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Scientia Horticulturae 125 (2010) 593–601

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l homepage: www.e lsev ier .com/ locate /sc ihor t i

he origin and dissemination of the cultivated almond as determined by nuclearnd chloroplast SSR marker analysis

. Zeinalabedinia, M. Khayam-Nekouia, V. Grigorianb, T.M. Gradziel c, P. Martínez-Gómezd,∗

Agriculture Biotechnology Research Institute of Iran (ABRII), Karaj, IranDepartment of Horticulture, University of Tabriz, Tabriz, IranDepartment of Plant Sciences, University of California at Davis, Davis, CA, USADepartment of Plant Breeding, CEBAS-CSIC, PO Box 164, E-30100 Murcia, Spain

r t i c l e i n f o

rticle history:eceived 1 March 2010eceived in revised form 6 May 2010ccepted 12 May 2010

eywords:omesticationrunus dulcisene flow

a b s t r a c t

Sixteen nuclear and 10 chloroplast SSR markers were evaluated for 40 almond genotypes including cul-tivated almond, 18 related species and 5 interspecific-hybrid populations. Results establish the value ofSSR (nuclear and chloroplast) markers for distinguishing different genetic lineages and characterize anextensive gene pool available to almond genetic improvement. Hierarchical analysis using integratednuclear and chloroplast DNA markers support Prunus fenzliana, a species native to the northeast Iran,as a probable ancestor of the cultivated almond. Results also established the importance of interspe-cific hybridization and subsequent genetic introgression in the development of cultivated almond anddemonstrate continuing value of an interspecific gene pool for modern cultivar improvement. Molecular

reedingenetic diversityolecular markersicrosatellites

SR

results implicate a dissemination of the cultivated almond from Asia to the Eastern Mediterranean andsubsequently the Western Mediterranean and the New World is supported by the molecular analysis ofregional germplasm.

© 2010 Elsevier B.V. All rights reserved.

uclear DNAhloroplast DNA

nterspecific hybridization

. Introduction

Over 20 species of wild almond are native to the xeric envi-onments of Western and Central Asia, many of which haveeen harvested for their edible kernels from prehistoric to mod-rn times (Gradziel, in press; Gradziel and Martínez-Gómez, inress; Grasselly, 1976b; Kester et al., 1991). Samples recoveredrom King Tut’s tomb as well as ancient shipwrecks in the East-rn Mediterranean show that the sweet almond [Prunus dulcisMiller) D.A. Webb, syn. Prunus amygdalus Batsch., Amygdalus com-unis L., Amygdalus dulcis Mill.] commerce was well established by

000 BCE, and appeared widely disseminated along trade routesstablished among these early Central Asian and Mediterranean

ivilizations (Gradziel, in press).

Species integrity has been maintained largely through geo-raphic isolation in the diverse and often marginal nativenvironments throughout central Asia since almond species arebligate outcrossers and readily hybridize with other species

∗ Corresponding author. Tel.: +34 968 396237; fax: +34 968 396 213.E-mail address: pmartinez@cebas.csic.es (P. Martínez-Gómez).

304-4238/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.scienta.2010.05.007

within the subgenus Amygdalus (Kester et al., 1991). Becausespecies native to the Asian centres of origin and diversity aremorphologically distinct from the cultivated almond, it is widelybelieved that the cultivated almond originated from interspecifichybridizations (Grasselly, 1976a,b; Ladizinsky, 1999; Browicz andZohary, 1996). Evreinoff (1958) first proposed that the cultivatedalmond arose by hybridization with Prunus fenzliana Fritsch. (nativeto the northeast Iran) and possibly other species such as Prunusbucharica Korchinsky or Prunus kuramica Korchinsky. Based onmorphology and habitat Ladizinsky (1999) identified P. fenzliana,a species native to northeast Iran as the most plausible ancestor.Grasselly (1976a), however, proposed P. kuramica as a probableancestor of cultivated almond because they coexist in cultivatedareas.

Natural hybridization between cultivated almond and nearbywild species has also been widely described (Grasselly, 1976b;Denisov, 1988) indicating that hybridization and subsequent geneintrogression continue into modern times. Recent studies by Socias

i Company (2002) have confirmed the original suggestion bySocias i Company and Felipe (1992) that the gene conferring self-compatibility in the Italian cultivar ‘Tuono’ was introgressed fromwild Prunus webbii (Spach) Vieh. following natural interspecifichybridizations.

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Archeological and historical evidence supports the introduc-ion of cultivated almond to the Eastern Mediterranean by theecond millennium BC (Gradziel, in press; Vavilov, 1930; Zoharynd Hopf, 1993). This evidence also supports extensive almondrade in the Eastern Mediterranean in the fourth century BC (Cerdá,973). Almond cultivation appears to have rapidly spread through-ut Mediterranean regions from Central Asia (Stylianides, 1976;arcía et al., 1988; Sorkheh et al., 2007) with the required mildinter and hot, dry summer climates and subsequently to simi-

ar climates in North and South America, and Australia (Kester etl., 1991; Kester and Gradziel, 1996; Gradziel, in press). Cultivationn the Mediterranean regions often involved mixed seedling pop-lations with selection for land races with local adaptedness andesired kernel characteristics. New World plantings initially exper-

mented with hundreds of diverse genotypes of Asian, Europeannd North African origins as well as hybrids between accessionsntil well adapted and desirable genotypes were selected (Wood,925). Elite selections were then clonally propagated onto peachootstock and widely disseminated so that a relatively few cultivars,ften of obscure origin, have come to dominate production.

Both sweet and bitter forms of the cultivated almond are grownommercially (Kester et al., 1991; Gradziel and Martínez-Gómez, inress). Although the sweet form (which is determined by a singleominant gene) (Dicenta et al., 2007; Sánchez-Pérez et al., 2008)redominates, bitter forms [sometimes classified as Prunus dul-is (Mill.) D.A. Webb var. ‘amara’ (DC.) Buchheim] continue to berown for the ‘amaretto’ or ‘cherry’ essence used in traditional andontemporary foods and beverages.

The analysis of genetic diversity and phylogenetic relation-hips among almond species and among almond cultivars based onabitat and morphology has proven difficult because of the exten-ive variability within species resulting from obligate out-crossingnd the resulting high genetic heterozygosity (Kester et al., 1991;radziel and Martínez-Gómez, in press). Consequently, DNA mark-rs have proven very useful in studies of genetic diversity and thelarification of certain questions on cultivar origin. The develop-ent of PCR-based DNA markers, such as simple sequence repeat

equences (microsatellites, SSRs), has created the opportunity foride genetic characterization of genotypes (Martínez-Gómez et al.,

007).SSR markers are multi-allelic, co-dominant genetic markers

ith a very high repeatability, and so are particularly suitableor phylogenetic studies because of their high polymorphism andbundance (Gupta et al., 1996). In the case of Prunus species, primerairs flanking nuclear SSRs were cloned and sequenced in differentpecies including peach, plum, cherry, apricot and almond (Ciprianit al., 1999; Testolin et al., 2000, 2004; Cantini et al., 2001; Aranzanat al., 2002; Dirlewanger et al., 2002; Messina et al., 2004; Xie etl., 2006). More recently primer pairs flanking chloroplast SSRsere cloned and sequenced in Prunus salicina by Ohta et al. (2005).

hese PCR markers (nuclear and chloroplast) have been previouslysed for the molecular characterization of almond genotypes andelated Prunus species (Martínez-Gómez et al., 2003a,b; Testolin etl., 2004; Zeinalabedini et al., 2007, 2008)

The objective of the present study was a clarification of the originf the cultivated almond and a preliminary mapping of its subse-uent dissemination throughout the Mediterranean and on to theew World as determined by nuclear and chloroplast DNA markernalysis.

. Materials and methods

.1. Plant material

Plant material assayed included 40 almond cultivars fromiverse growing regions (Table 1). In addition, different acces-

iculturae 125 (2010) 593–601

sions from 14 related species including P. bucharica (Korschinsky),P. davidiana [(Carr.) Franch], P. elaeagnifolia (Mill.), P. fenzliana(Fristch), P. gorki (Fristch), P. hausskenchetii (Schneider), P. lycioides(Spach), P. orientalis (Mill.) [syn. P. argentia (Lam)], P. kotschyi(Fritsch); P. persica [(L.) Batsch] (cultivated peach), P. scoparia(Spach), P. trichamygdalus (Hand.-Mazz.), P. vavilovi (Spach), andP. webbii [(Spach) Vieh.] were included in the study. Four culti-vated Prunus species P. armeniaca (L.), P. avium (L.), P. mandshurica[(Maxim.) Koehne] and P. salicina (Lindl) were also included as out-groups (Table 2). Finally, four populations of both presumed andknown interspecific origin were assayed including two mixed pop-ulations of P. scoparia and P. lycioides collected in the province ofEsfahan (Central Iran) and two directed interspecific crosses of P.dulcis (‘S5133’) × P. scoparia and P. dulcis (‘S5133’) × P. webbii cre-ated in the experimental field of CEBAS-CSIC at Murcia (South Eastof Spain) were also included in the study (Table 3).

2.2. DNA isolation

Total genomic DNA (nuclear and chloroplast) was isolated usinga modified procedure of the described by Doyle and Doyle (1989).Approximately 50 mg of young leaves were ground in a 1.5 mlEppendorf tube with 750 �l of CTAB extraction buffer (100 mMTris–HCl, 1.4 M NaCl, 20 mM EDTA, 2% CTAB, 1% PVP, 0.2% mer-captoethanol, 0.1% NaHSO3). Samples were incubated at 65 ◦C for20 min, mixed with an equal volume of 24:1 chloroform:isoamyl-alcohol, and centrifuged at 6000 × g (20 min). The upper phasewas recovered and mixed with an equal volume of isopropanolat −20 ◦C. The nucleic acid pellet was washed in 400 �l of 10 mMNH4Ac in 76% ethanol, dried, resuspended in 50 �l of TE (10 mMTris–HCl, 0.1 mm EDTA, pH 8.0), and incubated with 0.5 �g ofRNase-A at 37 ◦C for 30 min, to digest RNA. DNA was quantifiedusing a Biophotometer (Eppendorf, Barcelona, Spain).

2.3. Application of nuclear and chloroplast SSR markers

Extracted DNA was PCR-amplified using 16 pairs of primersflanking SSR sequences which were previously cloned andsequenced in peach (Cipriani et al., 1999; Testolin et al., 2000;Aranzana et al., 2002; Dirlewanger et al., 2002); and cherry (Cantiniet al., 2001). In addition, 10 pair of primers flanking chloroplast SSRsequences previously cloned and sequenced in P. salicina (Ohta etal., 2005) were included in the study (Table 4).

PCR reactions and analysis of products were performed accord-ing to the protocol optimized previously by Zeinalabedini et al.(2008). The reaction mixture in a final volume of 25 �l contained16 mM (NH4)2SO4, 67 mM Tris–HCl, pH 8.8, 0.01% Tween 20, 2 mMMgCl2, 0.2 �M of each primer, 0.1 mM of each dNTP, one unit of TaqDNA Polymerase (Ecogen S.R.L.), and 90 ng of genomic DNA. Cyclingparameters were: one cycle of 95 ◦C for 3 min; 35 cycles of 94 ◦C for1 min, annealing temperature for 1 min (see Table 4), and 72 ◦C for1 min; followed by 10 min at 72 ◦C. PCR reactions were carried outin a 96-well block Eppendorf Mastercycler.

Amplified PCR products were separated based on size differ-ences of the segregating alleles. If the difference was more than5 bp we used 3% Metaphor® agarose gel electrophoresis (Biowit-taker, Maine, USA) stained with ethidium bromide (0.5 �g/ml), andvisualized under UV light (Fig. 1). When the difference was lessthan 5 bp we used polyacrylamide gel electrophoresis (PAGE). Thisis relatively common in the case of dinucleotide and trinucleotiderepeat SSRs (Table 4) where only changes of a few nucleotides are

presented (Xie et al., 2006). PAGE was carried out denaturing thePCR products by adding 2.5 �l of the 95% formamide/bromophenolbuffer. Samples were loaded onto gel (6% polyacrylamide, 7.5 Murea) electrophoresis in 1 × TBE buffer at a current of 120 W andgel temperature of 50 ◦C. Results were visualized using silver stain-

M. Zeinalabedini et al. / Scientia Horticulturae 125 (2010) 593–601 595

Table 1Origin, pedigree and main agronomic characteristics of the 40 almond cultivars assayed.

Genotype Country Origin Pedigree Shell Flavour Flowering Self-compatibility

‘Achaak’ Tunisia Cultivar Unknown Semi-hard Sweet Early Self-incompatible‘All in one’ USA Release Np. × peach, BC5 Soft Sweet Late Self-compatible‘Antoneta’ Spain Release Ferragnès × Tuono Hard Sweet Late Self-compatible‘Ardechoise’ France Cultivar Unknown Soft Sweet Intermediate Self-incompatible‘Azar’ Iran Cultivar Unknown Semi-hard Sweet Late Self-incompatible‘Bonita’ Portugal Cultivar Unknown Hard Sweet Intermediate Self-incompatible‘Carmel’ USA Cultivar Unknown Soft Sweet Early Self-incompatible‘Chellaston’ Australia Cultivar Unknown Semi-hard Sweet Late Self-incompatible‘Cristomorto’ Italy Cultivar Unknown Hard Sweet Late Self-compatible‘Deserthiij’ Ukraine Cultivar Unknown Hard Sweet Late Self-incompatible‘Desmayo’ Spain Cultivar Unknown Hard Sweet Early Self-incompatible‘Ferrangnes’ France Release Cristomorto × Ai Soft Sweet Late Self-incompatible‘Genco’ Italy Cultivar Unknown Hard Slightly bitter Late Self-compatible‘Guara’ Spain Release Tuono (OP) Hard Sweet Late Self-compatible‘Jalstenskj’ Ukraine Cultivar Unknown Hard Sweet Late Self-incompatible‘Languedoc’ France Cultivar Unknown Soft Sweet Early Self-incompatible‘Lauranne’ France Release Ferragnès × Tuono Semi-hard Sweet Late Self-compatible‘Marcona’ Spain Cultivar Unknown Hard Sweet Early Self-incompatible‘Marta’ Spain Release Ferragnès × Tuono Hard Sweet Late Self-compatible‘Mashhad’ Iran Cultivar Unknown Semi-hard Sweet Mid Self-incompatible‘Mission’ USA Cultivar Unknown Semi-hard Sweet Late Self-incompatible‘MNNR-1’ Slovakia Cultivar Unknown Hard Sweet Late Self-incompatible‘Monagha’ Iran Cultivar Unknown Soft Sweet Early Self-incompatible‘Nonpareil’ (NP.) USA Cultivar Unkonwn Paper Sweet Middle Self-incompatible‘Primorskij’ Ukraine Release Princes × Nikitski Soft Sweet Extra-late Self-incompatible‘R1000’ France Release Tardy Np. × Tuono Semi-hard Sweet Late Self-incompatible‘Rabii’ Iran Cultivar Unknown Hard Sweet Intermediate Self-incompatible‘Ramillete’ Spain Cultivar Unknown Hard Sweet Early Self-incompatible‘S5133’ Spain Release Primorski (OP) Hard Sweet Extra-late Self-incompatible‘Sahand’ Iran Cultivar Unknown Hard Sweet Early Self-incompatible‘Shokofeh’ Iran Cultivar Unknown Soft Sweet Late Self-incompatible‘Shiraz-10’ Iran Cultivar Unknown Hard Sweet Intermiedate Self-incompatible‘Sladkoploda’ Slovakia Cultivar Unknown Hard Slightly bitter Late Self-incompatible‘Sonora’ USA Release Np. × Eureka Soft Sweet Early Self-incompatible‘Tardy Nonpareil’ USA Cultivar Mutant of Nonpareil Soft Sweet Very late Self-incompatible‘Texas-1’ USA Cultivar Mission × Nonpareil Soft Sweet Late Self-incompatible‘Tuono’ Italy Cultivar Unknown Hard Sweet Late Self-compatible‘Yalda’ Iran Cultivar Unknown Soft Sweet Late Self-incompatible‘Vama’ Czech Rep. Cultivar Unknown Hard Sweet Late Self-incompatible‘Zord’ Iran Cultivar Unknown Hard Sweet Late Self-incompatible

Table 2Prunus species assayed including related (cultivated and wild) almond species and cultivated Prunus species included as outgroup of subgenera Amygdalus.

Subgenera Section Species Accession Country Origin Pedigree

Amygdalus Euamygdalus P. bucharica Accession 1 Pakistan Wild spp. UnknownP. davidiana Accessions 1, 2 China Wild spp. UnknownP. eleagnifolia Accessions 1, 2, 3, 4, 5 Iran Wild spp. UnknownP. fenzliana Accessions 1, 2, 3, 4 Iran Wild spp. UnknownP. gorki Accessions 1, 2 Turkey Wild spp. UnknownP. kotschyi Accession 1 Iran Wild spp. UnknownP. orientalis Accession 1 Iran Wild spp. UnknownP. persica Babygold USA Cultivar UnknownP. persica Catherine France Cultivar UnknownP. persica Sudanel Spain Cultivar UnknownP. persica GF677 France Rootstock Peach × almondP. trichamygdalus Accessions 1, 2 Iran Wild spp. UnknownP. vavilovi Accessions 1, 2 Iran Wild spp. UnknownP. webbii Accessions 1, 2 Greece Wild spp. Unknown

Lycioides P. lycioides Accessions 1, 2, 3, 4, 5 Turkey Wild spp. UnknownSpartioides P. haussknechtii Accessions 1, 2, 3, 4, 5 Iran Wild spp. Unknown

P. scoparia Accessions 1, 2, 3, 4, 5 Iran Wild spp. Unknown

Cerasus Microcerasus P. avium Burlet USA Cultivar UnknownPrunus Armeniaca P. armeniaca Canino Spain Cultivar Unknown

P. armeniaca Pepito Spain Cultivar UnknownP. armeniaca Real Fino Spain Cultivar UnknownP. mandshurica Accession 1 China Wild spp. Unknown

Prunus P. salicina Blackamber USA Cultivar UnknownP. salicina Red Beaut USA Cultivar UnknownP. salicina Santa Rosa USA Cultivar Unknown

596 M. Zeinalabedini et al. / Scientia Horticulturae 125 (2010) 593–601

Table 3Observed heterozygosity (Ho) in the parental genotypes and the four mixed popu-lations assayed.

Plant material No. of accessions Ho

Populations from natural intermixingIsolated population

P. scoparia 7 0.234

Geographically mixed populationsP. scoparia (mixed with P. lycioides) 13 0.375P. lycioides (mixed with P. scoparia) 2 0.281

Directed interspecific hybridizationParental

P. dulcis (‘S5133’) 1 0.312P. webbii 2 0.289P. scoparia 1 0.307

iae

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On the other hand, genetic relationships were calculated

TDc

DescendantsP. dulcis (‘S5133’) × P. scoparia 8 0.555P. dulcis (‘S5133’) × P. webbii 19 0.390

ng kit from Promega (Promega Inc., Madison, USA). In both cases,1 Kb Plus ladder was assayed as DNA size marker (Sánchez-Pérezt al., 2006a).

.4. Data analysis

Polymorphic alleles were scored as present or absent (1/0). Bandcoring was analyzed using the GeneTools gel analysis softwareSYNGENE, Cambridge, UK). Expected genetic heterozygosity (He)as calculated as 1 − ∑

P2i

where Pi is the frequency of the ith alleleNei, 1973). Observed genetic heterozygosity (Ho) of each genotype

r SSR marker was calculated as the number of heterozygous geno-ypes divided by the total number of markers or genotypes. Powerf discrimination was calculated as PD = 1 − ∑

g2i

where gi is therequency of the ith genotype (Kloosterman et al., 1993). The level

able 4NA (nuclear and chloroplast SSRs) markers assayed in the characterization of almond gultivated outgroup species) (He – expected genetic heterozygosity, Ho – observed genet

Marker (locus) Reference SSR sequence structure Annealing

Nuclear SSR markersBPPCP010 Dirlewanger et al. (2002) (AG)8GG(AG)10 57BPPCP011 Dirlewanger et al. (2002) (CT)16 57BPPCP026 Dirlewanger et al. (2002) (AG)8GG(AG)6 57BPPCT007 Dirlewanger et al. (2002) (AG)22(CG)2(AG)4 57CPPCT008 Aranzana et al. (2002) (CT)6. . .(CT)7 62CPPCT022 Aranzana et al. (2002) (CT)28CAA(CT)20 57CPPCT030 Aranzana et al. (2002) (CT)30 50CPPCT033 Aranzana et al. (2002) (CT)16 50PCeGA025 Cantini et al. (2001) – 62UDP96003 Cipriani et al. (1999) (CT)11(CA)28 57UDP96005 Cipriani et al. (1999) (AC)16TG(CT)2CA(CT)11 57UDP96008 Cipriani et al. (1999) (CA)23 57UDP97402 Cipriani et al. (1999) (AG)17 57UDP98409 Testolin et al. (2000) (AG)19 57UDP98410 Testolin et al. (2000) (AG)23 50UDP98411 Testolin et al. (2000) (TC)16 57Mean – – –

Chloroplast SSR markersTPScp1 Ohta et al. (2005) (T)9 55TPScp2 Ohta et al. (2005) (ATAGAT)2TAGAT 55TPScp3 Ohta et al. (2005) (T)9 55TPScp4 Ohta et al. (2005) TAAAAT(TAAAA)2 55TPScp5 Ohta et al. (2005) (T)8 55TPScp7 Ohta et al. (2005) (A)8 55TPScp8 Ohta et al. (2005) (A)8 55TPScp9 Ohta et al. (2005) (TCAAA)3 55TPScp10 Ohta et al. (2005) (T)9 55TPScp11 Ohta et al. (2005) (G)8 55Mean – – –

Fig. 1. Metaphor agarose gels showing the allelic segregation of nuclear and chloro-plast SSR markers in the almond cultivars and the related Prunus species assayed.

of polymorphism was also estimated using PIC with the follow-ing equation: PIC = 1 − ∑

P2i

−∑∑

2PiP2j

where Pi and Pj are thefrequencies of the ith and jth alleles, respectively, at one locus.

with the unweighted pair group method using arithmetic aver-ages (UPGMA) cluster analysis obtained from the proportion ofshared alleles (Nei and Li, 1979). The bootstrap analysis run-ning 2000 iterations was employed to determine the sampling

enotypes (cultivars and new release) and relative Prunus species (wild almond andic heterozygosity, PD – power of discrimination, PIC – level of polymorphism).

temperature Number alleles Size (bp) He Ho PD PIC

16 120–194 0.89 0.47 0.95 0.8815 105–195 0.76 0.23 0.82 0.7413 77–194 0.84 0.31 0.70 0.8217 109–188 0.87 0.23 0.94 0.8610 153–200 0.84 0.21 0.86 0.8212 209–305 0.88 0.21 0.48 0.87

9 146–207 0.85 0.21 0.50 0.8312 129–200 0.83 0.28 0.91 0.8115 158–241 0.87 0.36 0.90 0.8618 74–178 0.92 0.47 0.95 0.9219 103–194 0.92 0.55 0.94 0.9217 128–183 0.87 0.35 0.66 0.8614 97–183 0.88 0.28 0.60 0.8718 114–181 0.90 0.31 0.82 0.9018 106–194 0.91 0.55 0.94 0.9017 103–200 0.89 0.26 0.95 0.8814.5 74–305 0.87 0.31 0.80 0.86

3 186–200 0.05 0.20 0.25 0.431 250 0.00 0.00 0.00 0.001 200 0.00 0.00 0.00 0.001 135 0.00 0.00 0.00 0.001 163 0.00 0.00 0.00 0.001 93 0.49 0.00 0.00 0.371 280 0.00 0.00 0.00 0.006 216–242 0.65 0.50 0.32 0.637 94–106 0.85 0.65 0.41 0.835 96–112 0.68 0.50 0.35 0.623.1 93–242 0.32 0.18 0.33 0.29

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ariance of the genetic similarities calculated from the data setsbtained with the different marker systems using the MEGA 3.1rogram.

. Results

Amplification was successful in almond and related speciessing nuclear SSR markers initially developed for different Prunuspecies. A high level of polymorphism was detected. The total num-er of alleles ranged from 9 to 19. The most polymorphic markeras UDP96005 with 19 alleles. An average number of 14.5 alleleser locus were found. The total number of polymorphic bands iden-ified was 232 with amplified band sizes ranging from 74 to 305 bp.everal alleles were found in different species. Amplification waslso successful with the 10 chloroplast SSR markers initially devel-ped in Prunus salicina. A lower level of polymorphism, rangingrom 1 to 7 alleles and 93 to 242 bp, was observed assays. As withuclear SSRs, several alleles were found in different species (Fig. 1nd Table 4).

Expected heterozigosity (He) at a locus increased with increas-ng number of alleles per locus. Expected heterozigosity in nuclearSR varied between 0.76 (Locus BPPCP011) and 0.92 (LocusDP96003 and UDP96005) with an average 0.87. The loci for whiche was maximal and minimal does not correspond with the highest

owest absolute number of alleles. This demonstrates that the abso-ute number of alleles is not the sole determinant of heterozigosityTable 4). Observed heterozigosity (Ho) of alleles in each nuclearSR locus varied between 0.21 (Locus CPPCT008, CPPCT022 andPPCT030) and 0.55 (Locus UDP96005 and UDP98410) with anverage of 0.31. For chloroplast SSR, Ho ranged from 0 to 0.65,ith an average of 0.18 in agreement with the lower polymor-hism found with these chloroplast markers as compared withuclear markers. Finally, the power of discrimination (PD) in eachuclear SSR locus varied between 0.48 (CPPCT022) and 0.95 (LocusPPCP010, UDP96003, UDP98411) with an average of 0.80, withhloroplast SSR data being lower in agreement with the less poly-orphism observed with a range between 0.0 and 0.41 and a mean

alue of 0.33. The polymorphism information content (PIC) variedrom 0.74 to 0.92 for nuclear SSRs and 0.0 and 0.83 in the evaluationf chloroplast SSRs (Table 4).

On the other hand, two well-supported main clusters areresent in the UPGMA generated dendrogram (Fig. 2). Cluster

contains the almond cultivars (A.1) and accession of P. fen-liana (A.2) with a high bootstrapping value around 60%, whileluster (B) contains accessions from related and outgroup Prunuspecies. Clustering in group (A1) is organized primarily accordingo the geographic origin, as with the traditional cultivars ‘Desmayo’nd ‘Ramillete’(Spain), ‘Cristomorto’ and ‘Tuono’ (Italy), ‘Monagha’nd ‘Shiraz’ (Iran), ‘Mission’ and ‘Sonora’ (USA), and ‘MNR-1’ and

Desertheiij’ (Eastern Europe) or the pedigree as with ‘Sonora’ ‘Alln one’, ‘Texas-1’, ‘Tardy Nonpareil’, ‘R100’ and the common parentNonpareil’. These clusters are supported in most cases with sig-ificant bootstrapping values higher than 50%. Cluster A2 containsnly the accessions of P. fenzliana. On the other hand, cluster B cane separated in four distinct subclusters with significant bootstrap-ing values higher than 50%, the first dominated by the species P.ausknechtii and P. eleagnifolia in Section Chameamygdalus Spach,he second dominated by the species P. scoparia in Section Spar-ioides Spach and P. lycioides in Section Lycioides Spach, the thirdontaining outgroup species, and the fourth containing cultivated

each P. persica.

Further support for the possibility of gene flow among speciess shown in Table 3 where an increase in the genetic variabilityobserved heterozygosity) is observed both in the geographically

ixed population and in interspecific crosses relative to the iso-

iculturae 125 (2010) 593–601 597

lated P. scoparia population and the P. scoparia, P. webbii and P.dulcis parents where known. The phenetic relationships for theseinterspecific-hybrid populations show strong support for progenyclustering (Fig. 3). In addition, the cultivated almond parent ‘S5133’clustered in a separated group from the other species as well astheir hybrid progeny. According to the genetic proximity of mostof assayed interspecific crosses, we have to not that there is rela-tively little confidence in most of the clusters obtained where onlya few bootstrap values are significant and exceed 50%. This wouldsuggest that this SSR analysis does not have the power to make thedistinctions in this type of plant material presented.

Finally, molecular results provide valuable insight to the patternof dissemination for cultivated almond. The clustering of culti-vars representative of 11 diverse regions of almond cultivation isshown in Fig. 4. In Eurasia, the closest genetic relationships areamong Spain, French, Italian and Iranian cultivars in comparisonwith Ukraine, Slovakian and Czech cultivars. Distinct clustering isalso apparent for Australian and North American, and Portugal andTunisia cultivars.

4. Discussion

Results confirmed the value of the SSR markers, particularlynuclear, in the study of the origin and dissemination of the plantspecies such as almond. In addition, the high level of polymor-phism as estimated by PIC with the extensive genetic diversityamong accessions at most loci, demonstrate that nuclear markersare useful for phylogenetic studies in Prunus species and cultivars.Our results confirmed that dinucleotide copy number in the Prunusspecies genome is high. A comparison of these results with thosefrom previous studies suggests that genetic erosion is probablynot extensive in wild species though the differences in number ofalleles per locus are also partly due to differences in sample size(Vanwaynsberghe, 2006).

The level of polymorphism observed in the analysis of nuclearSSR markers analyzing fragment with Metaphor® agarose andpolyacrylamide electrophoresis was similar to that reported usingsame markers by different authors in almond cultivars and relatedspecies (Martínez-Gómez et al., 2003a,b; Zeinalabedini et al., 2007,2008) and in other Prunus species such as peach, apricot andcherry (Testolin et al., 2000; Cantini et al., 2001; Dirlewangeret al., 2002; Aranzana et al., 2002; Sánchez-Pérez et al., 2005).The observed polymorphism of chloroplast SSR is also in agree-ment with the previous data of Ohta et al. (2005). The averagepolymorphism observed (He, Ho, PIC and PD) was lower in com-parison with nuclear SSR markers in agreement with the higherlevel of conservation of chloroplast DNA as described by previousresearchers (Badenes and Parfitt, 1995; Katayama and Uematsu,2005; Bouhadida et al., 2007).

Molecular results support, both nuclear and chloroplast in acomplementary way, a close evolutionary distance between thesespecies as originally suggested by Watkins (1976). The results alsoshow the high degree of homology for the SSR loci (nuclear andchloroplast) among Prunus species and so their transportabilityas reported by Cipriani et al. (1999), Decroocq et al. (2003) andMartínez-Gómez et al. (2003a). In addition, the similar order of SSRmarkers observed in different Prunus maps suggests a high levelof synteny within this genus (Aranzana et al., 2003; Sánchez-Pérezet al., 2007). Consequently, an advantage of microsatellites in thestudy of conservation genetics is the fact that primers developed

for one species are frequently applicable to related taxa.

On the other hand, almond cultivar clustering is in agreementwith previous finding in P. dulcis (Martínez-Gómez et al., 2003a;Sorkheh et al., 2007; Zeinalabedini et al., 2007; Gerald et al., 2009).Results show P. fenzliana as having the closest genetic relationship

598 M. Zeinalabedini et al. / Scientia Horticulturae 125 (2010) 593–601

F od wia ost sig

w(EbiPsc

sSo

ig. 2. Dendrogram obtained with the similarity Jacard coefficient pair group methnd 18 related species. Numbers below branches represent bootstrapping values. M

ith cultivated almond supporting the hypothesis of Ladizinsky1999) which was based mainly on morphological characteristics.vreinoff (1958) also suggested that the cultivated almond arosey hybridization with P. fenzliana and, along with Grasselly (1976a)

ncluded the possibility of hybridization with other species such as. bucharica. However, the different clustering of the single acces-ions of P. bucharica evaluated in this study does not support its

ontribution to cultivated almond.

Typically wild almond species such as P. fenzliana produce bittereeds which protects seeds from herbivores (Gradziel et al., 2001;orkheh et al., 2009). Individual trees in native populations of manyf these almond species, however, have been reported to produce

th arithmetical average clustering algorithm (UPGMA) for the 40 almond cultivarsnificant bootstrapping values (around 50% or more) were indicated in bold letters.

sweet kernels (Vavilov, 1930; Denisov, 1988). In addition, the muta-tion for sweet kernel is expressed as a dominant trait in almond asopposed to other Prunus species such as apricot or peach wherebitterness appears to be dominant (Dicenta et al., 2007; Negri etal., 2008; Sánchez-Pérez et al., 2008). Consequently, sweet kernelalmond types could have been readily selected during the earlystages of almond domestication (Zohary and Spiegel-Roy, 1975;

Gradziel, in press).

Given the ease of interspecific hybridization among species inthe subgenus Amygdalus (Gradziel et al., 2001; Kester et al., 1991),the presence of the same SSR alleles among morphologically dis-tinct species supports the possibility of natural gene flow between

M. Zeinalabedini et al. / Scientia Horticulturae 125 (2010) 593–601 599

F od witp st sign

s(tltg

uara

ig. 3. Dendogram obtained with the similarity Jacard coefficient pair group methopulations assayed. Numbers below branches represent bootstrapping values. Mo

pecies as has been described inside the cultivated almond speciesJackson and Clarke, 1991). This possibility is further supported byhe observed clustering patterns (Fig. 2). For example, the morpho-ogically distinct species P hausknechtii and P. eleagnifolia are notightly clustered but show a mixed order suggesting some level ofenetic mixing in their geographically overlapping habitats.

Natural interspecific hybridizations have previously been doc-mented in many regions of Europe and Asia where cultivatedlmonds are planted (Grasselly, 1976a; Denisov, 1988), and theesulting vigour typical of such hybrids has encouraged their uses invigorating rootstocks for cultivated almond (Sorkheh et al.,

h arithmetical average clustering algorithm (UPGMA) for the interspecific almondificant bootstrapping values (around 50% or more) were indicated by arrows.

2009). Incidences of more extensive exotic gene introgression tocultivated almond have also been proposed, most notably the sug-gestion by Godini (1977) and more recently Socias i Company andFelipe (1992) that the source of self-compatibility in the Italian cul-tivar ‘Tuono’ was wild P. webbii populations native to southern Italy.Molecular studies by Martínez-Gómez et al. (2003b) have recently

verified this proposal, providing unambiguous evidence for geneflow between wild and cultivated almond. Because the naturalrange of P. webbii extends from the Eastern Mediterranean throughthe Balkans where the cultivation of seedling almond orchards hasbeen carried out for centuries, additional instances of exotic gene

600 M. Zeinalabedini et al. / Scientia Hort

Fig. 4. Unrooted dendogram obtained with the similarity Jacard coefficient pairgr

flaatI(BotAM

siaidrSewc

tcftfitp

5

soitvIcrNA

169–173.Kester, D.E., Gradziel, T.M., Grasselly, C., 1991. Almonds (Prunus). In: Moore, J.N.,

roup method with arithmetical average clustering algorithm (UPGMA) among theepresentative almond cultivars assayed from the 11 different countries.

ow to almond may exist, awaiting only the precision of molecularnalysis to verify them. A recent evaluation of self-incompatibilitylleles in cultivars from Hungary by Halasz et al. (2008) indicatedhe presence of different alleles from those detected in ‘Tuono’.n this sense, many of the more than 30 named almond speciesGrasselly, 1976b; Kester et al., 1991; Kester and Gradziel, 1996;rowicz and Zohary, 1996) may not be true species but productsf such interspecific hybridization followed by geographic isola-ion in the often harsh dessert habitats typical of almond’s Centralsian centre of origin and diversity (Valizadeh and Ershadi, 2009;ousavi et al., in press).In addition to facilitating accurate analysis of gene flow among

pecies, molecular markers have proven effective in distinguish-ng different genetic lineages, evaluating the genetic heterozygositynd characterizing the inter-species variability available to breed-ng programs (Sánchez-Pérez et al., 2005, 2006b). Results alsoocument a rich source of new interspecific germplasm which iseadily transferred to cultivated almond (Gradziel et al., 2001).ánchez-Pérez et al. (2006b) have recently described mechanismsffective in promoting out-crossing with related apricot specieshich further facilitate exotic gene introgression to cultivated apri-

ot.Finally, molecular results support previous pathways of cul-

ivated almond dissemination based on historical, linguistic, andulinary relationships, with the introduction of cultivated almondrom Central Asia to the Eastern Mediterranean and subsequentlyo the Western Mediterranean regions, to North America andnally the South Hemisphere including South America and Aus-ralia (Kester et al., 1991; Kester and Gradziel, 1996; Gradziel, inress).

. Conclusions

Molecular results using nuclear and chloroplast DNA markersupport in a complementary way P. fenzliana as a probable ancestorf the cultivated almond. Results also established the importance ofnterspecific hybridization and subsequent genetic introgression inhe development of cultivated almond and demonstrate continuingalue for interspecific gene pool for modern cultivar improvement.n addition, molecular results evidenced a dissemination of theultivated almond from Central Asia (Iran) to the Eastern Mediter-

anean and subsequently to the Western Mediterranean regions, toorth America and finally the South Hemisphere including Southmerica and Australia.

iculturae 125 (2010) 593–601

Acknowledgements

This study was financed by projects “Mejora Genética delAlmendro” from the Spanish Ministry of Science and Technologyand the project “Relationship of genetic diversity in some culti-vated almond and related Prunus using SSRs” from an InternationalCollaboration between the Agricultural Biotechnology ResearchInstitute of Central Region of Iran (ABRICI) (Esfahan, Iran), the Uni-versity of Tabriz (Tabriz, Iran), and the CEBAS-CSIC (Murcia, Spain).

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