Genetic structure of the date palm ( Phoenix dactylifera ) in the Old World reveals a strong...

Genetic structure of the date palm (Phoenix dactylifera) in the Old Worldreveals a strong differentiation between eastern and western populations

Salwa Zehdi-Azouzi1,*, Emira Cherif1,2, Souhila Moussouni3, Muriel Gros-Balthazard2,4,Summar Abbas Naqvi5, Bertha Ludena2,6, Karina Castillo2, Nathalie Chabrillange2, Nadia Bouguedoura3,

Malika Bennaceur7, Farida Si-Dehbi3, Sabira Abdoulkader8, Abdourahman Daher8, Jean-Frederic Terral4,Sylvain Santoni9, Marco Ballardini10, Antonio Mercuri10, Mohamed Ben Salah11, Karim Kadri11,

Ahmed Othmani11, Claudio Littardi12, Amel Salhi-Hannachi1, Jean-Christophe Pintaud2 andFrederique Aberlenc-Bertossi2

1Universite Tunis El Manar, Faculte des Sciences de Tunis, Laboratoire de Genetique Moleculaire, Immunologie et Biotechnologie,Campus universitaire El Manar, 2092, Tunisia, 2IRD, UMR DIADE-F2F, DYNADIV, and EVODYN teams, 911 Av. Agropolis, BP

64501, 34394 Montpellier, Cedex 5, France, 3Universite des Sciences et de la Technologie Houari Boumediene (USTHB),Laboratoire de Recherche sur les Zones Arides (LRZA), BP 32 Bab Ezzouar–El Alia, 16111, Alger, Algeria, 4Institut des Sciencesde l’Evolution de Montpellier, UMR 5554, equipe Dynamique de la biodiversite, anthropo-ecologie, Place Eugene Bataillon, CC

065, 34095 Montpellier cedex 05, France, 5Institute of Horticultural Sciences, University of Agriculture, 38040 Faisalabad,Pakistan, 6School of Biology, Yachay-Tech, Yachay City of Knowledge, 100119 Urcuqui, Ecuador, 7Universite Oran1-Ahmed BenBella, Faculte des sciences de la nature et de la vie, Departement de Biologie, BP 1524 El Mnaouar, 31000 Oran, Algerie, 8ISV/

CERD, route de l’Aeroport, BP 486, Djibouti, 9INRA, UMR AGAP, 2 Place Viala, 34060 Montpellier, Cedex 1, France,10Consiglio per la Ricerca e la Sperimentazione in Agricoltura-Unita di Ricerca per la Floricoltura e le Specie Ornamentali (CRA-

FSO), Corso degli Inglesi 508, I-18038 Sanremo (IM), Italy, 11Centre Regional de Recherche en Agriculture Oasienne, 2260Degueche, Tunisia and 12Centro Studi e Ricerche per le Palme, Corso F. Cavallotti 113, 18038 Sanremo (IM), Italy

* For correspondence. E-mail [email protected] or [email protected]

Received: 7 January 2015 Returned for revision: 2 March 2015 Accepted: 13 April 2015

! Background and Aims Date palms (Phoenix dactylifera, Arecaceae) are of great economic and ecological valueto the oasis agriculture of arid and semi-arid areas. However, despite the availability of a large date palm germplasmspreading from the Atlantic shores to Southern Asia, improvement of the species is being hampered by a lack of in-formation on global genetic diversity and population structure. In order to contribute to the varietal improvement ofdate palms and to provide new insights on the influence of geographic origins and human activity on the geneticstructure of the date palm, this study analysed the diversity of the species.!Methods Genetic diversity levels and population genetic structure were investigated through the genotyping of acollection of 295 date palm accessions ranging from Mauritania to Pakistan using a set of 18 simple sequence repeat(SSR) markers and a plastid minisatellite.! Key Results Using a Bayesian clustering approach, the date palm genotypes can be structured into two differentgene pools: the first, termed the Eastern pool, consists of accessions from Asia and Djibouti, whilst the second,termed the Western pool, consists of accessions from Africa. These results confirm the existence of two ancientgene pools that have contributed to the current date palm diversity. The presence of admixed genotypes is alsonoted, which points at gene flows between eastern and western origins, mostly from east to west, following ahuman-mediated diffusion of the species.! Conclusions This study assesses the distribution and level of genetic diversity of accessible date palm resources,provides new insights on the geographic origins and genetic history of the cultivated component of this species, andconfirms the existence of at least two domestication origins. Furthermore, the strong genetic structure clearly estab-lished here is a prerequisite for any breeding programme exploiting the effective polymorphism related to eachgene pool.

Key words: Date palm, Arecaceae, genetic diversity, genetic structure, nuclear microsatellite, Phoenix dactylifera,plastid minisatellite, SSR markers.

INTRODUCTION

During the plant domestication process, the combined actionsof breeding, selection, migration and admixture have given riseto cultivated populations genetically and phenotypically distinct

from the ancestral gene pools (Grassi et al., 2003; Doebleyet al., 2006; Arroyo et al., 2006). During this process, humanshave notably selected traits linked to productivity, fruit qualityand fertility (Zohary et al., 2012). Knowledge of the population

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genetics and domestication history of cultivated species is ofgreat importance for the genetic improvement of crops relyingon the effective conservation and use of the germplasm repre-sented by the agrobiodiversity and the wild relativepopulations.

The date palm, Phoenix dactylifera, belongs to theArecaceae, and is the emblematic species of the oasis agricul-ture. These trees produce dates with high nutritional value andmaintain fertile areas of life in deserts. They are said to have athousand uses, are highly symbolic for Muslim, Christian andJewish religions, and have accompanied the development ofearly human societies. The historical distribution area extendsfrom Mauritania in the west to Pakistan in the east and north-western India (Pintaud et al., 2013). The date palm is also pre-sent in sub-Saharan Africa and has been introduced inCalifornia, Peru, Australia and other countries (Barrow, 1998).The date palm is one of the earliest cultivated fruit trees andone of the first plants pollinated by humans (Zohary andSpiegel-Roy, 1975). Sexual propagation of date palm has beencarried out since the Neolithic (Tengberg, 2003), but the result-ing progeny often has unpredictable characteristics because ofthe high heterozygosity rate of the species. Thus, by maintain-ing the genetic integrity of date palm cultivars, vegetative prop-agation of offshoots has been set up in palm groves mainlyto preserve the organoleptic traits of fruits which reduce thegenetic diversity of the germplasm in the cultivated areas.Moreover, offshoots are produced in limited numbers duringthe date palm’s life span (Zaid and de Wet, 2002;Bouguedoura, 2012).

Date palm is currently the main crop of the arid and semi-arid countries of North Africa and the Middle East. World datefruit production reaches almost 8 million tons, every year gen-erating millions of US dollars in benefit to local and nationaleconomies (FAOstat). However, date palm production hasshifted from traditional cultivation in rich and diverse agrosys-tems to intensive monocultures (Jain et al., 2011). This devel-opment has led to severe genetic erosion, with the loss ofcultivars and the overall impoverishment of date palm agrobio-diversity (Jain et al., 2011). Furthermore, date palm groves un-dergo biotic and abiotic stress, which has made it essential toset up breeding programmes to select tolerant varieties and en-rich the germplasm.

The conservation of date palm genetic resources, hence, hasbecome a critical issue for the development of date palm pro-duction and food security in desert and semi-desert areas. Aglobal evaluation of the genetic diversity of current date palmaccessions is therefore required, along with plans for the preser-vation of worldwide date palm germplasm.

Genetic studies using simple sequence repeat (SSR) markerswere first carried out for the analysis of the genetic diversity ofPhoenix dactylifera in Tunisia (Zehdi et al., 2004, 2012) and,since then, several studies have focused on the date palm ge-netic diversity in Sudan (Elshibli and Korpelainen, 2008),Oman (Al-Ruqaishi et al., 2008), Qatar (Ahmed and Al-Qaradawi, 2009; Elmeer et al., 2011), Iraq (Khierallah et al.,2011), Iran (Arabnezhad et al., 2012) and recently in the UAE(Chaluvadi et al., 2014). However, all these studies were basedon a relatively small number of accessions centred on countries,and consequently are not representative of the overall date palmgenetic diversity. A wider analysis of the date palm diversity is

required to unravel the genetic relationships between the geo-graphic groups distributed in the Old World from west to east,and to identify the potential backgrounds of genetic diversityuseful for breeding programmes and for the selection of adapt-ability traits to biotic and abiotic stress. Furthermore, the loca-tion of centres of date palm domestication still remains unclear.Recently, the existence of an eastern domestication centre hasbeen shown, but the question of the existence of other possibleorigins has not been fully resolved to date (Tengberg, 2012).Moreover, according to the domestication syndrome criteria ofMeyer et al. (2012), some morphological characters, that arelikely to have evolved from the ancestral wild date palm popu-lations to the selected cultivars, correspond essentially to differ-ences in branching, fruit and seed character. These criteriacorrespond largely to the traits generally selected in fruit crops(Doebley et al., 2006), but remain hypothetical since wild datepalm populations have not been characterized yet (Pintaudet al., 2013).

In order to study the genetic structure and the genetic diver-sity in date palm, we have assessed the polymorphism of SSRmarkers in date palm accessions to reveal the molecular basisof genetic diversity and the relationships among a large sampleof date palm accessions over a wide geographic distribution.We used nuclear microsatellite markers to examine the geneticdiversity and to provide a description of the genetic structurethat could be used to select genotype samples appropriate forfurther genetic association studies and for building a geneticcore collection. We also analysed a chloroplast minisatellite(CpSSR), which is a haploid marker with a lower mutation ratethan the nuclear SSRs (Provan et al., 1999), and thus evolvedifferently. Indeed, being maternally inherited, the chloroplastgenome provides us with information about the maternal originof the plant.

MATERIALS AND METHODS

Plant material

The sample included in this study consisted of 295 Phoenixdactylifera genotypes. The plants were collected from the geo-graphical distribution of the species to cover the greatest possi-ble genetic diversity, including accessions from traditionalwestern (from Mauritania to Egypt) and eastern (from Djiboutito Pakistan) cultivation areas (Fig. 1; Supplementary DataTable S1).

Ten groups were defined according to the geographic originof the studied date palm accessions. The number of accessionsper group ranged from 14 for the Mauritanian group to 50 forthe Algerian and Pakistan groups (Fig. 1).

DNA preparation

Total cellular DNA was extracted from young, silica gel-dried or lyophilized leaves of the 295 accessions using theTissueLyser and the DNeasy Plant Mini Kit (Qiagen SA,Courtaboeuf, France) or the PureLink Plant Total DNAPurification Kit (Invitrogen) according to the manufacturer’sprotocol. After purification, DNA concentrations were deter-mined using a GeneQuant spectrometer (Amersham Pharmacia

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Biotech, France). The quality was checked by agarose minigelelectrophoresis (Sambrook et al., 1989). The resulting DNAsolutions were stored at "20 #C.

Amplification and genotyping

The 18 SSR loci selected for this study included dinucleotiderepeats from an SSR genomic library (Billotte et al., 2004) and

from introns of genes coding for transcription factors (Ludenaet al., 2011), as well as tri-/hexanucleotide repeats from codingsequences identified by in silico mining (Aberlenc et al., 2014)in the whole date palm genome sequence (Al Dous et al.,2011). New primers for some of these loci were designed in thepresent study (Table1, Supplementary Data Table S2). In addi-tion to the 18 nuclear SSR loci, the plastid dodecanucleotideminisatellite identified in the intergenic spacer psbZ-trnfM(Henderson et al., 2006) was genotyped for all date palm

Mauritania

MoroccoAlgeria

Tunisia

Egypt

Iraq

Oman

EAU

Pakistan

N = 50

N = 18

N = 24

N = 34

N = 18

N = 28

N = 38

N = 50N = 21

N = 14

Djibouti

FIG. 1. Origin of the 295 date palm accessions classified into ten groups and two regions: I and II as defined according to their spatial and genetic proximity. RegionI, Mauritania, Algerian, Morocco, Tunisia and Egypt; region II, Djibouti, Oman, the UAE, Iraq and Pakistan. The colours correspond to the genetic clusters defined

by STRUCTURE analysis, with cluster I in green and cluster II in red.

TABLE 1. Genetic diversity obtained at 18 SSR loci in the 295 date palm accessions

Marker Reference NA NA,P Major allelefrequency

NG PIC Ho He FIS

mPdCIR010 Billotte et al. (2004) 15 6 0$260 45 0$821 0$815 0$787 –0$035mPdCIR015 Billotte et al. (2004) 13 6 0$341 35 0$775 0$750 0$747 "0$005*mPdCIR016 Billotte et al. (2004) 5 3 0$436 11 0$612 0$580 0$589 0$016mPdCIR025 Billotte et al. (2004) 16 5 0$300 47 0$781 0$793 0$722 "0$098mPdCIR032 Billotte et al. (2004) 13 5 0$342 36 0$774 0$758 0$743 "0$021mPdCIR035 Billotte et al. (2004) 11 3 0$519 24 0$603 0$527 0$609 0$135***mPdCIR057 Billotte et al. (2004) 10 4 0$639 26 0$536 0$492 0$469 "0$049mPdCIR063 Billotte et al. (2004) 15 4 0$347 32 0$726 0$637 0$647 0$016*mPdCIR078 Billotte et al. (2004) 27 5 0$158 94 0$888 0$806 0$810 0$005mPdCIR085 Billotte et al. (2004) 20 7 0$266 68 0$848 0$787 0$786 "0$001mPdIRD013 Aberlenc-Bertossi et al. (2014) 3 1 0$986 3 0$027 0$240 0$230 "0$040mPdIRD031 Aberlenc-Bertossi et al. (2014) 4 3 0$863 7 0$232 0$240 0$225 "0$064mPdIRD033 Aberlenc-Bertossi et al. (2014) 4 2 0$880 7 0$210 0$202 0$206 0$021mPdIRD040 Aberlenc-Bertossi et al. (2014) 5 3 0$651 10 0$438 0$485 0$451 "0$076PdAG1-ssr Ludena et al. (2011) 33 5 0$210 92 0$874 0$802 0$812 0$012PdAP3-ssr-F4 Accession number: KC188337 13 6 0$224 47 0$839 0$795 0$744 "0$068PdCUC3-ssr1 Accession number: HM622273 2 1 0$992 2 0$017 0$200 0$190 "0$050PdCUC3-ssr2 Accession number: HM622273 28 7 0$185 82 0$882 0$769 0$810 0$052*Overall 13$17 4$22 0$478 37$11 0$605 0$571 0$569 "0$014

NG, number of genotypes per locus; NA, number of alleles per locus; NA,P, number of alleles with a frequency >5 %; PIC polymorphic information content;He expected heterozygosity; Ho observed heterozygosity; FIS, fixation index values.

Exact test significant at *P< 0$05, ***P< 0$001.

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accessions to reveal the repeat number (three or four), definingthe chlorotype (Occidental and Oriental, respectively) in datepalm (Pintaud et al., 2010, 2013). This repeat number is linkedto other mutations in the chloroplast genome (Ballardini et al.,2013).

Amplification reactions were performed in a final volume of20 lL containing 15 ng of template DNA, 10% reaction buffer,5 pmol of each forward and reverse primer, 0$2 mM of eachdeoxynucleotide, 2 mM MgCl2 and 1 U of Taq polymerase(Sigma, USA). The forward primers were 5’ labelled with oneof three fluorescent compounds (6-FAM, NED or HEX) to en-able analysis with automated sequencers. PCR was carried outusing an Eppendorf Mastercycler pro vapo protect (AG,Hamburg, Germany) thermocycler. After 5 min at 94 #C, 35 cy-cles were performed with 30 s at 94 #C, 60 s at the annealingtemperature (depending on the locus) and 30 s at 72 #C, fol-lowed by a final extension step of 5 min at 72 #C. Amplifiedproducts were run on an ABI 3130XL Genetic Analyzer(Applied Biosystems, USA). Analysis and allele size scoringwere performed using the GeneMapper V3.7 software (AppliedBiosystems).

Genetic diversity analyses

PowerMarker 3.25 (Liu and Muse, 2005) was used to esti-mate the total number of alleles (NA), the number of alleleswith a frequency higher than 5 % (NA,P) major allele frequency,total number of genotypes (NG) and the polymorphic informa-tion content (PIC) at each locus. The GenAlEx 6.5 program(Peakall and Smouse 2006, 2012) was used to calculate the ob-served (Ho) and the expected (He) heterozygosities. The in-breeding coefficient (FIS) and the genetic differentiation (FST)were computed according to the formula of Weir andCockerham (1984) using GENEPOP 4.0 (Raymond andRousset, 1995). GENEPOP software was also used to estimatelinkage disequilibrium. v2 tests were used to determine the sig-nificance of the linkage disequilibrium (Weir, 1996). In addi-tion, the multilocus characterization of the sample was testedby the use of Ohta’s parameters (Ohta, 1982). DIT

2, DIS2, DST

2,D0IS

2 and D0ST2 are the total variance of linkage disequilibrium,

the components due to linkage within groups, allelic differentia-tion between pairwise loci, gametic differentiation betweenpairwise loci and linkage disequilibrium in the total sample,respectively.

Genetic structure analysis

To identify the population structure of the date palm collec-tion, we employed a model-based clustering algorithm imple-mented in the computer program STRUCTURE version 2.2(Pritchard et al., 2000). This algorithm identifies the optimalnumber of clusters (K) with different allele frequencies and as-signs portions of individual genotypes to these clusters. It as-sumes Hardy–Weinberg equilibrium and linkage equilibriumwithin clusters. The STRUCTURE algorithm was run withoutprevious information about the geographic origin of the acces-sions, using a model with admixture and correlated allele fre-quencies, and with ten independent replicate runs for each Kvalue (K ranging from 1 to 10). For each run, we used a burn-in

period of 10 000 iterations, and a post-burn-in simulation lengthof 1 000 000. The most probable number of clusters was as-signed by using the run with the maximum likelihood, whichwas validated with an ad hoc quantity based on the second-order rate of change in the log probability of data betweendifferent K values (Evanno et al., 2005). To obtain optimalalignment of the independent runs, the CLUMPP version 1.1software (Jakobsson and Rosenberg, 2007) was used withgreedy algorithms, 10 000 random input orders and 10 000 re-peats, to calculate the average pairwise similarity (H’) of runs.The output obtained was used as input by the clusterDISTRUCT version 1.1 visualization program (Rosenberg,2004).

Date palm genetic relationships between populations

The GenAlEx 6.5 program (Peakall and Smouse, 2006,2012) was used to calculate the number of alleles per locus andper group (NA) as well as the number of alleles with a fre-quency higher than 5 % (NA,P). PowerMarker 3.25 (Liu andMuse, 2005) was used to estimate the observed (Ho) and the ex-pected (He) heterozygosities, fixation index values (FIS) and theallelic richness of each group.

To generate hierarchical classifications, we calculated theshared allele distance (DAS) (Chakraborty and Jin, 1993) amongthe ten pre-defined populations. We used the obtained distancematrix to construct the dendrogram using the Neighbor–Joining(NJ) algorithm. Bootstrap values were computed over 10 000replications with the PowerMarker V3.25 software (Liu andMuse, 2005).

Multivariate analysis was performed to examine the relation-ship between accessions, using discriminant analysis of princi-pal components (DAPC) (Jombart et al., 2010), carried out onthe allele frequency matrix using the R software (R Core Team,2012).

The genetic differentiation generated by the 18 loci was esti-mated by calculating FST according to the formula of Weir andCockerham (1984) using GENEPOP 4.0, and Fisher’s method(Raymond and Rousset, 1995) was applied to test the signifi-cance of pairwise FST values. The analysis of molecular vari-ance (AMOVA) implemented in the GenAlEx 6.5 program(Peakall and Smouse, 2006, 2012) was conducted to estimatethe hierarchical differentiation at two levels: a country of originlevel and a region level, distinguishing two geographic regions:an eastern region and a western region. The FST significancewas determined by running 10 000 permutations.

RESULTS

SSR polymorphism

A total of 237 alleles were identified across the 18 nuclear SSRloci studied, varying from two (PdCUC3-ssr1) to 33 (PdAG1-ssr) per locus (Table 1). The number of alleles per locus with afrequency >5 % ranged from one (PdCUC3-ssr1 andmPdIRD013) to seven (mPdCIR85 and PdCUC3-ssr2). The av-erage number of alleles per locus was 13$17, but dropped to4$22 when rare alleles (i.e. with a frequency of <5 %) were re-moved. The major allele showed a highly variable frequency




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from one locus to another, ranging from 0$158 (mPdCIR078) to0$992 (PdCUC3-ssr1) (Supplementary Data Fig. S1). The num-ber of genotypes per locus varied from two (PdCUC3-ssr1) to94 (mPdCIR078), with an average of 37$11 and with a totalnumber of 668. The average PIC value for the 18 loci was0$605, and the most informative locus was mPdCIR78 (0$888).The Nei’s genetic diversity ranged from 0$190 (PdCUC3-ssr1)to 0$812 (PdAG1-ssr) with an average of 0$569 (Table 1), sug-gesting that the examined date palm germplasm has high levelsof polymorphism. Three of the 18 SSR loci displayed a signifi-cant heterozygosity deficit (P< 0$01) (mPdCIR035 andmPdCIR063 loci and PdCUC3-ssr2) and the mPdCIR015 locusdisplayed a significant heterozygosity excess (P< 0$01).

Variance components of linkage disequilibrium

A total of 153 two-locus pairs were analysed to estimate vari-ance components of linkage disequilibrium. The total varianceof the disequilibrium DIT

2 was of 0$0287. D0IS2 (0$0255) was

larger than D0ST2 (0$0001); in addition, the DST

2 (0$0015) valuewas larger than DIS

2 (0$0006), signifying that a greater propor-tion of total variance in disequilibrium resulted from deviationamong sub-groups rather than from within groups. Out of 153pairs of loci, 52 significant non-random associations were de-tected for SSR loci combinations assessed by the v2 test(Supplementary Data Table S3). As shown in Table 2, non-random association of the SSR alleles at particular variable lociwas mainly caused by limited migration and random process orgenetic drift. In fact, the dual relationship I (DIS

2<DST2;

D0IS2>D0ST

2) was only found for 11 loci pairs. However, thedual relationship II (DIS

2>DST2; D0IS

2>D0ST2), characteristic

of non-systematic disequilibrium, was found for all other pairs.

Model-based Bayesian clustering analysis

The model-based Bayesian clustering approach implementedin STRUCTURE (Pritchard et al., 2000) was used to investigatethe genetic structure of date palm according to the models withtwo clusters (K¼ 2) to six clusters (K¼ 6). The change rate inthe log likelihood between successive K values (DK) (Evannoet al., 2005) revealed a first level of clustering at K¼ 2 for thestudied date palm accessions (DK¼ 1428$11) (Fig. 2;Supplementary Data Fig. S2).

Based on the average pairwise similarity of individual as-signments across runs (H0) generated by CLUMPP for the tenSTRUCTURE runs, the highest similarity coefficient (H0) wasobserved for K¼ 2 (H0¼ 0$997), indicating the stability of theresults for this model (Fig. 2).

At K¼ 2, the date palm accessions were differentiated intotwo geographic clusters, the first one named the Western clusterconsisted of accessions from North Africa except those fromDjibouti, and the second cluster named the Eastern cluster con-sisted of the accessions originated from Asia and those fromDjibouti (Fig. 2). At K¼ 3, the accessions from Djibouti wereseparated from those collected in Asia and an admixture wasobserved in the Egyptian group. At K¼ 4, a fourth cluster wasidentified, including the Mauritanian and Algerian accessions.

At K¼ 5, the Egyptian accessions were separated from theother western accessions. At K¼ 6, the genetic structure of datepalm within two main gene pools was not modified since theaccessions of the sixth cluster were not consistently assignedand hence no meaningful additional cluster was observed(Fig. 2).

It is important to note that the Tunisian, Omanian and UAEgroups showed minimal admixture signal and were clearlymaintained for all K values. This allowed us to consider that theTunisian group is the most characteristic of the Western clusterdiversity, whereas the Omanian and the UAE groups are thebest representatives of the Eastern cluster.

Genetic diversity of the chloroplast minisatellite

Two haplotypes (242 and 254 bp), corresponding, respec-tively to three and four repetitions of the 12 bp minisatellite,were identified in the date palm accessions studied, correspond-ing to the two chlorotypes previously identified (Pintaud et al.,2013). Their frequencies were significantly different betweenthe two geographic regions (G-tests, P< 0$05). The Orientalchlorotype (254 bp) was present at a very high frequency ineastern date palms (0$965). A total of 139 individuals out of144 displayed this chlorotype and only five accessions had theOccidental chlorotype. The African date palms presentedthe two chlorotypes; however, the 242 bp haplotype (theOccidental chlorotype) is slightly more abundant, with an allelefrequency of 0$529 (80 samples out of 151), with a maximumvalue obtained for the Tunisian group (Fig. 3). It is important tonote that only the Occidental chlorotype was found in theTunisian samples collected on the islands of Kerkennah andDjerba, representing spontaneous populations (Ben Salah,2010; Pintaud et al., 2013).

Genetic structure and diversity between date palm geographicgroups

The genetic diversity levels within the ten defined geo-graphic groups measured by the estimators differed. In the

TABLE 2. Dual relationships and average values of significant disequilibrium coefficients for 32pairs of SSR loci

Dual relationships No. of locus pairs Ohta coefficient

DIS2 D0IS

2 DST2 D0ST

2 D0IT2

I. DIS2<DST

2; D0IS2>D0ST

2 41 0$0005 0$0285 0$0017 0$0001 0$0319II. DIS

2>DST2; D0IS

2>D0ST2 10 0$0010 0$0145 0$0008 0$0001 0$0173

All 52 0$0005 0$0255 0$0015 0$0001 0$0028




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western groups, Egypt showed the highest expected heterozy-gosity values, with 0$610, while the Mauritanian group had thelowest, with 0$476 (Table 3). The observed heterozygosity wasthe highest for Algeria (0$616) and the lowest for Mauritania(0$460). As the number of alleles observed in a group is highlydependent on the sample size, the allelic richness was computedfor each group and region (Table 3). The highest allelic richnesswas detected for the Egyptian group, with 5$904, while the low-est value was noted in Mauritania (4$166). When the easternand western regions were considered, the number of privatealleles was higher in the eastern region, whereas the valuesof allelic richness were equivalent between the two regions(Table 3).

The defined groups displayed substantial genetic differentia-tion since the average FST value was 0$107, ranging from 0$004for pairwise comparisons between the Oman and UAE groupsto 0$152 between the Oman and Tunisian groups (Table 4). Allpairwise FST values were significant at P< 10–4 (Table 4),

except for those observed between the UAE and Oman and be-tween Iraq and Pakistan. The same result was obtained withDAS distances, where the highest distances were obtained be-tween Tunisia and the two eastern groups: Oman and the UAE(Table 4).

Genetic relationships among the pre-defined date palmgroups were also assessed based on DAS genetic distances andthe NJ algorithm (Fig. 4). According to the bootstrap values,the ten date palm groups were classified into two clusters: clus-ter I, including Mauritania, Morocco, Algeria, Tunisia andEgypt and corresponding to the Western cluster, was clearlydistinguished from cluster II (or Eastern cluster), includingDjibouti, Oman, Iraq, the UAE and Pakistan groups, by a highbootstrap support of 80 % (Fig. 4).

The DAPC performed on allelic frequencies and with a pro-portion of conserved variance of 0$85 showed that the first axisdifferentiated the eastern from the western geographic groups(Fig. 5), and the second axis differentiated Mauritania and

K = 2H ′= 0·997

DK = 1428·11

K = 3H ′= 0·974DK = 0·850

K = 4H ′= 0·798DK = 1·097

K = 5H ′= 0·788DK = 3·853

K = 6H ′= 0·763DK = 3·552

Mau

ritan

iaM

oroc

co

Alge

ria

Tuni

sia

Egyp

t

Djib

outi

Om

an

UAE

Iraq

Pakis

tan

FIG. 2. Inferred population structure for K¼ 2 to K¼ 6 as the presumed number of sub-populations within the date palm collection, including 295 accessions. Thebar plot chart, generated by DISTRUCT, depicts classifications with the highest probability among assumed clusters in the date palm groups. Each individual is rep-resented by a vertical bar, partitioned into coloured segments representing the proportion of the individual’s genome in the K clusters. The date palm groups are sepa-

rated by a black line.




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1·0

0·9

0·8

0·7

0·6

0·5

0·4

0·3

0·2

Group

Fre

quen

cy

0·1

0

Maurita

nia

Moroc

co

Algeria

Tunisi

aEgy

pt

Djibou

tiUAE

Oman Iraq

Pakist

an

Haplotype 242Haplotype 254

FIG. 3. Frequencies of the occidental (red column charts) and oriental (green column charts) chlorotypes.

TABLE 3. Genetic diversity within geographic date palm groups and regions

Population Western region Eastern region

Maur 14 Mc 21 Al 50 Tn 38 Eg 28 All 151 Dj 18 Om 34 UAE 24 Iq 18 Pak 50 All 144

NA 4$167 5$944 7$111 5$833 6$944 9$000 5$833 5$611 5$722 5$167 8$111 10$722NA,P 3$000 4$000 4$278 3$833 4$667 4$167 3$778 3$722 3$889 3$722 3$944 3$833He 0$476 0$598 0$590 0$580 0$610 0$614 0$564 0$539 0$539 0$577 0$593 0$588Ho 0$460 0$606 0$616 0$579 0$566 0$582 0$559 0$570 0$583 0$590 0$583 0$578FIS 0$070 0$011 "0$033* 0$014 0$089*** 0$052 0$038* "0$043 "0$060 0$007 0$027 0$008Ar 4$166 5$423 5$422 4$783 5$904 5$140 5$343 4$676 5$037 4$818 5$629 5$100

Maur, Mauritania; Mc, Morocco; Al, Algeria; Tn, Tunisia; Eg, Egypt; Dj, Djibouti; Ir, Iraq; Om, Oman; Pak, Pakistan.NA, number of alleles per locus; NA,P, number of alleles with a frequency >5 %; He expected heterozygosity; Ho observed heterozygosity; FIS, fixation index

values; Ar, allelic richness.Exact test significant at *P< 0$05, ***P< 0$001.

TABLE 4. Geographic group pairwise comparisons

Maur Mc Al Tn Eg Dj Om UAE Iq Pak

Maur – 0$101 0$093 0$148 0$140 0$157 0$182 0$170 0$195 0$173Mc 0$066* – 0$064 0$057 0$084 0$165 0$201 0$213 0$180 0$169Al 0$059* 0$036* – 0$072 0$097 0$140 0$190 0$183 0$162 0$165Tn 0$093* 0$031* 0$037* – 0$124 0$257 0$276 0$275 0$254 0$251Eg 0$085* 0$040* 0$054* 0$065* – 0$115 0$191 0$187 0$133 0$137Dj 0$102* 0$087* 0$079* 0$136* 0$062* – 0$112 0$122 0$066 0$065Om 0$116* 0$114* 0$105* 0$152* 0$104* 0$070* – 0$ 040 0$042 0$037UAE 0$118* 0$119* 0$105* 0$151* 0$104* 0$080* 0$004 – 0$065 0$040Iq 0$119* 0$089* 0$085* 0$129* 0$067* 0$036* 0$042* 0$046* – 0$011Pak 0$104* 0$087* 0$089* 0$132* 0$072* 0$035* 0$023* 0$028* 0$006 –

DAS, genetic distances (above diagonal) and FST (below diagonal); global FST¼ 0$107.*P> 10–4.Maur, Mauritania; Mc, Morocco; Al, Algeria; Tn, Tunisia; Eg, Egypt; Dj, Djibouti; Ir, Iraq; Om, Oman; Pak, Pakistan.




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Egypt from the other western groups and Djibouti from theeastern ones. The observed group distribution reflected theirgeographic location; Egypt and Djibouti were found at an inter-mediate position between the two eastern and western regions.

Based on the AMOVA, the genetic variance was about 7 %among these two defined regions and 4 % among date palmpopulation groups (Table 5). The majority of total geneticvariance (89 %) was due to the variation within groups.

The number of accessions assigned to their geographic group(ten countries) was 204 (69$15 %) and the number of accessionsassigned to their region (western or eastern) was 286 (96$95 %)(Table 6).

DISCUSSION

Date palm germplasm is structured into two gene pools

The current study used both nuclear SSR and chloroplast micro-satellite genotyping to analyse the diversity and genetic

structure of date palm accessions collected in ten countriesfrom Mauritania to Pakistan.

A total of 295 accessions were analysed using 18 nuclearSSRs loci which revealed >200 alleles (approx. 13 alleles perlocus). This result highlights a high degree of genetic diversitywithin the studied date palm sampling and shows an average al-lele per locus significantly higher than that obtained in severalprevious studies focusing on a single country, such as Tunisia(9$71) (Zehdi et al., 2012), Iraq (8$54) (Khierallah et al., 2011)or Qatar (7$7) (Elmeer et al., 2011).

The analyses of the genetic structure of the date palm popula-tion, using the Bayesian clustering approach, NJ hierarchicalclassification and DACP, are clearly consistent with a geo-graphical structuring into two clusters. The first one, named theEastern pool, consists of the date palm accessions fromDjibouti, Oman, Iraq, the UAE and Pakistan groups, and thesecond, named the Western pool, consists of the remaining ac-cessions from Africa, including Mauritania, Morocco, Algeria,Tunisia and Egypt groups. This geographic divergence is highlysignificant (FST¼ 0$077, P¼ 0$001). Furthermore, the

UAE

Oman

100

56

62

94

0·1

Iraq

Pakistan

Djibouti

Egypt

Tunisia

Morocco

Algeria

Mauritania

79

88

FIG. 4. NJ clustering of geographic groups based on DAS genetic distance values, as well as the distribution of the genetic clusters within each of them. The colourscorrespond to genetic clusters defined by the STRUCTURE analysis, as reported in Fig. 1 with cluster I in green and cluster II in red (pie charts). The numbers next

to the nodes indicate bootstrap support percentages in 1000 pseudoreplicates.




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assignation percentage of accessions reached 69$15 % when theten pre-defined groups were considered and it increased up to96$95 % when the western and eastern regions were considered.In addition, DAS distances showed the highest values whencomparing Western vs. Eastern groups, in particular Tunisia vs.Oman or UAE.

These results are consistent with those of Cherif et al.(2013), that identified an east–west structure in male-specificmicrosatellite alleles using a set of male and female date palmgenotypes representative of the diversity of the species. The dis-tribution of Y haplotypes in western and eastern haplogroupsallowed two male ancestral paternal lineages to be traced

accounting for all known Y diversity in date palm (Cherif et al.,2013).

Al-Ruqaishi et al. (2008) also used microsatellite markers toanalyse the genetic diversity among genotypes of date palmfrom Oman, Bahrain and Iraq, and one Moroccan genotypeMedjool that appeared highly distinct from the Middle Eastaccessions.

DA eigenvalues

UAEOm

Pak

Iq

Dj

Eg

Al

Mc

Tn

Maur

FIG. 5. Discriminant analysis of principal components (DAPC) on SSR data of genotypes. The colours of the groups correspond to the geographic groups. Axes 1and 2 explain 83 % of the total variance (shaded vertical bars in the eigenvalue histogram). Maur, Mauritania; Mc, Morocco; Al, Algeria; Tn, Tunisia; Eg, Egypt;

Dj, Djibouti; Ir, Iraq; Om, Oman; Pak, Pakistan.

TABLE 5. Analysis of molecular variance (AMOVA) for the clus-ters of date palm accessions based on 18 SSR markers

Source of variation d.f. Sum ofsquares

Estimatedvariability

Percentagevariance

Among regions 1 138$471 0$398 7*Among groups 8 145$974 0$230 4*Within groups 580 3049$454 5$258 89*Total 589 3333$900 5$886

The region level considered the Western and Eastern clusters; the grouplevel included the ten geographic date palm groups.

*P< 0$001 based on 999 permutations.

TABLE 6. Pattern of assigned accessions per geographic groupand region

Region Group Total Assigned group Assigned region

Self-group Othergroup

Self-region Otherregion

Western Mauritania 14 11 3 14 0Morocco 21 11 10 19 2Algeria 50 39 11 49 1Tunisia 38 36 2 38 0Egypt 28 23 5 23 5

Eastern Djibouti 18 11 7 18 0Oman 34 24 10 34 0UAE 24 13 11 24 0Iraq 18 11 7 17 1Pakistan 50 25 25 50 0Total (%) 295 204 (69$15) 91 (30$85) 286 (96$95) 9 (3$05)




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Several genetic analyses performed on date palm previouslysuggested the existence of eastern and western gene pools(Pintaud et al., 2013). Elshibli and Korpelainen (2008) com-pared the genetic diversity of date palm from Sudan andMorocco and showed a significant differentiation betweenthem. Additionally, Zehdi et al. (2012) showed that the easterngroup was genetically different from the Tunisian groups.Arabnezhad et al. (2012) obtained the same result using a newset of SSR markers in a different date palm collection fromIran, Iraq and North Africa.

Similar outcomes were reported in genetic structuring analy-ses of other Mediterranean crops. Indeed, numerous geneticstudies on olive trees reported a genetic differentiation betweenwestern and eastern Mediterranean areas (Besnard et al., 2002,2007, 2013; Breton et al., 2006; 2009 Sarri et al., 2006;Haouane et al., 2011) and showed the structuring of theMediterranean olive germplasm into three main gene pools,strongly matching three distinct geographic areas, i.e. western,central and eastern Mediterranean regions. The genetic struc-ture of cultivated grapevine revealed three main genetic groups:cultivars from western regions, from the Balkans and EasternEurope, and from Eastern Mediterranean, Caucasus, Middleand Far East countries (Bacilieri et al., 2013). In apricots, theMediterranean germplasm was also structured into three maingene pools: Irano-Caucasian, North Mediterranean Basin andSouth Mediterranean Basin (Bourguiba et al., 2012). All thesestudies indicate that the main Mediterranean crops have morethan one cradle of genetic diversification, being strongly linkedto geographic distribution. In line with these conclusions, ourfindings in date palm revealed a deep genetic structuring arisingfrom two geographic gene pools.

Genetic history of cultivated date palm

The currently accepted hypothesis for date palm domestica-tion history is a single origin around the Persian Gulf, followedby the diffusion of the cultivated date palms toward the west upto Morocco and the east up to India (Munier, 1973). In the pre-sent study, however, the nuclear SSR markers have shown thatthe western accessions strongly differed from the eastern ones.Assuming a single eastern origin, this pattern of diversity wouldbe observed only in the case of a strong bottleneck during ex-pansion toward the west. This would have led to an importantgenetic drift and therefore a loss or a fixation of alleles in thewestern group that therefore would have displayed a very dif-ferent allelic pattern from that of the eastern group. However,the diversity levels observed in the Eastern and Western clus-ters appear similar, questioning the accuracy of the single originhypothesis, in accordance with previous morphometric data(Terral et al., 2012).

The strong geographic structure between the Eastern andWestern pools highlighted here suggests another hypothesis,which is the existence of two cultivation origins, one in the eastand one in the west. The Persian Gulf might indeed not havebeen the only domestication centre for date palms. North Africacould also have been a domestication centre, either independent(primary domestication) or after the introduction of genotypesfrom the Middle East and their crossing with local date palms(secondary domestication). The origin of this structuration is,

however, imperfectly known due to the still limited informationabout distribution and genetic structure of wild date palm popu-lations in the two diversification centres (Gros-Balthazard,2012).

The plastid data provide additional insights on this structure.The two previously identified chlorotypes, Oriental andOccidental (Henderson et al., 2006; Pintaud et al., 2010, 2013),were also recovered in the present study. Investigations on thechloroplast genome have shown that these two haplotypes un-derlie a strong genetic differentiation. Indeed, sequence align-ment of approx. 50 kb of an Occidental chloroplast from Elche,Spain and an Oriental chloroplast (cultivar Khalass) from theArabic peninsula revealed 18 SNPs along with a number ofother mutations, including microsatellites, indels and small in-versions (Scarcelli et al., 2011), while the comparison of twocomplete sequences of Oriental chloroplast genomes, of culti-vars Khalass and Aseel, resulted in only three SNP (Kahn et al.,2012).

The Eastern cluster showed a predominance of the Orientalchlorotype with a mean of 96$5 % and it reached 100 % in theDjiboutian, UAE and Omanian groups. The Western cluster,however, revealed both haplotypes but with a predominance ofthe Occidental chlorotype and with a maximum value in theTunisian group. The most parsimonious hypothesis is that eachcluster first had its own haplotype before population move-ments and anthropogenic actions resulted in the disseminationof the Oriental chlorotype in the Western cluster, and vice versato a lesser extent. The high level of genetic diversity and the co-existence of the two chlorotypes in the Western cluster on theother hand indicate that the diffusion of accessions from theeastern to the western region was more important.

The absence of the Occidental chlorotype in the Djiboutian,Omanian and UAE groups, as well as the absence of theOriental chlorotype in the Tunisian island’s samples strengthensthis hypothesis. However, this pattern could also be observed inthe case of a strong reduction of population size leading to ge-netic drift and/or selection of one or the other chlorotype.Therefore, the identification of wild populations and their geno-typing is necessary to test these hypotheses.

While the study of the nuclear genome using SSR markersrevealed that this component is highly structured between west-ern and eastern date palms, the previously identified chloro-types (Occidental and Oriental) were not always related to theregion, particularly in the west. Consequently, some groups pre-sent a western nuclear genome associated with the Orientalchlorotype, which is particularly the case in Mauritania andAlgeria. The chloroplast provides information concerning thematernal origin of the plant. This pattern could be observed iffemale genotypes were initially imported from the eastern tothe western regions and were subsequently pollinated by localmales (displaying the Occidental chlorotype) over generations,leading to new populations with a predominantly western nu-clear genome and Oriental chlorotype. This would be in accor-dance with the fact that date palm cultivation has always beenbased on the selection of female accessions.

In the STRUCTURE analysis based on microsatellites, wehave noted the presence of admix genotypes (33 out of the 295)(self-population assignment <0$85). These genotypes couldrepresent hybrids between eastern and western plants and aresignificant in the border area between Asia and Africa. This




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hypothesis has been confirmed by the presence of the twochlorotypes with similar frequencies in the Egyptian samples.This can be explained by the fact that Egypt is a strategic areacrossed by most nomads and pilgrims who imported date fruitsfrom the Middle East to the Maghreb and vice versa. However,the high frequency of the Oriental chlorotype in Mauritanianand Algerian date palms could also be the result of recent intro-duction or import of accessions.

Conclusions

The results presented here show that date palm displays highgenetic diversity and that the genetic variation is geographicallystructured. Indeed, nuclear SSRs have confirmed the existenceof two pools named Eastern and Western. Eastern accessionsare substantially different from the Western ones, suggestingthat they each have their own autochthonous origin. Therefore,this strong differentiation confirms the existence of at least twodomestication events. Northern Africa would also have been ei-ther a primary or a secondary domestication centre.

The present study gives a detailed picture of date palm ge-netic diversity across its traditional cultivation area in the OldWorld. This information can be used to establish core collec-tions useful in breeding strategies for agronomically interestingtraits such as fruit quality or resistance to biotic and abioticstress through genome-wide association studies.

SUPPLEMENTARY DATA

Supplementary data are available online at www.aob.oxford-journals.org and consist of the following. Table S1: date palmgermplasm accessions, region, code, sex, origin and chlorotype.Table S2: characteristics of the 18 microsatellite and the plastidminisatellite markers. Table S3: analysis of significant linkagedisequilibrium. Figure S1: histogram illustrating the frequencydistributions of alleles for each microsatellite among groups.Figure S2: DK plotted against K values.

ACKNOWLEDGEMENTS

We thank Abdelmajid Rhouma, Ali Zouba, Alain Borgel,Mohamed Kneyta and Djibril Sane for kindly providing datepalm leaf material. This work was financed by the AUFMeRSi project (6313PS001), the Tunisian Ministere del’Enseignement Superieur et de la Recherche Scientifique, theDirection Generale de la Recherche Scientifique et duDeveloppement Technologique (DGRSDT) in Algeria, theQatar National Research Fund (NPRP-EP X-014-4-001),ANR Phoenix and ANR Fructimedhis

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