u0026rsquo;, were also inferred as descendant of this parent pair, although their trio assignments did not reach the criteria required under our robustness thresholds (data not shown). These genealogical connections should be re-evaluated and confirmed in future analyses using higher-density SNP arrays or whole-genome sequencing.\u003c/p\u003e \u003cp\u003eIn the New World, as in the Central and Western Mediterranean, second-generation crosses appear to have contributed to the local diversification of olive cultivars. For instance, the South American variety \u0026lsquo;Azapa\u0026rsquo;, identified as an offspring of the parent pair \u0026lsquo;Lech\u0026iacute;n de Granada\u0026rsquo; \u0026times; \u0026lsquo;Gordal Sevillana\u0026rsquo; and likely introduced from the Iberian Peninsula, was found to act as a progenitor of several local cultivars in that region. Thus, throughout the history of olive cultivation in the New World, both the introduction and preservation of ancient Mediterranean cultivars\u0026mdash;some of which may have been lost or remain to be identified in their regions of origin\u0026mdash;together with the local selection and further propagation of seedlings, may have shaped the current olive diversity observed in that area [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is noteworthy that most of the cultivars identified as offspring of this parent pair belong to gene pool C and share the dominant E1.1 chlorotype, corroborating the bottleneck of diversity described for Western Mediterranean in other studies [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The high number of offspring attributed to this pair may reflect both the long-term coexistence of these founders and their high efficiency as crossing pair [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Since \u0026lsquo;Lech\u0026iacute;n de Granada\u0026rsquo; has the E1.1 chlorotype and \u0026lsquo;Gordal Sevillana\u0026rsquo; the E1.2, it implies that the latter usually had to act as male progenitor [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This fact could be explained by a high pollinic viability of \u0026lsquo;Gordal Sevillana\u0026rsquo;, and an optimal phenological overlapping between these two founder cultivars. Another possible explanation could be the easier dispersal of seeds from \u0026lsquo;Lech\u0026iacute;n de Granada\u0026rsquo; than form \u0026lsquo;Gordal Sevillana\u0026rsquo;, whose smaller fruit size allows better ingestion by bird species known to act as olive seed dispersers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eHybridization between local and introduced germplasm\u003c/h2\u003e \u003cp\u003eThe diverse origin of olive cultivars can also be observed at the scale of individual countries. In the case of Spain, which provided the best-represented germplasm in our study, the cultivars mainly grown in northern and north-eastern regions presented a broader diversity of origins respect to those from the south, which is in accordance with results obtained previously [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Many cultivars of these regions were traced back to diverse foreign ancestors or their first-degree relatives, resulting in a much more diversified network of relationships than in the south of the peninsula. \u0026lsquo;Gordal Sevillana\u0026rsquo;, \u0026lsquo;Toffehi Tataouine\u0026rsquo;, and \u0026lsquo;Safrawi\u0026rsquo; were found to be a progenitor in a significant number of trios of Northern and North-eastern Spain. Interestingly, \u0026lsquo;Lech\u0026iacute;n de Granada\u0026rsquo; was never inferred as a putative parent in cultivars of these region. This suggests an influx of cultivars linked to different historical periods and different civilizations with dominium in northern or southern coast of the Mediterranean. On the other hand, the autochthonous material also seems to have played an important role in this part of the Western Mediterranean. The high level of admixture observed in this area, together with the genetic similarity to local wild forms, led some authors to suggest an autochthonous influence in some cultivars [\u003cspan additionalcitationids=\"CR51 CR52\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. A cultivar that clearly exemplifies this admixture is \u0026lsquo;Farga\u0026rsquo;. Although \u0026lsquo;Farga\u0026rsquo; harbours the western E3.1 chlorotype, its nuclear genome is more similar to cultivars carrying the eastern E1 lineage, suggesting a cytoplasmic capture through backcrossing with cultivars originating from the Eastern Mediterranean basin [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. This can be better understood if we analyse the putative progenitors inferred for this cultivar in our study: \u0026lsquo;Patr\u0026oacute;n de Cab\u0026uacute;s\u0026rsquo; and \u0026lsquo;Safrawi\u0026rsquo;. There are two clues that suggest that \u0026lsquo;Patr\u0026oacute;n de Cab\u0026uacute;s\u0026rsquo; may represent autochthonous material. The first is that it was assigned to gene pool B, as were other cultivars known to be closely related to wild forms, such as \u0026lsquo;Dokkar\u0026rsquo; [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The second clue comes from its name: since \u0026ldquo;Patr\u0026oacute;n\u0026rdquo; means rootstock in Spanish, it may refer to local wild material that was grafted in the past. In contrast, \u0026lsquo;Safrawi\u0026rsquo; seems to be a cultivar introduced from the East by ancient civilizations, as mentioned above and in other studies [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Together, these findings support the hypothesis of hybridization between local and introduced germplasm and highlight \u0026lsquo;Farga\u0026rsquo; as a likely outcome of such genetic exchange.\u003c/p\u003e \u003cp\u003eFurthermore, \u0026lsquo;Farga\u0026rsquo; has been observed to be linked in the single parent-offspring duo to other 19 local cultivars of Northeastern Spain, suggesting that it acted as a focus of introgression and local diversification. In this context, it is worth noting that cytoplasmic male sterility (CMS) has been described in olive and shown to be maternally inherited and associated with the E3 chlorotype [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Androsterility within the \u0026lsquo;Farga\u0026rsquo; group had already been reported by Rojas-G\u0026oacute;mez \u003cem\u003eet al.\u003c/em\u003e, who proposed a potential founder effect [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. This observation is consistent with our results, since \u0026lsquo;Farga\u0026rsquo; carries the E3.1 chlorotype and several of its inferred descendants exhibit andro-sterility, such as \u0026lsquo;Carrasco\u0026rsquo;, \u0026lsquo;Cuquello de la Jana\u0026rsquo;, and \u0026lsquo;Rufina\u0026rsquo;. The co-occurrence of andro-sterility and the E3.1 haplotype therefore suggests that CMS could have been transmitted through the maternal line of \u0026lsquo;Farga\u0026rsquo;, further reinforcing its role as a cytoplasmic donor and as a centre of diversification in the Northeastern Iberian Peninsula.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePractical implications for breeding and germplasm management\u003c/h2\u003e \u003cp\u003eApart from shedding light on the diversification history of olive, the pedigree information inferred in this study may provide substantial value for breeding programs and germplasm management. In particular, we combined paternity results with information on stigmatic compatibility groups to deduce mating behaviours, which reinforced the robustness of trios inferred with incomplete information and expanded the knowledge already available on compatibility groups [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. This approach can improve the selection of inter-compatible genitors in future breeding programs. In this context, our analysis revealed the compatibility groups of cultivars of potential interest for modern high-density systems, such as \u0026lsquo;Arr\u0026oacute;niz\u0026rsquo;, \u0026lsquo;Rotondella di Melfi\u0026rsquo; and \u0026lsquo;Zeitoun Boubezzoula\u0026rsquo;. These cultivars could represent promising alternatives to the widely used \u0026lsquo;Arbequina\u0026rsquo;, contributing to the diversification of intensive olive cultivation and helping to reduce risks associated with a narrow genetic base. Also, pedigree information has resulted useful for knowing the origin of progenies obtained from open pollination, or progenies received in exchanges of plant material between collections but with incomplete or lost passport information. Thirdly, knowledge of pedigrees is also useful for assembling balanced sets of progenitors in terms of inbreeding. Otherwise, released cultivars may become susceptible to new biotic and abiotic stresses due to their genetic similarity [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Although inbreeding depression has not yet been documented in olive, it has been widely reported in other woody crops, where it leads to reduction in vigour, flower number and fruit set, increase in fruit abortion, lower seed germination and seedling survival, abnormal growth, and a loss of disease resistance [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Finally, the construction of a pedigree atlas may also have additional practical applications, as highlighted by other authors. For example, it can help optimize whole-genome sequencing by using key founders as reference points to estimate missing genetic data in other cultivars [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In addition, pedigree information can be exploited for the description and valorisation of cultivars, serving as a resource for storytelling in cultivar marketing and providing added value, as demonstrated in other woody crops [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFinally, paternity analysis should be seen as a systematic task that should be continuously updated as new cultivars are genotyped and included in the reference databases. The approach used here is based on a set of 96 informative EST-SNPs [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] (mean \u003cem\u003eH\u003c/em\u003e\u003csub\u003eO\u003c/sub\u003e = 0.501, mean Minimum Allele Frequency, MAF value, of 0.380). A substantial number of studies have demonstrated that SNP markers are as suitable for parentage analysis as classical markers, such as SSRs, and that a relatively small number of SNPs, from 60 to 200, with higher MAF values, are sufficient for most parentage analyses [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our results confirm that this set of 96 EST-SNPs markers provides robust pedigree inferences across a wide and diverse olive germplasm collection. Many of these inferences agree with those reported in previous studies using diverse sets of SSRs [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] or a larger set of SNPs [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Thus, although the resolution could be improved further through the use of whole-genome sequencing or higher-density SNP arrays, the cost-effective and standardisable nature of the current method makes it particularly suitable for systematic pedigree analyses applied in germplasm banks and breeding platforms.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe comprehensive analysis of paternity conducted here provides the first olive pedigree atlas, with insights into the spread of olive cultivars along the Mediterranean Basin, but also with practical tools for modern breeding programs. The results not only confirm previously suggested genealogical links but also uncover new key founder cultivars and robust parent-offspring relationships, shedding light on the historical processes that have shaped olive cultivation. Additionally, the pedigree atlas offers actionable information for breeding and germplasm management: it enables the identification of inter-compatible parents, the reconstruction of previously unknown pedigrees, and the design of balanced genitor sets to ensure genetic diversity while minimizing the risks of inbreeding. The methodological approach used in this study demonstrates the effectiveness of a practical set of SNP markers for a rapid and a continuous pedigree analysis, which can be widely applied in germplasm management and breeding programs. This study establishes a strong basis for future research and advancements in breeding programs, emphasizing the need for continuous updates to the olive pedigree atlas as new cultivars are discovered and characterized.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eNot applicable. This study does not involve human participants, animals, or clinical trials.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot aplicable\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe datasets and material analysed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis research was financially supported by the regional IFAPA projects PR.CRF.CRF201900.004 and PR.CRF.CRF202200.004, partially funded by European Agricultural Fund for Rural Development (EAFRD). The management, identification and conservation of WOGBC-ESP046 have also been financially supported by the contract CAICEM 23-76. FJ G-G has been supported by PR.CRF.CRF202200.004, partially funded by EAFRD. G.B. is supported by PatrimOlea and is a member of the CRBE laboratory, which is supported by the Laboratory of Excellence (LabEx) CEBA (grant ANR-10-LABX-25-01) and LabEx TULIP (grant ANR-10-LABX-0041), both managed by the French National Research Agency (ANR).\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026rsquo; contributions\u003c/h2\u003e\n\u003cp\u003eFG-G: Data curation, Formal analysis, Methodology, Resources, Software, Visualization, Writing\u0026ndash; original draft. RR-N: Conceptualization, Resources, Formal analysis, Funding acquisition, Methodology, Validation, Writing review \u0026amp; editing, Writing\u0026ndash; original draft. G-B: Formal analysis, Validation, Writing review \u0026amp; editing. IL: Data curation, Formal analysis, Writing review \u0026amp; editing. AB: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing\u0026ndash; review \u0026amp; editing. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThe authors are grateful to all the field and laboratory technicians for their work in the conservation and identification of accessions. The authors are also grateful for the EST-SNP genotyping support of staff at UPV/EHU\u0026mdash;Scientific Park Maria Goyri Biotechnology Center (Bizkaia, Spain), especially to Fernando Rendo. The authors also acknowledge the contribution of Pedro G\u0026oacute;mez G\u0026aacute;lvez for the support with R programming and are thankful to Christian Pinatel, Jean-Fr\u0026eacute;d\u0026eacute;ric Terral, and H\u0026eacute;l\u0026egrave;ne Lasserre for their contribution to the search for ancient varieties (especially \u0026apos;Filayre noir\u0026apos; in France) and for their encouragement to conduct further work at the regional level.\u003c/p\u003e\n\u003ch3\u003eSupplementary Data\u003c/h3\u003e\n\u003cp\u003eSupplementary data are available as online supplementary material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZohary D, Hopf M, Weiss E. Domestication of Plants in the Old World: The Origin and Spread of Domesticated Plants in Southwest Asia, Europe, and the Mediterranean Basin. Oxford University Press; 2012.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTerral J, Alonso N, Capdevila R, et al. 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Sci Hortic. 2011;133:23\u0026ndash;30. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.scienta.2011.10.001\u003c/span\u003e\u003cspan address=\"10.1016/j.scienta.2011.10.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"pedigree atlas, genealogy, breeding, cultivar diversification, inter-compatibility, founder cultivars","lastPublishedDoi":"10.21203/rs.3.rs-8524821/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8524821/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eOlive pedigree has been scarcely explored beyond domestication and diversification studies, even though it can be valuable for breeding programs and germplasm management. This study presents a new and comprehensive exploration of olive parentage relationships by combining a large dataset of 840 cultivars with a cost-effective and highly informative panel of 96 EST-SNP markers routinely applied at the World Olive Germplasm Bank of C\u0026oacute;rdoba. Parentage assignments were performed combining two approaches, SambaR and CERVUS, and using 13 seedlings of known crosses as validation controls.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThis strategy revealed 1,218 parent-offspring duos and 280 robust parents pair-offspring trios, involving more than 85% of the genotypes analysed. Four founder cultivars, \u0026lsquo;Gordal Sevillana\u0026rsquo;, \u0026lsquo;Lech\u0026iacute;n de Granada\u0026rsquo;, \u0026lsquo;Toffehi Tataouine\u0026rsquo;, and \u0026lsquo;Safrawi\u0026rsquo;, emerged as central nodes in the pedigree network, highlighting their crucial role in the diversification of olive cultivars across the Mediterranean Basin. \u0026lsquo;Gordal Sevillana\u0026rsquo;, in combination with \u0026lsquo;Lech\u0026iacute;n de Granada\u0026rsquo; and \u0026lsquo;Toffehi Tataouine\u0026rsquo;, contributed substantially to the origin of Western and, to a lesser extent, Central Mediterranean cultivars, while \u0026lsquo;Safrawi\u0026rsquo; acted as a key connector across the entire basin.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study provides the first olive parentage atlas, providing new insights into the diversification processes, but also a practical tool for the management of genetic resources. In particular, these results demonstrate that a small but informative SNP set can generate reliable pedigree information to identify compatible parents, resolve uncertain genealogies of cultivars of agronomic interest, reconstruct unknown pedigrees in open-pollination, and guide the selection of balanced parental sets for developing new cultivars.\u003c/p\u003e","manuscriptTitle":"An olive parentage atlas: founder cultivars, regional diversification, and implications for breeding programs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-13 18:07:08","doi":"10.21203/rs.3.rs-8524821/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-22T16:30:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-22T11:07:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81079364138366982967032988147663200592","date":"2026-01-14T20:55:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"112261811629081832957609607009888900757","date":"2026-01-14T11:52:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-12T13:47:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"133263488811108577870646047640370085542","date":"2026-01-12T09:40:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-09T13:30:37+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-09T13:03:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-09T11:59:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-09T11:53:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-01-05T21:09:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f390bff7-56b1-4acd-b5fd-172d05267f2d","owner":[],"postedDate":"January 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T16:13:59+00:00","versionOfRecord":{"articleIdentity":"rs-8524821","link":"https://doi.org/10.1186/s12870-026-08504-y","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2026-03-11 15:59:49","publishedOnDateReadable":"March 11th, 2026"},"versionCreatedAt":"2026-01-13 18:07:08","video":"","vorDoi":"10.1186/s12870-026-08504-y","vorDoiUrl":"https://doi.org/10.1186/s12870-026-08504-y","workflowStages":[]},"version":"v1","identity":"rs-8524821","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8524821","identity":"rs-8524821","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}