nuclear and chloroplast dna variability and phylogeny of iranian apples (malus domestica)

ORIGINAL ARTICLE

Nuclear and chloroplast DNA variability and phylogeny of Iranianapples (Malus domestica)

Abdollah Khadivi-Khub • Shahin Jahangirzadeh •

Elahe Ahadi • Samad Aliyoun

Received: 25 February 2013 / Accepted: 2 February 2014

� Springer-Verlag Wien 2014

Abstract This study was conducted to reveal genetic

diversity among 23 local apple genotypes using nuclear

(RAPD) and chloroplast DNA (PCR-cpRFLP) markers.

Eleven RAPD primers and four cpDNA primer combina-

tions were used in this study. RAPD primers produced a

total of 77 polymorphic fragments with an average of seven

bands per primer. The percentage of polymorphic bands

(68.14 %) showed the efficiency of used RAPD primers in

distinguishing all the genotypes considered. Genetic simi-

larity between studied genotypes varied from 0.38 to 0.72

and cluster analysis showed the abundant diversity, indi-

cating high intraspecific genetic variation between Iranian

apple genotypes. From the four universal chloroplast pri-

mer pairs, three primer pairs amplified the fragments and

their combinations showed polymorphic patterns and

revealed intraspecific chloroplast variation. The informa-

tion will facilitate germplasm identification, conservation

and new cultivar development.

Keywords Nuclear and chloroplast DNAs � Cluster

analysis � Genetic diversity � Phylogenetic relationships �Malus domestica

Introduction

Apple (Malus 9 domestica Borkh.) is a member of Rosa-

ceae and one of the most widespread and popular fruit trees

in the world (Janick et al. 1996). About 59 species and

7500 cultivars were identified in all over the world. Apple

is an ancient fruit crop in Iran (Janick et al. 1996) and there

is an extremely abundant germplasm resource for it. There

is a high level of genetic diversity in Iran’s cultivated apple

due to closely distance to apple origin in Central Asia.

Apple is an economically important fruit crop and has a

broad spectrum uses as food; most apples are eaten raw but

can be made into jellies, pies, cakes and puddings. They

can also be canned, juiced and optionally fermented to

produce apple juice, cider, vinegar and pectin (Jackson

2003).

Knowledge of genetic variation and phylogenetic rela-

tionship among genotypes is an important consideration for

classification, utilization of germplasm resources and

breeding. Traditionally, cultivar identification has relied on

morphological and agronomic characteristics of plant

materials. Although there is substantial intraspecific vari-

ation in vegetative traits, especially leaf and fruit charac-

ters, it is difficult to distinguish genotypes on their external

morphology alone. Further, phenotypic characters are

generally influenced by environmental factors and the

growth stage of the plant. In fruit trees, this requires a

lengthy and expensive evaluation during the whole vege-

tative growth. There is an additional problem to distinguish

among cultivars which come from the same cross. In such

cases, the relatively narrow range of variation of morpho-

logical traits limits cultivar identification, and different

methods must be used (Khadivi-Khub et al. 2008).

The development of randomly amplified polymorphic

DNA (RAPD) markers, generated by the polymerase chain

A. Khadivi-Khub (&)

Department of Horticultural Sciences, Faculty of Agriculture and

Natural Resources, Arak University, 38156-8-8349 Arak, Iran

e-mail: [email protected]; [email protected]

S. Jahangirzadeh

Department of Horticultural Sciences, Faculty of Agriculture,

University of Guilan, Rasht, Iran

E. Ahadi � S. Aliyoun

Department of Horticultural Sciences, Faculty of Agriculture,

University of Tehran, 31587 Karaj, Iran

123

Plant Syst Evol

DOI 10.1007/s00606-014-1007-y

reaction (PCR) using arbitrary primers, has provided a new

tool for the detection of DNA polymorphisms (Welsh and

McClelland 1990; Williams et al. 1990). The RAPD ana-

lysis has already proved to be valuable in apple genome

analysis. These markers have been used for identification

of apple cultivars (Koller et al. 1993; Mulcahy et al. 1993)

and apple rootstocks (Autio et al. 1998) and for paternity

analysis (Harada et al. 1993; Gardiner et al. 1996). Luis

et al. (2001) employed RAPD in discrimination and esti-

mation of genetic similarities among apples cultivars,

while Zhou and Li (2000) used RAPD for the phylogenetic

relationships of the closely related species of cultivated

apple. In most cases, data on genetic similarity obtained by

RAPD analysis matched classifications based on morpho-

logical traits in apple (Landry et al. 1994).

Chloroplast DNA (cpDNA) shows maternal inheritance

in most flowering plants, does not undergo recombination

and has a slow rate of evolution (Palmer 1987); therefore,

cpDNA is a good tool for tracing seed parents and maternal

ancestors. The detection of restriction fragment length

polymorphisms (RFLPs) in specific regions of cpDNA,

amplified by the polymerase chain reaction (PCR), has

been developed as a method for detecting variations in

cpDNA. There are many other horticultural plants whose

backgrounds are unclear, and PCR–RFLP analysis has

been used in horticultural cultivars of chrysanthemum

(Kishimoto et al. 2003), rose hybrids (Takeuchi et al. 1995)

and sweet cherry (Mohanty et al. 2001; Panda et al. 2003).

This method also has been used for analysis of apple

genotypes and phylogenetic relationships in wild and

domesticated apples (Coart et al. 2006).

In this study, we used RAPD and PCR-cpRFLP markers to

characterize nuclear and chloroplast genome in apple geno-

types from different regions of Iran, for both commercial and

selected clones from the breeding program. Molecular

markers were used to study the genetic variation and phylo-

genetic relationships among local apple genotypes.

Materials and methods

Plant material and DNA isolation

Twenty-three local apple genotypes from different geo-

graphical and/or agroclimatic locations in Iran were used in

this study (Table 1). These genotypes are representing

genetic diversity of three main regions of apple in Iran. We

selected these materials with high severity and accuracy. It

is necessary to note that these genotypes have high mor-

phological variation, and their resistance to chilling and

Table 1 The studied apple

genotype names, codes and their

regions

Genotype Region Code of genotype Origin Flavor

T3 Central Alborz T3 Iran Sweet

Ferdos Central Alborz Ferdos Iran Sweet

Shahrod10 Central Alborz Shah10 Iran Sweet

Shahrod19 Central Alborz Shah19 Iran Sweet

Golab-Damavand Central Alborz GolDa Iran Sweet

Golab-Paize Central Alborz GolPA Iran Sweet

Golab-Nemati Central Alborz GolNe Iran Sweet

Azerbaijan6 Azerbaijan Azer6 Iran Sweet



Beyghi Azerbaijan Beyghi Iran Sweet

Mahali-Urmia Azerbaijan Mahali Iran Sweet–sour

ME 3 Azerbaijan ME 3 Iran Sweet

ME 4 Azerbaijan ME 4 Iran Sweet

Ghara-Yarpagh Azerbaijan Ghara Iran Sweet

Khan-Almasi Azerbaijan Khan Iran Sweet

Meshkin-Shahr Azerbaijan Mesh Iran Sweet

SBA Central Zagros SBA Iran Sweet

Golab-Arak Central Zagros GolAr Iran Sweet

ShahreKord8 Central Zagros ShahKord8 Iran Sweet

Torsh-Kermanshah Central Zagros TorKer Iran Sour

Ghermez1 Central Zagros Gherm1 Iran Sweet

Zodras-Oghlid Central Zagros ZodOgh Iran Sweet

Granny-Smith Australia Granny Australia Sweet–Sour

A. Khadivi-Khub et al.

123

drought is different. Also, flowering and ripening times in

these genotypes are varied. Furthermore, one commercial

cultivar, Granny-Smith, was used as an outgroup. Young

leaves were collected, frozen in liquid nitrogen and subse-

quently stored at -20 �C until processed. Genomic DNA

(nuclear and chloroplast) was isolated from leaf samples

using a CTAB extraction method (Doyle and Doyle 1987).

The extraction buffer contained 2 % CTAB, 1.5 M NaCl,

20 mM EDTA, 100 mM Tris pH 8.0, 2 % soluble poly

vinyl pyrrolidone (PVP), and 2 % 2-mercaptoethanol. DNA

quality was examined by electrophoresis in 0.8 % agarose,

and DNA concentration was quantified on a GeneQuant

(Amersham Pharmacia Biotech).

RAPD analysis

For the RAPD reactions, 20 ng of DNA was used as tem-

plate in a final volume of 25 ll containing 1x reaction

buffer (20 mM (NH4)2SO4, 75 mM Tris–HCl pH 8.8,

0.01 % Tween 20), 1.9 mM MgCl2, 200 lM of each dNTP,

0.4 lM primer and 0.6 U Taq DNA polymerase, overlaid

with a drop of mineral oil. An extra reaction containing all

the components, plus water instead of template DNA, was

included in all the experiments. The DNA amplifications

were performed in a thermocycler (iCycler, Bio Rad Co.,

USA) as follows: 1 cycle of 5 min at 93 �C, 45 cycles of

1 min at 93 �C, 1 min at 36 �C, and 2 min at 72 �C (for

denaturing, annealing and primer extension, respectively).

The last cycle was followed by a final incubation for 10 min

at 72 �C, and the PCR products were stored at 4 �C before

analysis. The DNA amplification products were analyzed

by electrophoresis in 1.5 % agarose gels (Roche Co., Ger-

many) in 1x TBE buffer for 5 h at 3.5 V/cm. The gels were

stained for 20 min with ethidium bromide (1 lg/ml) and

destained in water for 15 min and photographed under UV

light, by a Gel Doc system (UVP, Bio Doc Co., USA).

Fragment length was estimated by comparison with stan-

dard size markers (100 base pair ladder).

Chloroplast analysis (PCR-cpRFLP)

Extracted DNAs were PCR-amplified using four universal

primer pairs for chloroplast DNA (K1K2, CS, HK and TF)

(Dumolin-Lapegue et al. 1997; Grivet et al. 2001). The

amplification of cpDNA regions was performed in 30 ll of

reaction mixture containing 30 ng of total DNA, 10 ng of

each primer pair, 200 lM of each four dNTPs, one unit

Taq DNA polymerase (recombinant, Fermentas, Canada),

2 mM of MgCl2 and 1X PCR buffer with KCl. The ther-

mocycle system used was Gene Amp PCR system 9700.

PCR was carried out as described by Mohanty et al.

(2001), using an initial cycle of 4 min at 94 �C, followed

by 30 cycles of 45 s at 94 �C, 45 s at 54.5 �C to 58.5 �C

and 2 or 3 min at 72 �C (annealing temperature and

extension time depending on the primer and the length of

the fragment to be amplified) and finally a 10 min exten-

sion at 72 �C. Two restriction enzymes (EcoRI and MseI;

Fermentas, Canada) were used for digesting 5 ll PCR

products in a volume of 30 ll containing 10 unit of

restriction enzyme following the manufacturer’s instruc-

tions (each reaction mixture was incubated for 10 h).

cpDNA restriction fragments were separated in 3 % aga-

rose gels cast in a Tris–borate-EDTA buffer (1 9), by

running at 70 V for 4 h, and after that, fragments were

visualized in UV light. Each mixture loaded in agarose gel

contained 10 ll of restriction mixture, 3 ll of loading

buffer and 2 ll Gel Red (Biotium, USA). The size markers

used for analyzing the size of polymorphic bands were

1 kb and 50 bp ladders (Fermentas, Canada).

Data analysis

Data were recorded as presence (1) or absence (0) of

amplified products from the examination of photographic

negatives. The total number of fragments scored, the number

of polymorphic fragments and the percentage of polymor-

phic fragments were determined for each primer used.

Genetic similarity based on RAPD and chloroplast data was

calculated by making a pairwise comparison between all

genotypes according to the Jaccard coefficient using the

Simqual module of NTSYS-pc software version 2.01e

(Rohlf 1998). Similarity matrices were compared using the

Mantel matrix-correspondence test (Mantel 1967) using

MxComp module in NTSYS-pc ver 2.1 (Rohlf 2000).

Bootstrap analysis with 1000 replicates was performed using

FreeTree software (version 0.9.1.50, Pavlicek et al. 1999) to

obtain the confidence of branches of the UPGMA and

Neighbor-joining (NJ) trees and validation of the dendro-

grams. Trees were viewed using Tree View program, version

1.6.6 (Page 1996). The bootstrap analysis estimates the

probability values (P values) for each cluster (the probability

of a true cluster at each edge of the dendrogram). Also, Dollo

Parsimony was used for phylogenetic relationships by PUAP

software. Genetic parameters such as total diversity (Ht),

average intraspecific diversity (Hs) and level of population

subdivision (GSTC) were estimated using POPGENE ver.

1.31 (Yeh et al. 1999) to determine Nei’s genetic diversity

(He) and Shannon’s information index (I) amongregions.

Results and discussion

RAPD analysis

Primer selection is essential for discrimination analysis.

Obviously, the more bands scored and plants studied, the

Nuclear and chloroplast DNA variability and phylogeny

123

higher the statistical significance of the calculation. About

100 bands should be enough to obtain statistically signifi-

cant results (Kocsis et al. 2005), however, Koller et al.

(1993) described 11 apple cultivars using only two RAPD

primers. Tancred et al. (1994) also used only two RAPD

primers to distinguish a new apple genotype from three

commercial apple cultivars. Autio et al. (1998) identified 15

apple rootstocks using only two RAPD primers, and Or-

aguzie et al. (2001) described 155 new and old apple

genotypes using only nine RAPD primers. In the present

study, one-hundred 10-mer primers, corresponding to

TIBMOLBIOL Co., Germany, were initially screened using

three genotypes from different regions to determine the

suitability of each primer for the study. Primers were

selected for further analysis based on their ability to detect

distinct, clearly resolved and polymorphic amplified pro-

ducts between genotypes. To ensure reproducibility, the

primers generating no, weak, or complex patterns were

discarded. In total, 11 primers out of 100 primers produced

clear and reproducible bands. Table 2 shows codes and

sequences of the used primers, total number of the ampli-

fication fragments (bands) resultant from apple samples and

the number of polymorphic fragments for each primer. A

total of 113 RAPD fragments were detected for the studied

genotypes with an average 10.27 bands per primer. Eleven

primers generated 77 polymorphic fragments with an

average 7 bands per primer, thus reflecting the high genetic

diversity within the germplasm, which distinguished

Granny-Smith cultivar from all the Iranian genotypes. Or-

aguzie et al. (2001) reported 42 polymorphic bands for 155

apple genotypes by nine RAPD primers and suggested high

variation and Bayan et al. (2007) reported 82 % polymor-

phism for Syrian local apples using RAPD, while Goulao

et al. (2001) stated that RAPD techniques revealed low level

of polymorphism percentage among apple cultivars.

The amplified fragments per primer ranged from seven

(for TIBMBD-16 primer) to 15 (for TIBMBC-20 primer)

(Table 2). Also, the polymorphic bands varied from four

(for TIBMBD-16 primer) to 10 (for TIBMBC-20 primer).

The relative number of polymorphic fragments to the total

number of amplified fragments ranged between 50 % for

TIBMBB-05 and TIBMBB-17, while TIBMBE-08 primer

showed the highest polymorphism (100 %) that agreed

with the finding of Khadivi-Khub et al. (2008) about this

primer in the study of sweet cherry. In the present study,

the average of polymorphism was 68.14 % for all primers.

Erturk and Akcay (2010) reported 180 polymorphic bands

in 11 apple genotypes by 38 RAPD primers with an

average of 40 % polymorphism. The PIC values for the

used primers were high in this study. The size of amplified

fragments ranged from 300 to 3,000 bp (Fig. 1) which

agreed with the findings of other apple studies by RAPD

(Koller et al. 1993; Mulcahy et al. 1993; Duneman et al.

1994; Autio et al. 1998; Luis et al. 2001). This polymor-

phism showed high genetic diversity in studied genotypes.

Also, previously, these set RAPD primers showed high

polymorphism and diversity in Prunus avium cultivars

(Khadivi-Khub et al. 2008), confirming the suitability of

these RAPD primers for analysis of diversification in fruit

trees that belong to Rosaceae family. In the present study,

studied genotypes were collected from different parts of

Iran, which might be the reasons for the higher percentage

polymorphism (68.14 %) in comparison with the results of

Goulao et al. (2001) about Malus domestica. It was pre-

viously shown that RAPD markers have great potential to

identify relationships among Malus domestica genotypes

(Koller et al. 1993; Mulcahy et al. 1993; Harada et al.

1993; Gardiner et al. 1996; Autio et al. 1998; Luis et al.

2001; Zhou and Li 2000). Some polymorphic bands pro-

duced by RAPD primers seemed to be unique for some

Table 2 List of RAPD primers,

their numbers of total and

polymorphic fragments and

percentage of polymorphism

used in this study

Primer Sequence 50 to 30 Total of

bands (a)

Polymorphic

bands (b)

Polymorphism

%(b/a 9 100)

TIBMBA-06 GGACGACCGT 8 5 62.50

TIBMBA-08 CCACAGCCGA 10 7 70.00

TIBMBA-20 GAGCGCTACC 12 9 75.00

TIBMBB-05 GGGCCGAACA 10 5 50.00

TIBMBB-17 ACACCGTGCC 14 7 50.00

TIBMBC-04 CCACGTGCCA 11 9 81.82

TIBMBC-12 CCTCCACCAG 9 6 66.67

TIBMBC-20 AGCACTGGGG 15 10 66.67

TIBMBD-16 GAACTCCCAG 7 4 57.14

TIBMBD-17 GTTCGCTCCC 8 6 75.00

TIBMBE-08 GGGAAGCGTC 9 9 100.00

Total 113 77

Mean 10.27 7.00 68.14


123

genotypes, indicated that nuclear genome of these geno-

types had some special differences due to mutations (Matus

and Hayes 2002). For example, one band from primer

TIBMBA-08 was unique for Zodras-Oghlid genotype. It

should be noted that this genotype is the most early-rip-

ening fruit between studied genotypes, and may be this

unique band have been related to this traits.

Genetic similarity values between studied genotypes

varied from 0.38 to 0.72 (Table 3), indicating high intra-

specific variation between Iranian apple genotypes. This is

in agreement with a formerly study on Iranian apple

genotypes by Gharghani et al. (2009) and Farrokhi et al.

(2011). They studied genotypes of other regions of Iran and

reported high genetic variation for them, so that Farrokhi

et al. (2011) reported 0.19-0.79 genetic similarity for Ira-

nian apples. Bayan et al. (2007) reported 0.43 to 0.76

diversity between studied Syrian local apples using RAPD,

while, Adebayo et al. (2009) reported 65-87 % genetic

similarity between Nigerian apples by RAPD and sug-

gested a narrow genetic base in the local Nigerian selection

of apple.

The highest genetic similarity was scored between two

genotypes from Central Alborz that included Golab-Da-

mavand and Golab-Paize (72 %) and followed by simi-

larity between two genotypes from Azerbaijan, included

Mahali-Urmia and Azerbaijan6 (71 %), while the lowest

genetic similarity was scored between Zodras-Oghlid from

Central Zagros and Granny-Smith as an outgroup (38 %).

Furthermore, the lowest genetic similarity between Iranian

genotypes was observed between ShahreKord8, from

Central Zagros, and Zodras-Oghlid, from Central Alborz

(46 %) and followed by similarity between ME 4, from

Azerbaijan, and Golab-Arak, from Central Zagros (49 %).

The UPGMA cluster analysis revealed genetic rela-

tionships among genotypes. The cophenetic correlation

coefficient (CCC) indicated high correlation (r = 0.93)

between the similarity matrix and the cophenetic matrix

(obtained from the UPGMA dendrogram), indicating a

good representation of the molecular relationships among

genotypes. The cophenetic correlation coefficient is con-

sidered to be a very good representative of the data matrix

in the dendrogram if it is 0.90 or greater (Romesburg

1990). When the dendrogram determining the rate of rel-

ativity between genotypes on the basis of coefficient of

similarity was examined (Fig. 2), it was observed that the

diversity ratio among the apple genotypes was high. The

dendrogram generated by RAPD analysis showed two

distinct clusters with strong bootstrapping value (100 %)

for these two clusters. The first main cluster included all

Iranian genotypes except Torsh-Kermanshah, while,

Granny-Smith, foreign cultivar, was placed in the second

main cluster. The first cluster was divided into five subcl-

usters but was supported by low to moderate bootstrap

values. This clustering was according to geographical

distribution so that genotypes collected from Central Za-

gros, Central Alborz and Azerbaijan showed high variation

based on their geographical region. These results agreed

with the finding of other studies about apple genotypes

(Royo and Itoiz 2004; Adebayo et al. 2009) that reported

correlation between geographical distribution and intra-

specific variation in apple. Some genetic similarities were

observed between genotypes of different regions. For

example, Azerbaijan14 genotype, from Azerbaijan region,

showed high similarity with Golab-Paize genotype from

Central Alborz. Beside, Ferdos genotype from Central

Alborz was placed with Azerbaijanian genotypes, indicat-

ing possibility of genotype migration or gene flow between

regions. This finding confirmed that the RAPD method can

solve one of the major problems of varietal identification in

pome fruits: the existence of homonyms and synonyms,

particularly with regard to cultivars that were cultivated for

centuries and are widely distributed (Borrego et al. 2002).

Previously, RAPD analysis was used successfully to

reveal genetic variations in many studies (Kocsis et al.

2005; Lisek et al. 2006; Koc et al. 2009). Notwithstanding

the limitations, RAPD markers proved to be a highly

Fig. 1 RAPD profile of 23 Iranian apple genotypes and Granny-Smith cultivar as an outgroup, produced by TIBMBE-08 primer


123

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123

effective and efficient method for the genetic analysis (Luo

et al. 2002; Ulanovsky et al. 2002). Large numbers of data

sets can be generated because different RAPD primers are

commercially available (Fanizza et al. 2000). Our results

confirmed that RAPD analysis can be used to differentiate

apple genotypes and confirmed the potential of RAPD

technology as a reliable, rapid and inexpensive screening

method to discriminate quince genotypes.

Chloroplast DNA analysis

In the present study, from the four universal chloroplast

primer pairs tested on apple studied genotypes (K1K2, CS,

HK and TF), only primer pair TF that amplified fragment

trnT- trnF in tobacco, did not amplify any band in studied

apple genotypes. Similar result was reported by Mohanty

et al. (2001) that used TF primer pairs on Prunus avium

having the same family with genus Malus and did not

obtain any amplification. Other primer pairs amplified the

fragments (Table 4). Chloroplast DNA size in apple has

been reported by Ishikawa et al. (1992) to digest this

molecule by restriction enzyme (BamHI) in Golden

Delicious, McIntosh and Red Delicious cultivars which

are, respectively, 143.4, 143.35 and 143.7 kb. We con-

sidered this molecule (143.48 kb) by averaging the

reported sizes. Therefore, the fragment that primers

amplified (5920 bp) by approximately 4.13 % of the

chloroplast genome was analyzed.

Cluster I

Shahrod19

Azerbaijan14

Golab-Arak

Golab-Paize24

46

Golab-Damavand

SBA48

40

Azerbaijan6

T3

Khan-Almasi

Zodras-Oghlid26

14

8

Shahrod10

Ghermez1

Ghara-Yarpagh

Golab-Nemati11

6

ME3

Meshkinshahr30

4

3

1

ME4

ShahreKord818

1

Azerbaijan7

Ferdos22

Beyghi

Mahali-Urmia34

15

51

Granny-Smith

Torsh-Kermanshah41

100

Cluster II

Fig. 2 Dendrogram of genetic

relationships between apple

genotypes, constructed by

UPGMA based on 11 RAPD

markers. Numbers in the

branches represent bootstrap

values


123

From six combinations of primer-restriction enzyme

used in this study for analysis of cpDNA, two combina-

tions, K1K2-MseI and CS-MseI, resolved monomorphic

fragments, and the region amplified by CS primer did not

have any site for EcoRI. The remaining combinations

showed polymorphic pattern. Combinations of K1K2-

EcoRI and HK-MseI had two sites for enzymes. HK-MseI

showed two polymorphic patterns but in combination with

K1K2-EcoRI having two sites; one of the fragments was

monomorphic and the other one was polymorphic

(Table 4). All of these polymorphic patterns resulted

because of insertion–deletion (indel) mutation between 20

and 50 bp ranges. Indel mutations in the combinations of

K1K2-EcoR I and HK- EcoRI are shown in Fig. 3.

This estimation illustrated that the genetic similarity

was varied. In some cases, studied genotypes showed

100 % similarity that confirmed the conservation of the

chloroplast genome within these genotypes (Palmer 1987).

According to polymorphism pattern, all studied genotypes

were placed into two main clusters with strong bootstrap-

ping value (100 %) (Fig. 4). Based on this clustering, in

some case, intraspecific chloroplast variation was observed

within genotypes. This could be due to the existence of

intraspecific cpDNA mutation, which has been previously

reported in sweet cherry (Mohanty et al. 2001) and apple

(Coart et al. 2006). Also, in some cases, genotypes of

different regions showed high chloroplast similarity. The

first main cluster was divided to two subclusters with low

to moderate bootstrapping values that most of Azerbaja-

nian genotypes were placed in this cluster. Also, the sec-

ond cluster was divided to two subclusters that Granny-

Smith cultivar showed high chloroplast similarity to Torsh-

Kermanshah genotype. These two apples have sour fruit

flavor.

Chloroplast DNA is known to be a very conservative

molecule (Palmer 1987), hence chances of detecting

intraspecific cpDNA polymorphism are low. But, in the

present study, intraspecific polymorphism was observed in

most cases. This result agreed with the finding of Coart

et al. (2006) that reported intraspecific chloroplast variation

in apple. But, Verbylait _e et al. (2006) evaluated chloroplast

of Maloideae and reported no intraspecific chloroplast

variation within studied species because of low studied

genotypes for each species.

The results of the present study demonstrated that,

although cpDNA is a very conservative cytoplasmic

molecule, variation exists therein to distinguish even at

the level of intraspecific cultivars. Intraspecific diversity

in plant cpDNA has been believed to be the result of

mutations, genetic drift, selection by man for particular

cpDNA-encoded characters (herbicide resistance), or the

Fig. 3 Restriction patterns in two chloroplast DNA primer-enzyme combinations HK-EcoRI (a) and K1K2-EcoRI (b) resolved on agarose gel

for chloroplast analysis

Table 4 Nei’s analysis of gene diversity in studied apple genotypes

based on chloroplast DNA

cpDNA

primers

Combination Digestion

pattern

Ht Hs Gst I

KIK2 K1K2-EcoRI KEA 0.50 0.49 0.03 0.68

CS K1K2-EcoRI KEB 0.50 0.49 0.03 0.68

HK HK- EcoRI HEA 0.39 0.30 0.23 0.67

TF HK- EcoRI HEB 0.39 0.30 0.23 0.67

HK-MseI HM1A 0.46 0.33 0.29 0.69

HK-MseI HM1B 0.46 0.33 0.29 0.69

HK-MseI HM2A 0.49 0.49 0.01 0.67

HK-MseI HM2B 0.34 0.29 0.15 0.61

HK-MseI HM2C 0.45 0.43 0.05 0.61

Mean – 0.44 0.38 0.14 0.67

St. Dev – 0.01 0.01 – 0.03

Ht: Total diversity; Hs: Average intraspecific diversity; GSTC: level

of population subdivision; I: Shannon’s index


123

introgression of a foreign cpDNA (chloroplast capture)

from related species during the early domestication of the

taxon and even in wild taxa (Harris and Ingram 1991).

Such introgression has been reported to be common for

many plant species (e.g. in Iris species; Arnold et al.

1992).

In some cases, genotypes of Central of Alborz region,

which is the nearest region to Azerbaijan, were beside

Azerbaijanian genotypes. This means that these genotypes

probably have been transferred from Azerbaijan that has a

vast area of culture and history of apple. Meanwhile, the

same maternal parent cannot be completely rejected

because introduction from other areas and selection for

better genotypes have been factors for transmission of

genotypes between regions from the past. One of the main

reasons for the absence of genetic structure among

Golab-Arak

Khan-Almasi

Ghara-Yarpagh

T330

77

Ghermez1

Golab-Paize83

40

ShahreKord8

Azerbaijan14

Ferdos24

88

17

47

Meshkinshahr

Azerbaijan6

Azerbaijan792

Shahrod10

Shahrod1990

65

32

21

Beyghi

ME390

Golab-Damavand

Mahali-Urmia85

48

Golab-Nemati

ME441

30

Granny-Smith

Torsh-Kermanshah53

SBA

Zodras-Oghlid98

13

20

100

Cluster I

Cluster II

Fig. 4 UPGMA dendrogram of studied apple genotypes based on chloroplast data. Numbers in the branches represent bootstrap values


123

different populations appears to be cytoplasmic gene flow

among them (that is, seeds migration). The migration rate

of seeds may be higher than expected (Powell et al. 1996).

Phylogenetic analysis revealed the evolution of studied

germplasm (not shown), so that Beyghi, ME3 and Mahali-

Urmia genotypes, from Azerbaijan, had the most close to

their ancestors, while, Ferdos and ShahreKord8 genotypes

showed the lowest close to their ancestors. Granny-Smith

cultivar and Torsh-Kermanshah genotype showed similar

phylogeny. It should be noted that Torsh-Kermanshah has

sour flavor like Granny-Smith.

Mean of total diversity (Ht), average intraspecific diver-

sity (Hs) and level of population subdivision (GSTC) calcu-

lated by POPGENE program were 0.44, 0.38 and 0.14,

respectively (Table 4). According to the genetic differenti-

ation among genotypes of different regions in apple

(GSTC = 0.14) and comparison of it by similar reports such

as in Prunus avium (GSTC = 0.08; Mohanty et al. 2001) and

Prunus spinosa (GSTC = 0.19; Mohanty et al. 2000) which

are from the same family with the genus Malus (Rosaceae), it

could be determined that chloroplast genetic differentiation

among regions in the present study (GSTC) is low and is much

Ferdos

T3

Ghara-Yarpagh

Khan-Almasi54

34

Azerbaijan14

ShahreKord817

4

3

Ghermez1

Golab-Arak27

1

ME3

Meshkinshahr25

Mahali-Urmia

Azerbaijan6

Azerbaijan764

22

Shahrod10

Shahrod1981

2

Golab-Paize

Golab-Damavand

Golab-Nemati47

25

1

1

Torsh-Kermanshah

SBA

Zodras-Oghlid82

16

3

Granny-Smith

Beyghi

ME424

6

100

Cluster I

Cluster II

Fig. 5 UPGMA dendrogram of

studied apple genotypes based

on integrating data of nuclear

and chloroplast DNA

(RAPD ? cpRFLP). Numbers

in the branches represent

bootstrap values


123

lower compared to forest species such as Quercus petraea

(GSTC = 0.90; Petit et al. 1993a), Fagus sylvatica

(GSTC = 0.83; Demesure et al. 1996), Argania spinosa

(GSTC = 0.60; El Mousadik and Petit 1996) and Alnus glu-

tinosa (GSTC = 0.87; King and Ferris 1998). Petit et al.

(1993b) reported that in some species the GSTN (genetic

differentiation among populations using nuclear markers) is

much lower than the GSTC. It could be deduced that cyto-

plasmic genomes which have inheritance from maternal

parents are relatively much more structured than the nuclear

genome because cytoplasmic gene flow is limited just by

seed dispersal (Powell et al. 1996).

Combined analyses of RAPD and cpDNA

The collection and characterization of local germplasm are

of primary importance for the prevention of their loss and

to allow the exploitation of materials that are potentially

useful for future purposes (Palmer 1987). Furthermore, the

characterization of collections of local germplasm can also

help in the clarification of some aspects concerning the

diffusion and evolution of crop plants. Therefore, a char-

acterization of local genotypes and cultivars of apple from

different Iran regions can help us to understand the path-

ways of dissemination and evolution of this species.

0.1

Azerbaijan6

Beyghi

ME422

Granny-Smith

Torsh-Kermanshah27

4

Golab-Damavand

Mahali-Urmia10

Golab-Nemati

Shahrod10

Shahrod1985

3

Ferdos

Azerbaijan7

Meshkinshahr7

2

ME3

ShahreKord814

Golab-Arak

Ghermez1

Khan-Almasi2

1

Ghara-Yarpagh

T314

1

Azerbaijan14

Golab-Paize9

SBA

Zodras-Oghlid81

4

100

Cluster I

Cluster II

Fig. 6 NJ phylogenetic

dendrogram of studied apple

genotypes based on integrating

data of nuclear and chloroplast

DNA (RAPD ? cpRFLP).

Numbers in the branches

represent bootstrap values


123

Accordingly, in the present study, 23 apple genotypes were

analyzed using two different types of markers, including

nuclear (RAPD) and chloroplast markers (PCR–RFLP).

The combined use of different types of markers from two

genomes that are likely to be unevenly affected by major

evolutionary forces, such as migration, selection, mutation,

genetic drift and recombination, offers the opportunity to

make inferences from the patterns of genetic diversity, and

thus to define a detailed evolutionary scenario and to fully

describe the genetic makeup of different groups of germ-

plasm (Provan et al. 2001). Our results showed that this

approach is very useful and informative. For instance, the

use of markers showing biparental vs uniparental trans-

mission allowed us to detect the structure of genetic

diversity in the apple. Similarly, we were able to observe

phylogenetic diversity in studied apples based on the

cytoplasmic (chloroplast). Overall, our results argue for a

co-ordinated multiple marker approach for germplasm

characterization, conservation and evaluation. Based on

data, studied apple genotypes showed a higher nuclear

variation (RAPD) than chloroplast variation.

The level of polymorphism revealed by nuclear (RAPD)

and chloroplast markers could determine the relationships

between the DNA profiles and the geographical distribution

of some genotypes. Chloroplast markers are more suitable

for this purpose due to the low rate of mutations and

recombination in the chloroplast genome (Provan et al.

2001) as well as because of their maternal inheritance in

Malus. Also, some cases of similarity were observed

between data based on RAPD and chloroplast data. For

instance, genotypes of Central Alborz region showed

similarity with Azerbaijanian genotypes according to these

two data. The estimation of correlation between matrices of

nuclear data (RAPD) and cpDNA data (r = 0.40) indicated

non-significant correlation between these data and agreed

with the finding of Horvath et al. (2008) that reported an

insignificant correlation coefficient between nuclear DNA

and chloroplast DNA data in Prunus cerasifera (cherry

plum).

Three dendrograms were constructed based on the

combined data of both markers (RAPD ? cpRFLP) using

UPGMA, NJ and Dollo Parsimony methods. The

Fig. 7 Dollo Parsimony phylogenetic dendrogram of studied apple genotypes based on integrating data of nuclear and chloroplast DNA

(RAPD ? cpRFLP). Numbers in the branches represent bootstrap values


123

advantage of combining different datasets is a compre-

hensive taxonomic picture, since each represents a distinct

level of taxonomic differentiation and confirmed view-

point of Sneller et al. (1997) in this case. The UPGMA

dendrogram produced based on combined data presented a

high resolution in separating the different apple genotypes

into two major clusters but with a high bootstrapping

value (100 %) (Fig. 5). The first cluster consists of three

subclusters. Subcluster I contained most genotypes from

Central Alborz. Subcluster II contained two subgroups, so

that subgroup I contained Azerbaijanian genotypes, sug-

gesting a possible genetic relatedness between them.

Another interesting feature was that Golab cultivars which

had much similarity in tree, flower, fruit, flowering and

ripening were placed in the same subgroup by the com-

bined analysis. Subcluster III was consisted of genotypes

of Central Zagros and showed high molecular genetic

similarity between them. Finally, the second major cluster

was consisted of two genotypes of Azerbaijan and

Granny-Smith. These two genotypes have green fruit skin

like Granny-Smith.

Phylogenetic dendrogram according to the integrating

data based on both data (RAPD ? cpRFLP) using NJ

method revealed the evolution of studied germplasm but

with low and moderate bootstrapping values (Fig. 6),

while, phylograms derived from Dollo parsimony analysis

performed using PAUP were similar to those produced by

NJ method in most cases with moderate to high boot-

strapping values (50–100 %) (Fig. 7), so that most of the

bootstrapping values were 50–80 % and some of them

were strong (80–100 %). In both dendrograms of NJ and

Dollo parsimony methods, most cases of similarities were

observed. For instance, genotypes Shahrod10, Shahrod19,

Azerbaijan7 and Azerbaijan6 were placed in the same

group. Also, T3, Ghara-Yarpagh and Khan-Almasi showed

close similarity based on both clusters. Furthermore, both

dendrograms detected close relationships between Mahali-

Urmia, ME3, Beyghi, ME4 and Granny-Smith and also

among ShahreKord8, Ferdos, Meshkinshahr and Azerbai-

jan14. Beside, these similarities were observed for other

genotypes. For the 24 apple genotypes analyzed in the

present study, there existed a theoretical expectation for the

clustering of genotypes within the dendrogram, based on

their botanical pedigree. Genetic similarity dendrograms

were constructed either using the results taken by RAPD or

cpRFLP analysis and by their combination. The prevailing

opinion over the past few years is that nuclear and chlo-

roplast genome-based approaches are complementary

methods to provide the most complete coverage of these

genomes. Hence, in the pedigree analysis, the combined

RAPD ? cpRFLP analysis was found to be the most

accurate system for the identification of genotypes from the

different regions.

Conclusion

We have demonstrated that old varieties are potential

sources of diversity in apples, in addition to the wild

species and current cultivars. Different apple cultivars and

local genotypes have been cultivated in Iran for a long

time, possibly for many centuries and are least frequently

grown in the southeastern, northeastern and central

regions and most widespread in the north and west of Iran.

It can be generalized that genotypes selected from these

regions most likely represent the majority of genotypes

cultivated or local varieties in Iran. The Azerbaijanian

genotypes did not completely separate from other samples,

and this could be due to migration of genotype by mostly

human activity or gene flow. The genotypes from this

collection will be used in the forthcoming cross-breeding

studies. The characterization results will be of great

importance on the selection of the parents and the com-

binations. Information on the origin of cultivated and its

phylogenetic relationship with the closely related species

is now becoming more and more important for future

apple breeding.

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nuclear and chloroplast dna variability and phylogeny of iranian apples (malus domestica)

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