Plant Cell Rep (2003) 22:71–78DOI 10.1007/s00299-003-0659-9

G E N E T I C S A N D G E N O M I C S

M. Martins · R. Tenreiro · M. M. Oliveira

Genetic relatedness of Portuguese almond cultivars assessedby RAPD and ISSR markers

Received: 23 January 2003 / Revised: 6 May 2003 / Accepted: 12 May 2003 / Published online: 24 June 2003� Springer-Verlag 2003

Abstract Randomly amplified polymorphic DNA(RAPD) and inter-simple sequence repeat (ISSR) markerswere used to analyse the genetic diversity of PortuguesePrunus dulcis cultivars and their relationship to importantforeign cultivars. Of the primers tested, 6 (out of 60)RAPD and 5 (out of 18) ISSR primers were selected fortheir reproducibility and high polymorphism. Out of 124polymerase chain reaction fragments that were scored,120 (96.8%) were polymorphic. All the plants could bediscriminated and constitute a very heterogeneous group.Five unidentified almond plants found in the region ofFoz C�a (north Portugal) and wild almond (P. webbii)from Italy and Spain were also included. Four maingroups of plants could be distinguished: P. dulciscultivars; one Foz C�a plant; P. webbii; and P. persica(outgroup). The segregating Foz C�a plant may representa feral individual or a hybrid between P. dulcis and P.webbii.

Keywords Genetic diversity · ISSR · Prunus dulcis Mill. ·Prunus webbii · RAPD

Abbreviations dNTP: Deoxynucleotide triphosphate ·CTAB: Cetyltrimethylammonium bromide ·ISSR: Inter-simple sequence repeats · PCR: Polymerasechain reaction · RAPD: Randomly amplified polymorphicDNA · RASTM: Regional Agricultural Services ofTr�s-os-Montes · TE: Tris-EDTA buffer ·UPGMA: Unweighted pair group method witharithmetical averages

Introduction

Almond, Prunus dulcis (Miller) D.A. Webb, syn. Prunusamygdalus Batsch apparently originated from one or morewild species that evolved in the deserts and lowermountain slopes of central and southwestern Asia (Mickeand Kester 1998). This fruit species is most closelyrelated to peach [Prunus persica (L.) Batsch], bothbelonging to the Prunoideae subfamily of the Rosaceae.Almond is an important tree crop (Kester et al. 1986) andit produces fruits of high commercial value. Almondcultivation in the Iberian Peninsula was intensified duringthe occupation by the Muslims but it is especially spreadthrough and well adapted to the whole Mediterraneanregion from which about 28% of the world production isobtained. In Portugal, almond is a traditional culture,mainly spread through the Algarve and Baixo Alentejo inthe south, and the Hot Land of Tr�s-os-Montes in thenorth. In traditional orchards, almond trees are still grownon marginal soils with no irrigation or much care,justifying their low productivity. Recently, large invest-ments have been made in modern orchards in the Tr�s-os-Montes region, resulting in more than 1,500 ha of newplanting areas (Cordeiro and Monteiro 2001). The imple-mentation of modern orchard management is increasingPortuguese almond production. However, the new or-chards are being established using cultivars originallyfrom France and Spain, mainly ‘Ferragn�s’, ‘Ferraduel’,‘Ferrastar’ and ‘Guara’, while Portuguese cultivars areless represented (Cordeiro and Monteiro 2001). Aiming topreserve national almond germplasm, Regional Agricul-

Communicated by P. Puigdom�nech

M. Martins ()) · M. M. OliveiraIBET/ITQB,Quinta do MarquÞs,2784-505 Oeiras, Portugale-mail: mmartins@itqb.unl.ptTel.: +351-21-4469647Fax: +351-21-4421161

M. Martins · R. Tenreiro · M. M. OliveiraDepartamento de Biologia Vegetal,Faculdade de CiÞncias de Lisboa,Campo Grande,1749-016 Lisbon, Portugal

R. TenreiroCentro de Gen�tica e Biologia Molecular,Faculdade de CiÞncias de Lisboa,Campo Grande,1749-016 Lisbon, Portugal

tural Services have recently started to establish somecollections. Portuguese cultivars with very good produc-tion and high fruit quality were identified, such as’Parada’, ’Casa Nova’ and ’Verdeal’. Breeding pro-grammes were also initiated in 2000 at RegionalAgricultural Services of Tr�s-os-Montes (RASTM), toassociate the high fruit quality of Portuguese cultivarswith the late flowering and self-fertility characters offoreign cultivars such as ’Lauranne’. Molecular studiesfor almond cultivar identification, characterisation andrelatedness are important for a better knowledge ofPortuguese cultivars, since these have been poorlycharacterised until now. Similar studies have beenperformed on foreign cultivars using several types ofmarkers: isozymes (Arulsekar et al. 1986; Hauagge et al.1987; Cerezo et al. 1989; Jackson and Clarke 1991; Arfflset al. 1994); restriction fragment length polymorphisms(Viruel 1995); randomly amplified polymorphic DNA(RAPD) (Resta et al. 1998; Bartolozzi et al. 1998; Martinset al. 2001; Ryan et al. 2001); microsatellites (Cipriani etal. 1999); inter-simple sequence repeats (ISSRs) andamplified fragment length polymorphisms (Martins et al.

2001). In our work, markers (RAPDs and ISSRs) based onthe polymerase chain reaction (PCR) have been devel-oped to establish the genetic diversity and relatednessamong Portuguese cultivars and some important foreigngenotypes. Five almond trees found in the region of Tr�s-os Montes (Foz C�a) showing certain similarities to wildalmond were also analysed and a comparison wasestablished with wild almond plants of Prunus webbiiobtained from Italy and Spain.

Materials and methods

Plant material

A total of 53 accessions were used in this study as follows: 40almond [P. dulcis (Miller) D.A. Webb, syn. P. amygdalus Batsch]cultivars from Portugal (30), Spain (2), France (3), Italy (2) and theUnited States (3); five almond plants from the Foz C�a region(north Portugal); six plants of P. webbii ; and two plants of P.persica, used as the outgroup (Table 1). Plant material was obtainedas follows: from RASTM, Regional Agricultural Services ofAlgarve, Instituto Superior de Agronomia, and directly from thefield in M�rtola and Tr�s-os-Montes, Portugal; from the University

Table 1 Accessions analysed

Plants Parental relations Sources Origin

‘Boa Casta’a, ‘Bonita’a, ‘Bonita de S¼o Braz’a,‘Casa Nova’a, ‘Colossala’, ‘Dona Virtude’a,‘Duro Amarelo’a, ‘Duro de Estradaa’, ‘DuroItaliano’, ‘Fura Sacoa’, ‘Gama’, ‘Jos� Dias’a,‘Mouriscaa’, ‘Paradaa’, ‘Pegarinhos’,‘Pestaneta’a, ‘Romeira’a, ‘Verdeal’a

Unknown RASTMb Portugal

‘Bico de Papagaio’, ‘Boa Casta’,‘Bonita de S¼o Braz’, ‘C�co do Prato’,‘Coelhinha’, ‘Duro de Estrada Grado’,‘Galamba’, ‘Paulino’, ‘Molar da Fuzeta’,‘Z� Sales’

Unknown RASAc Portugal

‘M�rtola’, ‘PenÞda’ Unknown M�rtola Portugal

‘Marcona’ Unknown RASAc Spain (region of Alicante)‘Guara’ Putative seedling selection

of unknown originServ Inv Agr (DGA)d Spain

‘Ferragn�s’ ‘Cristomorto‘�’A�’ RASTMb France (INRA, 1960)

‘Lauranne’, ‘Steliette’ ‘Ferragn�s‘x’Tuono’ Centre Mas Bov�, IRTAe France (INRA, 1978)

‘Cristomorto’, ‘Tuono’ Unknown Centre Mas Bov�, IRTAe Italy (region of Puglia)

‘Nonpareil’ Chance seedling obtained in1879 by Hatch

Centre Mas Bov�, IRTAe USA (California)

‘Tardy Nonpareil’ Mutant of ‘Nonpareil’ withaffected flowering time andgrowth habit

Centre Mas Bov�, IRTAe USA

‘Texas’ (or ‘Mission’) Seedling of ‘Languedoc’ Centre Mas Bov�, IRTAe USA (Texas)

P1, P2, P3, P4, P5 Unknown Tr�s-os-Montes Portugal (Foz C�a)

A2, A4, B2, B3, C4 (Prunus webbii) Seedlings University of Bari Italy

E1 (Prunus webbii) Seedling Serv Inv Agr (DGA)d Spain

‘Tasty Free’, ‘Rich Lady’ (Prunus persica) – ISAf USA

a Cultivar used in the prescreening of randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) primersb Regional Agricultural Services of Tr�s-os-Montes, Portugalc Regional Agricultural Services of Algarve, Portugald Servicio de Investigacin Agroalimentaria (DGA), Spaine Institut de Recerca I Tecnologia Agroalimentries, Spainf Instituto Superior de Agronomia, Portugal

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of Bari, Italy; and from Centre de Mas Bov�, IRTA, Tarragona andServicio de Investigacin Agroalimentaria (DGA), Zaragoza, Spain

DNA extraction

Total DNA was extracted from young leaves collected shortly afterbloom, following the method described by Doyle and Doyle (1987)modified by Weising et al. (1995) and adapted to almond asfollows: 1.5 g young leaves were ground in liquid nitrogen to a finepowder and extracted with cetyltrimethylammonium bromide(CTAB) hot extraction buffer [50 mM Tris-HCl, pH 8.0, 1.4 MNaCl, 20 mM EDTA, 2% (w/v) CTAB and 1% (v/v) b-mercap-toethanol]. The mixture was incubated at 60�C for 30 min, followedby two extractions with chloroform/isoamyl alcohol (24:1).Isopropanol was used to precipitate nucleic acids and the pelletobtained was dissolved in Tris-EDTA (TE) buffer (10 mM Tris-HCl, pH 8.0 and 1 mM EDTA, pH 8.0). RNA was removed bydigestion with deoxyribonuclease-free ribonuclease A. Remainingimpurities were extracted with chloroform. Total DNA wasprecipitated using sodium acetate and cold ethanol. The precipitatewas washed twice with 10 mM ammonium acetate in 76% ethanoland the pellet was dissolved in TE buffer.

The purified total DNA was quantified by gel electrophoresisand its quality verified by spectrophotometry. DNA samples werestored at 4�C. Two independent extractions were performed foreach sample.

RAPD and ISSR amplifications

A prescreening of 60 RAPD primers (OPA, OPB and OPC primersfrom Operon Technologies) and 18 ISSR primers [based onHantula et al. (1996) and Martin et al. (1998)] (Table 2) wasperformed using 15 Portuguese almond cultivars (noted in Table 1).

Six RAPD primers (OPA-07, OPA-08, OPA-10, OPA-11, OPA-16 and OPB-11) and five ISSR primers (IS03, IS06, IS16, IS17 andIS19), which combined high polymorphism with good repro-ducibility of the fragments generated were selected for screeningthe full set of accessions used in this study.

The protocol for RAPD analysis was adapted from that ofWilliams et al. (1990). PCR was performed in a volume of 25 mlcontaining 25 ng total DNA, 1�PCR buffer (Gibco-BRL), 2.0 mMMgCl2, 200 mM deoxynucleotide triphosphates (dNTPs), 1 mMdecamer oligodeoxynucleotide primer and 1 unit Taq DNA

polymerase (Gibco-BRL). The amplification reaction consisted ofan initial denaturation step at 94�C for 3 min, followed by 40 cyclesof 1 min at 94�C, 1 min at 36�C and 2 min at 72�C.

ISSR amplifications were performed in a volume of 40 mlcontaining 40 ng total DNA, 1�PCR buffer (Gibco-BRL), 2.0 mMMgCl2, 200 mM dNTPs, 1 mM oligodeoxynucleotide primer and2 units Taq DNA polymerase (Gibco-BRL). For primers IS03, IS17and IS19, a touchdown PCR was used as follows: an initialdenaturation step at 94�C for 3 min; 36 cycles of 45 s at 94�C; 45 sat specific annealing temperature and 1 min at 72�C; one lastextension step of 7 min at 72�C. The annealing was divided into11 cycles with touchdown decrements of 0.5�C, starting at 65�C(for primer IS03) or 60�C (for primers IS17 and IS19), followed by25 cycles at 60�C (for primer IS03) or 57ºC (for primers IS17 andIS19). For primers IS06 and IS16, the amplification consisted of aninitial denaturation step at 94�C for 3 min, 36 cycles of 30 s at94�C, 45 s at 60�C and 90 s at 72�C, followed by one last extensionstep of 5 min at 72�C.

Amplifications were performed in a Biometra (UNO-ther-moblock) thermocycler for both techniques.

At least two PCR amplifications were performed for eachsample with RAPD and ISSR primers to evaluate the reproducibil-ity of the bands obtained. DNA amplification fragments wereseparated in a 2% agarose gel using 1�Tris-Acetate-EDTA buffer,and stained with ethidium bromide. Gels were analysed with GelDoc 2000 software (Biorad, USA).

Data analysis

Only distinct, reproducible, well-resolved fragments, in the sizerange from 300 bp to 2.8 kb, were scored as present (1) or absent(0) for each of the RAPD and ISSR markers with the 53 accessions.Genetic similarity between pairs was estimated by the dice (SD)coefficient (Sneath and Sokal 1973), an algorithm that considersindividuals to be genetically similar only when they possess a bandin common. This approach seems to be more appropriate for agenetic character, since the lack of a common band should notimply a similarity in genetic terms. In parallel, the simple matchingsimilarity coefficient (SM) was also used for comparison withpreviously published data. This algorithm, used in several studies,considers individuals, which either possess or lack a band incommon, as a match.

Dendrograms were constructed first, independently for eachtype of molecular marker, RAPD or ISSR, using the scorablefragments. Dendrograms were compared and the congruence ofclustering produced with the two types of markers was assessed bythe Pearson correlation coefficient prior to the construction of afinal dendrogram using RAPD plus ISSR markers.

All dendrograms were constructed by cluster analysis basedupon the unweighted pair group method with arithmetical averages(UPGMA) of the BioNumerics software package, version 1.50(Applied Maths, Kortrijk, Belgium). To evaluate the fitnessbetween the original similarity matrix and dendrogram-derivedsimilarities, the cophenetic correlation was determined according toSneath and Sokal (1973).

Results and discussion

RAPD/ISSR analysis

The analysis of the prescreening data using 15 Portuguesealmond cultivars and 60 RAPD primers showed that3 primers failed to generate any amplification product, 17generated weak or ambiguous amplification products, and40 generated bright and reproducible amplification prod-ucts, from which 28 detected polymorphisms among thecultivars used. Six primers were chosen that had more

Table 2 ISSR primers tested in prescreening with 15 Portuguesealmond cultivars

Primer Sequencea

IS01 GTG(5)IS02 CA(8)IS03 GACA(4)IS04 CT(8)IS05 GT(8)IS06 GTGC(4)IS07 GAC(5)IS08 TG(10)IS10 GGAT(4)IS11 CA(8)RGIS12 AGC(6)TYIS13 AT(8)RGIS14 RG AT(8)IS15 YTA GC(6)IS16 DHB CGA(5)IS17 BDB ACA(5)IS18 DDB CCA(5)IS19 YHY GT(5)G

aY stands for C and T residues; B for C, G and T residues; D for A,G and T residues; H for A, C and T residues

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than 5 scorable bands/primer and generated more than75% of polymorphic bands.

From the prescreening assays with 15 Portuguesealmond cultivars and 18 ISSR primers, 2 failed togenerate any amplification product, 6 generated weak orambiguous amplification products, and 10 generatedbright and reproducible amplification products, fromwhich 9 detected polymorphisms among the cultivarsused. Five primers produced more than 6 scorable bands/primer, yielding over 67% of polymorphic bands, andwere selected.

Using the described DNA purification strategies andamplification conditions with the selected primers, goodand clear patterns could be obtained for the variouscultivars and accessions under study. Examples ofamplification patterns obtained by RAPD and ISSR indifferent accessions of P. dulcis are shown in Fig. 1a(RAPD) and b (ISSR).

When screening all the 53 accessions, 5–15 ampliconsper selected RAPD primer were scored, originating a totalof 63 fragments of which 96.8% were polymorphic. ForISSR, 6–19 amplicons were scored per selected primer

and from the 61 fragments obtained, 96.7% werepolymorphic. An average number of 10.5 and 12.2 bandsper selected primer was obtained for RAPD and ISSR,respectively. The integration of data obtained from thetwo techniques, using the 11 selected primers (Table 3),yielded a total of 124 reliable fragments, 120 of whichwere polymorphic (96.8%). Twenty fragments were onlyfound in the accessions of P. dulcis, 9 in the accessions ofP. webbii, and 10 in the accessions of P. persica. These39 fragments, representing ca. 32.5% of the polymorphicfragments, are putative species-specific markers.

In a study of genetic relatedness among 29 cultivars ofAmygdalus communis L. (P. dulcis) and 9 accessions of A.webbii Spach (P. webbii) Resta et al. (1998) used60 RAPD primers (Operon kits J, K and L). Theseauthors obtained a total of 470 bands with only 60.4%polymorphism and an average of 4.7 polymorphisms/primer, since all the primers originating scorable patterns(48 out of 60) were used in the study without furtherselection. From the 284 polymorphic bands, 7 bands wereconsidered specific for P. dulcis and 10 for P. webbii.These results indicate that only 17 out of the 284 (6.0%)

Fig. 1 a RAPD amplification pattern generated using primer OPA-10. b ISSR amplification pattern generated using primer IS03.Prunus dulcis cultivars are: 1 ‘Pegarinhos’ (Tr�s-os-Montes), 2‘Casa Nova’ (Tr�s-os-Montes), 3 ‘Duro Italiano’ (Tr�s-os-Montes),4 ‘PenÞda’ (M�rtola), 5 ‘Gama’ (Tr�s-os-Montes), 6 ‘Duro deEstrada Grado’ (Algarve), 7 ‘Boa Casta’ (Algarve), 8 ‘Galamba’(Algarve), 9 ‘Bonita de S¼o Braz’ (Algarve), 10 ’Molar da Fuzeta’

(Algarve), 11 ‘Z� Sales’ (Algarve), 12 ‘Guara’ (Spain), 13‘Verdeal’ (Tr�s-os-Montes), 14 ‘Colossal’ (Tr�s-os-Montes), 15‘Jos� Dias’ (Tr�s-os-Montes), 16 ‘Dona Virtude’ (Tr�s-os-Montes),17 ‘Fura Saco’ (Tr�s-os-Montes), 18 ‘Romeira’ (Tr�s-os-Montes),19 ‘Bonita’ (Tr�s-os-Montes), 20 ‘Mourisca’ (Tr�s-os-Montes), 21‘Pestaneta’ (Tr�s-os-Montes). M(a) 1 kb DNA ladder, M(b) 1 kbplus DNA ladder

Table 3 RAPD and ISSR se-quences used for the screeningof the 53 accessions, togetherwith the scorable and polymor-phic fragments obtained foreach primer

Method Primer Sequencea Fragments scored Polymorphic fragments

RAPD OPA-07 50-GAAACGGGTG 15 14OPA-08 50-GTGACGTAGG 9 9OPA-10 50-GTGATCGCAG 11 10OPA-11 50-CAATCGCCGT 9 9OPA-16 50-AGCCAGCGAA 5 5OPB-11 50-GTAGACCCGT 14 14

ISSR IS03 (GACA)4 15 15IS06 (GTGC)4 10 10IS16 DHB(CGA)5 6 4IS17 BDB(ACA)5 11 11IS19 YHY(GT)5G 19 19

a Y stands for C and T residues; B for C, G and T residues; D for A, G and T residues; H for A, C and Tresidues

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bands could be considered species-specific, which ismuch lower than 24.2% (29 out of 120) that we obtainedfor the same two species. Moreover, the 11 primers weselected produced an average of 10.9 polymorphisms/primer.

From the comparison of dendrograms obtained witheither RAPD or ISSR fragments, a value of 0.73 wasobtained for Pearson correlation coefficient (r value),indicating a good fit of both types of markers. The geneticsimilarity values obtained with both techniques wereequivalent and the dendrograms obtained were alsoanalogous. These data indicated that RAPD and ISSRmarkers were equally effective for our studies of almonddiversity, thus allowing the combination of results in onedendrogram.

Genetic relationships

The relatedness of the accessions studied was efficientlyestablished through the use of RAPD and ISSR markers.The selection of the 11 primers, based on the number andquality (high reproducibility and polymorphism) of themarkers obtained, proved to be an appropriate strategythat will facilitate future studies and make the wholeprocess more efficient in further analyses.

The dendrogram obtained by the UPGMA methodusing the total number of amplified RAPD plus ISSRfragments (124) had a cophenetic correlation of 0.90 andmost of the clusters and subclusters of the dendrogramhad values of cophenetic correlation higher than 0.80,giving a good degree of confidence in the associationsobtained for the accessions.

The dendrogram consists of four main clusters (Fig. 2),forming four groups of plants: P. dulcis cultivars (clusterI); P5 (cluster II); P. webbii (cluster III); and P. persica(cluster IV), the outgroup.

In the group of P. dulcis cultivars, minor clusters wereformed which contained Portuguese cultivars only, ormixtures with foreign cultivars indicating a close relationwith them. The genetic diversity of P. dulcis cultivars washigh; the estimated similarity coefficients, based on dicevalues (SD), ranged between 0.97 and 0.65 and it waspossible to discriminate all almond cultivars analysed.

Foreign cultivars were found to associate according totheir origin and genotype relatedness. This is a goodindication of the fitness of the results that were obtained,which is particularly important for the analysis ofPortuguese cultivars, since their genetic diversity andparental relations are unknown.

‘Tardy Nonpareil’ is usually considered to be a budsport mutation of ‘Nonpareil’, and in the study ofBartolozzi et al. (1998) using 37 RAPD markers (onlythe polymorphic ones), these two accessions could not beseparated. However, in our study based on 124 markers,we could clearly distinguish the two accessions, with a93.5% similarity level. Although a higher similarity mightbe expected, this result is in agreement with the number ofdifferences that were reported for ‘Tardy Nonpareil’ as

compared to ‘Nonpareil’: later flowering, lower produc-tivity, lower ramification and upright growth habit. In thecluster we obtained, ‘Nonpareil’ and ‘Tardy Nonpareil’are associated with several Portuguese cultivars and alsowith ‘Texas’ from the United States.

‘Ferragn�s’, ‘Cristomorto’, ‘Steliette’ and ‘Lauranne’grouped in the same cluster. ‘Ferragn�s’ was obtainedfrom a cross between ‘Cristomorto’�‘A�’ (Grasselly andDuval 1997), while ‘Steliette’ and ’Lauranne’ wereselected from the same cross of ‘Ferragn�s�’Tuono’(Grasselly and Duval 1997). As expected, ‘Steliette’ and‘Lauranne’, had a high genetic similarity (0.94).

The Spanish cultivar ‘Marcona’, originally from theAlicante region, is associated with two Portuguesecultivars, ‘Bonita’ and ‘PenÞda’. ‘Guara’ was foundassociated with ’Tuono’, segregating independently fromother almond cultivars. These varieties are phenotypicallyvery similar, and one of the possibilities for the origin of‘Guara’ is from a ‘Tuono’ seedling (R. Socias i Company,personal communication) obtained in Servicio de Inves-tigacin Agroalimentaria of Zaragoza. ‘Tuono’ is origi-nally from Italy (Puglia region) and considered to belongto the offspring of a possible ancient hybrid between P.dulcis and P. webbii (R. Socias i Company, personalcommunication). This possible origin could justify thesegregation of both cultivars.

The genetic variability of Portuguese almond cultivarswas identical to that of the P. dulcis cluster (SD=0.97–0.65), making Portuguese cultivars a very heterogeneousgroup.

For the American cultivars, Bartolozzi et al. (1998),showed in a dendrogram based on SM, similarity valuesranging from 1.00 to 0.62 (using only polymorphicmarkers), values quite similar to those we obtained withthe Portuguese cultivars (0.98–0.72) using the samesimilarity coefficient. The high variability of the geno-types may be justified by the way almond culture spreadin both countries, since Portuguese and American culti-vars were frequently originated by chance seedlings.

The development of this culture in Portugal was moresuccessful in Algarve (southern) and the Hot Land ofTr�s-os-Montes (northern), where the edaphoclimaticconditions were more propitious. In the dendrogram(Fig. 2), we can see that northern and southern cultivarsare in general mixed, indicating that no specific geo-graphical distribution was found among the Portuguesecultivars studied. Historically it is known that thecirculation of cultivars has occurred between these tworegions in Portugal, justifying our results.

The analysis of cultivars with the same or similarclassification from Algarve and Tr�s-os-Montes showedsome interesting results: ‘Duro de Estrada’ (Tr�s-os-Montes) and ‘Duro de Estrada Grado’ (Algarve) have thehighest similarity (0.97), probably corresponding tosynonymous cultivars due to improper labelling of oneof the accessions. ‘Boa Casta’ from Algarve and fromTr�s-os-Montes have a similarity coefficient of 0.94 thusrepresenting the same cultivar [they also have the same S-allele composition, S8S21 (Ma and Oliveira 2001; Certal

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et al. 2002)]. The two accessions of ‘Bonita de S¼o Braz’were considered to be homonyms (different genotypeunder the same name) since their similarity is only 0.81.This idea was reinforced by the determination of differentS-allele composition of both accessions (Sanchez et al.,unpublished). A plausible explanation for this result isthat the accession present in Tr�s-os-Montes collection is

derived from a seedling of ‘Bonita de S¼o Braz’ collectedin Algarve (originally from the S¼o Braz region).

The plants P1, P2, P3 and P4, from Foz C�a integratedclusters of P. dulcis cultivars, so they must correspond tounreported cultivars that are growing freely in the fieldhaving some phenotypic characteristics similar to wildplants.

Fig. 2 Dendrogram obtainedwith the similarity dice coeffi-cient and unweighted pair groupmethod with arithmetical aver-ages clustering algorithm for 51almond accessions and 2 Pru-nus persica cultivars (out-group). Cophenetic correlationvalues are indicated for eachcluster. ALG Algarve, Portugal;ME M�rtola, Portugal; TMTr�s-os-Montes, Portugal

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P5 is the only plant segregating independently from allother P. dulcis cultivars and also from P. webbii plants.The position of P5 on the dendrogram can be explained ifthis is a feral individual of almond or a hybrid between P.dulcis cultivars and a wild almond species. The firsthypothesis is more likely since P5 appears integrated inthe P. dulcis cluster when pairwise comparison isperformed using the simple matching coefficient similar-ity (data not shown), although this method enhances theimportance of the absence of bands in both accessions.However, when a dendrogram is made only for P5 withthe P. webbii group or with P5 and the P. dulcis group(data not shown) we find that P5 is equidistant from bothgroups (SD=0.55 or 0.57, respectively). Thus, the possi-bility of P5 being a hybrid could also be a feasibleexplanation, since in the Mediterranean region spontane-ous hybridisation between almond cultivars and wildspecies of almond, mainly P. webbii, has already beenreported (Godini 1979; Socias i Company 1990). More-over, P5 characteristics are intermediate between those ofP. webbii and P. dulcis cultivars and there were severalplants found in Foz C�a region appearing to havephenotypic characteristics of P. webbii (V. Cordeiro andA. Monteiro, personal communication).

All the plants of P. webbii formed a unique clusterwhere the wild plant from Spain associated with the fivewild plants from Italy. In our work, the wild species P.webbii is closely related to cultivated almond, withgenetic similarity values SD=0.55 and SM=0.68. Further,Resta et al. (1998) found a close relationship of P. webbiito P. dulcis cultivars, obtaining in their studies a geneticsimilarity SM=0.65 between these species.

P. persica was used as an outgroup since, in the genusPrunus, this species is closely related to almond. In thisstudy the similarity between peach and almond wasSD=0.36 and SM=0.56. This value is higher than thosefound in previous studies where only RAPD markers wereused: SM=0.42, in the study of Bartolozzi et al. (1998) andSM=0.50 according to Warburton and Bliss (1996). In thesecond case they were studying peach genetic diversityand almond was the outgroup.

The great diversity found among Portuguese cultivarsand the interesting germplasm of Foz C�a plants supportsthe idea that Portugal has a valuable source of almondgenes to be exploited in breeding programmes. Theestablishment of genetic relatedness and molecular char-acterization of Portuguese almond cultivars is fundamen-tal as an informational basis for such programmes. Thesetechniques will also be useful for the organisation andconservation of world almond collections.

Acknowledgements Part of this research was supported by PraxisXXI through PhD grant BD/11323/97 provided to M.C. Martinsand the research projects PRAXIS 3/3.2/HORT/2143/95 andPRAXIS/PCNA/P/BIO/72/96. Prof. Peter Lindley is gratefullyacknowledged for the English revision of the manuscript. We alsogratefully acknowledge Ing. Vitor Cordeiro, Ing. E. LeopoldoFerreira, Prof. Cristina Oliveira, Prof. Angelo Godini, Prof. RafaelSocias i Company and Dr. Ignasi Batlle, for providing the plantmaterial.

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