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Scientia Horticulturae 152 (2013) 61–69

Contents lists available at SciVerse ScienceDirect

Scientia Horticulturae

journa l h o me page: www.elsev ier .com/ locate /sc ihor t i

enetic relationships between local North African apricot (Prunus armeniaca L.)ermplasm and recently introduced varieties

edia Bourguibaa,b,d,∗, Bouchaib Khadarib,c, Lamia Krichena, Neila Trifi-Faraha, Ali Mamounie,amia Trabelsi f, Jean-Marc Audergond

Faculté des Sciences de Tunis, Laboratoire de Génétique Moléculaire, Immunologie et Biotechnologie, Campus Universitaire, 2092 El Manar, TunisiaINRA, UMR 1334 Amélioration Génétique et Adaptation des Plantes (AGAP), F-34398 Montpellier, FranceCBNMED, UMR 1334 AGAP, F-34398 Montpellier, FranceINRA Centre PACA – UR1052 GAFL, Domaine St Maurice, BP94, 84143 Montfavet Cedex, FranceINRA, UR Amélioration des Plantes et Conservation des Ressources Phytogénétiques, Meknès, MoroccoUniversité de Blida, Chaire d’arboriculture, Blida, Algeria

r t i c l e i n f o

rticle history:eceived 13 October 2012eceived in revised form 2 January 2013ccepted 22 January 2013

eywords:pricotayesian analysisene flow

a b s t r a c t

Graft and seed propagated apricots in North Africa (Algeria, Morocco and Tunisia) are identified asbeing from the southwestern Mediterranean gene pool. However, recently introduced varieties fromnorthern Mediterranean areas are also encountered in this region. Do gene exchanges occur betweenlocal apricot and introduced varieties? We addressed this question by analyzing the genetic structure of183 accessions using 24 nuclear microsatellite markers and a model-based Bayesian clustering method.We classified these accessions into 4 genetic clusters: cluster 1 from Morocco, cluster 2 from northern,central and southern Tunisia which were all propagated by grafting, cluster 3 from the oasis region inTunisia and the Messaad region in Algeria, and finally, cluster 4 (green) included accessions belonging

enetic structureicrosatellites

to the northern Mediterranean gene pool. These accessions were more diversified and geneticallydifferentiated with respect to local North African apricot. The results revealed the presence of admixedaccessions between the local gene pool and introduced varieties, mainly in seed propagated populations,thus highlighting the presence of gene exchanges between northern and southern Mediterraneancountries. Our findings should be useful for improving the management and conservation of Maghrebian

.

apricot genetic resources

. Introduction

Apricot (Prunus armeniaca L.) is one of the most importantruit crops in the Mediterranean Basin, a major apricot produc-ion region in the world (over 50% of global production; FAOSTAT,010). Apricot was probably initially domesticated in China, whereild apricots are found (Bailey and Hough, 1975). The first domes-

icated forms would then have been disseminated through centralsia and the Irano-Caucasian area (Vavilov, 1992). This latter is con-idered as a secondary center of apricot diversification because ofts presumed intermediate geographic position between the mainreas where domesticated apricots and their wild relatives are

rown (Faust et al., 1998). Several works based on molecular dataupported that most common European genotypes originated in

∗ Corresponding author at: Faculté des Sciences de Tunis, Laboratoire de Géné-ique Moléculaire, Immunologie et Biotechnologie, Campus Universitaire El Manar,092 El Manar, Tunisia. Tel.: +216 70 860 432; fax: +216 71 872 600.

E-mail address: hediabourguiba@hotmail.com (H. Bourguiba).

304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.scienta.2013.01.012

© 2013 Elsevier B.V. All rights reserved.

Central Asia, reached Europe through the Caucasian and spreadover a large area (Zhebentyayeva et al., 2003; Pedryc et al., 2009).

A recent study on apricot domestication and disseminationwithin the Mediterranean Basin showed that in this region apri-cot is structured within three main gene pools, correspondingto the Irano-Caucasian, northern Mediterranean and southernMediterranean areas (Bourguiba et al., 2012a). It has also beendemonstrated that starting from the Irano-Caucasian area, apricotspread to the Mediterranean Basin through two main dissem-ination routes: the first one through countries north of theMediterranean Sea and the second one through North Africancountries (Bourguiba et al., 2012a).

The Maghreb region (Algeria, Morocco and Tunisia) in NorthAfrica is characterized by original apricot genetic resources,with the coexistence of traditional local varieties propagated bygrafting and populations specific to oasis agroecosystems that arepropagated by seeds. Apricot landraces in the Maghreb region

are currently under severe genetic erosion as a consequenceof the urbanization and farmers’ switch from apricot to moreeconomically profitable crops. For instance, Krichen et al. (2009)reported that in Tunisia, only 19 apricot varieties have been

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ecovered among the 47 previously identified by Valdeyron androssa-Raynaud (1950). Hence, assessment of apricot geneticiversity in North Africa is crucial for the management of apricotenetic resources in order to improve protection and conser-ation programs. Recently, Bourguiba et al. (2012b) revealedhat in the Maghreb region apricot is clustered according tooth their country of origin and mode of propagation. Graft andeed-propagated apricots share a common gene pool originatingrom the Irano-Caucasian area, as reported by Bourguiba et al.2012a). The presence of southern European accessions in the

aghreb region was evidenced in different studies using bothsozymes and molecular markers (Badenes et al., 1996; Hagent al., 2002). However, no significant gene exchanges have beenevealed between northern and southern Mediterranean countriesBourguiba et al., 2012a). We still have no clear view on the geneticelationships between local apricot germplasm in North Africa andntroduced varieties from the ‘North Mediterranean’ gene pool.

Here, we investigated gene exchanges between the local apri-ot gene pool in North Africa and varieties recently introduced fromhe ‘North Mediterranean’ gene pool. For this purpose, we aimedo: (i) test for the presence of an underlying genetic structure in

sample of 183 North African apricot accessions using a set of4 nuclear SSR markers dispersed over the Prunus genome and aodel-based Bayesian clustering method; (ii) quantify the neu-

ral genetic diversity and differentiation available among identifiedenetic clusters; and (iii) examine the relationships between localpricot germplasm and introduced varieties.

. Materials and methods

.1. Plant material

North African apricot accessions consisting of 80 accessionsrom Tunisia, 69 from Morocco and 34 from Algeria were ana-yzed. Except for 7 Moroccan accessions collected from an exitu germplasm collection maintained at the National Institute ofgronomic Research of Meknes (Morocco), all the studied apricotccessions were collected in situ from the main apricot croppingreas in each country, with: (i) Messaad in Algeria; (ii) Dadès Val-ey, Drâa Valley, Moulouya Valley and Ziz Valley in Morocco; (iii)orth (Ras Jbel and Testour), Centre (Kairouan, Mahdia and Sfax),outh (Gabes and Jerba) and the Oasis (Gafsa, Midess, Tameghza,egache, Nefta and Tozeur) in Tunisia.

Apricots from the northern, central and southern Tunisia, as wells the collection at INRAM Meknes in Morocco, were propagatedy grafting, while all the remaining accessions were propagated byeeds (Table 1).

.2. DNA extraction and genotyping procedure

Genomic DNA extraction, selection of the 24 microsatellite locised for genotyping as well as the PCR amplifications were asescribed by Bourguiba et al. (2012b).

.3. Genetic diversity analysis

Genetic diversity parameters for each SSR locus were calculateds the total number of alleles, the number of rarest alleles (fre-uency less than 5%) and the observed (Ho) and expected (He) het-rozygosities using GENETIX 4.05 (Belkhir et al., 2004). The numberf genotypes was computed using the PowerMarker program (Liund Muse, 2005). FSTAT 2.9.3 software (Goudet, 2001) was used to

ompute the allelic richness corresponding to the estimated num-er of alleles standardized to the smallest number of genotypesccording to El Mousadik and Petit (1996). Wright’s F-statisticsere estimated according to the formula of Weir and Cockerham

culturae 152 (2013) 61–69

(1984) using the GENEPOP 4.0 program (Raymond and Rousset,1995a). The significance of pairwise Fst values was assessed byFisher’s exact probability test (Raymond and Rousset, 1995b). Fac-torial correspondence analysis (FCA) was performed with GENETIXto obtain a synthetic representation of the genetic variability of thestudied accessions according to their genetic origin.

2.4. Model-based Bayesian clustering analysis

To infer the genetic structure of the apricot material from NorthAfrica, a Bayesian model-based clustering method implementedin the STRUCTURE 2.2 program (Pritchard et al., 2000) based onSSR data was used. The analysis was carried out without priorinformation concerning the geographic origin of the accessions.STRUCTURE was run using a model with admixture and correlatedallele frequencies, with the assumed number of genetic clusters (K)ranging from 1 to 10, with 10 independent replicate runs for each Kvalue. Each run involved a burning period of 100 000 iterations, anda post-burning simulation length of 1 000 000. The run showing thehighest posterior probability of data was considered to assign themost probable number of clusters K. Statistical parameters definedby Evanno et al. (2005) based on the rate of change in the log proba-bility of data between successive K values were used to confirm theexact estimation of the most likely number of clusters K. Q-matrixvalues for individual runs for each K were analyzed by the CLUMPP1.1 program (Jakobsson and Rosenberg, 2007). Graphical represen-tation of clustering results was performed with DISTRUCT software(Rosenberg, 2004).

3. Results

3.1. SSR polymorphism

The analysis of the 183 apricot accessions using 24 SSR locirevealed 191 alleles (Table 2). The number of alleles detected perlocus ranged from 2 (BPPCT001) to 16 (UDP98-409) with an aver-age of 7.95 alleles (Table 2). There were 108 alleles (56.54%) withfrequencies equal to or less than 5%, which were considered as rarealleles (Table 2). The expected heterozygosity (He) at individualloci ranged from 0.144 (AMPA119) to 0.870 (UDP98-409), with amean of 0.593, revealing high polymorphism among the studiedapricot accessions. The observed heterozygosity (Ho) ranged from0.120 (AMPA109) to 0.710 (UDP98-409), with an average of 0.504(Table 2).

3.2. Model-based Bayesian clustering analysis

Using model-based Bayesian clustering, the estimated log prob-ability of the data (ln Pr(X|K)), given the assumed number ofancestral populations K, was highest at K = 4 to K = 6. However, anad hoc quantity based on the second order rate of change of the like-lihood function with respect to K (�K), as defined by Evanno et al.(2005), showed that the best model is at K = 2 (Fig. 1). Taking thehighest similarity coefficient H′ and the statistics of Evanno et al.(2005) into account, the models at K = 2 and K = 4 were consideredthe best to depict the genetic structure of apricot in North Africa.

A first overview at K = 2 indicated a clear separation betweengrafted (red) and seed (blue) propagated accessions (Fig. 1). Accord-ing to the model at K = 4, apricot genotypes were assigned tofour genetically different clusters identified by STRUCTURE anal-ysis: cluster 1 (red) included accessions from Morocco, cluster 2

(blue) consisted of apricots originating from northern, central andsouthern Tunisia which were all propagated by grafting, cluster 3(yellow) grouped accessions from the oasis region of Tunisia andthe Messaad region in Algeria, and finally cluster 4 (green) included

H. Bourguiba et al. / Scientia Horticulturae 152 (2013) 61–69 63

Table 1List of apricot accessions and their assignment to the clusters identified in this study.

Accession name Accession code Geographical site Geographical region Country of origin Clusterb

Canino Al.04 Messaad Messaad Algeria 4Louzi Rouge Al.05 Messaad Messaad Algeria 4Rosé de corail Al.07 Messaad Messaad Algeria 4Rosé de Ménaa Al.08 Messaad Messaad Algeria NAPaviot rouge Al.09 Messaad Messaad Algeria 4Boulida Al.11 Messaad Messaad Algeria 4Rouge du Roussillon Al.12 Messaad Messaad Algeria NAMechmech hlou Al.14 Messaad Messaad Algeria NAMechmech laghdech Al.16 Messaad Messaad Algeria NANail Al.17 Messaad Messaad Algeria 3Abyad el imlak Al.18 Messaad Messaad Algeria 3Laouzi greffé Al.19 Messaad Messaad Algeria 4Pêcher rouge Al.21 Messaad Messaad Algeria 3Arbi V1 Al.22 Messaad Messaad Algeria 3Arbia nadir Al.23 Messaad Messaad Algeria 3Mahalat el djoundi Al.24 Messaad Messaad Algeria 3Louzia greffé Al.25 Messaad Messaad Algeria 4Arbi kd Al.26 Messaad Messaad Algeria 3Mouzemèche Al.28 Messaad Messaad Algeria NAKahf Al.29 Messaad Messaad Algeria NAMessaad greffé Al.30 Messaad Messaad Algeria NALouizi rouge Al.31 Messaad Messaad Algeria 3Arbi nadir Al.32 Messaad Messaad Algeria 3Saafi arbi Al.33 Messaad Messaad Algeria NAEl Maghreb Al.34 Messaad Messaad Algeria 3Hamrai Al.35 Messaad Messaad Algeria NAMoutaakhir Al.36 Messaad Messaad Algeria NAEl Bakria Al.39 Messaad Messaad Algeria 3Douk el kamel Al.40 Messaad Messaad Algeria 3Hamrai greffé Al.43 Messaad Messaad Algeria 3Chems el massa Al.45 Messaad Messaad Algeria 3Naila Al.46 Messaad Messaad Algeria 3Saïb ennahdha Al.47 Messaad Messaad Algeria 3Ikhtiyar ettayeb Al.48 Messaad Messaad Algeria NABoumalen J3 L1V6 Boumalen Dadès Morocco 1Kalaat Meggouna G6 L2V6 Kalaat Meggouna Dadès Morocco 4Kalaat Meggouna G5 L3V7 Kalaat Meggouna Dadès Morocco NABoumalen A4 L3V9 Boumalen Dadès Morocco 1Boumalen A2 L5V11 Boumalen Dadès Morocco 1Boumalen A1 L6V10 Boumalen Dadès Morocco 1Kalaat Meggouna G8 L6V9 Kalaat Meggouna Dadès Morocco NAKalaat Meggouna G1 L1V10 Kalaat Meggouna Dadès Morocco 1Kalaat Meggouna G2 L1V11 Kalaat Meggouna Dadès Morocco 3Boumalen A3 L2V8 Boumalen Dadès Morocco 1Kalaat Meggouna G7 L4V9 Kalaat Meggouna Dadès Morocco NABoumalen KH1 L7V6 Boumalen Dadès Morocco 1Kalaat Meggouna G9 L8V8 Kalaat Meggouna Dadès Morocco 1Boumalen J2 L8V9 Boumalen Dadès Morocco 1Kalaat Meggouna G4 L9V3 Kalaat Meggouna Dadès Morocco 1Agdez A7 L1V7 Agdez Drâa Morocco 4Agdez C1 L3V11 Agdez Drâa Morocco 1Skoura SKT1 L4V6 Skoura Drâa Morocco NASkoura SKH1 L4V7 Skoura Drâa Morocco 1Skoura SKT5 L7V11 Skoura Drâa Morocco 1Agdez A5 L7V8 Agdez Drâa Morocco 1Agdez A8 L1V8 Agdez Drâa Morocco 4Skoura SKH2 L1V9 Skoura Drâa Morocco 1Skoura SKH3 L3V8 Skoura Drâa Morocco 1Agdez A6 L4V10 Agdez Drâa Morocco NASkoura SKH4 L8V10 Skoura Drâa Morocco 1Agdez A4 L8V12 Agdez Drâa Morocco 1Agdez IG1 L8V15 Agdez Drâa Morocco 4Canino 2 L3V6 Canino 2 INRAMa Morocco 4Géli L4V5 Géli INRAMa Morocco 4Marouch 3 L6V1 Marouch 3 INRAMa Morocco 4Khorb L6V5 Khorb INRAMa Morocco 1Gmat L7V1 Gmat INRAMa Morocco 1Mans L7V2 Mans INRAMa Morocco NAMarouch 1 L8V4 Marouch 1 INRAMa Morocco 1Missour V4 L5V4 Missour Moulouya Morocco NAGuersif 2 L6V11 Guersif Moulouya Morocco 1Outat Elhaj 1 L6V8 Outat Elhaj Moulouya Morocco NAMissour V17 L7V4 Missour Moulouya Morocco NAMissour V3 L8V5 Missour Moulouya Morocco NAOutat Elhaj 7 L4V8 Outat Elhaj Moulouya Morocco 4Outat Elhaj 6 L5V10 Outat Elhaj Moulouya Morocco NA

64 H. Bourguiba et al. / Scientia Horticulturae 152 (2013) 61–69

Table 1 (Continued )

Accession name Accession code Geographical site Geographical region Country of origin Clusterb

Missour V2 L5V6 Missour Moulouya Morocco NAOutat Elhaj 2 L5V8 Outat Elhaj Moulouya Morocco 4Outat Elhaj 8 L7V7 Outat Elhaj Moulouya Morocco 4Missour V12 L8V1 Missour Moulouya Morocco 1Missour V15 L8V7 Missour Moulouya Morocco NAMissour V22 L9V1 Missour Moulouya Morocco 1Jorf 7 L1V3 Jorf 7 Ziz Morocco 1Goulmima AJG2 L1V5 Goulmima Ziz Morocco 1Rich RK1 L2V1 Rich Ziz Morocco 4Jorf 6 L2V5 Jorf 6 Ziz Morocco 1Rich 4 L3V1 Rich Ziz Morocco 1Goulmima RG1 L3V2 Goulmima Ziz Morocco 1Rtil 5 L4V2 Rtil 5 Ziz Morocco 1Goulmima GR1 L4V3 Goulmima Ziz Morocco 1Goulmima GM1 L4V4 Goulmima Ziz Morocco 1Goulmima Gay2 L5V2 Goulmima Ziz Morocco 1Goulmima Gay3 L5V3 Goulmima Ziz Morocco 1Rtil 3 L5V5 Rtil 3 Ziz Morocco 1Goulmima AJG1 L1V1 Goulmima Ziz Morocco 1Jorf 8 L1V4 Jorf 8 Ziz Morocco 1Rich 3 L2V3 Rich Ziz Morocco 4Rich 3 L2V4 Rich Ziz Morocco 1Rtil 1 L3V3 Rtil 1 Ziz Morocco NARtil 2 L3V4 Rtil 2 Ziz Morocco 4Rtil 4 L4V1 Rtil 4 Ziz Morocco 4Goulmima Gay1 L5V1 Goulmima Ziz Morocco NARich RT1 L8V3 Rich Ziz Morocco 4H’midi Tu12C Ras Jbel North Tunisiaa Tunisia 2Bouk Hmed Tu13A Ras Jbel North Tunisiaa Tunisia 2Faggoussi Tu14C Ras Jbel North Tunisiaa Tunisia NAAddadi Ahmar Tu15 Ras Jbel North Tunisiaa Tunisia 2Om Youness Tu16 Ras Jbel North Tunisiaa Tunisia NAAranji Tu17A Ras Jbel North Tunisiaa Tunisia NAOud Rhayem Tu18A Testour North Tunisiaa Tunisia 2Bedri Ahmar Tu19A Testour North Tunisiaa Tunisia 2Bouthani Tu20C Testour North Tunisiaa Tunisia 2Oud Hmida Tu21A Testour North Tunisiaa Tunisia 2Oud Tijani Tu22B Testour North Tunisiaa Tunisia NAOud Nakhla Tu23B Testour North Tunisiaa Tunisia 2Oud Salah Ben Salem Tu25B Testour North Tunisiaa Tunisia 2Oud Gnaa Tu27 Testour North Tunisiaa Tunisia 2Chechi Bazzaa Tu28D Testour North Tunisiaa Tunisia NAChechi Horr Tu29 Testour North Tunisiaa Tunisia 4Bouk Hmed Akhal Tu34B Ras Jbel North Tunisiaa Tunisia 2Oud El Haj Tahar Tu70 Testour North Tunisiaa Tunisia NAOud Aouicha Tu71 Testour North Tunisiaa Tunisia 2Chechi Khit El Oued Tu10A Kairouan Centre Tunisiaa Tunisia 4Bayoudhi Tu11B Kairouan Centre Tunisiaa Tunisia 4Baccour Tu1C Kairouan Centre Tunisiaa Tunisia 2Bedri Tu1G Gabès Centre Tunisiaa Tunisia 2Khad Hlima Tu2C Kairouan Centre Tunisiaa Tunisia 4Bangui Tu31 Mahdia Centre Tunisiaa Tunisia 2Fourati Tu38B Sfax Centre Tunisiaa Tunisia NAFourati Tu4B Kairouan Centre Tunisiaa Tunisia NAAmor El Euch Tu5C Kairouan Centre Tunisiaa Tunisia 2Messelmani Tu6A Kairouan Centre Tunisiaa Tunisia 2Zalouzi Tu72 Sfax Centre Tunisiaa Tunisia 2Zbidi Tu7C Kairouan Centre Tunisiaa Tunisia 2Chechi Dhraa Tammar Tu9 Kairouan Centre Tunisiaa Tunisia 4Bedri Tu48A Gabès South Tunisiaa Tunisia 2Ben Souileh Tu53A Gabès South Tunisiaa Tunisia 2Bedri Louzi Tu57A Gabès South Tunisiaa Tunisia 2Thaleth Tu58B Gabès South Tunisiaa Tunisia 2Thani Tu59A Gabès South Tunisiaa Tunisia 2Bou Khobza Tu62A Jerba South Tunisiaa Tunisia 2Mazouzi 65 Tu65B Jerba South Tunisiaa Tunisia 2Bedri Louzi 67 Tu67 Jerba South Tunisiaa Tunisia 2Chechi Tu68 Jerba South Tunisiaa Tunisia NAMazouzi 69 Tu69 Jerba South Tunisiaa Tunisia NAAgrégé de Messelmani Tu6B Sfax South Tunisiaa Tunisia 2Bargoug 40A Tu40A Gafsa Gafsa oasis Tunisia 3Bargoug 40B Tu40B Gafsa Gafsa oasis Tunisia 3Bargoug 40E Tu40E Gafsa Gafsa oasis Tunisia 2Bargoug 40G Tu40G Gafsa Gafsa oasis Tunisia 3Bargoug 40H Tu40H Gafsa Gafsa oasis Tunisia NABargoug 40I Tu40I Gafsa Gafsa oasis Tunisia 3Bargoug 40J Tu40J Gafsa Gafsa oasis Tunisia 3

H. Bourguiba et al. / Scientia Horticulturae 152 (2013) 61–69 65

Table 1 (Continued )

Accession name Accession code Geographical site Geographical region Country of origin Clusterb

Bargoug 40K Tu40K Gafsa Gafsa oasis Tunisia 3Bargoug 40M Tu40M Gafsa Gafsa oasis Tunisia 3Bargoug 40N Tu40N Gafsa Gafsa oasis Tunisia 3Agrégé de Baccour Tu41A Gafsa Gafsa oasisa Tunisia 2Bedri Thani Tu48G Gafsa Gafsa oasisa Tunisia 2Kasserine 2 TuK2 Gafsa Gafsa oasisa Tunisia NABargoug 42A Tu42A Tozeur Other oasis Tunisia 3Bargoug 42B Tu42B Tozeur Other oasis Tunisia 3Bargoug 42C Tu42C Tozeur Other oasis Tunisia 3Bargoug 42G Tu42G Tozeur Other oasis Tunisia 3Bargoug 42H Tu42H Tozeur Other oasis Tunisia 3Bargoug 42B Tu43B Nefta Other oasis Tunisia 3Bargoug 43C Tu43C Nefta Other oasis Tunisia NABargoug 43D Tu43D Nefta Other oasis Tunisia 3Bargoug 43F Tu43F Nefta Other oasis Tunisia 3Bargoug 44A Tu44A Degache Other oasis Tunisia 3Bargoug 44B Tu44B Degache Other oasis Tunisia 3Bargoug 44C Tu44C Degache Other oasis Tunisia 3Bargoug 44D Tu44D Degache Other oasis Tunisia NABargoug 44E Tu44E Degache Other oasis Tunisia 3Bargoug 44F Tu44F Degache Other oasis Tunisia 3Bargoug 44G Tu44G Degache Other oasis Tunisia 3Bargoug 44H Tu44H Degache Other oasis Tunisia 3Bargoug 45B Tu45B Tamaghza Other oasis Tunisia 3Bargoug 45C Tu45C Tamaghza Other oasis Tunisia 3Bargoug 46B Tu46B Midess Other oasis Tunisia 3Bargoug 46C Tu46C Midess Other oasis Tunisia 3Bargoug 46D Tu46D Midess Other oasis Tunisia 3Bargoug 46E Tu46E Midess Other oasis Tunisia 3Variété de Mahdia Tu47 Midess Other oasisa Tunisia NA

NA, genotype not assigned to a genetic cluster (assignment level defined at 70%).a Grafted propagated accessions; all the remaining material were seed propagated.b Clusters identified using STRUCTURE analysis.

Table 2Diversity parameters calculated for 24 SSR markers among 183 apricot accessions.

Locus Allele size range (bp) Total allele number Rare allele numbera He Ho

AMPA100b 196–218 7 3 0.681 0.551AMPA101b 188–212 6 2 0.585 0.497AMPA105b 182–218 9 4 0.687 0.644AMPA109b 193–227 6 4 0.169 0.120AMPA116b 115–141 10 5 0.733 0.617AMPA119b 098–112 5 3 0.144 0.134BPPCT001c 116–118 2 0 0.412 0.415BPPCT004c 178–209 9 5 0.756 0.612BPPCT008c 107–129 9 4 0.782 0.699BPPCT017c 161–209 6 3 0.611 0.568BPPCT025c 147–161 6 3 0.564 0.464BPPCT030c 136–150 6 2 0.734 0.639BPPCT038c 127–149 12 9 0.715 0.579BPPCT040c 128–144 4 2 0.371 0.360CPPCT006d 175–203 9 4 0.790 0.623CPPCT022d 234–274 13 9 0.726 0.623CPPCT030d 147–185 11 6 0.733 0.704CPPCT033d 137–161 8 6 0.377 0.289CPPCT034d 187–207 9 7 0.519 0.459Ma014ae 136–142 3 1 0.470 0.377Ma040ae 207–253 11 8 0.656 0.502UDP97-402f 118–144 8 6 0.507 0.464UDP98-409f 122–164 16 10 0.870 0.710UDP98-412g 083–115 6 2 0.636 0.448

Mean – 7.95 4.5 0.593 0.504

Total – 191 108 – –

He, expected heterozygosity; Ho, observed heterozygosity.a Observed alleles with frequencies of less than 5%.b Hagen et al. (2004).c Dirlewanger et al. (2002).d Aranzana et al. (2002).e Yamamoto et al. (2002).f Cipriani et al. (1999).g Testolin et al. (2000).

66 H. Bourguiba et al. / Scientia Horticulturae 152 (2013) 61–69

Fig. 1. Inferred North-African apricot population structure obtained using STRUCTURE software, where K is the potential number of genetic clusters that may occur in theo us gec ps art

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structured genetic diversity among the identified clusters accord-ing to a continuum (Fig. 2). In fact, clusters 1 (‘Morocco’) and 3(‘Oasis of Tunisia/Algeria) were separated from clusters 2 (‘Graftedpropagated accessions of Tunisia’) and 4 (‘North Mediterranean’) by

-1.5-1-0.500.511.52

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Fig. 2. Plot based on the first and second axes of factorial correspondence analysis

verall sample of individuals. Each vertical line represents one individual multilocluster 2 in blue, cluster 3 in green and cluster 4 in yellow. Apricot geographic grouhis figure legend, the reader is referred to the web version of the article.)

ccessions identified as belonging to the ‘North Mediterranean’ene pool (Bourguiba et al., 2012a).

Overall, 142/183 accessions (77.5%) were clearly assigned withore than 70% of the assignment probability (Table 1 and Fig. 1).

he remaining 41 accessions (22.5%) of the sample were consid-red as having admixed ancestry (Table 3). The admixture waslearly observed in apricots from the northern Tunisia (6 acces-ions; 31.5%), Messaad (11 accessions; 32.3%) and Moulouya Valley7 accessions; 53.8%) groups (Table 3).

Among the admixed accessions, the majority (25/41; 60.9%)ere admixed with cluster 3; consisting of apricots from Tunisian

ases and Messaad region in Algeria, as shown by their highssigned probability value for cluster 3 ≥ 0.170 (Table 3). Most ofhe mixed inferred ancestry was between clusters 1 and 2 (6), clus-ers 1 and 3 (10) and clusters 2 and 3 (6), thus highlighting thexistence of genetic relationships within local gene pools in theaghreb region. Regarding cluster 4, 19 (46.3%) were admixed with

his cluster, which consisted of apricots from the ‘North Mediter-anean’ gene pool, as shown by their high assigned probabilityalue for cluster 4 ≥ 0.177 (Table 3). For the Messaad region in Alge-ia, most of the mixed inferred ancestry accessions were betweenlusters 3 and 4.

.3. Genetic diversity and differentiation among clusters

Except for cluster 4, the three genetic clusters (clusters 1–3)dentified by STRUCTURE analysis could be related to a specific geo-raphical region, as mentioned in Table 4. In fact, cluster 1 wasncountered principally in the geographical regions of Morocco,luster 2 in the northern, central and southern regions of Tunisia,nd cluster 3 in both the Algerian region of Messaad and theunisian oasis regions (Table 4). However, the ‘North Mediter-anean’ gene pool could not be associated with a specific region

ince it was mainly represented in Algeria, Morocco and northernnd central Tunisia.

The genetic variation within each apricot genetic cluster wasssessed to study their level of variability (Table 5). The mean

notype. Genetic clusters are represented by different colors, with cluster 1 in red,e separated by a vertical black line. (For interpretation of the references to color in

number of alleles ranged from 3.95 (cluster 2) to 4.87 (cluster 3).The allelic richness was highest for cluster 4 (4.58) and lowest forcluster 2 (3.89). The expected heterozygosity (He) ranged from0.450 (cluster 1) to 0.555 (cluster 4), indicating that cluster 4 hadthe highest genetic variation level (Table 5).

The four genetic clusters were significantly differentiated, asreflected by the high global Fst value of 0.145 (Table 6). Pairwise Fstvalues among the identified clusters were significant, ranging fromFst = 0.080 (between clusters 1 and 3) to 0.233 (between clusters1 and 4; Table 6). The ‘North Mediterranean’ gene pool (cluster 4)was the most differentiated as compared to the three remainingclusters, and as shown by the Fst values, which ranged from 0.112to 0.233.

The two-dimensional factorial correspondence analysis (FCA)scatter plot coordinates for the first and second axes, which respec-tively explained 6.84% and 4.24% of the total variance, highlighted

(FCA) coordinates showing genetic differentiation among the four defined apricotgenetic clusters with filled lozenges representing accessions from Morocco (cluster1), times accessions from both Algeria and the oases of Tunisia (cluster 3), squaresgrafted propagated accessions from Tunisia (cluster 2) and filled triangles North-Mediterranean accessions (cluster 4).

H. Bourguiba et al. / Scientia Horticulturae 152 (2013) 61–69 67

Table 3Propagation mode, geographic origin and probability of assignment of the 41 admixed ancestry accessions identified by STRUCTURE analysis.

Accession name Geographic region Propagation mode Assignment probability

Cluster 1 Cluster 2 Cluster 3 Cluster 4

Rosé de Ménaa Messaad Seed 0.158 0.022 0.410 0.410Rouge du Roussillon Messaad Seed 0.500 0.030 0.367 0.103Mechmech hlou Messaad Seed 0.045 0.518 0.390 0.047Mechmech laghdech Messaad Seed 0.011 0.043 0.457 0.489Mouzemèche Messaad Seed 0.013 0.015 0.395 0.577Kahf Messaad Seed 0.020 0.040 0.605 0.335Messaad greffé Messaad Seed 0.282 0.028 0.634 0.056Saafi arbi Messaad Seed 0.025 0.038 0.412 0.525Hamrai Messaad Seed 0.300 0.221 0.165 0.314Moutaakhir Messaad Seed 0.015 0.022 0.523 0.440Ikhtiyar ettayeb Messaad Seed 0.011 0.072 0.322 0.595Kalaat Meggouna G5 Dadès Seed 0.054 0.025 0.637 0.284Kalaat Meggouna G8 Dadès Seed 0.585 0.056 0.309 0.050Kalaat Meggouna G7 Dadès Seed 0.021 0.053 0.617 0.309Skoura SKT1 Drâa Seed 0.564 0.056 0.015 0.365Agdez A6 Drâa Seed 0.695 0.145 0.084 0.076Mans INRAM Graft 0.365 0.020 0.562 0.053Missour V4 Moulouya Seed 0.468 0.028 0.475 0.029Outat Elhaj 1 Moulouya Seed 0.530 0.043 0.386 0.041Missour V17 Moulouya Seed 0.652 0.170 0.033 0.145Missour V3 Moulouya Seed 0.420 0.006 0.544 0.030Outat Elhaj 6 Moulouya Seed 0.513 0.447 0.023 0.017Missour V2 Moulouya Seed 0.434 0.530 0.020 0.016Missour V15 Moulouya Seed 0.440 0.055 0.490 0.015Rtil 1 Ziz Seed 0.483 0.043 0.454 0.020Goulmima Gay 1 Ziz Seed 0.630 0.220 0.025 0.125Faggoussi North Tunisia Graft 0.055 0.671 0.194 0.080Om Youness North Tunisia Graft 0.088 0.639 0.170 0.103Aranji North Tunisia Graft 0.035 0.360 0.022 0.583Oud Tijani North Tunisia Graft 0.083 0.261 0.555 0.101Chechi Bazzaa North Tunisia Graft 0.052 0.668 0.103 0.177Oud El Haj Tahar North Tunisia Graft 0.022 0.585 0.042 0.350Fourati Centre Tunisia Graft 0.066 0.374 0.526 0.034Fourati Centre Tunisia Graft 0.182 0.674 0.016 0.013Chechi South Tunisia Graft 0.068 0.312 0.037 0.583Mazouzi 69 South Tunisia Graft 0.104 0.310 0.031 0.555Bargoug 40H Gafsa oasis Seed 0.283 0.011 0.016 0.690Kasserine 2 Gafsa oasis Graft 0.289 0.019 0.587 0.105Bargoug 43C Other oasis Seed 0.203 0.483 0.010 0.304Bargoug 44D Other oasis Seed 0.016 0.674 0.031 0.279Variété de Mahdia Other oasis Graft 0.008 0.512 0.474 0.006

Bold values represented accessions admixed with cluster 4 (‘North-Mediterranean’) as shown by their high assigned probability value for cluster 4 (≥0.177), in comparisonto the other clusters.

Table 4Apricot accessions from the 11 different geographical groups assigned to the four clusters identified by STRUCTURE analysis.

Geographic groups Accession number Assigned accession numbera Cluster 1 Cluster 2 Cluster 3 Cluster 4

N % N % N % N %

Messaad 34 23 0 0 0 0 16 70 7 30Dadès Valley 15 12 10 84 0 0 1 8 1 8Drâa Valley 13 11 8 73 0 0 0 0 3 27Moulouya Valley 13 6 3 50 0 0 0 0 3 50Ziz Valley 21 19 14 74 0 0 0 0 5 26INRAM 7 6 3 50 0 0 0 0 3 50North Tunisia 19 13 0 0 12 92 0 0 1 8Centre Tunisia 13 11 0 0 7 64 0 0 4 36South Tunisia 11 9 0 0 9 100 0 0 0 0

apgr

4

f

Gafsa oasis 13 11Other oasis 24 21

a Under the assignment probability of P > 70%.

xis 1. In addition, as previously illustrated by the genetic diversityarameters, the FCA demonstrated that the ‘North Mediterranean’ene pool (cluster 4) was the most diversified as compared to theemaining clusters (Fig. 2).

. Discussion

Native traditionally cultivated apricot accessions originatingrom different geographical regions of Algeria, Morocco and Tunisia

0 0 3 27 8 73 0 00 0 0 0 21 100 0 0

in North African region were investigated for genetic structureusing 24 nuclear microsatellite loci and a model-based Bayesianclustering method.

Except for one genetic cluster (cluster 4), apricots from NorthAfrica were classified into three genetic clusters according to their

mode of propagation. Bayesian and multivariate analyses showed adistinction between grafted (cluster 2) and seed propagated acces-sions (clusters 1 and 3). Similar findings were previously obtainedwhen focusing on only Tunisian apricots (Bourguiba et al., 2010).

68 H. Bourguiba et al. / Scientia Horticulturae 152 (2013) 61–69

Table 5Genetic diversity parameters within each genetic cluster identified by STRUCTURE analysis.

Cluster size Mean allele number Allelic richnessa He Ho

Cluster 1 38 4.79 4.43 0.450 0.372Cluster 2 31 3.95 3.89 0.540 0.534Cluster 3 46 4.87 4.52 0.535 0.510

sadik a

CafitMa(enam

sAsribeaafccgiisAidtwtaTawrpcoftg

e

TPT

Cluster 4 27 4.58

a Standardized to the smallest number of genotypes (n = 27) according to El Mou

ompared to the global genetic differentiation, lower Fst valuesmong the three clusters (clusters 1–3) were observed, thus con-rming their common genetic base. It was previously demonstratedhat apricot in the Maghreb region belonged to a common ‘South-

editerranean Basin’ gene pool, which was identified through study of apricot genetic resources in the Mediterranean BasinBourguiba et al., 2012a). However, in that study, only a few vari-ties were considered, and the surveys were limited to a smallumber of apricot cropping areas. Here, a larger set of North Africanpricot varieties was studied, which was considered as being theost representative of the local available variability.Cluster 4 identified by STRUCTURE analysis included 27 acces-

ions derived from different geographical regions in North Africa.mong them, 7 were involved in a previous study of apricot genetictructure in the Mediterranean Basin (Bourguiba et al., 2012a). Theesults demonstrated that these 7 apricot genotypes, which orig-nated from Drâa Valley, Moulouya Valley and northern Tunisia,elonged to the ‘North-Mediterranean Basin’ gene pool (Bourguibat al., 2012a). Therefore, we assumed that cluster 4 consisted ofccessions belonging to the ‘North-Mediterranean Basin’ gene pool,nd had probably been introduced into North African countriesrom southern Europe. As compared to the three apricot geneticlusters characteristic of local North African apricot germplasm,luster 4 had more diversified genetic variability, as shown by theenetic diversity parameters and the multivariate analysis find-ngs. In addition, this cluster was the most genetically differentiatedn comparison with the three other identified clusters. Hence, wehowed that this cluster was clearly distinct from local Northfrican apricot germplasm, thus indicating that apricots were

ntroduced into this region from European countries. Such intro-uctions could be attributed to different historical events. In fact,he planting materials were brought by Andalusian immigrantsho established themselves in North African countries following

heir mass eviction from Spain in the early 17th century (Carrautnd Crossa-Raynaud, 1974; Valdeyron and Crossa-Raynaud, 1950).hus, these apricots could be derived from that original stockfter a few centuries of cropping, with seed propagation being aell-established practice and recommended for some soils. More

ecently, the French colonization period could also explain theresence of introduced varieties from Europe in North Africa. Theonnection between two apricot gene pools was also evidenced inther geographical regions as reported by Halász et al. (2010). Inact, Hungarian and Turkish apricots shared S-alleles confirminghat some Hungarian apricot accessions were issued from Turkish

ermplasm.

Does gene flow occur between these recently introduced vari-ties and local North African apricots? The model-based Bayesian

able 6airwise genetic differentiation among the four genetic clusters identified by STRUC-URE analysis (global Fst = 0.145).

Cluster 1 Cluster 2 Cluster 3

Cluster 2 0.157* 0Cluster 3 0.080* 0.109* 0Cluster 4 0.233* 0.112* 0.194*

* P < 0.001.

4.58 0.555 0.520

nd Petit (1996).

method was efficient for detecting hybrid individuals with differentnumbers of loci and under different hybridization scenarios (Falushet al., 2003). Focusing on the admixed genotypes highlighted by theSTRUCTURE analysis, the results revealed that about 46.3% (19/41accessions) were admixed with cluster 4. These genotypes werelocated mainly in Messaad, Moulouya Valley and northern Tunisiaregions located in the northern of the Maghreb region, correspond-ing to areas where apricot from southern European countries havebeen recently introduced. Accessions from cluster 4 could not beassigned to a specific geographical region as they were derived fromdifferent apricot cropping regions in North Africa (Messaad, DadèsValley, Drâa Valley, Moulouya Valley, Ziz Valley, INRAM, northernTunisia and central Tunisia), which correspond to the main areaswhere admixed accessions were encountered. As expected, wenoted a highly significant proportion of admixed accessions derivedfrom seed propagated populations (68.3%) since gene flow can onlyoccur when farmers propagate their apricots through seedlings.However, we observed a similar proportion of admixed accessionsamong graft (24%) and seed propagated apricots (21.7%), thus sup-porting the assumption that graft and seed propagated apricotsshare a common gene pool, as previously suggested in Tunisia(Khadari et al., 2006; Bourguiba et al., 2010) and the Maghreb region(Bourguiba et al., 2012a).

5. Conclusions

In agreement with a recent study on the apricot genetic struc-ture in the Mediterranean Basin (Bourguiba et al., 2012a), thepresent study demonstrated that the studied North African apri-cot varieties consisted of local material, including graft and seedpropagated accessions, having the same genetic base and belongingto the ‘South-Mediterranean Basin’ gene pool. Our findings identi-fied apricot varieties recently introduced from southern Europe aswell as accessions derived from admixtures between a local genepool and varieties introduced both via ancient and recent events,thus highlighting that gene flow has occurred in apricot croppingregions in North Africa. We have developed a molecular databasethat should facilitate the set up and management of core apricotgermplasm collections in the North African region, which probablycorresponds to a secondary center of diversification of this species.

Acknowledgments

The authors would like to thank Dr. Sylvain Santoni and A.Weber for their help in microsatellite genotyping. This work wassupported by a bilateral Franco-Tunisian initiative within theframework of the CMCU project (05G0904), with a helpful assis-tance from the Tunisian Ministère de l’Enseignement Supérieur et dela Recherche Scientifique (Project Lab B02). B. Khadari was supportedby Agropolis Fondation FruitMed no. 0901-007.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.scienta.2013.01.012.

Horti

R

A

B

B

B

B

B

B

C

C

D

E

E

F

F

F

G

from an enriched genomic and cDNA libraries. Mol. Ecol. Notes 2,

H. Bourguiba et al. / Scientia

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