Comparison of SSR polymorphisms using automated capillary sequencers,

and polyacrylamide and agarose gel electrophoresis: Implications for the

assessment of genetic diversity and relatedness in almond

R. Sanchez-Perez a, J. Ballester b, F. Dicenta a, P. Arus b, P. Martınez-Gomez a,*a Departamento de Mejora Vegetal, CEBAS-CSIC, P.O. Box 164, E-30100 Espinardo, Murcia, Spain

b Departament de Genetica Vegetal, IRTA, Laboratori de Genetica Molecular Vegetal, CSIC-IRTA, Crta, Cabrils s/n, 08348 Cabrils, Barcelona, Spain

Received 18 July 2005; received in revised form 10 January 2006; accepted 2 February 2006

Abstract

In this work we have performed a comparative study of the utilization of three electrophoresis separation methods for the analysis of peach SSR

polymorphism in almond and its implications in the assessment of genetic diversity and relatedness. Automated SSR detection and polyacrylamide

gel electrophoresis were the most efficient methods and would be able to resolve allelic variation at a finer scale than the electrophoresis in

MetaPhor1 agarose. Moreover, automated electrophoresis detection is much more expensive in terms of cost of equipment and cost of the analysis.

In addition, SSR detection using polyacrylamide gel electrophoresis showed similar results than automated sequencing, although it is more time-

consuming and toxic than electrophoresis in agarose gels. Discrepancies among polyacrylamide and automated capillary, and MetaPhor1 were

observed when differences in SSR polymorphisms were between 1 and 5 bp. While the use of MetaPhor1 agarose appears less indicated for

genotype characterization, this technique may be the most convenient in other applications, i.e. mapping of population, due to its lower cost and

easier routine application. The utilization MetaPhor1 agarose produced a slightly different clustering of genotypes in spite of the big differences

observed in the DNA fingerprinting. However, bootstrap values using MetaPhor1 agarose were lower indicating a lower accuracy of this SSR

polymorphism analysis method.

# 2006 Elsevier B.V. All rights reserved.

Keywords: Prunus; Molecular markers; Genetic relationships; Germplasm

www.elsevier.com/locate/scihorti

Scientia Horticulturae 108 (2006) 310–316

1. Introduction

Simple sequence repeat sequences (i.e. microsatellites, SSR

markers) are good molecular markers for genetic diversity

studies in a wide range of plants. Because of their high

polymorphism, abundance, and codominant inheritance, they

are well suited for the assessment of genetic diversity within

crop species (Gupta et al., 1996; Wunsch and Hormaza, 2002a).

In the case of Prunus species, primer pairs flanking SSRs have

been cloned and sequenced in different species including

peach, apricot, cherry, and almond (Wunsch and Hormaza,

2002a; Aranzana et al., 2003; Martınez-Gomez et al., 2003a).

SSR markers have been used in Prunus species for the

molecular characterization and identification of cultivars

(Cipriani et al., 1999; Testolin et al., 2000; Hormaza, 2002;

* Corresponding author. Tel.: +34 968 396 200; fax: +34 968 396 213.

E-mail address: pmartinez@cebas.csic.es (P. Martınez-Gomez).

0304-4238/$ – see front matter # 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.scienta.2006.02.004

Wunsch and Hormaza, 2002b; Martınez-Gomez et al., 2003b;

Ahmad et al., 2004; Sanchez-Perez et al., 2005) and rootstocks

(Serrano et al., 2002), and the elaboration of genetic linkage

maps (Aranzana et al., 2003). In these assays, different

methods for the separation and detection of the DNA amplified

fragments have been used. Polyacrylamide gel electrophoresis

with radioactive and silver staining was the first method used

in the analysis of the PCR amplified fragment of DNA

(Cipriani et al., 1999). MetaPhor1 agarose gel electrophoresis

is a method used as alternative to the polyacrylamide due to

its cheaper cost and easier application to routine (Morgante

et al., 2001). More recently, new methods for the PCR analysis

of SSR polymorphisms have been developed based on the

utilization of automated sequencer capillary electrophoresis

(Aranzana et al., 2003; Ahmad et al., 2004). However, to date a

comparison of the efficiency of these methods for the

separation and analysis of the DNA fragments and the

implications in the assessment of genetic diversity and

relatedness have been not performed.

R. Sanchez-Perez et al. / Scientia Horticulturae 108 (2006) 310–316 311

The objective of this work was to compare the relative

efficiency of automated sequencer capillary electrophoresis,

and polyacrylamide and MetaPhor1 agarose gel electrophor-

esis for the analysis of peach SSR polymorphism in almond

and its implications in the assessment of genetic diversity and

relatedness.

2. Material and methods

2.1. Plant material and DNA isolation

The plant material assayed included 21 almond (Prunus

dulcis (Mill.) D.A. Webb) cultivars from different origins

(Table 1). Total genomic DNA was isolated using the

procedure described by Doyle and Doyle (1987). Approxi-

mately 50 mg of young leaves were ground in a 1.5 ml

Eppendorf tube with 750 ml of CTAB extraction buffer

(100 mM Tris–HCl, 1.4 M NaCl, 20 mM EDTA, 2% CTAB,

1% PVP, 0.2% mercaptoethanol, 0.1% NaHSO3). Samples

were incubated at 65 8C for 20 min, mixed with an equal

volume of 24:1 chloroform–isoamyl alcohol, and centri-

fuged at 6000 � g (20 min). The upper phase was recovered

and mixed with an equal volume of isopropanol at �20 8C.

The nucleic acid pellet was washed in 400 ml of 10 mM

NH4Ac in 76% ethanol, dried, resuspended in 50 ml of

TE (10 mM Tris–HCl, 0.1 mM EDTA, pH 8.0), and

incubated with 0.5 mg of RNase-A at 37 8C for 30 min, to

digest RNA.

Table 1

Pedigree, origin and main agronomic characteristics (shell hardness, self-compatib

Cultivar Origin Pedigree Shell hardness

Achaak Tunisia Unknown Semi-Hard

All in one USA Almond � peach Soft

Antoneta Spain Ferragnes � Tuono Hard

Ardechoise France Unknown Soft

Bonita Portugal Unknown Hard

Chellastone Australia Unknown Semi-Hard

Desmayo Spain Unknown Hard

Ferragnes France Cristomorto � Aı Semi-Hard

Garrigues Spain Unknown Hard

Genco Italy Unknown Hard

Glorieta Spain Primorskii � Cristomorto Hard

Guara Spain Open poll. of Tuono Hard

Lauranne France Ferragnes � Tuono Semi-Hard

Marcona Spain Unknown Hard

Marta Spain Ferragnes � Tuono Hard

Masbovera Spain Primorskii � Cristomorto Hard

Mission USA Unknown Semi-Hard

Nonpareil USA Unknown Soft

Primorskii Ukraine Princes 2077 � Nikitski 53 Soft

R-1000 France Tardy Nonpareil � Tuono Semi-Hard

Tuono Italy Unknown Hard

Means values of heterozygosity

a Heterozygosity level of the almond cultivars using automated sequencer cap

MetaPhor1 agarose electrophoresis (MAGE).

2.2. PCR amplification

Extracted almond genomic DNA was PCR-amplified using

six pair of primers flanking SSR sequences, previously cloned

and sequenced in peach (Prunus persica (L.) Basch) by

different authors (Table 2). PCR reactions were performed in a

25 ml volume and the reaction mixture contained 16 mM

(NH4)2SO4, 67 mM Tris–HCl pH 8.8, 0.01% Tween-20, 2 mM

MgCl2, 0.2 mM of each primer, 0.1 mM of each dNTP, one unit

of Taq DNA Polymerase (Ecogen S.R.L.), and 90 ng of

genomic DNA. Cycling parameters were: one cycle of 95 8Cfor 3 min; 35 cycles of 94 8C for 1 min, 57 8C for 1 min, except

for the SSR primer CPPCT 022, for which the annealing

temperature was 50, and 72 8C for 1 min; followed by 10 min at

72 8C. PCR reactions were carried out in a 96-well block

Eppendorf Mastercycler.

2.3. PCR product analysis

Amplified PCR products were analyzed by Automated

Sequencer Capillary Electrophoresis (ASCE) using PRISM

ABI1 Genetic Analyzer (Applied Biosystems, Foster City,

California, USA). The size standard used in the sequencer was

Gene ScanTM 500 RoxTM using in the analysis the software

GeneScan 3.7 (Applied Biosystems).

On the other hand, SSR products were separated using

polyacrylamide gel electrophoresis (PAGE). PCR products

were diluted and mixed with a formamide/bromophenol blue

loading buffer. Samples were denatured at 93 8C for 5 min,

ility, and flowering date) of the almond cultivars assayed

Self-compatibility Flowering date Heterozygosity levela

ASCE PAGE MAGE

Self-incompatible Early 0.83 0.83 0.83

Self-compatible Intermediate 1.0 1.0 1.0

Self-compatible Late 1.0 1.0 1.0

Self-incompatible Intermediate 0.67 0.67 0.50

Self-incompatible Intermediate 0.67 0.67 0.67

Self-incompatible Late 0.83 0.83 0.83

Self-incompatible Early 1.0 1.0 0.67

Self-incompatible Late 0.67 0.67 0.50

Self-incompatible Intermediate 0.67 0.67 0.67

Self-compatible Late 1.0 1.0 1.0

Self-incompatible Late 0.67 0.67 0.50

Self-compatible Late 1.0 1.0 0.67

Self-compatible Late 0.83 0.83 0.83

Self-incompatible Intermediate 0.67 0.67 0.67

Self-compatible Late 0.50 0.50 0.17

Self-incompatible Late 0.83 0.83 0.83

Self-incompatible Intermediate 0.83 0.83 0.83

Self-incompatible Intermediate 1.0 1.0 0.83

Self-incompatible Late 1.0 1.0 1.0

Self-compatible Late 0.83 0.83 0.83

Self-compatible Late 1.0 1.0 0.67

0.83 0.83 0.74

illary electrophoresis (ASCE), polyacrylamide electrophoresis (PAGE) and

R. Sanchez-Perez et al. / Scientia Horticulturae 108 (2006) 310–316312

Table 2

Peach simple sequence repeat (SSR) markers assayed and polymorphism obtained in the almond cultivars assayed using automated sequencer capillary

electrophoresis (ASCE), polyacrylamide electrophoresis (PAGE) and MetaPhor1 agarose electrophoresis (MAGE)

SSR marker Reference Linkage Heterozygosity Power of discrimination N8 of alleles Size

Groupa ASCE PAGE MAGE ASCE PAGE MAGE ASCE PAGE MAGE Range (bp)

pchgms3 Sosinski et al. (2000) G1 0.95 0.95 0.67 0.89 0.89 0.83 10 10 7 168–212

BPPCT007 Dirlewanger et al. (2002) G3 0.90 0.90 0.76 0.92 0.92 0.85 10 10 5 124–158

CPPCT005 Aranzana et al. (2002) G4 0.90 0.90 0.90 0.93 0.93 0.84 11 11 6 122–158

MA27a Yamamoto et al. (2002) G6 0.76 0.76 0.76 0.93 0.93 0.90 12 12 6 114–146

MA40a Yamamoto et al. (2002) G6 0.81 0.81 0.57 0.93 0.93 0.83 12 12 8 196–261

UDP96005 Cipriani et al. (1999) G1 0.81 0.81 0.81 0.92 0.92 0.91 9 9 8 125–158

Mean values 0.86 0.86 0.75 0.92 0.92 0.86 10.7 10.7 6.7

a Linkage group according to Aranzana et al. (2003).

loaded onto sequencing gel (6% polyacrylamide, 7.5 M urea)

electrophoresis in 1� TBE buffer at a constant current of

120 W. Results were visualized using silver staining kit from

Promega (Promega Inc., Madison, USA) following the manu-

facturer’s instructions. A 10 bp Plus DNA Ladder (Invitrogen

Life Technologies, Barcelona, Spain) was used as molecular

size standard.

Finally, amplified PCR products were separated by 3%

MetaPhor1 Agarose Gel Electrophoresis (MAGE) (Biowit-

taker, Maine, USA) gel (1� TBE buffer) at 5 V/cm, stained by

ethidium bromide (0.5 mg/ml), and visualized under UV light.

A 10 bp Plus DNA Ladder (Invitrogen Life Technologies,

Barcelona, Spain) was also used as molecular size standard.

2.4. Data analysis and genetic relatedness

Polymorphic alleles were scored as present or absent (0/1)

calling these alleles with letters starting with the letter ‘‘a’’ for

the smaller one. Band scoring was analyzed using GeneTools

gel analysis software of SYNGENE (Beacon House, Nuffield

Road, Cambridge, UK).

Heterozygosity of each SSR marker was calculated as the

number of heterozygous cultivars divided by the total number

of cultivars. Power of discrimination of each SSR marker was

calculated as PD ¼ 1�P

g2i where gi is the frequency of the

ith genotype. In addition, heterozygosity of almond cultivars

was calculated as the number of heterozygous loci for a

given cultivar divided by the total number of loci assayed.

Mean character difference distances were calculated for

all pairwise comparisons with the MEGA test (Kumar et al.,

2001), which was used to construct neighbor-joining (NJ)

dendrograms depicting the phenetic relationship among geno-

types. Relative support for the branches in each dendrogram

was assessed with 2000 replicates of NJ bootstrap.

3. Results

3.1. Polymorphism and heterozygosity of SSR markers

Amplification was successful in almond with the different

SSR markers initially developed for peach and a high level of

polymorphism was detected in all of them. The number of

presumed alleles revealed by the SSR analysis was similar in

the case of the utilization of ASCE and PAGE (from 9 to 12)

(Table 2).

The analysis of DNA fingerprinting using MAGE revealed a

lower level of polymorphism, from 5 to 8 alleles. Size range of

amplified bands was between 114 and 261 bp for all the

analysis methods. A total of 64 polymorphic bands were scored

using ASCE and PAGE, and 40 bands using MAGE. The mean

number of alleles per locus was 10.7 with ASCE and PAGE, and

6.7 with MAGE (Table 2).

On the other hand, direct-count heterozygosities of SSR

markers were higher in the case of the ASCE and PAGE

methods in comparison with MAGE in concordance with the

higher number of allele revealed. They ranged from 0.57, for

the SSR marker MA40a (using MAGE) to 0.95 for pchgms3

(using ASCE and PAGE), with average of 0.86 (ASCE and

PAGE), and 0.75 (MAGE) for all loci studied. However,

power of discrimination was similar with the tree methods

assayed in spite of the big differences observed in the

number of alleles revealed. The average of this parameter for

all loci was 0.92 for ASCE and PAGE and 0.86 for MAGE

(Table 2).

3.2. DNA fingerprinting and heterozygosity of almond

cultivars

Table 3 shows the results on molecular identification of the

21 almond cultivars examined. Size differences detected

between alleles at a locus ranged from 2 bp (observed in all

the SSR markers assayed) to 12 bp (MA40a). All primer pairs

produced a maximum of two bands per cultivar, in accordance

with the diploid constitution of this species. Fingerprinting

obtained using ASCE and PAGE was similar. However,

differences between these two methods and MAGE in the

score of bands were very important in concordance with the loss

of polymorphism observed with the use of MAGE in

comparison with ASCE and PAGE. Discrepancies among

MAGE, ASCE and PAGE were observed when size differences

in SSR fragments were between 1 and 5 bp in most of cases. In

these situations the interpretation of band between MAGE and

the other two methods were different.

On the other hand, heterozygosities of almond cultivars

were higher with the use of ASCE and PAGE in comparison

with MAGE in concordance with the higher number of alleles

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Table 3

Comparison between the DNA fingerprinting obtained in each almond cultivars using the peach simple sequence repeat (SSR) sequences and analyzing the DNA polymorphisms using automated sequencer capillary

electrophoresis (ASCE), polyacrylamide electrophoresis (PAGE) and MetaPhor1 agarose electrophoresis (MAGE)

Almond cultivars pchgms3 BPPCT007 CPPCT005 MA27a MA40a UDP96005

ASCE PAGE MAGE ASCE PAGE MAGE ASCE PAGE MAGE ASCE PAGE MAGE ASCE PAGE MAGE ASCE PAGE MAGE

Achaak 212 jj gg 124/145 ae ac 142/152 eh ce 122/138 di be 226/234 fi de 127/158 bi ag

All in one 176/188 eg ce 145/153 ei cd 122/130 ab ab 128/140 fj ce 212/228 bg bd 130/140 ce bd

Antonieta 170/174 bd ab 124/149 ag ad 122/154 ai ae 118/134 bh bd 226/246 fj df 135/140 de cd

Ardechoise 174/176 de bb 143 dd cc 130/138 bc bc 114/118 ab ab 222 ee cc 127/155 bh ag

Bonita 168/196 ah ae 124 aa aa 130/142 be bc 118/126 be ac 230 hh dd 125/140 ae ad

Chellastone 172/204 ci af 132/158 bj be 130/138 bc bc 114 aa aa 214/220 cd bc 147/158 gi fh

Desmayo 170/174 bd ab 143/145 de cc 142/152 eh ce 138/146 il ef 228/230 gh dd 125/140 ae ad

Ferragnes 174 dd bb 149/151 gh dd 122/138 ac ac 120 cc bb 226/246 fj df 140/155 eh dg

Garrigues 174/184 df bd 143/145 de cc 130/142 be bc 126 ee cc 222/258 ek cg 125 aa aa

Genco 174/188 dg cd 134/153 ci bd 122/154 ai ae 128/132 fg cd 212/230 bh bd 130/143 cf be

Glorieta 174 dd bb 143/151 dh cd 122/156 aj af 140 jj ee 226/228 fg dd 127/155 bh ag

Guara 170/172 bc aa 124/149 ag ad 138/154 ci ce 134/140 hj de 226/230 fh dd 135/155 dh cg

Lauranne 170/174 bd ab 124/151 ah ad 138 cc cc 118/132 bg bd 226/246 fj df 135/140 de cd

Marcona 170/174 bd ab 132/147 bf bc 130/158 bk bf 118/126 be bc 261 ll hh 140 ee dd

Marta 172/174 cd bb 124/151 ah ad 138 cc cc 140 jj ee 226/230 fh dd 155 hh gg

Masbovera 174 dd bb 143/151 dh cd 122/150 ag ae 140/144 jk ef 196/226 af ad 127/155 bh ag

Mission 168/188 ag ae 132/149 bg bd 130/140 bd bc 140/144 jk ef 228 gg dd 143/158 fi eh

Nonpareil 168/170 ab aa 132/149 bg bd 144/154 fi de 114/120 ac ab 212/258 bk bg 155/158 hi gh

Primorskii 174/188 dg cd 143/149 dg cd 150/156 gj ef 140/144 jk ef 196/228 ag ad 127/158 bi ah

R-1000 168/172 ac ab 124/132 ab ab 144/154 fi de 120/134 ch bd 212/226 bf bd 155 hh gg

Tuono 170/172 bc bb 124/149 ag ad 138/154 ci ce 134/140 hj de 226/230 fh dd 135/155 dh cg

Allele Size (pb)a a = 168, b = 170, c = 172,

d = 174, e = 176, f = 184,

g = 188, h = 196, i = 204;

j = 212

a = 124, b = 132, c = 134,

d = 143, e = 145, f = 147,

g = 149, h = 151, i = 153,

j = 158

a = 122, b = 130, c = 138,

d = 140, e = 142, f = 144,

g = 150, h = 152, i = 154,

j = 156, k = 158

a = 114, b = 118, c = 120,

d = 122, e = 126, f = 128,

g = 132, h = 134, i = 138,

j = 140, k = 144, l = 146

a = 196, b = 212, c = 214,

d = 220, e = 222, f = 226,

g = 228, h = 230, i = 234,

j = 246, k = 258, l = 261

a = 125, b = 127, c = 130,

d = 135, e = 140, f = 143,

g = 147, h = 155, i = 158

a Calculated using PRISM ABI1 genetic analyzer.

R. Sanchez-Perez et al. / Scientia Horticulturae 108 (2006) 310–316314

Fig. 1. Dendrogram obtained by NJ cluster analysis based on the mean character difference distances among almond cultivars using SSR markers and analyzing the

DNA by automated sequencer capillary electrophoresis (ASCE), polyacrylamide electrophoresis (PAGE) and MetaPhor1 agarose electrophoresis (MAGE). Numbers

in the branches represent bootstrap values. The scale bar represents simple matching distance.

detected by these two methods. These differences were very

important in the case of cultivar ‘Marta’ with a heterozigosity

level of 0.17 using MAGE and 0.50 with ASCE and PAGE.

Mean values for all cultivars were 0.83 using ASCE and

PAGE, and 0.74 with MAGE. These heterozygosities ranged

between 0.17 (detected by MAGE) for ‘Marta’, and 1 for ‘All

in one’, ‘Antoneta’, ‘Desmayo’, ‘Genco’, ‘Guara’, ‘Nonpar-

eil’ and ‘Primorskii’ (with ASCE and PAGE) (Table 1).

3.3. Genetic relatedness and clustering of almond cultivars

Almond NJ dendrogram was similar in the case of the

utilization of ASCE and PAGE. However, the analysis of DNA

fingerprinting using MAGE produced a slightly different NJ

dendrogram. Almond cultivars clustered into different groups

mainly according to their pedigree. No apparent clustering by

agronomic characteristics was observed. Bootstrap values of NJ

dendrogram obtained with the utilization of ASCE and PAGE

were higher than those obtained using MAGE (Fig. 1).

Regarding the NJ dendrogram obtained with the utilization

of ASCE and PAGE, there is strong support (bootstrap values

higher than 60%) for clustering of ‘Tuono’ and ‘Guara’; self-

compatible cultivars ‘Genco’ and ‘All in one’, and the

descendants of ‘Primorskii’, ‘Masbovera’ and ‘Glorieta’. Other

relationships moderately supported (bootstrap values higher

than 40%) are ‘Tuono’ and ‘Guara’ and ‘Marta’ (descendants of

‘Tuono’); ‘Primorskii’ and its descendants ‘Masbovera’ and

‘Glorieta’; cultivars ‘Achaak’ and ‘Desmayo’; and ‘Ferragnes’

and its descendants ‘Lauranne’ and ‘Antoneta’. A weak

relationship (bootstrap values lesser than 40%) was observed

in the rest of the almond cultivars assayed. In the case of NJ

dendrogram obtained with the utilization of MAGE strongly

supported clusters were similar although with lower bootstrap

values with the exception of the cluster of ‘R-1000’ and

‘Nonpareil, and ‘Ardechoise’ and ‘Garrigues’ (Fig. 1).

4. Discussion

4.1. Polymorphism and heterozygosity of SSR markers

Overall, the results of the obtained polymorphism suggest a

high degree of transportability between peach SSR in almond.

These results agree with reports by Cipriani et al. (1999),

Serrano et al. (2002), and Martınez-Gomez et al. (2003b) on the

successful utilization of these markers in different Prunus

species.

However, important differences in the methods used in the

analysis of SSR polymorphisms were observed in the number

of alleles revealed and the heterozygosity level of the markers,

being ASCE and PAGE the most informative methods with a

loss of polymorphism with the use of MAGE. In this sense,

heterozygosities of SSR markers were higher in the case of the

ASCE and PAGE in comparison with MAGE in concordance

with the higher number of allele revealed.

4.2. DNA fingerprinting and heterozygosity of almond

cultivars

Automated SSR detection using ASCE and PAGE are the

most efficient methods and would be able to resolve allelic

R. Sanchez-Perez et al. / Scientia Horticulturae 108 (2006) 310–316 315

variation among the almond cultivars at a finer scale in

comparison with MAGE. Moreover, ASCE detection is much

more expensive than the other two methods in term of cost

of equipment and cost of the analysis. In addition, PAGE is

more time-consuming and toxic than MAGE. On the other

hand, SSR detection using ASCE and PAGE can complicates

the identification of microsatellite alleles, mainly due to the

presence of ladder-like stutter patterns (Wunsch and Hormaza,

2002b).

SSR analysis using ASCE and MAGE, enable to detect a

higher number of SSR alleles, gave a higher heterozygosity

level in the almond cultivars assayed. In accordance with these

results, level of SSR heterozygosity described by Martınez-

Gomez et al. (2003b) in a wide range of almond cultivars using

the same MetaPhor1 agarose was 0.56, on average, for all

the cultivars studied. Results showed a higher level of

heterozygosity in almond cultivars (0.74 on average using

MAGE) in comparison with the results obtained with a simi-

lar set of peach SSR markers in peach cultivars (0.22) using

the same MetaPhor1 agarose by Martınez-Gomez et al.

(2003b). These results confirm the narrow genetic base

observed in peach when compared to the great diversity

observed in the almond cultivars probably due to mating-

system differences: selfing in peach and out crossing in

almond, as already found studying isoenzyme systems (Byrne,

1990).

4.3. Genetic relatedness and clustering of almond cultivars

DNA fingerprinting originated a phylogenetic dendrogram

similar in the case of ASCE and PAGE, while the analysis with

MAGE produced a slightly different NJ dendrogram.

However, the big differences observed in the DNA finger-

printing did not affect strongly the relationships observed in

both dendograms.

In general, relationships strongly and moderately supported

were similar in both cases, although bootstrap values of NJ

dendrogram obtained with the utilization of ASCE and PAGE

were higher than those obtained using MAGE, indicating the

larger number of observed bands with a higher polymorphism.

These results also indicate a higher accuracy of these SSR

polymorphism analysis methods (ASCE and PAGE) in

comparison with MAGE in the studies of genetic relatedness

in almond.

5. Conclusions

Results establish the value of SSR markers for identifica-

tion and distinguishing different genetic lineages and chara-

cterize a large gene pool available for almond breeding

programmes, because of their high polymorphism, abundance,

and codominant inheritance. In these studies, the analysis

of amplified PCR products by MetaPhor1 Agarose Gel

Electrophoresis (MAGE) has to be completed by the auto-

mated sequencer capillary electrophoresis (ASCE) or the

polyacrylamide gel electrophoresis (PAGE) analysis due to its

lower level of accuracy.

The DNA fingerprinting obtained by ASCE or PAGE could

be of great importance for a certification programme to protect

the new releases, particularly in the cases of species such as

fruit crops that involve vegetative propagation. For routine

SSR genotype analysis, for example in mapping of popula-

tions, MAGE is the most convenient method when DNA

amplified fragments differed in 5 bp or more, due to its lower

cost and easier routine application. Finally, the application of

SSRs for Marker Assisted Selection was described by Testolin

(2003), who indicated the possibility of finding SSR markers

near the encoding genome sequences. However, further

studies of SSR markers in progenies segregating for

agronomic traits have to be performed for the development

of genetic maps and markers associated with genes or QTLs

involved in the inheritance of agronomic traits.

Acknowledgements

This work has been financed by the project AGL2001-1054-

C03-01 (‘‘Mejora Genetica del Almendro’’) from the Spanish

Ministry of Science and Technology.

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