Impact of Mapped SSR Markers on the Genetic Diversityof Apricot (Prunus armeniaca L.) in Tunisia

Hedia Bourguiba & Lamia Krichen &

Jean-Marc Audergon & Bouchaib Khadari &Neila Trifi-Farah

Published online: 23 February 2010# Springer-Verlag 2010

Abstract The impact of mapped microsatellites on thestudy of genetic diversity of Tunisian apricot accessionswas assessed. The genetic variability of 47 traditionalapricot cultivars originating from several areas in Tunisiawas investigated with 32 polymorphic microsatellite lociselected for their location throughout the eight linkagegroups of Prunus genome. The higher polymorphism andgreater transportability of these markers among Prunusspecies were proved by the expected heterozygosity (He=0.56) and Shannon’s index of diversity (I=1.05), indicatingthat Tunisian apricot germplasm maintained a substantiallevel of genetic diversity. According to their geographicalorigin, the genetic differentiation among groups (north,center, and south; Fst=0.04) was lower, while the gene flowamong groups was consequent (Nm=4.79), attesting anarrow genetic background of apricot in the country. Bothunweighted pair-group method with arithmetic meandendrogram, based on Nei’s genetic distances and factorialcorrespondence analysis, separated northern cultivars fromcentral and southern cultivars, revealing the same molecular

basis of apricot material in the Center and the South ofTunisia. These results revealed the efficiency of mappedmarkers for genetic variability measurements compared torandomly ones, however, no advantage was observed consid-ering the genetic relationships among studied accessions.

Keywords Genetic diversity . Linkage groups .Mappedmarkers . Microsatellites .Prunus armeniaca L.

AbbreviationsAFLP Amplified fragment length polymorphismDNA Deoxyribonucleic acidFCA Factorial correspondence analysisLG Linkage groupPCR Polymerase chain reactionsSSR Simple sequence repeatUPGMA Unweighted pair-group method with arithmetic

mean

Introduction

Apricot, Prunus armeniaca L., is cultivated in Tunisia inseveral areas throughout the country. It is adapted to diverseclimatic conditions: from the sub-humid climate of northernareas to the Saharan climate in the southern oasian zones.According to propagation method, Tunisian apricot materialmay be divided into two main groups: oasian localaccessions propagated by seed and traditional local culti-vars propagated by grafting. An important variabilityamong Tunisian apricot landraces is revealed by morpho-logical characters and pomological descriptions (Valdeyronand Crossa-Raynaud 1950; Crossa-Raynaud 1960; Carrautand Crossa-Raynaud 1974; Krichen 2001; Krichen et al.

H. Bourguiba : L. Krichen :N. Trifi-Farah (*)Laboratoire de Génétique Moléculaire,Immunologie et Biotechnologie, Faculté des Sciences de Tunis,Campus universitaire,2092 El Manar, Tunisiee-mail: Neila.Trifi@fst.rnu.tn

J.-M. AudergonINRA, UR 1052 Génétique et Amélioration des Fruits etLégumes, Domaine,Saint-Maurice,81143 Montfavet, France

B. KhadariINRA, UMR 1098 Développement et Amélioration des Plantes,2 Place Viala,34060 Montpellier cedex 1, France

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2006a). Many of these apricot cultivars are empiricallyselected mainly for their fruit qualities, conserved andclonally propagated by farmers. These landraces cultivarsare currently under severe genetic erosion as a consequenceof the urbanization and the farmer’s change of apricot tomore economically profitable crops. Thereby, the molecularcharacterization of the genetic variability of these apricotaccessions was assessed with the aim of protecting theTunisian apricot germplasm. Several molecular markershave been developed that allow the identification and studyof the genetic diversity of Tunisian apricot accessions, suchas amplified fragment length polymorphism (AFLPs) andsimple sequence repeats (microsatellites or SSRs; Krichenet al. 2006b, 2008).

Because of their abundance, codominant Mendelianinheritance, and high level of polymorphism, microsatellitemarkers were widely used for the improvement of newbreeding programs. Genomic SSR markers have beendeveloped in almost every cultivated fruit species of Prunus,including peach (Cipriani et al. 1999; Sosinski et al. 2000;Aranzana et al. 2003; Dirlewanger et al. 2004), Japaneseplum (Mnejja et al. 2004), almond (Mnejja et al. 2005), andapricot (Lopes et al. 2002; Decroocq et al. 2003; Messina etal. 2004). Previous studies used microsatellite primersdeveloped for Japanese plum (Prunus salicina) and almond(Prunus dulcis) and selected randomly without knowledgeof the map positions. Five microsatellites primers permittedto discriminate among 54 Tunisian cultivars with 100%accuracy and were exploited to establish an identificationkey used to resolve confusions in nomenclature (homonymsand synonyms; Krichen et al. 2006b).

In recent years, genetic linkage maps have beenconstructed for several Prunus species such as almond(Joobeur et al. 2000), Prunus davidiana (Decroocq et al.2005), and apricot (Lambert et al. 2004; Dondini et al.2007) as well as for interspecific almond × peach crossesproviding among others, the Prunus reference map codedas “TxE” (“Texas” × “Earlygold”; Joobeur et al. 1998;Aranzana et al. 2003). In this paper, the choice of micro-satellites loci was determined by the position of anchor SSRloci in the “TxE” reference map in comparison with thoselocated in the existing apricot genetic linkage maps(Lambert et al. 2004; Dondini et al. 2007). In this context,SSRs markers were selected according to their distributionthroughout the Prunus genome to ensure that most regionsof the genome are represented in the data sets used as wellas their polymorphism in Tunisian apricot accessions. It isworth noting that the considered mapped microsatellitesmarkers were used for the first time to characterize thegenetic variability of traditional apricot cultivars in Tunisia.For that purpose, thirty-two single-locus SSR markerscovering the whole Prunus genome were chosen to amplifyDNA from 47 apricot grafted accessions, considered as the

most representative cultivars of the apricot variability inTunisia on the basis of morphological and AFLP markers(Krichen 2007).

The aims of this work were to: (1) assess thepolymorphism and transportability of SSR primers obtainedfrom other Prunus plants in Tunisian apricot accessions; (2)reveal the molecular basis of genetic diversity and relation-ships of Tunisian cultivars; (3) estimate the impact of usingmapped SSR markers on the pattern of diversity of Tunisianapricot germplasm, by comparison with previous studiesinvolving randomly markers.

Materials and Methods

Plant Materials

A total of 47 domesticated cultivars propagated by graftingwere collected from nine areas of apricot cultivation inTunisia. These areas were Ras Jbel and Testour in the north,Kairouan, Mahdia and Sfax in the center and Gabes, Jerba,Gafsa, and Midess in the south (Fig. 1). Surveyed areaswere the sub-humid bioclimatic zone in the north to the aridinferior bioclimatic zone in the south. Based on theinformation provided by Krichen (2007), these apricotcultivars were recognized as the most representativetraditional accessions of the local variability using morpho-logical characters and AFLP markers.

DNA Extraction and Primer Screening

Genomic DNA was extracted from fresh young leavesaccording to the maxi-prep procedure described by Ber-natzky and Tanksley (1986). Genotyping was conductedwith a set of 32 SSR primers selected for their position asthey are distributed throughout the eight linkage groups ofPrunus genome (Table 1).

PCR Amplification and Electrophoresis

Polymerase chain reactions (PCR) were carried out in a20 µl reaction mix containing 20 ng of template DNA,2 mM of MgCl2, 4 pmol of the reverse primer and 1 pmolof the forward primer, 0.2 mM of each deoxynucleotidetriphosphate, and 1 unit of Taq polymerase (Sigma).Reverse primers were unlabeled while forward primerswere labeled on their 5′ end using one of the followingfluorescent compounds: FAM, NED, or HEX, to enableanalysis on automated sequencers. Amplifications wereperformed on a MasterCycler, ep gradient S (Eppendorf)with the following conditions: 5 min initial denaturationstep at 94°C followed by 35 cycles of amplification at 94°Cfor 30 s, T° annealing (depending on the locus) for 1 min,

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and 72°C for 1 min. This was ended by a final extensionstep at 72°C for 10 min.

PCR products were detected using capillary electropho-resis on an ABI prism 3130xl automatic DNA Sequencer(Applied Biosystems). Samples were prepared by mixing3 µl of diluted PCR products with 7.85 µl formamide and0.15 µl Rox-labeled GenSize 400HD. Fragment sizing wasperformed using the GENEMAPPER V3.7 software(Applied Biosystems).

Data Analysis

XLSTAT version 2009 software (http://www.xlstat.com)was employed to assess Pearson correlation matrix in orderto study the effect of linkage groups on the genetic diversityof Tunisian apricot germplasm. The information of micro-satellite loci was estimated by allele and genotype numberper locus and polymorphic information content (PIC) usingPowerMarker V3.25 software (Liu and Muse 2005). Theobserved and expected heterozygosity values (Ho and He)were calculated by GENETIX 4.05 program (Belkhir et al.1996–2004). Wright’s analysis of hierarchical F-statistics(Fis and Fst) was estimated according to the formula ofWeir and Cockerham (1984) using GENEPOP 4.0 program(Raymond and Rousset 1995). The POPGENE 1.31software (Francis and Yang 1993) served as a tool tocompute Shannon’s index of diversity (I) (Shannon andWeaver 1949), gene flow parameter (Nm) estimated fromthe formula Nm=0.25(1/Fst-1) by Nei (1978), and chi-square test of distribution of SSR allele frequency amongstudied accessions. In addition, genetic identity (GI) andgenetic distance (GD) were calculated according to Nei(1972) between groups of cultivars related to their

geographical origin. The UPGMA cluster analysis basedon Nei’s (1972) genetic distances was performed to furtherreveal the genetic relationships among the apricot acces-sions by TREEVIEW program (Page 1996). Factorialcorrespondence analysis (FCA) was performed withGENETIX software to provide a synthetic representationof the genetic variability of considered accessions.

Results

Microsatellite Selection and Linkage Groups Correlation

In order to have a homogeneous distribution throughout theeight linkage groups (LG) of Prunus genome, 32 micro-satellite primers were selected for their polymorphism inapricot cultivars and their location in different existingPrunus maps (Joobeur et al. 1998; Aranzana et al. 2003;Dirlewanger et al. 2004, 2006; Dondini et al. 2007). Thenumber of chosen loci varied from two for LG1 and LG8 toseven for LG6 with a mean value of four loci by linkagegroup (Table 1).

To detect potential effect of the selected markersgenomic distribution on the diversity patterns, a Pearsoncorrelation matrix assessed linear dependence betweenevery pair of the eight linkage groups (Table 2). Amongall the linkage groups, only LG7 containing CPPCT022,CPPCT033, and CPSCT042 markers showed no significantassociation with the other linkage groups. The significantpositive correlation indicative of a direct relationshipbetween the linkage groups on the genetic diversity ofstudied cultivars was clearly shown by LG1 and LG2, aswell as by LG5 and LG6, and altogether by LGs3, 4, and 5.

Jerba (8)

Mahdia (4)

Mediterranean Sea

Midess (7)

N

Gabes (9)

Ras Jbel (1)

Testour (2)

Kairouan (3)

Sfax (5) Gafsa (6)

L I BYA

A L G E R I A

(1) Addadi Ahmar; Aranji; Bouk Hmed; Bouk Hmed Akhal; Faggoussi; H’midi; Om Younes (2) Bedri Ahmar; Bouthani; Chechi Bazza; Chechi Horr; Oud Aouicha; Oud El Haj Tahar; Oud Gnaa; Oud Hmida; Oud Nakhla; Oud Rhayem; Oud Salah Ben Salam; Oud Tijani (3) Amor El Euch; Baccour; Bayoudhi; Bou Herra; Chechi Dhraa Tammar; Chechi Khit El Oued; Fourati; Khad Hlima; Messelmani; Zbidi (4) Bangui (5) Fourati; Zalouzi (6) Baccour; Bedri Thani; Kasserine 2 (7) Variety of Mahdia (8) Bedri Louzi 67; Bou Khobza; Chechi; Mazouzi 65; Mazouzi 69 (9) Bedri; Bedri Louzi; Ben Souileh; Louzi Thani; Thaleth; Thani

Fig. 1 Map of Tunisia showingthe nine prospected sites and thegeographical origin of the 47characterized apricot cultivars

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These latter showed a strong negative association with LG8(CPPCT006 and UDP98-409) expressing an importantopposite relationship between them (Table 2).

Microsatellite Polymorphism

Forty-seven apricot cultivars were genotyped with 32apricot SSRs. Polymorphic bands were generated for allthe used primers. Alleles were clearly differentiated usingthe capillary electrophoresis sequencer. Altogether, 152alleles were identified in the set of 47 apricot cultivars withan average of 4.75 alleles per locus, ranging from tenalleles for the primer UDP98-409 to two alleles for theprimers BPPCT001, BPPCT040, and Ma014a (Table 3). Atotal of 262 genotypes were identified with an average of8.18 genotypes and a maximum of 21 genotypes for theprimer UDP98-409. The PIC showed that CPSCT039 is the

most polymorphic primer (0.79) and AMPA119 the leastpolymorphic one (0.04). The mean value of PIC for thetotal number of locus was 0.50 (Table 3). Results indicatedthe effectiveness of selected microsatellite markers to detectmolecular polymorphism, thus, they can be used forperforming genetic diversity studies on Tunisian apricot.

Genetic Diversity

The expected heterozygosity (He) for individual loci rangedfrom 0.04 for AMPA119 to 0.82 for CPSCT039, with anaverage value of 0.56, thus revealing a wide geneticdiversity in Tunisian apricot germplasm (Table 3). Thetwo loci AMPA119 and CPSCT039 were previouslydescribed as the least and the most polymorphic loci,respectively (Table 3). The mean value of the observedheterozygosity (Ho) for all loci was 0.52, varying between

Locus Linkage group Repeat motif Sequence reference

CPPCT034 1 (CT)14 Aranzana et al. (2002)

AMPA109 1 (TG)11(AG)9 Hagen et al. (2004)

CPSCT021 2 (GA)15 Mnejja et al. (2004)

BPPCT030 2 (AG)25 Dirlewanger et al. (2002)

BPPCT004 2 (CT)22 Dirlewanger et al. (2002)

BPPCT001 2 (GA)27 Dirlewanger et al. (2002)

AMPA116 2 (GA)10 Hagen et al. (2004)

CPDCT033 3 (CT)18 Mnejja et al. (2005)

AMPA119 3 (TA)9 Hagen et al. (2004)

AMPA101 3 (TC)11(AC)12 Hagen et al. (2004)

UDP97-402 4 (AG)17 Cipriani et al. (1999)

CPSCT039 4 (GA)18 Mnejja et al. (2004)

CPDCT045 4 (GA)16 Mnejja et al. (2005)

BPPCT040 4 (GA)14 Dirlewanger et al. (2002)

CPSCT011 5 (CT)21 Mnejja et al. (2004)

CPSCT006 5 (CT)15 Mnejja et al. (2004)

CPDCT016 5 (GA)19 Mnejja et al. (2005)

BPPCT038 5 (GA)25 Dirlewanger et al. (2002)

BPPCT017 5 (GA)28 Dirlewanger et al. (2002)

AMPA105 5 (AG)11 Hagen et al. (2004)

UDP98-412 6 (AG)28 Testolin et al. (2000)

Ma040a 6 (GA)17 Yamamoto et al. (2002)

Ma014a 6 (GA)21 Yamamoto et al. (2002)

CPPCT030 6 (CT)30 Aranzana et al. (2002)

BPPCT025 6 (GA)29 Dirlewanger et al. (2002)

BPPCT008 6 (GA)36 Dirlewanger et al. (2002)

AMPA100 6 (AG)12 Hagen et al. (2004)

CPSCT042 7 (GA)10 Mnejja et al. (2004)

CPPCT033 7 (CT)16 Aranzana et al. (2002)

CPPCT022 7 (CT)28CAA(CT)20 Aranzana et al. (2002)

UDP98-409 8 (AG)19 Cipriani et al. (1999)

CPPCT006 8 (CT)16 Aranzana et al. (2002)

Table 1 Characteristics of thethirty-two selected SSRsprimers and their linkage groupposition

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0.04 (AMPA119) and 0.82 (CPPCT022 and CPSCT039).The greater part of loci showed no significant differencebetween the expected and observed heterozygosity. Amongthe 32 SSR markers studied, only six loci (AMPA100,BPPCT004, CPDCT016, CPPCT006, CPSCT011, andUDP98-412) showed a significant heterozygosity deficit,while the locus CPPCT022 exhibited a significant hetero-zygosity excess (Table 3). These latter, independent fromtheir linkage groups location, could be implicated incharacters affected by selective pressures. Shannon’sinformation index (I) averaged 1.05, with a maximum of1.86 for AMPA119 and a minimum of 0.11 for UDP98-412(Table 3).

For the Wright’s fixation index, the Fst values calculatedat the loci assayed ranged from 0.001 (BPPCT038) to 0.08(CPSCT011), with an average of 0.04. The mean value ofgene flow (Nm) was estimated from Fst averaged 4.79(Table 3). The observed low level of Fst value indicating ahigh level of gene exchanges could be due to either therelative proximity of geographical areas of the studiedaccessions in Tunisia or the human impact. In spite of thesame location at linkage group 5, the gene flow at locusBPPCT038 (158.72) was extremely higher than Nm atlocus CPSCT011 (2.55). In addition, to assess the contri-bution of all SSR alleles frequency among the definedgroups corresponding to the three geographical origins ofapricot cultivation areas (north, center, and south), the chi-square test demonstrated a significant homogenous distri-bution of alleles at all loci among the geographical groupsexcept for the following loci: AMPA100, BPPCT004,CPDCT016, CPPCT006, CPPCT022, CPSCT011, andUDP98-412 (Table 4) which expressed a significant excessor deficit of heterozygosity.

Genetic Relationships Between Cultivars

To elucidate genetic relationships among Tunisian apricotcultivars, a dendrogram was constructed using the UPGMAanalysis of Nei’s genetic distances (1972) over all 32 SSRloci. The dendrogram classified the 47 studied cultivars intotwo major clusters at 0.16 dissimilarity coefficient (Fig. 2).

Cluster I, which included seven cultivars from Kairouanand Testour, was divided into two subclusters (I.1 and I.2).Subcluster I.1 contained two accessions (“Oud Tijani” and“Chechi Bazza”) from Testour and subcluster I.2 comprisedof five cultivars originated from Kairouan (“Bayoudhi,”“Chechi Dhraa Tammar,” “Chechi Khit El Oued,” and“Khad Hlima”) and Testour (“Chechi Horr”). Cluster IIcontained the remaining cultivars (40 accessions) and couldbe divided into five subgroups (II.1 to II.5), when thedissimilarity coefficient was equal to 0.14. The subgroupsII.1 and II.2, each of them with eight cultivars, werecomposed mainly by cultivars from the northern areas ofapricot cultivation corresponding to Testour and Ras Jbel,respectively. The subgroup II.3 separated the cultivar“Kasserine 2” located in Gafsa from the remaining cultivarsand the subcluster II.4 included the two accessions of thecultivar “Baccour” (from Kairouan and Gafsa, respective-ly). The subcluster II.5 contained 21 cultivars from bothcentral and southern prospected zones of apricot cultivationareas (Fig. 2).

Geographic Group’s Variation

The obtained genetic relationships between cultivars,depending on the cultivars geographical origin (north,center, and south), revealed no significant correlation withthe environmental variables as expressed by the Mantel(1967) test (r=0.91, P value>0.05). In addition, the studyof genetic variation focused on these three groups showed acomparable intra-group molecular diversity with an approx-imately similar mean heterozygosity value ranging from0.49 (central group) to 0.56 (northern group; data notshown).

The two-dimensional scatter plot of factorial correspon-dence analysis coordinates for the first and second factors,which reveal 11.59% and 8.80% of variance, respectively,showed a structured genetic diversity between the consid-ered cultivars according to their geographical origin(Fig. 3). Six accessions from the seven cultivars of thecluster I (“Bayoudhi,” “Chechi Khit El Oued,” “ChechiDhraa Tammar,” “Khad Hlima,” “Chechi Horr,” and “Oud

LG1 LG2 LG3 LG4 LG5 LG6 LG7 LG8

LG1 1.0

LG2 0.74 1.0

LG3 0.50 0.59 1.0

LG4 0.41 0.58 0.83 1.0

LG5 0.46 0.63 0.73 0.84 1.0

LG6 0.43 0.68 0.48 0.57 0.78 1.0

LG7 0.24 0.45 0.15 0.19 0.36 0.62 1.0

LG8 −0.29 −0.27 −0.80 −0.87 −0.82 −0.46 −0.15 1.0

Table 2 Pearson correlationmatrix among the eight linkagegroups (LG) of Prunus genome

Bold values were different from0 at a significant level P=0.05

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Tijani”) were clearly differentiated from the other studiedaccessions according to the first factor. These cultivarsmight be considered as genetically different from theremaining diversity. The rest of the apricot cultivars wereseparated according to the second factor of the FCA in twogroups: cultivars from the northern region are located in thepositive side while both cultivars from the central andsouthern regions are situated in the negative side of theFCA plot (Fig. 3).

Moreover, the estimation of genetic identity and geneticdistance according to Nei (1972) between the three apricotgeographical groups showed that the highest geneticidentity as well as the lowest genetic distance existedbetween central and southern groups (0.93 and 0.07,respectively), indicating that these two geographical regionsof apricot cultivation contained the most similar geneticbackground (Table 5). The study of specific allelespresented in each group revealed that among the 152 alleles

Table 3 Genetic diversity, genetic differentiation, and gene flow of the Tunisian apricot cultivars

Locus Number ofalleles

Number ofgenotypes

Range of allelefrequency

PIC He Ho Fis P value I Fst Nm

AMPA100 6 16 0.04–0.31 0.75 0.79 0.65 0.17 0.006a 1.48 0.07 3.07

AMPA101 4 9 0.06–0.45 0.60 0.67 0.72 −0.07 0.192 1.20 0.05 4.28

AMPA105 5 11 0.07–0.44 0.67 0.72 0.72 −0.00 0.225 0.54 0.00 55.99

AMPA109 3 3 0.02–0.92 0.13 0.14 0.14 −0.05 1.000 1.42 0.05 4.72

AMPA116 5 11 0.01–0.32 0.66 0.72 0.68 0.06 0.052 0.66 0.04 5.27

AMPA119 3 3 0.01–0.98 0.04 0.04 0.04 −0.00 1.000 1.86 0.06 3.72

BPPCT001 2 3 0.38–0.61 0.36 0.47 0.55 −0.16 0.357 1.42 0.04 5.60

BPPCT004 7 13 0.02–0.38 0.67 0.72 0.63 0.12 0.005a 1.11 0.03 6.23

BPPCT008 7 12 0.01–0.45 0.68 0.72 0.70 0.03 0.139 0.30 0.05 4.72

BPPCT017 5 7 0.01–0.47 0.58 0.65 0.76 −0.17 0.231 1.44 0.03 6.17

BPPCT025 3 4 0.01–0.57 0.38 0.50 0.44 0.11 0.451 1.52 0.06 3.83

BPPCT030 4 9 0.08–0.37 0.64 0.70 0.65 0.06 0.131 1.17 0.01 14.84

BPPCT038 4 7 0.01–0.42 0.58 0.66 0.55 0.16 0.029 0.73 0.00 158.72

BPPCT040 2 3 0.23–0.76 0.29 0.36 0.42 −0.17 0.414 1.26 0.06 3.60

CPDCT016 3 4 0.03–0.62 0.39 0.49 0.12 0.74 0.000a 0.76 0.01 18.54

CPDCT033 4 7 0.01–0.70 0.41 0.46 0.43 0.06 0.599 0.84 0.06 3.65

CPDCT045 3 3 0.07–0.80 0.30 0.33 0.38 −0.16 0.827 0.61 0.02 11.56

CPPCT006 8 13 0.01–0.41 0.71 0.75 0.70 0.07 0.002a 1.63 0.08 2.72

CPPCT022 6 11 0.01–0.34 0.68 0.73 0.82 −0.12 0.007 0.55 0.07 2.98

CPPCT030 6 14 0.02–0.45 0.67 0.71 0.72 −0.01 0.541 0.91 0.01 13.93

CPPCT033 5 5 0.02–0.87 0.22 0.23 0.25 −0.07 1.000 1.63 0.08 2.75

CPPCT034 5 6 0.01–0.50 0.44 0.55 0.59 −0.07 0.223 1.34 0.08 2.80

CPSCT006 4 4 0.02–0.78 0.32 0.35 0.42 −0.18 0.778 0.68 0.02 10.52

CPSCT011 5 8 0.03–0.57 0.55 0.60 0.34 0.44 0.000a 1.16 0.08 2.55

CPSCT021 4 7 0.06–0.54 0.56 0.62 0.59 0.05 0.203 1.14 0.04 4.93

CPSCT039 7 16 0.03–0.27 0.79 0.82 0.82 −0.00 0.040 1.79 0.01 14.37

CPSCT042 5 7 0.01–0.47 0.44 0.55 0.53 0.04 0.673 0.92 0.04 5.11

Ma014a 2 3 0.22–0.77 0.28 0.35 0.31 0.09 0.673 0.53 0.05 4.44

Ma040a 6 9 0.02–0.59 0.51 0.57 0.42 0.26 0.035 0.72 0.01 13.48

UDP97-402 4 5 0.01–0.67 0.36 0.46 0.57 −0.25 0.270 1.11 0.07 2.91

UDP98-409 10 21 0.01–0.36 0.76 0.80 0.74 0.07 0.038 1.11 0.04 5.78

UDP98-412 5 8 0.02–0.56 0.53 0.59 0.38 0.36 0.000a 0.11 0.01 13.31

Mean 4.75 8.18 0.05–0.55 0.50 0.56 0.52 0.04 0.317 1.05 0.04 4.79

PIC polymorphism information content, He expected heterozygosity, Ho observed heterozygosity, Fis and Fst Wright’s analysis of hierarchical F-statistics, I Shannon’s index of diversity, Nm gene flow parametera Significant heterozygosity deficit

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identified, 15 alleles were specific to northern region and16 alleles were specific to both central and southern regions(data not shown).

Discussion

All 32 mapped SSR primer pairs were selected based ontheir distribution along the map, found to be highlytransferable across different genotypes, and served well infingerprinting analysis of Tunisian apricot cultivars. Inaddition, the results obtained confirmed the effectiveness ofusing mapped SSRs in variability measurements. The 32

mapped SSR markers used for the 47 traditional apricotcultivars revealed the identity of 152 alleles and 262genotypes. These values were higher than the 103 allelesand 155 genotypes reported by Krichen et al. (2006b) using26 randomly SSR primers for 54 Tunisian apricot cultivarsof which the 47 cultivars included in this study.

The exploitation of the eight linkage groups to analyzethe genetic diversity of the Tunisian material expressed asignificant positive linear dependence between differentpairs of the linkage groups: LG1 and LG2, LG5 and LG6,and altogether LGs3, 4, and 5, while an opposite one wasdetected between these latter linkage groups and LG8. OnlyLG7 seems to be independent from the other linkagegroups.

In addition, the average number of alleles per locus was4.75, which appears to be higher than previously studies onapricot cultivars. Effectively, Sanchez-Pérez et al. (2005)obtained a mean value of 3.9 using 17 polymorphic SSRsfor Spanish apricot cultivars and Romero et al. (2003)reported a mean value of 3.1 for 40 apricot cultivars with16 microsatellites. However, this number was lower thanthe 7.64 observed by Zhebentyayeva et al. (2003) in 74native apricot accessions (European, Irano-Caucasian,Chinese, and Central Asian accessions) with 14 poly-morphic SSRs. The expected average heterozygosity for the32 loci was 0.56 higher than the mean value reported withpeach SSRs by Romero et al. (2003) in apricot (0.46) andlower than the mean value reported by Zhebentyayeva et al.(2003; 0.64). Thus, the Tunisian traditional apricot cultivarsshowed a relatively high level of polymorphism ascompared with the Mediterranean materials. Moreover,our results confirmed the presence of a decreasing gradientof genetic diversity from East to West the Mediterraneanarea, which may be explained by bottleneck effects relatedto historical introduction and apricot propagation in Tunisiaby numerous seedling events (Hagen et al. 2002; Khadari etal. 2006).

The genetic relationships of the studied Tunisian apricotgermplasm were approached by UPGMA dendrogrambased on Nei’s genetic distances (1972). Two major clustersdistinguished seven cultivars (15%), originated from Kair-ouan and Testour, from the remaining cultivars (85%),which could be related to the existence of two apricotgenetic origins in Tunisia. The major cluster II containedfive subgroups structured according to the geographicorigin of the material, within order, cultivars from Northof Tunisia in subgroups II.1 and II.2 and cultivars fromboth Center and South of the country in subgroups II.3,II.4, and II.5. The cultivar “Kasserine 2” originated fromGafsa was clustered in subgroup II.3 and separated from theother southern cultivars, probably due to an ancientselection of this cultivar from introduced French material(Carraut and Crossa-Raynaud 1974). Although they were

Table 4 Chi-square test of distribution of SSR allele frequencyamong the Tunisian apricot cultivars

Locus Chi-square Degree of freedom Probability

AMPA100 36.61 15 <0.01a

AMPA101 9.56 6 >0.05

AMPA105 12.88 10 >0.05

AMPA109 0.25 3 >0.05

AMPA116 15.05 10 >0.05

AMPA119 0.01 3 >0.05

BPPCT001 1.2 1 >0.05

BPPCT004 142.7 21 <0.01a

BPPCT008 26.84 21 >0.05

BPPCT017 11 10 >0.05

BPPCT025 1.99 3 >0.05

BPPCT030 8.65 6 >0.05

BPPCT038 10.64 6 >0.05

BPPCT040 1.48 1 >0.05

CPDCT016 36.46 3 <0.01a

CPDCT033 2.58 6 >0.05

CPDCT045 2.46 3 >0.05

CPPCT006 68.87 28 <0.01a

CPPCT022 36.75 15 <0.01a

CPPCT030 10.83 15 >0.05

CPPCT033 0.91 10 >0.05

CPPCT034 6.67 10 >0.05

CPSCT006 3.23 6 >0.05

CPSCT011 56.56 10 <0.01a

CPSCT021 8.2 6 >0.05

CPSCT039 34.08 21 <0.05

CPSCT042 3.57 10 >0.05

Ma014a 0.39 1 >0.05

Ma040a 19.91 15 >0.05

UDP97-402 4.11 6 >0.05

UDP98-409 57.78 45 >0.05

UDP98-412 28.16 10 <0.01a

a Significant chi-square test

584 Plant Mol Biol Rep (2010) 28:578–587

located in two different regions, Kairouan (center) andGafsa (south), the two accessions of “Baccour” cultivarwere regrouped in the same subcluster II.4, explaining theirnarrow genetic basis and the existence of variety populationwhile they were expected to be propagated by grafting. Theobtained genetic relationships were in agreement with data

based on unmapped SSR markers as reported by Krichen2007. Similar results were also shown using the factorialcorrespondence analysis which agreed with the separationof apricot cultivars of cluster I from the remaining studiedaccessions. These findings supported previous studies ontheTunisian apricot accessions based on morphological

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

-2 -1 -0.5 0 0.5Factor 1 (11.59%)

Fac

tor

2 (8

.80%

)

-1.5

Fig. 3 Diversity of the 47 apricot cultivars evaluated with 32microsatellites. The first plane (factors 1/2) of the factorial correspon-dence analysis (FCA) shows 20.39% of the total variation (squares

indicate cultivars from the northern region, filled circles indicatecultivars from the central region, filled triangles indicate cultivarsfrom the southern region)

Chechi Dhraa Tammar (K)

0.1

Chechi Bazza (T) Bayoudhi (K) Chechi Horr (T)

Chechi Khit El Oued (K) Khad Hlima (K) Oud El Haj Tahar (T) Bouthani (T) Oud Hmida (T) Oud Nakhla (T) Oud Gnaa (T) Variety of Mahdia (Mi) Oud Rhayem (T) Bedri Ahmar (T) Om Younes (R) Bou Herra (K) Bangui (Ma) Bouk Hmed (R) Faggoussi (R) Bouk Hmed Akhal (R) H’midi (R) Addadi Ahmar (R) Kasserine 2 (Gf) Baccour-Kairouan (K) Baccour-Gafsa (Gf) Bedri-Gabès (Gb) Bedri Thani (Gf) Bedri Louzi (Gb) Aranji (R) Oud Salah Ben Salem (T)Ben Souileh (Gb) Fourati-Sfax (S) Oud Aouicha (T) Zalouzi (S) Fourati-Kairouan (K) Amor El Euch (K) Mazouzi 65 (J) Chechi (J) Mazouzi 69 (J) Thaleth (Gb) Louzi Thani (Gb) Thani (Gb) Bedri Louzi 67 (J) Bou Khobza (J) Messelmani (K) Zbidi (K)

Oud Tijani (T)

I

II

I.2

II.1

II.2

II.3

II.4

II.5

I.1 Fig. 2 Dendrogram obtained byUPGMA cluster analysis basedon Nei’s genetic distances(1972) among cultivars studied.In parenthesis, the origin ofclustered accessions with: RjRas Jbel, T Testour, K Kairouan,Ma Mahdia, S Sfax, Gf Gafsa,Mi Midess, Gb Gabes, and JJerba

Plant Mol Biol Rep (2010) 28:578–587 585

characters, showing that cultivars of cluster I wereespecially characterized by a rectangular-plate fruit shapeand a white fruit flesh color (Krichen 2007). Moreover, thepresence of continuum genetic diversity between northerncultivars and both central and southern ones revealed thesame genetic background of apricot cultivars in the centerand the south of the country.

The observed genetic identity and genetic distancebetween apricot cultivar groups according to their geo-graphical origin (north, center, and south) indicated a highdegree of similarity among the defined groups. Further-more, cultivars originating from the central region weregenetically close to the southern cultivars (GI=0.93 andGD=0.07). Taking into account the average of Fst values, alow level of genetic differentiation between apricot geo-graphic groups was detected in comparison with valuesreported among accessions of Central Europe (Maghuly etal. 2005). These results suggested that the genetic back-ground of cultivars from the northern, central, and southernareas of apricot culture in Tunisia was close. The sameresults were revealed also by Khadari et al. (2006), whichshowed that the Tunisian apricot cultivars represented adistinct group with a narrow genetic basis.

In this study, among the 32 SSRs used, significantgenetic variation among groups could be found at onlyseven microsatellite loci, which were independent fromtheir position on Prunus genome and presented, expect forone (CPPCT022), a significant heterozygosity deficit. As aresult, the level of gene flow, based on Fst, between thethree apricot geographic groups was 4.79, indicatingthe presence of a substantial gene exchange between them.The observed gene flow of the Tunisian apricot cultivarswas considered high in comparison to gene flow of apricotaccessions from Central Europe (0.18; Maghuly et al.2005). The main way of gene exchanges could be humanintroduction of occasional seedling events. In fact, ex-change of interesting cultivars among the different areas ofapricot cultivation occurred in Tunisia and was mostimportant between the center and the south, especially inKairouan which represents a recently fruit trees cultivatedarea comparing to Sfax, Gabes, and Gafsa (Krichen 2007).Moreover, the study of specific alleles showed that northerncultivars contained the highest number of specific alleles

(15), suggesting that this area of apricot cultivationharbored the highest genetic diversity. Consequently,northern cultivars in Tunisia must be given as a best choicefor apricot conservation and breeding programs.

In conclusion, to characterize the molecular pattern ofdiversity of Tunisian apricot germplasm, we can assumethat using relatively the same set of traditional cultivars, thechoice of selected genetically mapped SSR markers providemore reliable diversity measurements. However, no differ-ence was demonstrated considering the genetic relation-ships obtained among studied accessions.

Acknowledgments This work was partially funded by the “InstitutFrançais de Coopération” (project CMCU 05G0904) and by theTunisian “Ministère de l’Enseignement Supérieur et de la RechercheScientifique” (Project Lab B02). The authors would like to thank Mr.Sylvain Santoni (SupAgro Montpellier, France) for his involvement inmicrosatellite genotyping. Authors kindly acknowledge Dr. A. Hadidi(National Germplasm Resources Laboratory, U.S. Department ofAgriculture, ARS, Beltsville, MD 20705-2350, USA) for his fruitfulcomments and for checking the language on the manuscript.

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