Genetic diversity and relationships of wild and cultivated olives at regional level in Spain

Scientia Horticulturae 124 (2010) 323–330

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Genetic diversity and relationships of wild and cultivated olives at regional level in Spain ˜ oz-Diez b,1, Luciana Baldoni c, Zlatko Satovic d, Diego Barranco b Angjelina Belaj a,1,*, Concepcion Mun a

Centro IFAPA ‘‘Alameda del Obispo’’, Avda. Menendez Pidal s/n 14083, Cordoba, Spain Departamento de Agronomı´a, University of Cordoba, Campus Universitario de Rabanales, Edf. Celestino Mutis, Ctra Madrid-Ca´diz, Km 396, 14080 Co´rdoba, Spain c CNR - Istituto di Genetica Vegetale, Via Madonna Alta 130, 06128 Perugia, Italy d Faculty of Agriculture, University of Zagreb, Svetosimunska 25, 10000 Zagreb, Croatia b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 12 October 2009 Received in revised form 28 December 2009 Accepted 7 January 2010

Genetic diversity and relationships between local cultivars and wild olive trees from three important Spanish olive-growing regions, Andalusia (South), Catalonia and Valencia (from Eastern Mediterranean Coastal area), were studied by means of eight SSR loci. Distinct allelic composition and heterozygosity levels were found in wild olive populations and cultivars. The observed patterns of genetic variation revealed: a) the independent clustering of Andalusian wild olives in a separate gene pool, b) the belonging of wild populations and most cultivars from Catalonia to another gene pool, c) the joined clustering of Andalusian and a set of Valencian cultivars in a third gene pool, and d) clustering of wild individuals from Valencia to the three different gene pools. These results suggest that wild populations of Andalusia may represent true oleasters, the ones from Catalonia may be feral forms derived from cultivar seed spreading, while the population of Valencia seems to be the most admixed one. The significant differentiation between Andalusian and most Catalonian cultivars is indicating an independent selection of olive cultivars in the two regions. The detection of a certain wild genetic background in some Catalonian and Valencian cultivars and the similarity found between wild and cultivated forms may suggest the use of local wild trees in olive domestication. The proposed scenario for the development of olive cultivars in Andalusia includes an empirical selection of outstanding local wild genotypes followed by various generations of crosses and various replanting campaigns, as well as possible introductions of ancestral cultivars. Therefore, our findings would lead us to support the hypothesis that the current diversity found in Spanish olive cultivars may be regionally differentiated and due to both, autochthonous and allochthonous origin. The information obtained in this work gives insights into the genetic resources of the main olive producing country, demonstrating that wild olive populations and local cultivars both represent potential sources of useful variability for olive breeding programs. ß 2010 Elsevier B.V. All rights reserved.

Keywords: SSR Olea europaea Cultivar Genetic relationships Wild relatives

1. Introduction Spain, the main olive producing country, has a very rich olive genetic heritage, represented by both, the cultivated form (Olea europaea subsp. europaea var. europaea) and the wild trees, also known as oleasters (Olea europaea subsp. europaea var. sylvestris). In an exploratory survey about 272 different cultivars were identified in Spain (Barranco and Rallo, 2000). Most of them are of unknown origin and were most probably selected locally for extensive cultivation under dry conditions. The richness of olive germplasm in Spain has been evidenced by molecular studies carried out either exclusively on Spanish cultivars (Sanz-Corte´s

* Corresponding author. Tel.: +34 957 016035; fax: +34 957 016043. E-mail address: [email protected] (A. Belaj). 1 The first two authors contributed equally to this work. 0304-4238/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2010.01.010

et al., 2001; Belaj et al., 2004) and/or together with other Mediterranean cultivars (Besnard et al., 2001; Belaj et al., 2002, 2003; Dı´az et al., 2006; Sarri et al., 2006). However, the renovation of the olive orchards over the last 20 years, has favoured the use of a few well-known cultivars such as ‘Picual’, ‘Arbequina’ and ‘Hojiblanca’, which now dominate the Spanish olive-growing areas. In addition, in the near future, these varieties may also be displaced by new cultivars obtained by breeding (Rallo et al., 2008). Due to this selection, the use of local cultivars has become very rare or nonexistent, leading to a risk of their extinction. Therefore, the study of olive germplasm could provide sound background knowledge to safeguard and to implement strategies for its conservation, to select genotypes that are better adapted to certain environmental conditions, to use this genetic material for breeding purpose and to clarify the history of olive domestication. Wild olives represent a distinctive element of the Mediterranean flora of the Iberian Peninsula (Vargas and Kadereit, 2001;

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A. Belaj et al. / Scientia Horticulturae 124 (2010) 323–330

Rubio de Casas et al., 2002, 2006), where two distinct forms have been recognised: true oleasters (wild forms present in undisturbed areas) and feral forms occurring in secondary habitats such as the disturbed areas or abandoned fields and deriving from seedlings of cultivated clones or from hybridisation between oleasters and cultivars (Zohary and Hopf, 1994; Lumaret et al., 2004). Archeological and paleobotanical findings testify human exploitation of oleasters in Spain (4500–4300 BC) before the appearance of olive stones sized as the cultivated ones (Terral et al., 2004; Rodrı´guezAriza and Montes Moya, 2005). In recent years, various studies have focused on the distinction between true oleasters from feral forms and on evaluating the genetic variation of wild olive populations and their relationships with cultivars (Lumaret and Ouazzani, 2001; Besnard et al., 2002; Lumaret et al., 2004; Breton et al., 2006, 2008; Rubio de Casas et al., 2006; Belaj et al., 2007; Besnard et al., 2007). Most of these studies have dealt with wild and/or cultivated populations sampled throughout the Mediterranean, while in other works very restricted areas have been studied (Vargas and Kadereit, 2001; Bronzini de Caraffa et al., 2002; Baldoni et al., 2006; Hannachi et al., 2008). The majority of the above mentioned studies did not have a direct implication on conservation of wild olive genetic resources and their use for breeding purposes. In this sense, the studies performed in Australia (Guerin et al., 2003) represent the unique cases of the use of wild olive material for olive breeding purposes. The present research is part of an ongoing project aimed at prospecting and collecting Spanish wild olive populations, evaluating the genetic variability, ex situ conserving and studying their agronomic performance for future use in olive breeding programs. The first stage of this project consisted in collecting and characterizing wild plant material from the main olive-growing areas (Belaj et al., 2007). In the present study we wanted to go further by comparing the wild olive populations from Andalusia (South Spain), Catalonia and Valencia (Eastern Spain) with local cultivars in order to: 1) evaluate the genetic diversity found in wild and cultivated trees from these three olive-growing regions using SSR markers, 2) investigate the level of genetic differentiation and relationships between wild olives and local cultivars and 3) analyse the population genetic structure of wild and cultivated olives. The results of this study will provide us useful information for olive breeding programs as well as to devise sampling strategies for future collection of olive germplasm. 2. Materials and methods

Table 1 Size ranges, number of alleles (Na), observed (HO) and expected heterozygosity (HE) and Polymorphic Information Content (PIC) for eight microsatellite loci used in 158 olive accessions. Locus

Size range

Na

HO

HE

PIC

DCA3 DCA9 DCA16 DCA18 EMO-03 UDO99-019 UDO99-039 UDO99-043

227–255 160–213 122–222 158–182 203–218 98–161 139–190 165–220

11 23 32 12 10 6 16 22

0.78 0.94 0.95 0.92 0.78 0.30 0.49 0.76

0.75 0.92 0.91 0.89 0.82 0.51 0.86 0.91

0.73 0.91 0.91 0.88 0.81 0.47 0.85 0.91

16.5 6 32

0.74 0.30 0.95

0.82 0.51 0.92

0.81 0.47 0.91

Mean Min Max

The regions of Andalusia, Catalonia and Valencia are distinguished by different agronomical landscapes; the former being characterized by the most intensive and extended area of olive cultivation in the world and also a place where large areas of oleaster forests are present; in contrast, in the regions of Catalonia and Valencia, olive cultivation is less intensive, wild olive forests are less extensive and the majority of wild trees occur near cultivated areas. Most of wild trees in Catalonia and Valencia were sampled near cultivated areas (around 3–4 km) and only a few isolated trees were found at very high altitudes and near pine forests. Most of the wild olives collected in Catalonia were around 10 years old. 2.1.2. Cultivars Fifty-one cultivars from the three olive-growing regions under study were included in the analysis; their number ranged from four to 16 per each province. In Andalusia, the cultivated plants originated from the provinces of Cadiz, Cordoba, Jaen and Seville, whereas for the two Eastern regions, cultivars growing in Valencia and Catalonia were included. These cultivars are referred as main, local and typical (Barranco and Rallo, 2000) for these provinces and are rarely found elsewhere. All plant samples were derived from the World Germplasm Bank of IFAPA (The Andalusian Institute of Agricultural and Fishery Research) at the Centre ‘‘Alameda del Obispo’’ in Cordoba, which represents the main repository of olive genotypes in Spain and have been previously identified by means of various descriptors (Belaj et al., 2001, 2003). Considering their clonal origin (Baldoni et al., 2006) only one sample for each cultivar was analysed.

2.1. Plant material A total of 158 samples of wild (107) and cultivated (51) olive trees from Andalusian, Catalonian and Valencian olive-growing regions in Spain were studied by means of eight microsatellite loci (Table 1 and Fig. 1). The sample size ranged from 14 to 16 for the wild populations and from four to 15 per each province for the cultivated material (Table 2). 2.1.1. Wild olives In Andalusia, wild olive populations were sampled in the provinces of Cadiz, Cordoba, Jaen and Seville. In Catalonia two wild populations were collected in the province of Tarragona (Tarragona 1 and Tarragona 2), where most wild olives occur (J. Tous, personal communication); Tarragona 1 includes individuals sampled in Baix Ebre-Montsia (southern Tarragona) and Tarragona 2 samples were collected in Priorat (northern Tarragona). The two sampling sites in Catalonia were situated close to each other but they represent different edaphoclimatic zones (J. Tous, personal communication). In Valencia region, sampling of wild olives was performed in Valencia province.

Fig. 1. Sampling sites of wild olive populations and cultivation areas of the cultivars analysed.

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Table 2 Genetic diversity of wild olive populations (W) and groups of local cultivars (C) representing different provinces in three Spanish olive-growing regions (Andalusia—A, Catalonia—Ct and Valencia—V). Status

Region

Population/ province

No.

Cultivar names

Nav

Nar

W W W W W W W

A A A A V Ct Ct

Cadiz Seville Cordoba Jaen Valencia Tarragona 1 Tarragona 2

16 15 14 15 16 16 15

– – – – – – –

9.75 9.12 9.00 9.75 9.00 9.25 6.75

4.01 4.03 4.28 4.19 4.06 3.84 3.41

C

A

Cadiz

4.00

C

A

Seville

C

A

Cordoba

7

C

A

Jaen

6

C

V

Valencia

7

C

Ct

Catalonia

Amargoso; Chesna; Palomino; Verdial de Ca´diz ˜ ivano Negro; Manzanilla Can del Piquito; Buidiego; Dulzal de Carmona; Morona; Rapasayo; Gatuno; Pavo; Pico Limon; Lechı´n de Sevilla; Manzanilla de Sevilla; Gordal Sevillana ˜ o; Imperial; Ocal; Henden Manzanilla Prieta; Nevadillo Negro; ˜ o de la Sierra; Carrasquen Hojiblanca Limoncillo; Jabaluna; Manzanilla de Jaen; Verdala; Royal de Cazorla; Picual Villalonga; Alfafara; Changlot Real; Vera; Genovesa; Sollana; Callosina Canetera; Figueretes; Patronet; Vallesa; Llumeta; Menya; Corbella; Farga; Rojal; Verdiell; Blanqueta; Arbequina; Morrut; Sevillenca; Empeltre

4 12

15

W C P

HO

HE

4 6 1 2 3 4 1

0.73 0.62 0.75 0.71 0.73 0.79 0.62

0.76 0.77 0.82 0.80 0.78 0.76 0.68

3.46

1

0.88

0.62

4.62

3.20

1

0.80

0.65

3.62

3.00

0

0.82

0.61

3.50

2.89

0

0.81

0.57

5.25

3.54

0

0.79

0.65

7.50

3.84

1

0.78

0.76

8.95 4.75

3.97 3.32

57 3

0.71 0.80

0.79 0.70

0.01

0.01

0.01

Npa

Nav: Average number of alleles per locus; Nar: Allelic richness; Npa: Number of private alleles; observed (HO) and expected heterozygosity (HE); P: P-value of the tests for differences between groups of wild and cultivated populations for Nar, HO, and HE.

2.2. PCR reactions and SSR analysis Eight microsatellite primers, DCA3, DCA9, DCA16, DCA18 (Sefc et al., 2000), EMO-03 (De la Rosa et al., 2002), UDO99-019, UDO99039 and UDO99-043 (Cipriani et al., 2002) were used for the analysis (Table 1). Five of these loci are included in the list of best SSR markers for cultivar fingerprinting (Baldoni et al., 2009) while the other three were selected for their high polymorphism and discrimination capacity in other studies (Sefc et al., 2000; Cipriani et al., 2002; De la Rosa et al., 2002) and also showing high levels of genetic diversity parameters in cultivated and wild material (Sarri et al., 2006; Belaj et al., 2007). Total DNA was extracted from mature leaves collected from the upper part of trees following the protocol described by De la Rosa et al. (2002). The amplifications were carried out in 20 ml volumes, containing 2 ng genomic DNA, 1 supplied PCR buffer (Biotools, Spain), 200 mM of each dNTP (Roche), 0.25 U of Taq DNA polymerase (Biotools, Spain) and 0.2 mM of forward (fluorescently labelled) and reverse primers. The PCRs were carried out in a thermal cycler GeneAmp PCR system 9600 (Applied Biosystems) with an initial denaturation at 94 8C for 5 min, followed by 35 cycles of 94 8C for 20 s, the annealing temperature 50 8C for 30 s and 72 8C for 30 s, and final extension at 72 8C for 7 min. PCR products were separated using an automatic capillary sequencer (ABI 3130 Genetic Analyzer Applied Biosystems/HITACHI) at the Unit of Genomics of the Central Service for Research Support of the University of Co´rdoba (Spain). The software Genescan version 3.7

and Genotyper 3.7 from Applied Biosystems were used for sample analysis. Three reference samples were used in all runs. 2.2.1. Data analysis PowerMarker V3.23 (Liu, 2002) software package was used to calculate genetic diversity parameters (the average number of alleles per locus, Na, the observed heterozygosity HO and the expected heterozygosity or gene diversity, HE) per microsatellite locus and per population. Polymorphism Information Content (PIC) per each locus was also calculated (Botstein et al., 1980). The allelic richness (Nar), as the measure of allele number per locus independent of sample size, was calculated by the FSTAT v. 2.9.3.2 program package (Goudet, 1995, 2002); whereas the number of private alleles (Npr) was assessed by MICROSAT (Minch et al., 1997). FSTAT was also used to test the significance of differences in average values of Nar, HO, and HE between the groups of populations classified according to their status (wild vs. cultivated) (10000 permutations, two-sided test of the null hypothesis of no difference between groups). The proportion-of-shared-alleles distance (Dpsa; Bowcock et al., 1994) between pairs of individuals was calculated using MICROSAT (Minch et al., 1997). The distance matrix was subjected to the analysis of molecular variance (AMOVA) approach (Excoffier et al., 1992) using ARLEQUIN version 2.000 (Schneider et al., 2000). The analysis of molecular variance (AMOVA) was performed to examine the partition of microsatellite diversity (1) between groups of populations/groups of local cultivars pooled according to

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their status (wild vs. cultivated), among populations/groups of local cultivars within groups and within populations/groups of cultivars considering all data, as well as (2) considering Southern populations/groups of local cultivars only (Andalusia), and (3) Eastern populations/groups of local cultivars (Valencia and Catalonia). Pairwise comparisons between wild olive populations and local cultivars examined with AMOVA resulted in values of fST that were equivalent to the proportion of the total variance that is partitioned between two populations/groups. This approach rather than the calculation of Wright’s F-statistics has been applied because a group of local cultivars cannot be considered as a population. The significance of f-statistics was obtained nonparametrically by 106 permutations. In order to graphically represent genetic relationships among individual olive trees, a Factorial Correspondence Analysis (FCA) was carried out using Genetix 4.05 (Belkhir et al., 2004). A model-based clustering method was applied to multilocus microsatellite data to infer genetic structure and to define the number of clusters (gene pools) in the dataset using the Structure 2.1 software (Pritchard et al., 2000; Falush et al., 2003). Given a value for the number of clusters, this method assigns individual genotypes from the entire sample to clusters in a way in which linkage disequilibrium (LD) is maximally explained. Ten runs of structure were performed by setting the number of clusters (K) from 1 to 14 (one more than the number of sampled populations). Each run consisted of a burn-in period of 200,000 steps followed by 106 MCMC (Monte Carlo Markov Chain) replicates, assuming an admixture model and correlated allele frequencies. No prior information was used to define the clusters. The choice of the most likely number of clusters (K) was carried out by calculating an ad hoc statistic DK based on the rate of change in the log probability of data between successive K values, as described by Evanno et al. (2005).

Fig. 2. Factorial Correspondence Analysis (FCA) of 158 olive trees. Each individual genotype is indicated by coloured symbols while the ones outlined in black represent population centroids.

Tarragona 1 (1.8% alleles each). Wild olives possess all but three alleles detected exclusively in the olive cultivars. Contrasting patterns of HO and HE values were observed between wild and cultivated olives. On average, the groups of local cultivars showed significantly higher levels of observed heterozygosity than did the wild populations (P < 0.01); whereas the HE values were greater in wild populations than in groups of local cultivars (P < 0.01).

3. Results 3.1. Genetic diversity estimates in wild and cultivated olives

3.2. Genetic differentiation and relationships between wild and cultivated olives

A total of 132 alleles were found across the eight markers, the number of alleles per locus ranging from 6 (UDO-19) to 32 (DCA16), with a mean value of 16.5 alleles per locus (Table 1). Overall HO values per marker ranged from 0.30 to 0.95, with a mean value of 0.74, which was lower than the HE values (0.51–0.92 and a mean value of 0.82). All microsatellite loci displayed high values of PIC (from 0.47 to 0.91), permitting the identification of all the individuals analysed. The allelic richness (Nar, Table 2) was significantly higher in wild than in cultivated olives (3.974 vs. 3.324; P < 0.01). From the 132 alleles found, 57 were exclusively detected in wild olive populations, being the Andalusian ones those showing the highest number of specific alleles (Table 2). The wild populations with most private alleles were Seville (2.8%), followed by Ca´diz and

Fig. 2 represents the genetic relationships among individual olive trees defined by the first two axes of the FCA, which accounted for 27.3% and 19.3% of the total inertia, respectively. Along the first axis wild individuals from Andalusia (South) plotted separately from cultivars (either from Southern or Eastern Spain), as well as from the majority of wild individuals from Valencia and Catalonia (East). Cultivated and wild olives from Catalonia clustered separately from Andalusian and the majority of Valencian cultivars along the second axis, while wild olives from Valencia were the most scattered in the middle position. The AMOVA analysis (Table 3) showed that most of total genetic

Table 3 AMOVA analysis for the partitioning of microsatellite diversity: (1) between groups of populations/groups of local cultivars pooled according to their status (wild vs. cultivated), among populations/groups of local cultivars within groups and within populations/groups of cultivars considering all data, as well as (2) considering Southern populations/groups of local cultivars only (Andalusia), and (3) Eastern populations/groups of local cultivars (Valencia and Catalonia). Analysis

Source of variation

df

% Total variance

f-Statistics

P(f)

(1) All data

Between groups (wild vs. cultivated) Among populations/within groups Within populations

1 11 145

0.03 0.03 0.33

8.08 7.85 84.07

fCT = 0.08 fSC = 0.09 fST = 0.16

<0.001 <0.001 <0.001

(2) Southern populations/ groups of local cultivars (Andalusia)

Between groups (wild vs. cultivated) Among populations/within groups Within populations

1 6 81

0.08 0.01 0.33

18.55 1.86 79.60

fCT = 0.21 fSC = 0.02 fST = 0.19

<0.001 0.001 <0.001

(3) Eastern populations/ groups of local cultivars (Valencia and Catalonia)

Between groups (wild vs. cultivated) Among populations/within groups Within populations

1 3 64

0.01 0.04 0.34

2.17 9.34 92.83

fCT = 0.02 fSC = 0.09 fST = 0.07

0.800 <0.001 <0.001

Variance components

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327

Table 4 Pairwise proportion of shared allele distance (upper diagonal) and AMOVA’s fST values (lower diagonal) among the olive populations.

1 2 3 4 5 6 7 8 9 10 11 12 13

Status

Region

Province

1

W W W W W W W C C C C C C

A A A A V Ct Ct A A A A V Ct

Cadiz Seville Cordoba Jaen Valencia Tarragona 1 Tarragona 2 Cadiz Seville Cordoba Jaen Valencia Catalonia

0.011ns 0.023ns 0.037ns 0.074** 0.141** 0.168** 0.218** 0.247** 0.247** 0.271** 0.170** 0.167**

2

3

0.373

0.448 0.402

ns

0.022 0.030ns 0.061* 0.117** 0.175** 0.196** 0.236** 0.233** 0.254** 0.151** 0.145**

0.004ns 0.016ns 0.085** 0.124** 0.116* 0.151** 0.164** 0.188** 0.091* 0.106**

4

5

6

7

0.422 0.397 0.361

0.485 0.439 0.356 0.386

0.552 0.497 0.489 0.485 0.436

0.617 0.647 0.546 0.570 0.460 0.494

0.025ns 0.113** 0.137** 0.142* 0.165** 0.175** 0.154** 0.099* 0.130**

0.084** 0.092** 0.059ns 0.106** 0.114** 0.128** 0.020ns 0.063*

0.132** 0.165** 0.225** 0.235** 0.241** 0.141** 0.044ns

0.152* 0.237** 0.234** 0.260** 0.128* 0.051ns

8 0.768 0.730 0.603 0.625 0.517 0.617 0.528 0.036ns 0.086ns 0.120ns 0.046ns 0.063ns

9

10

11

12

13

0.728 0.688 0.551 0.552 0.446 0.604 0.597 0.238

0.736 0.717 0.609 0.611 0.504 0.643 0.557 0.321 0.263

0.754 0.720 0.613 0.575 0.490 0.626 0.632 0.359 0.281 0.301

0.664 0.645 0.542 0.571 0.391 0.573 0.522 0.307 0.315 0.336 0.386

0.619 0.568 0.510 0.557 0.393 0.374 0.372 0.447 0.505 0.504 0.555 0.427

0.077ns 0.064ns 0.042ns 0.165**

0.090ns 0.073ns 0.158**

0.111ns 0.208**

0.052ns

6

Status—W (Wild); C (Cultivated); Region—A (Andalusia); Ct (Catalonia); V (Valencian); Pairwise significance of fST values after 10 permutations. ‘**’ Significance at the 0.1% probability level; ‘*’ Significance at the 1% probability level, and ‘ns’ Not significant.

diversity was attributable to differences between individuals within populations/groups of local cultivars (84.07%). However, the highly significant f-values between groups according to their status (wild olives and cultivars), as well as among populations/ groups of local cultivars within each group, suggested the existence of genotypic differentiation. When the geographical criterion was used, highly significant differences between wild populations and groups of local cultivars were obtained in the Southern group (Andalusia). The proportion of diversity attributable to differences between wild and cultivated olives in the Eastern group (Valencia and Catalonia) had negative values (fCT = 0.02; p = 0.80), indicating a complete absence of genetic structure. The majority of tests for pairwise genetic differentiation among populations/groups of local cultivars was significant (Table 4). The comparison among wild populations revealed a strong similarity among Andalusian wild populations. In contrast, the three Eastern wild populations (Valencia, Tarragona 1 and Tarragona 2) were significantly different from each other and from all Andalusian wilds, except for the Valencian population, which did not significantly differ from the populations of Cordoba and Jaen (fST values 0.016 and 0.025, respectively). Regarding the cultivated olives, no significant differences were found among cultivars from the South (Andalusia) grouped by their provinces of origin, as did cultivars from the East (Valencia and Catalonia).

3.3. Genetic structure of wild and cultivated olives Using the model-based approach of STRUCTURE, the estimates of the log-likelihood of the data, conditional on a given number of clusters, ln P(XjK), were obtained for each of the ten independent runs for each K (from K = 1–14; hereby referred as gene pool). The highest DK value (53.55) resulted for K = 3, suggesting that the most likely number of gene pools in the dataset was three. The proportions of membership of each individual in each gene pool were calculated (Fig. 3; Electronic Supplementary Material Table S1). The gene pool A was predominant across all Andalusian wild populations. The typical population representing the gene pool A was Cadiz, with the average proportion of genome assigned to this gene pool at P = 93%. Gene pool B was found mostly in two wild populations as well as in the group of local cultivars from Catalonia, with the average proportion ranging from P = 84% in wild population Tarragona 2, and P = 73.7% in the group of local cultivars from Catalonia, to P = 58%, in wild population Tarragona 1. The third gene pool (C) occurred in cultivated olives from Andalusia, as well as in the group of local cultivars from Valencia. The group of local cultivars from Jaen had the highest average proportion of genome assigned to this gene pool (96.2%); whereas those from Valencia had the lowest one (69.5%). The average admixture of gene pools within sampled populations/groups of cultivars, expressed as the proportion of genome from a given sample assigned to the best-scoring gene pool, PMAX, in

Fig. 3. Structure of cultivated and wild olive populations. Each individual tree is represented by a single vertical line divided into three colours. Each colour represents one gene pool, and the length of the coloured segment shows the individual’s estimated proportion of membership in that gene pool. White lines separate populations that are labelled below the figure.

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the sample was 77.8%, ranging from 41.1% (wild olives from Valencia in gene pool A) to 96.2% (the group of local cultivars from Jaen in gene pool C). The wild population from Valencia was the most admixed one, containing individuals assigned to each of the three gene pools with the proportion of membership higher than 75% (Table S1). As shown in Fig. 3, the estimated proportions of membership for each individual in each of the three gene pools obtained by STRUCTURE revealed that some individuals were assigned to a gene pool different from the population/group of cultivars they originally belong. For example, three wild olives from Jaen had more than 75% of their genome derived from the gene pool C; whereas the group of local cultivars from Valencia belong predominantly to gene pool C. Similarly, two Catalonian olive cultivars, ‘Sevillenca’ and ‘Patronet’, belong to gene pool C (with the proportion of membership of 81.2% and 80.4%, respectively); they were therefore more related to Andalusian cultivars than to the rest of Catalonian ones. 4. Discussion 4.1. SSR variability in wild and cultivated olives In the present study, abundant allelic variation and high genetic diversity values were observed in wild and cultivated olives in the main Spanish olive producing regions. Previous studies have also reported high diversity levels of wild and cultivated trees of Western Mediterranean (Lumaret et al., 2004; Baldoni et al., 2006; Breton et al., 2006; Belaj et al., 2007; Besnard et al., 2007; Hannachi et al., 2008) and have identified the Iberian Peninsula as one of the most diverse regions (Rubio de Casas et al., 2006). The values of observed and expected heterozygosity detected at both, wild populations and cultivated olive groups were in general higher than those previously reported for these two forms, when analysed by means of different molecular markers (Sefc et al., 2000; Cipriani et al., 2002; De la Rosa et al., 2002; Lumaret et al., 2004; Breton et al., 2006; Sarri et al., 2006). Distinct allelic composition and heterozygosity values have been observed between cultivated and wild olives. Higher HE values, in accordance with significantly higher allelic richness and higher number of private alleles, were detected in wild populations in comparison to cultivars. While, in contrast to previous studies (Lumaret et al., 2004), cultivated olives showed significantly higher levels of observed heterozygosity than did the wild populations. These results may indicate a considerable loss of genetic diversity in olive during its domestication given that most traditional cultivars could have resulted from a restricted number of founding individuals (Lumaret et al., 2004; Erre et al., 2009). The continuous crossing of these individuals among themselves, as well as with introduced cultivars followed by selection of genotypes with better agronomic performance, could have led to the increase of observed heterozygosity, than maintained by vegetative propagation (Besnard et al., 2001; Sanz-Corte´s et al., 2001; Belaj et al., 2002; ˜ oz-Dı´ez, 2008; Erre et al., 2009). Mun 4.2. Genetic structure of wild olive populations The occurrence of genuine wild olive populations in a few isolated areas of native Mediterranean forests and the ability to discriminate true oleasters from putative hybrids (feral forms) have been previously reported in olive (Lumaret and Ouazzani, 2001; Lumaret et al., 2004; Baldoni et al., 2006; Breton et al., 2006; Besnard et al., 2007). All analysed parameters have indicated a clear differentiation of Andalusian wild olives from cultivars of this area, supporting the hypothesis that they may represent true oleasters. Among the Andalusian wild populations, that of Cadiz is the one with the

highest genome proportion assigned to the wild gene pool probably due to the geographic isolation of these plants from cultivated trees. Wild olive is a common component of the natural flora of this province, where, in contrast with the rest of Andalusia, the area of olive cultivation is very small. In contrasts to what observed in Andalusia, in Catalonia, the Structure, FCA and AMOVA analyses have evidenced high similarities between wild and cultivated olives, giving space to the hypothesis that they represent feral forms deriving from cultivar seed spreading. The close relationship with cultivated trees, previously used as a criterion to identify the presence of feral forms (Bronzini de Caraffa et al., 2002), the geographic proximity of the majority of wild trees from the cultivated fields and their young age, are striking features of wild populations from Catalonia supporting their feral origin. But it is also possible that climatic and geographical conditions and the traditional use of wild trees as rootstocks for cultivars propagation (J. Tous, personal communication) could have contributed to the deforestation of original wild olive trees in this area. AMOVA analysis has shown a significant difference between the wild populations of Tarragona 1 and Tarragona 2. According to Structure analysis, a high proportion (11 alleles, 31.2%) of the genome of the population Tarragona 1 was shared with the Andalusian wild gene pool and four private alleles were found in the Tarragona 1 population. This may indicate that some trees of Tarragona 1 population may be genuine oleasters sharing a common origin with the Andalusian ones. Previous studies have evidenced a common genetic base of genuine wild populations of Western Mediterranean even over great distances (Baldoni et al., 2006; Belaj et al., 2007). In contrast, the Tarragona 2 population seems to mainly represent the trees that originated directly from segregating seedlings of cultivated clones. The wild population from Valencia was the most admixed one with individuals assigned to three different gene pools. Furthermore, most individuals of this population plotted together and showed no significant differentiation with wild Andalusian populations. These facts could be explained by the interaction of different phenomena: a) part of the individuals of the Valencian population represent genuine wild olives that may belong to the Andalusian lineage, b) some individuals may be feral forms that originated directly from segregating seedlings of local cultivated clones, and c) feral individuals should have derived from the hybridization between local cultivated clones and adjacent true oleasters because they share with the other wild trees several alleles that are completely absent in the cultivated material. Other cases of complex origin of wild genotypes at regional level have been observed in Umbria (Italy) (Baldoni et al., 2006; Belaj et al., 2007) and Corsica (Bronzini de Caraffa et al., 2002). 4.3. Genetic structure and origin of cultivars The Structure and FCA analyses revealed that the majority of Spanish cultivars clearly clustered according to their regional origin. These results are in complete agreement with previous studies (Sanz-Corte´s et al., 2001; Belaj et al., 2003, 2004), as well as in representative samples from the Mediterranean basin (Besnard et al., 2001; Belaj et al., 2002; Dı´az et al., 2006). Significant differentiation between Andalusian and most of the cultivars from Catalonia is indicating an independent and local selection of olive cultivars in the two regions (Belaj et al., 2004), as reported in other studies for other Western Mediterranean cultivars (Bronzini de Caraffa et al., 2002; Lumaret et al., 2004; Breton et al., 2006; Sarri et al., 2006; Besnard et al., 2007). In this sense, the contribution of the local wild forms seems more evident in Catalonia and Valencia. The detection of a certain wild genetic background in the cultivars ‘Empeltre’ (Catalonia) and ‘Vera’ (Valencia) may suggest the former

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use of local wild trees in olive domestication (Erre et al., 2009). At the same time, besides a possible feral origin of the wild populations, the similarity found between wild and cultivated trees of these areas may as well suggest an autochthonous origin of cultivars. The envisaged scenario for the development of olive cultivars in Andalusia seems more complex. This might included an empirical selection of outstanding local wild genotypes as suggested by the common genetic base of Andalusian olive cultivars (Barranco and Rallo, 2000; Belaj et al., 2004) and the fact that the wild olives possess almost all of the alleles found in cultivars. The initial selection could have been followed by various generations of crosses between local wilds and cultivars introduced from abroad, combined further with other exchanges of cultivars and various replanting campaigns, thus contributing to enhance the diversification between wild and cultivated forms. A possible allochthonous introduction of ancestral cultivars and their intensive intercrossing and local selection of welladapted productive progenies, should also be considered. Similar conclusions were envisaged in a study of ancient cultivated and wild ˜ oz-Dı´ez, 2008). Based on the olive trees from Andalusia (Mun dissimilarity found between wild and cultivated plant material (Bronzini de Caraffa et al., 2002; Baldoni et al., 2006), the hypothesis of allochthonous origin of olive cultivars from Sardinia, Sicily and Corsica has been postulated. Finally, the assignment of most cultivars from Valencia and two cultivars from Catalonia (‘Sevillenca’ and ‘Patronet’) into the Andalusian cultivated gene pool, as well as the clustering of two cultivars from Cadiz (‘Amargoso’ and ‘Verdial de Ca´diz’) to the Catalonian gene pool are probably due to the human displacement of olive cultivars to different sites (Belaj et al., 2004). 5. Conclusions Our findings, other than confirming the complexity of olive cultivars origin, allowed us to support different routes of cultivars development in different Spanish areas of cultivation. And the current diversity found in Spanish olive cultivars may be explained by a continuous rearrangement of the genetic base involving local selection and genetic material exchange. The results obtained in Andalusia indicate that wild olive genetic resources of this area represent a clearly differentiated gene pool either from cultivars of the same area and wild olive populations of other regions. In addition, the presence of high morphological and agronomical variability in wild olive populations of this area has also been evidenced (A. Belaj, unpublished data). Wild olives of Andalusia may represent an interesting gene pool for olive breeders, bearing in mind that some important agronomic traits, such as resistance to pathogens and parasites, low plant vigour and adaptation to adverse environments, are rarely found in cultivated germplasm. Previous reports (Colella et al., 2008) have evidenced the potentiality of oleaster populations as a useful source of genetic variability for breeding cultivated olives. However, the use of feral olive populations may also be another alternative for olive breeding (Guerin et al., 2003). Based on the results of this study, future prospecting and collection of wild olive trees, new crosses between wild and cultivated trees as well as the establishment of ex situ wild olive collections will be carried out. Finally, the study of local cultivars has been useful to recognize the high genetic diversity of regional resources for their use in breeding programs and to understand the extent of gene flow between wild and cultivated forms. Acknowledgments The authors are grateful to the Agentes de Medio Ambiente of Junta de Andalusia (Spain) and to J. Tous, J.F. Hermoso, V. Cabus, F. Prats, J. Franch, and L. Sanchez for their help with collecting plant

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material. We thank R. de la Rosa, K.R. Tobutt, B. Sutherland, L. Rallo, and J. Tous for their contribution to the improvement of the manuscript. The present study was partly financed by the INIA project RF 2006-0017-C02. A. Belaj has got a post doctoral INIA contract (Subprograma DOC-INIA).

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