Substantial genetic diversity in cultivated Moroccan olive despite a single major cultivar: a paradoxical situation evidenced by the use of SSR loci
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- Khadari, B., Charafi, J., Moukhli, A. et al. Tree Genetics & Genomes (2008) 4: 213. doi:10.1007/s11295-007-0102-4
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To assess the genetic diversity in Moroccan cultivated olive, Olea europaea L. subsp. europaea, we performed molecular analysis of olive trees sampled in four geographic zones representing all areas of traditional olive culture. The analysis of 215 trees using 15 simple sequence repeat (SSR) loci revealed 105 alleles distributed among 60 SSR profiles. The analysis of chloroplast deoxyribonucleic acid polymorphism for these 60 olive genotypes allowed to identify four chlorotypes: 42 CE1, one CE2, nine COM1 and eight CCK. Among the 60 SSR profiles, 52 corresponded to cultivated olive trees for which neither denomination nor characterisation is available. These local olive genotypes displayed a spatial genetic structuring over the four Moroccan geographic zones (northwest, north centre, Atlas and southwest), as pairwise Fst values ranged from 0.0394 to 0.1383 and varied according to geographic distance. As single alleles detected in local olive were also observed in Moroccan oleaster populations, results suggest that plant material was mainly selected from indigenous populations. The assumption that Picholine marocaine cultivar is a multi-clonal cultivar was not supported by our data because we found a single genotype for 112 olive trees representing 31 to 93% of the olives sampled locally in the 14 different areas. Picholine marocaine and the few other named cultivars do not seem to belong to the same gene pools as the unnamed genotypes cultivated only locally. The situation is paradoxical: a substantial genetic diversity in Moroccan olive germplasm, probably resulting from much local domestication, but a single cultivar is predominant.
KeywordsOlea europaea L.SSR genotypingDNA chloroplast polymorphismSpatial genetic structureLocal olive domestication
Olive, Olea europaea L. subsp. europaea; is a Mediterranean long-lived tree cultivated since ancient times for oil and preserved fruit. Two forms are recognised within this outcrossing species, cultivated olive, var. europaea, which is clonally propagated by cuttings and grafting, and wild olive or oleaster, var. sylvestris, reproduced from seeds. These two forms co-exist in most Mediterranean regions and are fully inter-fertile. Archaeological data suggest that cultivated olive was derived from oleaster through vegetative multiplication of individuals presenting interesting traits such as fruit size and oil content (Zohary and Hopf 2000). Hence, the path from wild to domesticated status may have been very short and may still be occurring. Moreover, as olive is a very-long-lived species, we cannot exclude that individuals and genotypes reaching very far back into the origins of domestication may still be found.
The structuring of genetic diversity in cultivated olive is expected to be complex in Morocco. Indeed, it is cultivated under widely divergent ecological conditions, and the history of olive cultivation in the country is long and intricate. Phylogeographic studies have shown that wild olive pre-existed cultivation in the western Mediterranean and that genetic material of western origin has been incorporated into local cultivars (Besnard et al. 2002). Moreover, the analysis of fossil charcoal shows that wild olive was actively collected and consumed by humans in Spain and southern France during the Neolithic period (6,000–8,000 years b.p.; Terral and Arnold-Simard 1996). Olive domestication in the Western Mediterranean could have occurred contemporarily or only slightly later than in the Near-East, i.e. during the 5,700–5,500 b.p. period (Zohary and Spiegel-Roy 1975). Despite its early occurrence in Morocco, olive cultivation expanded mainly during the Roman period (Lenoir and Akerraz 1984). In addition to olive trees selected locally, cultivars originating from the eastern Mediterranean were introduced successively into Morocco by different civilisations. Molecular data suggest that some western cultivars result from hybridisation between eastern cultivars and local genotypes (Besnard et al. 2002). Today, in Morocco, olive is still cultivated in a number of areas presenting highly contrasted ecological conditions (Loussert and Brousse 1978). Historical aspects and the occurrence of contrasting ecological conditions should have favoured genetic diversification within Moroccan cultivated olive.
Surprisingly, a single cultivar, named Picholine marocaine, predominates in Moroccan olive orchards (Loussert and Brousse 1978). The name ‘Picholine marocaine’ alludes to the French cultivar ‘Picholine du Languedoc,’ which is, however, genetically distinct; this denomination likely originates in the marketing of Moroccan olives in southern France during the 1960s and the 1970s (M. Rozier, personal communication). Only a few other cultivars (Bouchouk, Bouchouika, Fakhfoukha, Hamrani and Meslala) are known, and they are cultivated in restricted areas (Maestratti 1922; Tornézy 1922). Aside these cultivars, cultivated trees displaying atypical traits can also often be observed in orchards. Whether these trees correspond to unnamed cultivars is an open question.
Enzyme polymorphism analysis at eight loci of a large sample originating from traditional olive cultivation areas showed that 172 trees (52.4%) displayed the same multi-loci genotype and belonged to the Picholine marocaine cultivar (Ouazzani et al. 1996). This suggests that the cultivar Picholine marocaine corresponds to a single clone. Nevertheless, in later work by the same authors, it is suggested that Picholine marocaine may be genetically heterogeneous (Ouazzani et al. 2002; Idrissi and Ouazzani 2003; Essadki et al. 2006). Other workers have evidenced phenotypic diversity suggesting that Picholine marocaine could be a mix of closely related clones (Boulouha et al. 1992; Lansari and Tahri Hassani 1996). Alternatively, we could suggest that Picholine marocaine is an old monoclonal cultivar for which somatic mutations may have led to some genotypic and phenotypic diversification.
Recently, several sets of microsatellite (simple sequence repeat [SSR]) loci have been isolated from olive deoxyribonucleic acid (DNA) and identified as efficient tools for olive genetic studies (Sefc et al. 2000; Rallo et al. 2000; Carriero et al. 2002, Cipriani et al. 2002; De La Rosa et al., 2002; Diaz et al. 2006). Compared to previous markers as isozymes, random amplification of polymorphic DNA and amplified fragment length polymorphism, microsatellite markers are more reproducible because they are locus specific and more genetically informative because of their multi-allelic states. However, very few genetic studies using microsatellite markers and a population genetics perspective have been published (Khadari et al. 2003; Baali-Cherif and Besnard 2005; Breton et al. 2006).
To assess the genetic diversity of Moroccan-cultivated olive and to clarify the genetic basis of Picholine marocaine, we performed a molecular analysis of a broad sample of trees originating from the main traditional areas of olive cultivation. We used a large array of olive SSR loci (Sefc et al. 2000; Carriero et al. 2002, Cipriani et al. 2002).
By analysing nuclear DNA SSR and chloroplast DNA polymorphism, our aims were: (1) to assess within-cultivar variation in Picholine marocaine, (2) to characterise all sampled olive genotypes and (3) to study the genetic structure of Moroccan olive germplasm.
Materials and methods
Plant material sampling
Local forms and minor cultivars according to areas and to geographic zones
Area of sampling
Local olivea, b
Number of local oliveb
1, 2, 3 and 4
5, 7 and 9
Fakhfoukha, Bouchouk Laghlid, Bouchouk Rkik
Bouchouk (2), Bouchouika (2), local forms no. 6 (1) and 8 (1)
11, 12, 14, 15 and 16
Local forms no. 6 (3), 10 (1) and 13 (4)
17, 18 and 19
Bouchouk (2), Meslala (3) and local form no. 13 (1)
20, 21 and 22
Bouchouika (3), Meslala (3) and local form no. 8 (1)
Beni Mellal (6)
23, 24, 25
Bouchouika (1) and local form no. 10 (2)
Marrakech Agdal (7)
26 to 38
Local form no. 8 (1)
Marrakech Menara (8)
39 (3)e, 40 (6), 41, 42 (2), 43, 44 and 45
Local forms no. 8 (3) and 10 (2)
Imine Tanounte (10)
51 (2) and 52
We sampled 215 trees from known traditional cultivars (Bouchouk, Bouchouika, Bouchouk Laghlid, Bouchouk Rkik, Hamrani, Fakhfoukha, Meslala), from the denomination Picholine marocaine, also including trees that had a local denomination as well as cultivated forms displaying specific traits but for which the farmers had no denomination. Our sampling strategy was based on basic phenotypic observations and on information from local farmers and technicians. Based on basic morphological descriptions (fruit and leaf size and shape, vigour etc.), we performed sampling with two aims: (1) We sampled plant material from very old trees displaying the Picholine marocaine phenotype to assess the genetic diversity within this cultivar, and (2) we collected samples from all trees displaying distinctive morphological traits to characterise as many as possible other cultivated olive genotypes. On average, approximately half of the samples were Picholine marocaine (Table 1). Furthermore, we analysed the five clones (Haouzia, Menara, K26, M26 and S19) selected at the Moroccan Agronomic Research Institute (Boulouha 1984; Boulouha et al. 1992). Leaves were always collected from tree canopy, even in the case of old trees.
DNA was extracted following Besnard et al. 2000 with modifications. About 1% polyvinylpyrrolidone (PVP 40.000) was added to the 2× cetyl trimethyl ammonium bromide buffer. The DNA solution was purified using the Dneasy Plant Mini Kit (Qiagen).
Selection of informative SSR loci was performed among three sets of SSR primers: 15 DCA (Sefc et al. 2000), 10 GAPU (Carriero et al. 2002) and 30 UDO primer pairs (Cipriani et al. 2002) on a sub-sample of 20 Moroccan olive trees: one Picholine marocaine, five traditional cultivars and one tree displaying a distinctive phenotype from each of the 14 sampling zones.
According to the annealing temperature and to the size range of alleles detected, five combinations of three SSR primer pairs were defined for SSR genotyping using the ABI 3100 sequencer. Amplification reactions were performed in a final volume of 20 μl in the presence of 20 ng template DNA, 4 pmol reverse primer and 1 pmol forward primer, 0.2 mM of each deoxynucleotide, 2 mM MgCl2, and 1 U Taq polymerase (Sigma). The forward primer was 5′ labeled with one of the three fluorophores (6FAM, NED or HEX). Polymerase chain reaction (PCR) was carried out using a PTC 100 thermocycler (MJ Research). After 5 min at 94°, 30 cycles were performed with 30 s at 94°C, 30 s at either 50 or 56°C and 30 s at 72°C, followed by a final extension step of 5 min at 72°C. Amplified products were detected on a ABI prism 3100 Genetic Analyser. Samples were prepared by adding 3 μl diluted PCR products to 6.875 μl formamide and 0.125 μl GenSize HD 400 Rox. Analyses were performed using the GeneScan 3.1 and Genotyper 2.5 softwares (Applied Biosystems).
DNA chloroplast polymorphism
To determine the chlorotype in the olive germplasm, two SSR loci (ccmp5 and ccmp7; Weising and Gardner 1999) and the QR fragment restricted by HaeIII and TaqI (Dumolin-Lapègue et al. 1997) were used according to the procedure described by Besnard et al. 2002. SSR genotyping was performed with the ABI 3100 sequencer as mentioned.
SSR data were scored and verified using Genescan and Genotyper softwares. Genetic differentiation among olive populations and its significance were estimated by computing pairwise Fst and exact tests (Raymond and Rousset 1995a, b).
Genetic relationships between olive genotypes were studied on the basis of a similarity matrix using the proportion of alleles (Nei and Li 1979). A phenogram was drawn based on the unweighted pair group method with arithmetic mean algorithm using the NTSYS-pc program ver. 2.11a (Rohlf 2000). Furthermore, a factorial correspondence analysis was performed using the Genetics 4.0 software (Belkhir 1999).
Three molecular criteria were used to select the 15 SSR loci: (1) clear amplified DNA fragments, (2) non-ambiguous scoring data following easy multiplexing primer pairs for Abi 3100 sequencer genotyping and (3) efficient detection of polymorphism in Moroccan olive (sub-sample of 20 trees) and French germplasm (unpublished data). These SSR loci revealed a total of 105 alleles ranging from two at the UDO 12 locus to 17 at the DCA 17 locus, with a mean of seven alleles per locus. The highest frequencies exceeding 66% were observed for the alleles 213, 89 and 146 at the loci GAPU 71A, UDO34 and UDO44, respectively. Among the 105 alleles, 21 were detected only once at the loci DCA3, DCA5, DCA 17, GAPU 59, GAPU 71A, UDO6, UDO14, UDO34 and UDO36. Repeated PCR amplifications and genotyping using the sequencer ABI 3100 showed complete reproducibility of the scored SSR data and particularly for alleles detected with the lowest frequency.
Number and size range of alleles and observed heterozygosity for each of the 15 SSR loci
Identifying molecular profiles and genotype diversity
Distinct SSR alleles between the standard Picholine marocaine genotype and the closely related genotypes
SSR loci with all detected alleles
141, 143 145, 147 149, 151 153, 155 159, 162
243, 254 263, 266
90, 94 100, 104 120
103, 105 107, 113 115, 121 125, 143 147, 161 165, 169 175, 179 185, 191 195
Picholine marocaine (112)
Local olive 22 (1)
Local olive 8 (6)
Local olive 10 (5)
Local olive 41 (1)
Local olive 23 (1)
Focussing on the three SSR loci distinguishing Picholine marocaine from the closely related SSR profiles by three to six alleles (Table 3) and assuming that no variation occurred in the flanking regions of the microsatellites, the variation between alleles corresponded to one, two and five dinucleotide repeat for the DCA 17, UDO 14 and DCA 15 loci, respectively (Table 3). We assumed that the genotype repeated 112 times corresponded to an original Picholine marocaine type while the highly similar SSR profiles (observed in one to six copies) could correspond to the observation of somatic mutations within Picholine marocaine. According to this assumption, mutated alleles would have been obtained by a gain of one and two dinucleotide repeat units for the DCA 17 and UDO 14 loci, respectively, and by a loss of five dinucleotide repeat units for the DCA 15 locus (Table 3).
Each of the seven following cultivars displayed a specific SSR profile: Bouchouk, Bouchouika, Bouchouk Laghlid, Bouchouk Rkik, Fakhfoukha, Hamrani and Meslala. However, the cultivar pairs Bouchouk/Bouchouk Rkik, Bouchouika/local olive no. 45 and Fakhfoukha/Meslala were differentiated by a single allele (Fig. 3). Hence, very similar genotypes were only observed for a group of genotypes close to Picholine marocaine and for pairs of known cultivars.
Among the 60 SSR profiles, 52 corresponded to cultivated olive trees for which neither denomination nor characterisation are available. Except for four of them, each of these genotypes was only observed within one sampling area. From 1 to 13, such different local cultivated olive genotypes were found per sampling area, and their abundance ranged from 7 to 68% with a mean of 36% (Fig. 1 and Table 1). In contrast, the Picholine marocaine genotype was found in all areas in frequencies ranging from 31 to 93% (Fig. 1). Among the four other widespread olive genotypes, two (local olives no. 8 and 10) clustered with Picholine marocaine (Tables 1 and 3, Fig. 3). Each of the seven minor cultivars was distinguished from Picholine marocaine by 15 to 22 alleles. Except for Hamrani, Bouchouk and Bouchouk Rkik, these cultivars clustered with Picholine marocaine (Fig. 3).
DNA chloroplast polymorphism
Using the two PCR restriction fragment length polymorphism combinations QR/TaqI and QR/HaeIII, and the ccmp5 and ccmp7 microsatellite polymorphisms on the 60 olive SSR profiles, we detected four chlorotypes: 42 CE1, 1 CE2, 9 COM1 and 8 CCK as defined by Besnard et al. 2002. Chlorotypes COM1 and CCK are characteristic of the west Mediterranean and were observed in 17 local olives, whereas Picholine marocaine, the seven minor cultivars and 36 local genotypes displayed the CE1 chlorotype (Fig. 3), which originates from the Eastern Mediterranean.
Genetic diversity within and among geographic zones
We separated the 14 sampling areas into four geographic zones and analysed the genetic composition of specific local olives and minor cultivars, excluding the Picholine marocaine genotype (Fig. 1, Table 1).
Fst pairwise between olive groups
This is the first SSR genetic study on olive germplasm from the southwest Mediterranean area at a fine scale. The SSR loci used in this study were carefully selected among three sets of primer pairs developed on olive (Sefc et al. 2000; Carriero et al. 2002; Cipriani et al. 2002). In addition to molecular criteria, we used the following genetic criteria to check for the quality of our SSR data: (1) most of the alleles were detected in the genotypes analysed, (2) most of the 21 single alleles were also observed in oleaster populations (Khadari, unpublished data) and (3) Mendelian segregation was confirmed in the two crosses, Olivière × Arbequina and Picholine marocaine × Picholine du Languedoc for the alleles at ten loci (unpublished data, Charafi et al., submitted). The number of alleles per locus determined in our study (seven) is slightly lower than the values obtained for Mediterranean cultivar populations (9.7; Breton et al. 2006) and for oleaster populations from south Morocoo and Algeria (9.4; Baali-Cherif and Besnard 2005) indicating that the Moroccan olive displays a substantial genetic diversity despite of the limited germplasm.
Most of the genotypes determined in our study correspond to olive material present in only one sampling area and for which farmers proposed neither denomination nor characterisation (48 genotypes among the 52 determined in this study). Whatever the geographic zone, this olive material was called ‘Zitoun beldi’ by local people, which translates as ‘the common local olive.’ These findings are similar to those by Ouazzani et al. 1996 based on the analysis of 328 trees using enzyme polymorphism at eight loci. More intensive sampling in all traditional olive areas will probably reveal more local genotypes.
These local genotypes of cultivated olive present clear geographic genetic structuring, a feature previous analyses did not allow to evidence (Ouazzani et al. 1996). Pairwise Fst values varied according to geographic distance. For instance, northwest olive and north centre olive were less genetically differentiated (Fst = 0.0394) than northwest and southwest (Fst = 0.1383). Pairwise genetic differentiation for seven Mediterranean oleaster populations assessed by microsatellite markers varied from 0.0686 to 0.186 with a mean of 0.115 (Breton et al. 2006). Compared to these results, the Moroccan olive germplasm is relatively highly structured. Furthermore, local olive genotypes were classified into two clearly defined clusters: a northern cluster and an Atlas and southwestern cluster (Figs. 3 and 4). These results suggest that the locally cultivated olive trees are the result of local selection with strong introgression by local oleaster populations or their local domesticated derivatives. Indeed, the very clear spatial structuring of genotypes observed in Figs. 3 and 4 cannot be explained by cultivar introductions. In agreement, 17 of the 52 local olive genotypes displayed the two chlorotypes COM1 and CCK characteristic of the west Mediterranean chlorotype (Besnard et al. 2002). On the other hand, the other genotypes displayed east Mediterranean chlorotypes, suggesting that the local genotypes resulted from introgressions between the original local gene pool and introduced cultivars. Strikingly, Picholine marocaine and the seven minor cultivars, i.e. all denominated cultivars, shared the CE1 and CE2 cytoplasms, characteristic of the east Mediterranean maternal lineages. Hence, we have no clear data that would allow rejecting the hypothesis that all the named cultivars were introduced from the eastern Mediterranean.
Despite of the use of 15 SSR loci, 112 of the 215 trees sampled from all areas (52%) displayed the same SSR profile. This is very similar to the findings reported by Ouazzani et al. 1996 using eight enzymatic loci, as a single genotype was observed for 52.4% of the 328 olive trees analysed. Obviously, the widely cultivated Picholine marocaine is a genetically very homogeneous entity. Nevertheless, comparatively to enzymatic markers, the use of SSR markers allows finer insights into the nature of the cultivar. As it is highly discriminant, SSR genotyping allows excluding the widespread hypothesis that Picholine marocaine is a mixture of closely related genotypes (Boulouha et al. 1992; Lansari and Tahri Hassani 1996; Ouazzani et al. 2002). There is, however, a small group of genotypes that very closely resemble Picholine marocaine particularly the two local olives no. 8 and 22, which differentiated by only four and three SSR alleles, respectively (Table 3). As the same deviant SSR profiles were obtained when the samples were re-amplified, we can safely conclude that these genotypes do not differ sufficiently to result from crosses between Picholine marocaine and another genotype or even from Picholine marocaine selfing. Because the early multiplication of the Picholine marocaine genotype may date back to the roman period, there may have been sufficient time for somatic mutations to arise. Assuming that no variation occurred in flanking sequences of microsatellites, we considered that Local olive no. 22 and no. 8 derived from Picholine marocaine genotype by a gain of one and two dinucleotide repeat units for the DCA 17 and UDO 14 loci, respectively, and by a loss of five dinucleotide repeat units for the DCA 15 locus (Table 3). Because we have neither analysed the sequence of the flanking regions of the microsatellites nor defined precisely phenotypic variation among these presumed clones, we can only suggest that these genotypes derive from Picholine marocaine by somatic mutations. Similarly, it is very obvious in Fig. 3 that beyond the case of Picholine marocaine, only genotypes that have a cultivar denomination can be very similar to another genotype. We suggest also that in each of these cases, the two genotypes belong to a single clone and have diverged through somatic mutation. Moreover, these genotypes, in the same way as Picholine marocaine, are apparently differentiated from the basic groups constituted by the local unnamed cultivated genotypes. These results point to the need of additional research to validate the somatic mutation hypothesis as the origin of the genotypes closely related to Picholine marocaine.
Hence, we get a global image of (1) a large availability of genotypes of local origin, but (2) only very few genotypes reach the cultivar status, (3) these cultivars may be sufficiently old to have accumulated detectable somatic mutations and (4) it is not yet solved whether these cultivars are of local origin or were introduced from other countries.
Indeed, Picholine marocaine is not restricted to Morocco. It is known as Sigoise in Western Algeria and as Canivano Blanco in the South of Spain (Besnard et al. 2001). Closer investigation of the distribution of somatic mutations may help infer the origin of this cultivar. A striking feature of olive cultivation in Morocco is the prevalence of this single cultivar because other olive-growing countries grow several main cultivars (Loussert and Brousse 1978). For instance, in Tunisia, Chemlali, Chetoui and Meski are the major cultivars from south to north (Trigui and Msallem 1995). In Spain, 17 main cultivars are grown depending on the region (Barranco 1995). This observation should be associated with the observation that no named cultivar could be linked to the local gene pool. We may wonder whether this should be attributed to historical factors such as a very rapid expansion of commercial olive cultivation in Morocco under Roman times or whether genotypes belonging to the local gene pool lacked some appreciated agronomic traits.
We whish to thank Drs. B. Boulouha and N. Ouazzani who provided some olive samples for the study. All molecular genotyping was performed in the laboratory: “Atelier de marquage moléculaire,” UMR DGPC, which has a convention with the “Conservatoire Botanique National Méditerranéen de Porquerolles” for fruit genetic resources study. We are grateful to S. Santoni and D. Bru for their support in SSR genotyping. During the whole study, the encouragement and help of Dr. F. Boillot and Prof. F. Dosba were decisive. Comments on an earlier version of the manuscript from J.I. Hormaza and F. Kjellberg and on the revised one from Ph. Chatelet were greatly appreciated.