Genetic identity and relationships of Iranian apple ... - Springer Link

Genet Resour Crop Evol (2009) 56:829–842 DOI 10.1007/s10722-008-9404-0

RESEARCH ARTICLE

Genetic identity and relationships of Iranian apple (Malus 3 domestica Borkh.) cultivars and landraces, wild Malus species and representative old apple cultivars based on simple sequence repeat (SSR) marker analysis Ali Gharghani Æ Zabihollah Zamani Æ Alireza Talaie Æ Nnadozie C. Oraguzie Æ Reza Fatahi Æ Hassan Hajnajari Æ Claudia Wiedow Æ Susan E. Gardiner Received: 25 November 2008 / Accepted: 22 December 2008 / Published online: 24 January 2009 Ó Springer Science+Business Media B.V. 2009

Abstract In order to shed light on the role of Iran in apple evolution and domestication, we chose to investigate the relationships of a collection of 159 accessions of wild and domesticated apples including Iranian indigenous apple cultivars and landraces, selected wild species, and old apple scion and rootstock cultivars from different parts of the world. The majority of the wild species belonged to M. sieversii, which is widely believed to be the main maternal wild ancestor of domestic apples, from Kazakhstan and A. Gharghani (&) Z. Zamani A. Talaie R. Fatahi Department of Horticultural Science, University of Tehran, 31587-11167 Karaj, Iran e-mail: [email protected] A. Gharghani Department of Horticultural Sciences, Faculty of Agriculture, University of Shiraz, PO Box 7144165186, Shiraz, Iran e-mail: [email protected] N. C. Oraguzie Department of Horticulture & Landscape Architecture, IAREC, Washington State University, 24106N Bunn Road, Prosser, WA 99350, USA H. Hajnajari Horticulture Department, Seed and Plant Improvement Institute (SPII), 31585-4119 Karaj, Iran C. Wiedow S. E. Gardiner The Horticulture and Food Research Institute of New Zealand Ltd (HortResearch), Private Bag 11030, Palmerston North 4442, New Zealand

M. orientalis, which is one of the probable minor ancestors of domestic apples, from Turkey and Russia located on the east and west of Iran, respectively. The accessions were assigned into six arbitrary populations for the purpose of generating information on genetic parameters. Nine simple sequence repeat (SSR) loci selected from previous studies in apple were screened over DNA extracted from all the accessions. Results showed that all SSR loci displayed a very high degree of polymorphism with 11–25 alleles per locus. In total, there were 153 alleles across all loci with an average of 17 alleles per locus. The SSR allelic data were then used for estimation of population genetic parameters, including genetic variation statistics, F-statistics, gene flow,genetic identity, genetic distance and then cluster analysis using POPGENE 1.32 software. The F-statistics and gene flow in particular, showed that there was more intra-population than between population variation. The genetic identity and genetic distance estimates, and the dendrogram generated from the un-weighted pair group arithmetic average (UPGMA) method of cluster analysis showed that the Iranian cultivars and landraces were more closely related to M. sieversii from Central Asia (east of Iran) and M. orientalis native to Turkey and Russia than to other accessions of Malus species. Also, the old apple cultivars from different parts of the world have a closer genetic relationship to M. sieversii, M. orientalis and the Iranian apples, than to other wild species. Based on these results, we suggest that the Iranian apples may occupy an intermediate position between the

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domesticated varieties and wild species. We propose that Iran could be one of the major players in apples’ domestication and transfer from Central Asia to the western countries. Keywords Apple rootstock cultivars Apple scion cultivars Genetic diversity Genetic relationship Iranian apples Malus spp M. orientalis M. sieversii Microsatellite

Introduction Apple is the fourth most important fruit crop worldwide after citrus, grapes, and banana and it is the most ubiquitous and well-adapted of the temperate fruit crop species. It is grown in areas ranging from high latitude regions of the world where temperatures may reach -40°C, to high elevations in the tropics where two crops may be grown in a single year (Janick et al. 1996). The cultivated apple (Malus 9 domestica Borkh.) is not a simple taxonomic group and includes all the cultivated types in the genus Malus Mill. In the 1920s, Vavilov (1926) traveled through Central Asia and found large wild stands of M. sieversii in specific localities and proposed that the region could be a center of origin of the domesticated apple. Another early study based on morphological characteristics suggested that several Malus species, including M. sylvestris Miller, M. prunifolia (Willd.) Borkh., and M. baccata (L.) Borkh., were involved in the origin and/or domestication of the cultivated apple (Rehder 1940). Other studies based on random amplified polymorphic DNA (RAPD), isozyme and simple sequence repeat (SSR) markers have demonstrated the importance of M. sieversii (Ledeb.) Roem., M. orientalis Uglitz., and M. mandshurica (Maxim.) V. Kom. in the evolution of domestic apples (Dunemann et al. 1994; Lamboy et al. 1996; Hokanson et al. 1998). The currently most widely accepted theory, based mainly on morphological and molecular evidence, points to series Malus (Dunemann et al. 1994; Forsline et al. 1994; Morgan and Richards 1993) and specifically to M. sieversii (Ledeb.) Roem. from Central Asia as the most likely maternal ancestor

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Genet Resour Crop Evol (2009) 56:829–842

(Robinson et al. 2001; Harris et al. 2002). Recently, Coart et al. (2006) suggested that M. sylvestris may be the main maternal wild progenitor of domestic apples, based on chloroplast diversity data. However, based on isozyme marker research, Wagner and Weeden (2000) believed that M. sylvestris may be in the genetic background of cider apples selected in Spain, France and Britain, rather than of dessert apples. Although the true origin of cultivated apples is still very contentious, it is worthwhile to note that apple cultivation moved west-ward very early in the history and spread north along the various branches of the Silk trade route. For instance, apple cultivation was found in Greece before the ninth century B.C., and was later spread by the Romans throughout the Mediterranean and central Europe (Ponomarenko 1987). However, the question remains of how apple reached the West from central Asia? Because apples do not propagate easily from cuttings, a likely explanation is the introduction of seed carried in saddle bags by caravans along the trade routes, passing through the fabled cities of Bukara and Samarkand to Persia, perhaps facilitated by seed germination in horse droppings. Propagation from root suckers could also be a possibility (Juniper 1999). Another explanation is that grafting technology, dating from as early as 3800 years ago (Harris et al. 2002), may have evolved with Persia as an intermediary stop (Janick 2005). According to Janick (2005), Persia is the source of many fruit species from Central Asia and China. Despite this historical antecedent and the close proximity of Persia (Iran) to Central Asia, widely believed to be the centre of origin of domestic apple and home to M. sieversii, Iran has never really been recognized to have played a part in the evolution and domestication of apple. In order to shed more light on the role of Iranian region in apple evolution and domestication, we investigated the relationship of Iranian indigenous apple cultivars and landraces with selected wild species as well as old apple scion and rootstock cultivars sourced from other parts of the world. The majority of the wild apples belonged to M. sieversii from Kazakhstan and M. orientalis from Turkey and Russia located on the east and west of Iran, respectively. We believe this is the first study to examine the genetic relatedness of Iranian germplasm with the wild Malus species and domesticated apple varieties.


Materials and methods Plant materials A total of 159 accessions including 54 wild Malus species sourced from the USDA-ARS genetic resource unit in Geneva, USA, 57 old apple scion cultivars and rootstocks representing varieties developed in different parts of the world from 1,600–1,950 and 48 named varieties from different apple growing regions in Iran representing the genetic diversity available in the Iranian apple germplasm, was used in this study (Table 1). The 39 old scion cultivars were maintained at the apple repository of HortResearch, Havelock North, New Zealand, while the rootstocks, particularly the Malling and Malling-Merton series types, were obtained from either the apple repositories at HortResearch, or from the USDA-ARS genetic resources unit. The old cultivars were selected from provenances that had a strong relationship with Iran during the last few centuries and therefore may have been instrumental in the dissemination of Iranian germplasm. These countries include England, Russia, France, Germany, Spain, Netherlands, Denmark, USA, Australia and New Zealand. The majority of the wild species belonged to M. sieversii from Kazakhstan and M. orientalis from Turkey and Russia. We assigned individuals arbitrarily into populations to facilitate comparisons as follows: Iranian cultivars and landraces = Population one; old apple scion cultivars = Population 2; M. sieversii accessions = Population 3; M. orientalis accessions = Population 4; the rest of the accessions of wild species = Population 5; rootstocks = Population 6. DNA isolation and choice of SSR markers DNA extraction was performed as described by Gardiner et al. (1996). Isolated DNA was quantified following a protocol developed in the Gardiner laboratory as follows. The gDNA concentration was estimated on a 0.9% agarose gel with different concentrations of the Lambda DNA marker (Invitrogen New Zealand Limited, Auckland). The choice of SSR loci was based on the degree of polymorphism and applicability over a range of Malus species (Liebhard et al. 2002). We selected nine that belonged to different apple linkage groups for this

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study to ensure independence among loci (Table 2). The normalized nomenclature for all loci with prefix ‘CH’ was obtained from Liebhard et al. (2002), while NZmsEB146613 and NZmsEB134379 are described by Celton et al. (2008). Polymerase chain reaction amplification Polymerase chain reaction (PCR) was carried out in a 15 ll volume containing 20 mM Tris-HCl pH 8.0, 0.2 mM of each dNTP, 2 mM MgCl2, 0.01 mM of forward primer, 0.15 mM of reverse primer, 0.15 mM of M13 labeled primer (Fluorescent dyes FAM, NED and VIC (Applied Biosystem, Foster City, CA, USA)), 0.25 unit of PlatinumTM Taq DNA polymerase (Invitrogen) and 3 ng of genomic DNA. PCR amplifications were performed in a Hybaid MBS Satellite 0.5 G Thermal Cycler with the same program for all SSR reactions under the following conditions: an initial denaturation at 94°C for 2.5 min was followed by four cycles of 94°C for 30 s, 65°C for 1 min, and 72°C for 1 min, where the annealing temperature was reduced by 1°C per cycle; these initial cycles were followed by 30 cycles of 94°C for 30 s, 60°C for 1 min, 72°C for 1 min. A final 5 min extension at 72°C was included (Celton et al. 2008). Differently labeled amplification products were mixed together in the following ratio; 4 ll FAM, 2 ll NED, 2 ll VIC and 2 ll of double distilled water. Then 2 ll aliquots were mixed with 2 ll of ET-900 Rox internal size standard (Pharmacia Amersham, Freiburg, Germany) and denatured for 3 min at 94°C. The reaction product was loaded on an ABI 377 sequencer (PE Applied Biosystems). The size of the alleles was calculated based on internal standards using GeneScan (version 2.0) software (PE Applied Biosystems). To synchronize the allele detection process across individuals and loci, we identified the last stable peak on the chromatogram (there were more than one peak because of presence of stutter bands) as an allele. Individuals with only one band at a locus were treated as being homozygous at that locus. Data analysis The SSR alleles identified in the individuals used in this study were used to calculate population genetic parameters, including genetic variation statistics,

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Table 1 Apple accessions, country of origin and arbitrary populations to which each was assigned Pop. No.

Accession Name

Country of Origin

Pop. No.

Accession Name

Country of Origin

1

Akhlamad-e Deraz

Iran

2

Northern Spy

–

1

Soltani-e Shabestar

Iran

2

Hawthornden

UK

1

Azayesh

Iran

2

Close

USA

1

Glab-e Kohanz (Ka. Ab.)

Iran

2

Mr Fitch

New Zealand

1

Gharaghach

Iran

2

Guldborg

Denmark

1

Mashhad-e Nouri

Iran

2

Alkmene

Germany

1

Mashhad

Iran

2

Lady Williams

Australia

1

Asali

Iran

2

Granny Smith

Australia

1

Zinati

Iran

3

M. sieversii (PI 596280)

Uzbekistan

1

Heidar Zadeh

Iran

3


Tajikistan

1

Ahar-e 2

Iran

3


Kazakhstan

1

Kompouti

Iran

3


Kazakhstan

1

Haji-e Karaj

Iran

3


Kazakhstan

1

Ardebil-e 2

Iran

3


Kazakhstan

1 1

Kowli-e Mahallat Sheikh Ahmad

Iran Iran

3 3

M. sieversii (PI 633802) M. sieversii (PI 633803)

Kazakhstan Kazakhstan

1

Ghermez-e Rezaeieh

Iran

3


Kazakhstan

1

Paeizeh-e Mashhad

Iran

3


Kazakhstan

1

Khorsijan

Iran

3


Kazakhstan

1

Zonouz-e Marand

Iran

3


Kazakhstan

1

Nayan Arangeh

Iran

3


Kazakhstan

1

Dirres-e Mashhad

Iran

3


Kazakhstan

1

Morabbaei

Iran

3


Kazakhstan

1

Narsib-e Mashhad

Iran

3

M. sieversii var. kirghisorum (PI 633798)

Kyrgyzstan

1

Shishehei-e Tabriz

Iran

3


Kazakhstan

1

Golab-e Isfahan (Ka. Ab.)

Iran

3


Kazakhstan

1

Ghandak-e Kashan

Iran

3

M. sieversii FORM 35 (PI 613967)

Kazakhstan

1

Paeizeh Zard-e Mashhad

Iran

3

M. sieversii (01P22)

Kazakhstan

1

Akhlamad-e Mashhad

Iran

3

M. sieversii (3563.q)

Kazakhstan

1 1

Shafiei Golden-e Karaj

Iran Iran

3 4

Okanagan (PI 148709) M. orientalis (PI 633819)

– Soviet Union

1

Engelisi-e Shiraz

Iran

4

M. orientalis (PI 633820)

Soviet Union

1

Golshahi

Iran

4


Soviet Union

1

Golab-e Sahneh

Iran

4


Soviet Union

1

Boshghabi-e Balkhi

Iran

4


Soviet Union

1

Shahyad (Semirom)

Iran

4


Turkey

1

Soltani(Semirom)

Iran

4


Turkey

1

Golshahi(Semirom)

Iran

4


Turkey

1

Sib Torsh-e-Zoudres (Semirom)

Iran

4


Turkey

1

Shafi Abadi (Semirom)

Iran

4


Turkey

1

Iran

5

M. bhutanica (PI 633808)

–

1

Sib Torsh-e Boshghabi (Semirom) Golab-e Isfahan (Semirom)

Iran

5

M. bhutanica (PI 633811)

–

1

Arous (Semirom)

Iran

5

M. domestica (PI 633825)

–

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Table 1 continued Pop. No.

Accession Name

Country of Origin

Pop. No.

Accession Name

Country of Origin

1

Sib Torsh-e Paeizeh (Semirom)

Iran

5


–

1

Shafi Abadi (Daneshkadeh)

Iran

5


–

1

Shabihe Soltani

Iran

5

M. kansuensis (Bat.) Schneid. (PI 633809)

China

1

Abbasi (Semirom)

Iran

5

M. kansuensis (Bat.) Schneid. (PI 633810)

China

1

Gami Almasi

Iran

5

M. ombrophila Hand.-Mazz. (PI 596281)

China

2

Cox Orange Pipin

UK

5

M. prattii (Hemsl.) Schneid. (PI 633804)

China

2

Beauty of Bath

UK

5

M. prattii (Hemsl.) Schneid. (PI 633813)

China

2

Red Delicious

USA

5

M. prunifolia (PI 613845)

China

2

Jonathan

USA

5

M. sylvestris (PI 619168)

–

2

Nonpareil

France

5

M. sylvestris (PI 633824)

–

2

Antonovka

Russia

5

M. toringo Sieb. (PI 613806)

China

2

Api Rose

France

5


China

2

Belle Bonne

UK

5


China

2 2

Calville blanc d’Hiver Camoesa de Llobregat

France Spain

5 5

M. transitoria (Batal.) Schneid. (PI 633805) China M. transitoria (Batal.) Schneid. (PI 633806) China

2

Colonel Vaughn

UK

5

M. 9 asiatica (PI 613835)

China

2

Cout Pendu Plat

France

5

M. zhaojiaoensis Jiang (PI 633816)

China

2

Devonshire Quarrenden

UK

5

M. zhaojiaoensis Jiang (PI 633817)

China

2

Flower of Kent

UK

5

Simcoe (PI 148709)

–

2

Golden Pippin

UK

6

CG202

–

2

Rhode Island Greening

USA

6

M9

–

2

Ribston Pippin

UK

6

Mac9

–

2

Roter Eiserapfel

Germany

6

M8

–

2

The Doctor

Denmark

6

M6

–

2

Brabanste BelleFleur

Netherlands

6

M4

–

2

Emperor Alexander

Russia

6

M3

–

2

Cambrigde Pippin

UK

6

M26

–

2

Lady Sudeley

UK

6

M25

–

2

Newtown Pippin

USA

6

M2

–

2 2

Nonnetit Bastard Red Astrachan

Denmark Russia

6 6

M15 M12

– –

2

Reinette arbree d’Auvergne

France

6

M11

–

2

Panenske

Czech Rep.

6

M10

–

2

Magdeburgski Podzni

Czech

6

M13

–

2

Kosikove

Czech Rep.

6

MM106

–

2

Golden Delicious (Smoothee)

USA

6

Mer793

–

6

MM111

–

F-statistics, gene flow, and genetic identity and distance using POPGENE 1.32 software (Yeh et al. 1997, http://www.ualberta.ca/*fyeh/download.htm2). The number of alleles observed per locus (no) and

effective number of alleles per locus (ne) was computed as described by Kimura and Crow (1964). Observed heterozygosity (Ho), expected heterozygosity (He) (Nei 1973) and Shannon’s index of

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Table 2 Allelic diversity of the microsatellite loci scored in 159 apple accessions Original publication

This study

Primer

Repeat type

Range of sizes (bp)

H

No. of loci

a

perf

6

a

8

186–226

0.80

c

comp

25

186–250

sloc

6

7

108–154

0.73

sloc

15

122–178

sloc

a a

imp

3

6

106–216

0.78

sloc

20

200–244

sloc

imp

11

8

158–196

0.86

sloc

17

164–220

sloc

a

perf

2

10

158–190

0.87

sloc

22

174–222

sloc

a

imp

9

7

111–149

0.84

sloc

16

136–176

sloc

a

perf

5/10

15

133–177

n.d.

d

15

154–188

sloc

CH03d07 CH03d12 CH03e03 CH04a12 CH05e03 CH05c07 CH02a08

LG

No. of alleles

sloc

mloc

No. of alleles

Range of sizes (bp)

No. of loci

b

–

14

–

–

–

sloc

12

144–200

sloc

b

–

5

–

–

–

sloc

11

161–209

sloc

NZmsEB146613 NZmsSeb134379

a

Liebhard et al. 2002

b

Celton et al. 2008

c

single locus

d

multi locus

diversity (I) (Shannon and Weaver 1949) were calculated to determine the diversity in the populations. It was assumed that the gene frequency within a population was under Hardy-Weinberg equilibrium. A hierarchical estimation of Wright’s (1978) F-statistics was also performed to measure heterozygosity within populations (FIS) and between populations (FIT). Fixation index (FST) was estimated as well according to Wright (1978). Gene flow was estimated from FST according to Nei (1973) as follows: Nm = 0.25(1 - FST)/FST. The genetic identity and distance between populations across all loci was estimated using Nei (1978) unbiased genetic distance coefficient. To further elucidate the relationships between populations, Nei’s unbiased measure of genetic distance (Nei 1978) was used in cluster analysis based on the un-weighted pair group arithmetic average method (UPGMA) to generate a dendrogram.

CH03d07. Across the populations, no ranged from 7.22 for M. orientalis to 11.11 for other wild species. The ne ranged from 3.47 for CH03e03 to 11.27 for CH03d07 with an average of 6.8 alleles per locus (Table 3). Across the populations, ne ranged from 4.26 for rootstock accessions to 6.95 for other Malus species. The Ho values ranged from 0.45 for CH05e03 to 0.87 for CH03d07, with an average of 0.63 across loci (Table 3). Ho values ranged from 0.51 for M. orientalis to 0.68 for old apple cultivars (Table 3). The He values ranged from 0.71 for CH03e03 to 0.91 for CH03d07, with an average of 0.83 overall loci (Table 3). The range of He values was from 0.76 for apple rootstocks to 0.86 for other Malus species (Table 4). The I values also showed an identical trend, with an average of 2.17 across all loci, a maximum of 2.72 for CH03d07, and a minimum 1.63 for NZmsEB134379. This parameter ranged from 1.62 for M. orientalis to 2.07 for other wild species across the populations (Table 3).

Results Fixation indices and heterozygosity measures Allelic variation and genetic diversity The nine SSR primer pairs (Table 2) screened across the 159 apple genotypes resulted in 153 alleles, with an average of 17 alleles per locus (Table 3). The no ranged from 11 for NZmsEB134379 to 25 for

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A summary of Wright’s (1978) F-statistics including measures of heterozygosity deficiency within population (FIS), between populations (FIT), and fixation index (FST) is reported in Table 4. Iranian landraces and old cultivars showed a negative FIS value


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Table 3 A summary of genetic parameters across single sequence repeat loci and apple germplasm accessions I

Ho

He

8.83

2.43

0.77

0.89

7.42

2.47

0.45

0.86

25

11.27

2.72

0.87

0.91

16

9.74

2.42

0.86

0.90

304 308

17 20

6.53 3.47

2.23 1.88

0.66 0.47

0.85 0.71

CH02a08

296

15

5.80

2.11

0.49

0.83

NZmsEB146613

284

12

4.12

1.65

0.50

0.76

NZmsEB134379

314

11

4.04

1.63

0.61

0.75

Mean

303

17

6.80

2.17

0.63

0.83

4.58

2.74

0.38

0.17

0.072

95

10.67

5.20

1.84

0.63

0.78

Old cultivars

78

10.89

5.28

1.84

0.68

0.78

M. sieversii

40

9.33

5.72

1.80

0.62

0.77

M. orientalis

18

7.22

5.14

1.62

0.51

0.76

Wild species Rootstocks

37 35

11.11 7.67

6.95 4.26

2.07 1.63

0.60 0.67

0.86 0.76

Locus

Sample Size

na

CH03d12

314

15

CH05e03

298

22

CH03d07

302

CH05c07

308

CH04a12 CH03e03

St. Dev

ne

Population Iranian landraces

na = Observed number of alleles ne = Effective number of alleles I = Shannon’s Information index Ho = Observed heterozygosity He = Expected heterozygosity

indicating heterozygosity excess at two of the loci (CH03d07 and CH05c07), M. sieversii showed heterozygosity excess at one locus only (CH03d12), M. orientalis at two loci (CH03d07 and CH03e03), other Malus species at none of the loci, and apple rootstock accessions at four loci (CH03d07, CH03d12, CH05c07 and NZmsEB146613). With an estimated mean of 0.197 over all loci, all populations showed positive FIS values indicating heterozygosity deficiency ranging from 0.109 for apple rootstock accessions to 0.298 for M. orientalis. The FIS for the loci across the whole population ranged from 0.023 for CH03d07 to 0.401 for CH05e03. The mean FIT value for the whole population was 0.269 and ranged from 0.083 for CH03d07 to 0.460 for CH02a08. The overall FST range was from 0.059 for CH03d07 and CH03d12, to 0.153 for NZmsEB146613 with a mean of 0.087.

The Nm for all loci was 2.94, with the highest values, 3.97 and 3.95, for loci CH03d12 and CH03d07, respectively, and the lowest, 1.38 and 1.51, for loci NZmsEB146613 and CH03e03, respectively. Genetic distance among populations Nei’s unbiased genetic distance and genetic similarity matrices were estimated over all SSR markers (Table 5). The genetic distance varied from 0.111 for old apple cultivars versus rootstocks, to 0.833 for M. orientalis versus other Malus species, with an average value of 0.490. Based on the mean genetic distances, Iranian landraces and other wild Malus species populations showed the lowest (0.365) and highest (0.713) distances, respectively, from the rest of the populations. Genetic identity data agree with the genetic distance estimates with a high genetic identity of

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123

0.087

2.94 2.94

0.269 0.460

0.108

2.07

0.096

0.074

3.13

1.38

0.078

0.197 0.383 0.027

0.153

0.276

0.219

0.186 -0.112

0.364

0.506 0.227

0.271

0.274

0.298

0.109 0.375 -0.072

0.004

0.185

0.588

0.252

0.681

0.117

0.218

0.729

0.185

0.016

0.533 0.033

0.160

0.080 -0.105

0.202

0.202 0.050 0.406 -0.098

0.352

CH02a08

0.895 between the old cultivar and rootstock populations. The Iranian landraces had a genetic identity of 0.819, 0.763, 0.770 and 0.718 with M. sieversii, M. orientalis, old apple cultivars and apple rootstocks, respectively. Another interesting result is the high genetic identity between M. orientalis and M. sieversii (0.812) and the poor identity between M. orientalis and most of the other populations. To further elucidate the relationships between populations, Nei’s unbiased measure of genetic distance matrices was used in cluster analysis based on the UPGMA algorithm to construct a dendrogram (Fig. 1). The dendrogram showed that Iranian germplasm is much closer to M. sieversii and M. orientalis than to the other populations. Interestingly, the old apple scion cultivar and rootstock populations were in the same clade, suggesting a possible co-evolution between the two. The wild Malus species population was separated clearly from other populations.

Discussion

3.67 3.85 1.51 3.95 Nm

3.97

0.064 0.061 0.142 0.059 FST

0.059

0.440

0.401 0.196

0.239 0.352

0.198

0.083

0.061 0.023 FIS

FIT

Over all

0.115

0.306 0.009 0.523 -0.132 Rootstocks

-0.099

0.532 0.418 0.235 0.235 Wild species

0.059

0.317 0.130 -0.143 -0.023 M. orientalis

0.186

0.428

0.183 0.389

0.160 0.249

0.126 0.165 M. sieversii

0.088 -0.065 Old cultivars

-0.109

-0.042

0.240

0.200

0.072

0.639

Allelic variation and genetic diversity

Iranian landraces

CH03d07

FIS over loci

CH03d12

CH03e03

CH04a12

CH05e03

CH05c07

NZmsEB146613

NZmsEB134379

Mean


Population

Table 4 A summary of Wright’s (1978) F-statistics including measures of heterozygosity deficiency within population (FIS), between populations (FIT), fixation index (FST) and gene flow (Nm)

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All SSR loci analyzed in this study displayed a high degree of polymorphism with 11 to 25 alleles per locus. More alleles and wider size ranges were observed for the CH series of markers than previously reported by Liebhard et al. (2002). This difference could probably be attributed to the number of accessions and diversity of germplasm used in the present study. In the case of Liebhard et al. (2002), a limited set of only cultivated apple varieties and Malus species was used. Also, microsatellite locus CH02a08 was recorded as being multi-locus by Liebhard et al. (2002), but in our study was found to be a single locus. We were only able to detect one or two alleles in each individual. The na and ne values were low for loci NZmsEB146613 and NZmsEB134379 when compared to other SSR markers, probably because of the tetra base pair interval of putative alleles in these EST markers. When all the different apple accessions/ populations were compared based on allelic variation parameters, the Iranian landraces displayed as much diversity as the old apple cultivars. The most diverse population was the wild Malus species, which seems logical considering the number of Malus species


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Table 5 Genetic identity (above diagonal) and genetic distance (below diagonal) estimates between populations across all loci based on Nei (1978) Pop ID*

Iran landraces

Old cultivars

M. sieversii

M. orientalis

Wild species

Rootstocks

Iran landraces

****

0.770

0.819

0.763

0.467

0.718

Old cultivars

0.262

****

0.628

0.494

0.538

0.895

0.665

M. sieversii

0.200

0.465

****

0.812

0.478

0.601

0.668

M. orientalis

0.270

0.706

0.209

****

0.435

0.484

0.597

Wild species

0.761

0.619

0.738

0.833

****

0.539

0.492

Rootstocks

0.331

0.111

0.509

0.726

0.618

****

Mean Over all

0.365

0.432

0.424

0.549

0.713

0.459

Mean

Over all 0.707

0.647 ****

0.629

0.490

****

Fig. 1 A phenogram based on the un-weighted pair group arithmetic average (UPGMA) clustering method depicting the relationships of the apple accession used in this study

grouped in this population. M. orientalis and the rootstock populations were less diverse than other populations probably due to the limited number of accessions in these two populations. The na = 17 obtained across populations was greater than the na = 8.7 obtained by Liebhard et al. (2002), who used a limited number of cultivars; the na = 12.3 by Pereira-Lorenzo et al. (2007), who studied both nonnative and local apple cultivar populations from three regions in Spain; and, the na = 7.17, 6.4 and 11.7, for wild, ornamental and domesticated apple populations, respectively, by Coart et al. (2003) in Belgium. This difference can be attributed to the higher number of accessions and diversity of germplasm used in the present study compared to previous studies. The He estimates obtained in this study were higher for some loci, including CH03d07 (0.91), CH05c07 (0.90) and CH03d12 (0.89), but slightly lower for others,

including CH03e03 (0.71), CH04a12 (0.85), and CH05e03 (0.86) when compared to those reported by Liebhard et al. (2002) (Table 3). The different populations had relatively similar He values, except for the wild species population, which had a significantly higher value (He = 0.86) than the others. In comparison with other studies, the mean He = 0.83 observed in our study was slightly higher than those reported by Larsen et al. (2006) (He = 0.778), Pereira-Lorenzo et al. (2007) (He = 0.80), and Coart et al. (2003) (He = 0.721 and 0.775 for wild and ornamental apple populations, respectively). However, it was slightly lower for domesticated apple populations (He = 0.841), which were also included in the apple study by Coart et al. (2003). This difference can be attributed to the number of accessions and diversity of germplasm used in this study compared with other studies. The situation was

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quite different for Ho. With a mean of 0.63, Ho was higher for some loci, but lower for others in our study when compared to those reported by Liebhard et al. (2002) (Table 3). In terms of the different populations in our study, the Ho was relatively similar. The smaller Ho (0.51) observed in M. orientalis could be due to small sample size of 10 individuals in this population compared to *20 individuals in each of the other populations. The Ho in our study was lower than those reported by Larsen et al. (2006) (Ho = 0.769), Pereira-Lorenzo et al. (2007) (Ho = 0.83), and Coart et al. (2003) (Ho = 0.679, 0.729 and 0.749 for wild, ornamental and domesticated populations, respectively). The relatively high level of null alleles observed in this study may partly explain the shortage of heterozygotes in this study (Bruford et al. 1998), although it is unlikely that each locus has null alleles at high frequencies. A more plausible reason for the shortage of heterozygotes could be non-random mating in both the wild Malus species and the domesticated accessions which potentially could lead to the Wahlund effect (Lowe et al. 2004). In general, all populations showed a relatively high level of genetic diversity (ne = 6.8, He = 0.83, and I = 2.17). The wild Malus species showed the highest level of genetic variation with ne, He, and I of 6.95, 0.86, and 2.07, respectively, whereas the rootstock population exhibited the lowest variation with ne, He, and I of 4.26, 0.76, and 1.62, respectively. Based on the genetic diversity data, Iranian cultivars and landraces would be considered to be as diverse as the old apple cultivars and M. sieversii accessions (Table 3). Fixation indices and heterozygosity measures The FIS is the mean reduction in heterozygosity of an individual due to non-random mating within a subpopulation. It is a measure of the extent of inbreeding within subpopulations and can range from -1 (all individuals heterozygous) to ?1 (no observed heterozygotes) (Wright 1978). For all populations a positive inbreeding coefficient was observed (mean FIS = 0.197), while a positive inbreeding coefficient was observed across loci. These results are not in agreement with those obtained by Larsen et al. (2006) for Danish populations of M. sylvestris, where FIS = -0.002 indicated random mating. Our findings contrasted with those by Pereira-Lorenzo et al.

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(2007) on non-native and local apple cultivar populations in Spain (FIS = -0.088) suggesting a slight excess of heterozygotes in this study. However, our results were very similar to those obtained by Coart et al. (2003) in Belgium, who reported FIS = 0.105, 0.126 and 0.058 for wild, ornamental and domesticated populations, respectively. The often reported cause of positive values for inbreeding coefficients is the presence of null alleles (Bruford et al. 1998). Null alleles were found in all loci examined in our study, which may partly explain the shortage of heterozygotes. It is unlikely that each locus has null alleles at high frequencies. A more plausible reason for the shortage of heterozygotes could be non-random mating in both the wild Malus species and the domesticated accessions potentially leading to the Wahlund effect (Lowe et al. 2004). The fixation index (FST) is the mean reduction in heterozygosity of a subpopulation (relative to the total population) due to genetic drift among subpopulations. It is a measure of the extent of genetic differentiation among subpopulations and can range from 0 (no differentiation) to 1 (complete differentiation, i.e., subpopulations fixed for different alleles) (Wright 1978). As expected for an out-crossing tree species (Hamrick and Godt 1990), a low or moderate overall differentiation among populations was observed (FST = 0.087). This value was considerably larger than the one for the comparison between wild and domesticated apples (FST = 0.011), and somewhat larger for comparisons between wild and ornamental apples (FST = 0.060) (Coart et al. 2003), non-native and local apple cultivars (FST = 0.058), and local cultivars from three regions (FST = 0.059) (Pereira-Lorenzo et al. 2007). However it was much smaller than the FST for comparisons between three wild populations of wild apricots in the Ily Valley of West China (FST = 0.137) (Tian-Ming et al. 2007). Low differentiation among populations means that most of the genetic variation is maintained within populations rather than between populations, probably due to more admixtures between or among populations. The overall fixation index (FIT) is the mean reduction in heterozygosity of an individual relative to the total population. It combines contributions from non-random mating within demes (FIS) and effects of random drift among demes (FST), and it ranges from -1 (all individuals heterozygous) to ?1


(no observed heterozygotes) (Wright 1978). For all populations a positive overall fixation index (FIT) was observed (mean FIT = 0.269). A positive overall fixation index was observed across all loci, too, indicating heterozygosity deficiency. The reasons for this are largely the same as that described for FIS above. Gene flow is the movement of genes within and between populations (Lowe et al. 2004). In this study, the level of gene flow based on FST (Nei 1973) was moderate to high (Nm = 2.94), which confirms the supposition of little genetic differentiation among populations. This means that the high level of genetic diversity maintained within each population is less susceptible to genetic drift. These results are in agreement with those reported by Tian-Ming et al. (2007) for three populations of wild apricots in the Ily Valley of West China (Nm = 2.684). Genetic distance among populations Based on genetic identity and genetic distance (Nei 1978) estimates as well as the dendrogram generated by UPGMA cluster analysis based on Nei’s genetic distance estimates (Table 5; Fig. 1), the Iranian cultivars and landraces have the closest relationship to M. sieversii (the widely believed principal wild maternal ancestor of domesticated apples) from Central Asia east of Iran, and to M. orientalis, one of the probable minor ancestors of domesticated apples, native to Turkey and Russia. It should be noted that native M. orientalis populations are present on the slopes of the mountains in the apple producing regions in the north and west of Iran (Rechinger 1963–2000). Likewise, the old apple cultivars from different parts of the world have a fairly close genetic relationship with M. sieversii, M. orientalis and the Iranian populations. The other wild Malus species did not show a close genetic relationship with the domesticated apple accessions (including Iranian, old cultivars and rootstock populations), and with the other two species, M. sieversii and M. orientalis. Based on genetic identity results, the rest of the wild Malus species appeared much closer to old cultivars (0.538) and rootstocks (0.539) than to Iranian landraces (0.467), M. sieversii (0.478) and M. orientalis (0.435) (Table 5; Fig. 1). This wild species group consisted of a number of species, some of which are

839

believed to be putative minor ancestors of the domestic apple (Coart et al. 2006; Rehder 1940; Ponomarenko 1991). However, due to the limited accessions of each of these species in this study (see Table 1), it is difficult to make any conclusions regarding their involvement in the origin and domestication of domesticated varieties. Interestingly, the domesticated old apple cultivars from different parts of the world grouped together with rootstock clones irrespective of the morphological differences between them (Table 5; Fig. 1). This could be due to the long co-evolution of scion and rootstock varieties in apple cultivation and history. The close grouping of M. sieversii and M. orientalis is not surprising, probably due to close geographical distribution of these species around the Central Asian region (Rechinger 1963–2000) with Iran acting as a bridge between them. It is possible that these two populations have been consciously or unconsciously admixed through human intervention, or by animals migrating along the silk trade route. The morphological, biochemical and molecular variation within wild apples of central Asia (M. sieversii) indicates that the earliest selections of domesticated apples could have come directly from M. sieversii, without the involvement of other species (Forsline et al. 2003). However, later hybridizations could have been important in the creation of new cultivars carrying economically important characteristics. For instance, M. orientalis found in the western sections of the trade routes in the Russian Caucasus as well as Turkey and North and west of Iran (Rechinger 1963–2001) does not have the diversity of fruit quality, but may have contributed other valuable traits, such as later blooming, adaptation to a wider array of habitats, and capacity for longer storage potential. Other potential contributing parents may include M. sylvestris, the European crabapple bearing small astringent, greenish-yellow fruits, native to an area from Britain across Europe to the Balkans, as well as M. baccata and some of its subspecies or natural hybrids (M. mandshurica, M. prunifolia, and M. 9 asiatica Nakai ex Matsumura) on the eastern side of the trade routes (Forsline et al. 2003). These theories are supported to some extent by the genetic groupings obtained in our study, where M. orientalis and M. sieversii grouped much more closely with the Iranian germplasm as well as old apple cultivars. To interpret these results, it may be necessary to note

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ancient Iran’s (Persia’s) location and history particularly, the critical period in terms of apple germplasm exploration and exchange. It may be that the introduction of domesticated apples to the west could have occurred with Persia as the intermediary location from Central Asia. This might have been possible due to the close proximity and interaction between Iran (Persia) and Central Asia dating back many centuries, as well as to the interaction between the Persian Empire and the West since the 7th century B.C. (Pigoloskaya et al. 1965; Lunde 1988). The Iranian germplasm appears to be an intermediate group between the domesticated group (old cultivars) and Malus sieversii. The weaker relationship between M. orientalis (and to some extent M. sieversii) and the old apple cultivars in comparison to the Iranian germplasm may be due to later hybridizations of apple germplasm exported to the west with other species, in particular M. sylvestris. This later introduction of M. sylvestris in the pedigree of domesticated varieties presumably contributed to the dilution of the contribution of M. sieversii and M. orientalis as reported by Coart et al. (2006). The conflicting results on maternal or paternal heritage of domesticated varieties (M. sieversii being the main maternal progenitor (Forsline et al. 2003; Harris et al. 2002), and M. sylvestris being the main maternal ancestor (Coart et al. 2006)) could be explained by the theory of reciprocal hybridizations of imported apples (mainly elite M. sieversii from Central Asia or Persia, or hybrids of M. sieversii and M. orientalis from Persia or Asia Minor) and native wild species (mainly M. sylvestris). Either group (that is, imported species or native species) could have been the maternal or paternal parent at that time. It is important to note that the Iranian apple genetic resources are located along the central Silk trade route. These genetic resources are diverse for a wide range of morphological and pomological characters due to minimal selection pressure, and some of the accessions could qualify as new cultivars in their own right, or they could be used as parents in a breeding program (Hajnajari 2006; Damyar et al. 2007). Also, the Iranian apple germplasm is relatively similar in morphology and fruit quality attributes to M. sieversii and to some extent M. orientalis (Dzhangaliev 2003; Rechinger 1963–2000). Therefore, we propose that Iran could be one of the major players in apples’ domestication and their transfer

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from Central Asia to the western countries. This view-point is supported by Khoshbakht and Hammer (2006) who identified north of Iran based on ethnobotanical data as a probable evolutionary centre for a number of tree fruit (including Malus orientalis, Prunus species and Ficus carica L. (Moraceae)) and shrub species. The Iranian apple germplasm has a huge potential for use in genetic improvement programs for development of novel apple types (Damyar et al. 2007) as have been the case with M. sieversii and M. orientalis (P. Forsline pers comm.). Recently, the Ministry of Agriculture in Iran developed a task force program for collection, evaluation, conservation and utilization of this valuable apple germplasm (Hajnajari 2006; Damyar et al. 2007). We suggest that a reference collection that represents the diversity within the core collection is identified to minimize genetic erosion and redundancy in the collection, and preserved for use in breeding.

Conclusion Our genetic parameter estimates showed that there is more intra-population than between population variation in apple, and that this is consistent with the life history and mating characteristics of apples. The closer geographic and genetic affinity of Iranian germplasm to M. sieversii (from Kazakhstan) and M. orientalis (from Turkey and Russia) suggests that Iranian apples could have arisen from these two species. The Iranian germplasm appears to be an intermediate group between the domesticated group (old cultivars) and the wild species. Based on these results we propose that Iran could be one of the major players in apples’ domestication and their transfer from Central Asia to the western countries. Acknowledgements We thank the Ministry of Science, Research and Technology of the Islamic Republic of Iran and the University of Tehran for financial assistance towards travel to and living costs in New Zealand. We also thank HortResearch New Zealand, in particular, the genome mapping laboratory in Palmerston North, for providing the facilities to do this research. We are grateful to Dr Phil. Forsline at the USDA-ARS, Plant Genetic Resource Unit, Cornell University, Geneva, NY, USA. for providing leaf samples from the wild Malus species and Hossein Gharaghani, a masterate student at the College of Agriculture, University of Tehran, for preparing and sending DNA samples from some Iranian accessions.


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