Characteristics and Transfer Ability of New Apple EST-Derived SSRs to Other Rosaceae Species

8/8/2019 Characteristics and Transfer Ability of New Apple EST-Derived SSRs to Other Rosaceae Species

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Characteristics and transferability of new apple

EST-derived SSRs to other Rosaceae species

Ksenija Gasic Yuepeng Han Sunee Kertbundit Vladimir Shulaev Amy F. Iezzoni Ed W. Stover Richard L. Bell Michael E. Wisniewski Schuyler S. Korban

Received: 19 March 2008 / Accepted: 19 November 2008 / Published online: 11 December 2008

Springer Science+Business Media B.V. 2008

Abstract Genic microsatellites or simple sequence

repeat markers derived from expressed sequence tags

(ESTs), referred to as ESTSSRs, are inexpensive to

develop, represent transcribed genes, and often have

assigned putative function. The large apple (Malus 9

domestica) EST database (over 300,000 sequences)

provides a valuable resource for developing well-

characterized DNA molecular markers. In this study,

we have investigated the level of transferability of 68

apple ESTSSRs in 50 individual members of the

Rosaceae family, representing three genera and 14species. These representatives included pear (Pyrus

communis), apricot (Prunus armeniaca), European

plum (P. domestica), Japanese plum (P. salicina),

almond (P. dulcis), peach (P. persica), sour cherry

(P. cerasus), sweet cherry (P. avium), strawberry

(Fragaria vesca, F. moschata, F. virginiana,

F. nipponica, and F. pentaphylla), and rose (Rosa

hybrida). All 68 primer pairs gave an amplification

product when tested on eight apple cultivars, and for

most, the genomic DNA-derived amplification product

matched the expected size based on EST (in silico)

data. When tested across members of the Rosaceae,

75% of these primer pairs produced amplification

products. Transferability of apple ESTSSRs acrossthe Rosaceae ranged from 25% in apricot to 59% in the

closely related pear. Besides pear, the highest trans-

ferability of these apple ESTSSRs, at the genus level,

K. Gasic Y. Han S. Kertbundit S. S. Korban (&)

Department of Natural Resources and Environmental

Sciences, University of Illinois, Urbana, IL 61801, USA

e-mail: [email protected]

Present Address:

K. Gasic

Department of Horticulture, Clemson University,

Clemson, SC 29634, USA

Present Address:

Y. Han

Wuhan Botanical Garden, Chinese Academy of Sciences,

Moshan, 430074 Wuhan, Peoples Republic of China

V. Shulaev

Virginia Bioinformatics Institute, Virginia Tech.,

Blacksburg, VA 24061, USA

A. F. Iezzoni

Department of Horticulture, Michigan State University,

East Lansing, MI 48824, USA

E. W. Stover

National Clonal Germplasm Repository, U.S. Department

of Agriculture-Agricultural Research Service

(USDA-ARS), Davis, CA 95616, USA

Present Address:

E. W. Stover

U.S. Horticultural Research Laboratory,

Fort Pierce, FL 34945, USA

R. L. Bell M. E. Wisniewski

Appalachian Fruit Research Station, U.S. Department

of Agriculture-Agricultural Research Service

(USDA-ARS), Kearneysville, WV 25430, USA

123

Mol Breeding (2009) 23:397411

DOI 10.1007/s11032-008-9243-x


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was observed for strawberry and peach/almond, 49 and

38%, respectively. Three markers amplified in at least

one genotype within all tested species, while eight

additional markers amplified in all species, except for

cherry. These 11 markers are deemed good candidates

for a widely transferable Rosaceae marker set provided

their level of polymorphism is adequate. Overall, thesefindings suggest that transferability of apple EST

SSRs across Rosaceae is varied, yet valuable, thereby

providing additional markers for comparative mapping

and for carrying out evolutionary studies.

Keywords Expressed sequenced tags (EST)

Rosaceae Simple sequence repeats (SSR)

Transferability

Introduction

Simple sequence repeats (SSRs) or microsatellites are

regions of DNA wherein a few bases are tandemly

repeated. These are ubiquitous in both prokaryotes and

eukaryotes, and can be found both in coding and non-

coding regions. Markers based on SSRs are the markers

of choice in genetics and breeding studies due to their

multi-allelic nature, codominant inheritance, high

abundance, reproducibility, transferability over geno-

types and extensive genome coverage. Two classes ofSSR markers are recognized based on their origin:

genomic, developed from enriched DNA libraries, and

genic or expressed sequence tags (EST)-SSRs, derived

from EST sequences originating from the expressed

region of the genome (Arnold et al. 2002; Chagne et al.

2004). The latter are relatively inexpensive to develop,

represent transcribed genes which often have assigned

putative function, and are found to be significantly more

transferable across taxonomic boundaries than

traditional genomic SSRs (Arnold et al. 2002; Chagne

et al. 2004; Kuleung et al. 2004; Pashley et al. 2006).These advantages out balance putative disadvantages

of EST-SSR like lower levels of polymorphism

(Silfverberg-Dilworth et al. 2006).

The Rosaceae family encompasses more than

3,000 species among which are herbs, trees, shrubs,

and climbing plants. Some of these species include

economically important crops such as fruit trees

(apples, pears, cherries, and peaches, among others),

soft fruit crops like strawberry, or cultivated flowers

(roses). However, there is a significant discrepancy in

the amount of genomic data available among mem-

bers of the Rosaceae. Some have extensive genomic

data in terms of molecular marker maps, EST and

gDNA sequences (apple, peach); while, others have

rather little genomic information available (plum,

sour cherry). Most of the work in rosaceous specieshas centered on the construction of genetic linkage

maps and development of molecular markers, such as

SSRs (Stockinger et al. 1996; Gianfranceschi et al.

1998; Maliepaard et al. 1998; Cipriani et al. 1999;

Liebhard et al. 2002; Wang et al. 2002; Aranzana

et al. 2003a; Clarke and Tobutt 2003; Esselink et al.

2003; Graham et al. 2004; Folta et al. 2005;

Dirlewanger et al. 2006; Silfverberg-Dilworth et al.

2006; Sargent et al. 2006, 2007; Hibrand-Saint Oyant

et al. 2008; Weebadde et al. 2008; Woodhead et al.

2008). Several reports have focused on SSR devel-opment and their transferability across the Rosaceae

(Yamamoto et al. 2001, 2004; Dirlewanger et al.

2002; Decroocq et al. 2003, 2004; Mnejja et al. 2004;

Dondini et al. 2007; Sargent et al. 2007; Vendramin

et al. 2007). There are also few reports on compar-

ative mapping and synteny assessment among

Rosaceae species (Dirlewanger et al. 2002, 2004).

In addition to the extensive number of genetic and

genomic Rosaceae studies, there are a few open

access web sites that provide information on avail-

able markers in apple (Gianfranceschi and Soglio2004) (http://www.hidras.unimi.it/index.html) and in

Rosaceae (Jung et al. 2008) (http://www.bioinfo.wsu.

edu/gdr/).

Malus and Prunus are the best characterized genera

and havethe largest ESTcollectionsamong all members

of the Rosaceae family (Newcomb et al. 2006; Gasic

et al. 2007; http://www.bioinfo.wsu.edu/gdr/projects/

prunus/unigeneV3/index.shtml). The apple EST data-

base ([300,000 ESTs) provides a valuable resource for

developing well-characterized DNA molecular markers

(Guilford et al. 1997; Silfverberg-Dilworth et al. 2006;Igarashi et al. 2008). However, little attention has been

paid to thepotentialtransfer of appleESTSSRs to other

Rosaceae relatives. In this study,we present a new set of

68 apple SSRs, developed from publicly available

Malus EST sequences. All these SSRs have been eval-

uated for their level of polymorphisms in eight apple

cultivars and their transferability to 50 individual

members of the Rosaceae family, representing four

genera and 14 species.

398 Mol Breeding (2009) 23:397411

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http://www.hidras.unimi.it/index.htmlhttp://www.hidras.unimi.it/index.htmlhttp://www.bioinfo.wsu.edu/gdr/http://www.bioinfo.wsu.edu/gdr/http://www.bioinfo.wsu.edu/gdr/http://www.bioinfo.wsu.edu/gdr/projects/prunus/unigeneV3/index.shtmlhttp://www.bioinfo.wsu.edu/gdr/projects/prunus/unigeneV3/index.shtmlhttp://www.bioinfo.wsu.edu/gdr/projects/prunus/unigeneV3/index.shtmlhttp://www.bioinfo.wsu.edu/gdr/projects/prunus/unigeneV3/index.shtmlhttp://www.bioinfo.wsu.edu/gdr/http://www.bioinfo.wsu.edu/gdr/http://www.hidras.unimi.it/index.html


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Materials and methods

Plant material and DNA extraction

A total of 58 genotypes belonging to four genera and 14

species of the Rosaceae were used (Table 1). Leaf

tissues for DNA extraction from these different geno-types were collected from several sources. Apple and

rose leaves were collected from trees and potted plants

located at the University of Illinois at Urbana-Cham-

paign pomology farm and greenhouse, respectively;

pear and peach samples were collected from trees

located at the USDA-ARS Kearneysville, WestVirginia

farm; apricot, almond, European and Japanese plum

samples werecollected from trees at the National Clonal

Germplasm Repository (Davis, CA; http://www.ars.

usda.gov/main/site_main.htm?modecode=53-06-20-00);

and cherry leaf tissues were collected from treeslocated at the Michigan State Universitys Clarksville

Horticultural Experiment Station, Clarksville, Michigan.

Apple, rose, peach, and almond DNA were extracted

using the Qiagen plant DNA mini-kit (Qiagen Inc.,

Valencia, CA). Apricot,European plum, Japaneseplum,

and cherry DNA were extracted using the CTAB

method as described by Stockinger et al. (1996).

EST-SSR selection, amplification and validation

Apple ESTSSRs used were randomly picked fromthe Genomic Facility, University of California-Davis

(Davis, CA) web site (http://cgf.ucdavis.edu/home/).

This database contains an analysis of public expres-

sed sequence tags (ESTs) from Malus (160,620

ESTsanalysis performed in October, 2004). All

ESTs are grouped as either contigs or singletons, and

analyzed for the presence of SSRs. SSR repeat type

and length, and suggested forward and reverse primer

information is provided.

Each PCR reaction was performed in 15 ll of total

volume consisting of: 19 Taq polymerase buffer; 1.5of 50 mM MgCl2; 0.2 mM each of dATP, dCTP,

dGTP, and dTTP; one unit of Taq DNA polymerase

(New England Biolabs); 0.2 lM of each of forward

and reverse primers; and 50 ng of template DNA.

Following initial denaturation at 94C for 2 min, the

PCR reaction was carried out for 4 cycles under the

following conditions: denaturation at 94C for 30 s,

annealing at 65C for 1 min (lowered by 1C per

cycle until 60C), and extension at 72C for 1 min;

then, for 30 cycles under the following conditions:

denaturation at 94C for 30 s, annealing at 60C for

1 min, and extension at 72C for 1 min. The final

extension was carried out at 72C for 5 min.

EST-SSR validation was first performed using

eight apple cultivars, and PCR products were sepa-

rated on 4% high resolution agarose E-Gels

(Invitrogen, Carlsbad, CA). A total of 68 ESTSSRs,

randomly picked, were then evaluated for amplifica-

tion in all Rosaceae genotypes, except for sweet and

sour cherry accessions wherein a subset of 30 EST

SSRs, showing amplification products in other Ros-

aceae genotypes, were used. PCR products were

separated by electrophoresis using 3.0% Metaphor-

agarose (Cambrex BioScience, Rockland Inc.) in

19 TBE buffer, stained with ethidium bromide

(0.8 mg/ml) and visualized using UV light. This

allowed for a resolution of 2% which is equivalent tothe resolution of polyacrylamide gels (48%).

Results and discussion

Amplification of ESTSSRs in apple

A total of 149 primer pairs, originating from singleton

ESTs, were selected from a collection of 2,041 apple

ESTSSRs that were detected in 160,620 apple ESTs

(CGF, Genomic Facility, UC Davis, CA; http://cgf.ucdavis.edu/home/). However, of these 2,041 apple

ESTSSRs, only 1,279 had long enough flanking

sequences for primer design; primer pairs for this

complete set of ESTSSRs are available on our Apple

ESTIMA website (http://titan.biotec.uiuc.edu/apple/

resources.shtml).

For the 149 selected primer pairs, these were tested

using gDNA of 8 (7 diploid and 1 triploid) apple

cultivars/selections in order to assess their amplifica-

tion and polymorphism in different apple genotypes

(Table 1; Fig. 1). These apple genotypes were chosenbecause of their previous use as sources of EST

sequences (GoldRush and Royal Gala), as major

founders in breeding programs (Golden Delicious

and Royal Gala), commercial value (Fuji, Hon-

eycrisp, and Jonagold), or their use in our own

breeding program (CO-OP 16 and CO-OP 17).

Amplification products were observed with 92%

(135/149) of these primer pairs. Among these primer

pairs, 30 (22.2%) gave an amplification product

Mol Breeding (2009) 23:397411 399

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http://www.ars.usda.gov/main/site_main.htm?modecode=53-06-20-00http://www.ars.usda.gov/main/site_main.htm?modecode=53-06-20-00http://cgf.ucdavis.edu/home/http://cgf.ucdavis.edu/home/http://cgf.ucdavis.edu/home/http://titan.biotec.uiuc.edu/apple/resources.shtmlhttp://titan.biotec.uiuc.edu/apple/resources.shtmlhttp://titan.biotec.uiuc.edu/apple/resources.shtmlhttp://titan.biotec.uiuc.edu/apple/resources.shtmlhttp://cgf.ucdavis.edu/home/http://cgf.ucdavis.edu/home/http://cgf.ucdavis.edu/home/http://www.ars.usda.gov/main/site_main.htm?modecode=53-06-20-00http://www.ars.usda.gov/main/site_main.htm?modecode=53-06-20-00


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Table 1 Plant material used for marker validation and cross-species transferability

Species Individuals tested Ploidy level Origin

Maloideae

Malus 9 domestica Fuji 29 Japan

GoldRusha

29 USA

Golden Delicious 29 USA

Honeycrisp 29 USA

Jonagold 39 USA

Royal Galab 29 USA

CO-OP 16 29 USA

CO-OP 17 29 USA

Pyrus communis var. caucasica 29

P. communis Abate Fetel 29 France

Ba Li Hsiang 29 China

Bartlett 29 Europe

Klemtanka 29

Shinseiki 29 Japan

Rosoideae

Fragaria CA67.2014 (149) 59

F. vesca ssp. californica Goat Rocks CA 29 USA

F. vesca ssp. californica 29 USA

F. vesca ssp. vesca KY-18 29

F. pentaphylla #1 29 China

F. moschata 69 Russia

F. niponnica J71 29 Japan

F. virginiana ssp. virginiana KY-09 89

Rosoideae Rosa hybrida Carefree Beauty 49 USA

Grand Gala 49 France

R. chinensis minima Red Sunblaze 29 France

Prunoideae Subgenus Prunophora

Prunus armeniaca Luizet 29 France

Santa Clara Sweet 29 USA

Csegled De Mamut 29 Hungary

Moniqui 29 Unknown

P. domestica French 69 Unknown

Precoce Prolifique 69 Unknown

Early Laxton 69 UKLaxtons Blue Tit 69 UK

Jefferson 69 USA

P. salicina Oushi-nakate 29 Japan

Sumomo 29 Unknown

Laetitia 29 Unknown

Redgold 29 South Africa

Burmosa 29 USA

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larger than that expected from EST (in silico) data,

suggesting the presence of an intron in genomic

sequences. In general, EST-SSR markers produced

high-quality banding patterns (Fig. 1).

Overall, 119 markersrepresenting *88% of the

total number of primer pairs with amplification

yielded strong and clear bands in apple; 14 primer

pairs gave single amplification products in apple;

while, 105 markers yielded complex amplification

with more than one allele and locus. Among the latter

group, two to six alleles have been detected in diploid

apple cultivars (Table 2), thus indicating amplification

Fig. 1 Amplification of sixESTSSRs in eight apple

cultivars: M, 1 kb

molecular DNA standard;

lanes 1, Fuji; 2,

GoldRush; 3,

HoneyCrisp; 4,

Jonagold; 5,Royal Gala;

6, Golden Delicious; 7,

CO-OP 17; and 8, CO-OP 16

Table 1 continued

Species Individuals tested Ploidy level Origin

Prunoideae Subgenus Amygdalus

Prunus dulcis Eureka 29 Unknown

Profuse 29 Unknown

Tarragona 29 Spain

Lanquedoc 29 Unknown

Ardechoise 29 Romania

P. persica Suncling 29 USA

Baby gold 5 29 USA

Redhaven 29 USA

Sugar giant 29 China

Prunoideae Subgenus Cerasus

Prunus avium Emperor Francis 29 Unknown

PMR-1 29 USA

Stella 29 Canada

Bing 29 USA

NY54 29 Germany

P. cerasus Montmorency 49 France

Reinische Schattenmorelle 49 Germany

Ujfehertoi f}urt}os 49 Hungary

Cigany 59 49 Hungary

Erdi Jubileum 49 Hungary

a Derived from the cross CO-OP 17 9 Golden Deliciousb

Derived from the cross Kids Orange Red 9 Golden Delicious

Mol Breeding (2009) 23:397411 401

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of one or more homeologous loci, and suggesting that

their primer sites are well conserved. This, in turn, will

support the higher likelihood of their successful

transferability to other Rosaceae species. Therefore,

amplification of these complex ESTSSRs has been

also evaluated across Rosaceae.

In this study, the amplification frequency acrossthe subfamily Maloideae has revealed that 59% of

apple ESTSSRs amplified in pear (Table 3); while,

both Pieratoni et al. (2004) and Yamamoto et al.

(2004) have reported amplification of*80% of apple

SSRs in two European pear populations and one

European 9 Japanese pear population, respectively

(Table 3). However, these observed differences in

amplification frequencies are not substantially differ-

ent as the high similarity between apple and pear

genomes allows for genomic SSRs to be just as

transferable as genic SSRs.

Transferability of apple ESTSSRs to other

Rosaceae species

A set of 68 randomly selected ESTSSRs (Table 2),

that were polymorphic in eight apple cultivars/

selections, were evaluated using genomic DNA of

40 genotypes belonging to four Rosaceae genera,

including Pyrus (6 accessions), Fragaria (8 acces-

sions), Rosa (3 accessions), and Prunus (23accessions) (Table 1). Overall, 75% (51/68) of the

tested ESTSSRs successfully amplified a PCR

product(s) of the approximate size expected for a

homologous gene in at least one of the Rosaceae

genera screened (Table 3). As expected, the highest

transferability (62%) was observed in the closely

related pear (Pyrus communis) in which the majority

of apple ESTSSRs were true to the in silico size and

showed amplification patterns similar to those

observed in apple. This indicated that primer binding

sites between these two closely related rosaceousgenera, Malus and Pyrus, were fairly well conserved

(Table 3; Figs. 2, 3). This high level of transferability

of ESTSSRs was similar to those previous findings

wherein apple SSRs were also reported to be capable

of identifying polymorphism and detecting genetic

diversity in pear (Yamamoto et al. 2001, 2004).

In this study, a high level of transferability of

apple ESTSSRs was observed in Fragaria, wherein

48% of apple ESTSSRs were successfully amplified

in at least one of the Fragaria accessions/species

tested (Table 3). Sargent et al. (2007) reported

similar transferability, 56%, of gene-specific markers

developed in Fragaria to two other rosaceous genera,

apple and cherry, and demonstrated their applicability

for comparative mapping between rosaceous subfam-

ilies. The transferability of apple ESTSSRs tomembers of the genus Rosa was also among the

least successful as 28% of ESTSSRs were amplified

in at least one of the three rose cultivars analyzed

(Table 3). Among those primer pairs producing

amplification products, half were of the expected

size for homologous genes (Table 2; Fig. 3); while,

the other half produced additional bands to those

detected in apple (Fig. 2). Recently, transferability of

Rosaceae genomic SSRs from Prunus (peach), Malus

(apple), and Fragaria (strawberry) to Rosa (rose) was

reported (Hibrand-Saint Oyant et al. 2008). It wasfound that transferability of peach and apple genomic

SSRs to rose was low, 17 and 8%, respectively;

while, that ofFragaria SSRs was high (76%). In this

study, the observed higher transferability of apple

ESTSSRs to strawberry and rose is attributed to

differences in the origin of SSRs; i.e., genic versus

genomic.

Overall, transferability of apple ESTSSRs to

members of the Prunus genus was similar to that

observed for Fragaria as 56% of ESTSSRs suc-

cessfully amplified PCR product(s) of the sizeexpected for a homologous gene in at least one

member of the three Prunus subgenera (Table 3). The

frequency of transferability ranged from 25% in the

subgenus Armeniaca to 38% in the subgenus

Amygdalus (Table 3). Apple ESTSSRs were suc-

cessfully amplified in 14 members of the subgenus

Prunophora, represented by apricot, and European

and Japanese plums, with an average of 40%; with

the highest frequency of transferability (35%)

observed for Japanese plum (Table 3). Substantial

transferability of apple EST-SSR to apricot andEuropean plum was also noted, 25 and 29%, respec-

tively (Table 3). Previously, Decroocq et al. (2003)

reported that apricot EST-SSR primers successfully

amplified polymorphic alleles only in closely related

species of Rosaceae, and were capable of distin-

guishing among genotypes of the European plum

(Decroocq et al. 2004). Similarly, most Japanese

plum genomic SSRs produced strong amplification of

putative homologous products in peach (85%) and

402 Mol Breeding (2009) 23:397411

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Table2

Amplificationof51EST-SSRmarkersineightapplecultivars

ESTIDa

Repeat

motif

Expe

cted

size(bp)

b

Observed

size(bp)c

Numberof

markers

d

Number

ofallelese

Forwardprimer

Reverseprim

er

CN490224

(GAC)6

198

215:234

2

2

ACCTTGGATTGAG

GTTGCAC

CAATTCCTAAACGAGGACGC

CN491513

(GAC)6

144

33

1

1

ACCTTGGATTGAG

GTTGCAC

TCAAACCA

AAACCAAGCTCA

CN495233

(TA)9

267

240270

2

3

AAGGAGAGAAGA

GAGGGAGGA

CATCAAGC

GAGGTTCTGACA

CN495362

(CT)9

108

700950

2

2

CCAGCACAAAGCTCTCTTCC

AAATTGCG

ATCCTTCAGGTG

CN849428

(AG)11

190

190210

2

3

CAGAGCTTTCAAC

TCGCACA

GGCTTGGA

TCTCCTTTAGGG

CN851797

(AGA)6

269

670766

2

3

CATAACTGCAGCA

GAAGAAGACA

CCGGTTAC

TTCCAACCAAGA

CN854771

(AAT)10

236

50257:261

2

3

AATTGGGGTGAATGTGCTTC

AAATTTCTCCCTCCACACCC

CN856811

(AT)9

235

235350

2

4

CAAGGCTCAAATT

TCCTTGC

TGGGTTCTTCAAATTCCAGC

CN857442

(TG)17

264

350500

2

3

AGGGCCTTGGGCT

AGTTTTA

ATACACAC

CCACACGTGCAT

CN857658

(AT)9

107

100210

4

4

CAAGGCTCAAATT

TCCTTGC

TGCATATG

TCCATTGAACGC

CN862287

(AG)16

139

100200

3

5

CCACCACAACCAC

CACTGTA

CAAGCTCC

CAACTTTCAAGC

CN862645

(CT)9

152

141:147:150

2

3

AGCCTCTGATTTC

TCCACCA

TGTTTCGCAGATCAAGATGC

CN871441

(GA)16

155

151:175:200

2

3

AGTCTGGTCAAAA

CGCAACC

GCTCGGTG

CATATAGAAGGC

CN876284

(AGA)6

102

102

1

1

CAGCGAGGAGAAGGAAATTG

GTTCCAGA

ACTTCACGCCAT

CN884552

(TCT)6

214

214

1

1

CCACCACCACCAA

GTTTACC

TCAGCTCTCGGTCGGTATCT

CN889061

(TC)22

273

268:272500

2

2

ATCCTTAAGCGCT

CTCCACA

ATTGCGAG

CAAATCGGTATC

CN890747

(TC)11

249

249350

2

3

CCACCACTTTTTCTCCCAAA

AGTCCGAG

TTCTCCGAGTCA

CN890770

(AT)9

242

140250

3

4

CCAACACAATGGAAAAGATCA

CCTACGGA

GATAGGGCAGAG

CN896269f

(CAG)6

280

250700

4

6

ATCTGTACGGCGG

AGAGAGA

AGATGGAA

ATGTGAGGCGAG

CN896931

(AG)14

270

200375

2

4

AAGGGAATCTCTC

TGCCCAT

AAGGGACA

GGGAGGCTAAAA

CN904664

(GAA)6

141

141

1

1

CCAGAAACATCACCACAACG

TGAGACGG

TGAGTGGAACAG

CN906052

(ACC)6

289

250350

2

4

CCACCAGGACCACCACTACT

ACTCCCTCCCTGGTTCTTGT

CN907352f

(GA)21

252

181:189700

3

4

ATAGAGGGACAGGGACAGGG

GGGCTTGT

TTGTTTTCTCCA

CN908484

(AG)12

152

150275

2

3

CAGGCGCCATTTT

TAGAGAG

GGAGTGGC

GAATTAGCTGAG

CN910353

(TC)9

251

200350

2

4

ATGCCCTTTTGCTTTCACAC

GAAGCACA

GAATCACGCAAA

CN910642

(GA)10

154

150766

2

4

CATATACGAAGTT

TGGTGAGGG

GAGATTGA

CGAGGTTGGCAT

CN911135

(CAG)6

231

250350

2

3

AGCGATAAAGGCTAGGGAGC

GCAGGGTT

CTGCTTCAAAAG

CN913979

(TC)14

130

125160

3

5

CAGCCTTCTGTTCCTCTCTCTC

GAAATCGA

TTAGGCGATGGA

CN917587

(TCC)6

299

250350

2

3

CAAATTCCAAAAC

TCCCACG

GCTTGTAG

GACTCGAGGACG

CN918509

(CT)10

173

150250

3

4

CAACAGTCTCACG

CCAAGAA

GGGTGGCG

AATCTAAAGACA

CN919347

(CCT)8

242

200350

2

3

CCATCCTCAACTC

AGTCCGT

ACTGATAT

GGGTTTGGAGCG

CN921650

(AG)9

293

320375

2

2

ACCAGGAAGACGATGGTGAC

TGACGGAA

ATACCCATGGAC

Mol Breeding (2009) 23:397411 403

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Table2

continued

ESTIDa

Repeat

motif

Expe

cted

size(bp)

b

Observed

size(bp)c

Numberof

markers

d

Number

ofallelese

Forwardprimer

Reverseprim

er

CN930910

(TC)19

297

297:307400

2

3

AAATCAAAGCCATTCCAACG

CAAGTAGT

TGAACGGCAGCA

CN937679

(ACC)6

110

100150

2

3

ACCAAAAGCGAACACCCATA

AGAGTGGA

AAGGGGGACAGT

CN943340

(CCA)6

146

140:143150

2

3

AAGCACAGCTTGG

AGCACTT

GACTTTCCAATCGTGACCGT

CN948075

(ATAC)6

267

300

1

1

CAAATACAAACACAAACACAAACAA

AAGGAATG

GAGAAGCCGTTT

CN948094

(AG)11

269

238:267:271

2

3

AAACACCCTTCAT

TCATCCG

TCGAGCTT

GTTTCTCGGTCT

CN948828

(ACC)6

263

270350

2

2

AGGTTCTACGCAG

CTTCCAA

GATCGGTT

CGAATGATGGTT

CN949077

(CT)14

270

258:268

2

2

AAATTCCCCTTCTCTCTCTTCC

CGGCTAGG

GTTAGGGTTAGG

CN949371

(TC)9

111

111

1

1

ATCCCCAATCCCT

TTACCAG

CACGAGGC

TCTTTCTTGCTT

CN996647f

(GTG)6

228

200250

2

3

CAGAGCTCAGAGCAGTGTGG

GCTTCAAT

CCGAAGAAGCAC

CO051724

(TCT)6

243

243:307500

3

3

ACCTGCACTTGGG

ATGTTTC

CAAGGGGA

CATGCATTGACT

CO067206

(GCT)7

219

214:223350

2

3

AAAAGTGGTAACGACGACGG

AGCTTAGC

TCAGCCGATAGC

CO068229

(TTTA)5

272

250275

1

2

AAAACATTTGCAG

GTGGAGC

CCCAGCAA

TTCCATAGCTTC

CO414802

(GGA)7

142

135:138

1

2

AAGAGGAGATGGTGGTGGTG

TTCGAGAT

GGGAAATGGAAG

CO416273

(CT)9

284

284

1

1

CAAAAATCCAGAATACTCTCTCTCTC

TCCTCGAG

ATTTTTCACGCT

CO576662

(CT)12

295

50350

3

4

CACCAGCTCCCTT

AGACTCG

ATGCGAGA

TTTTTCTGTGGG

CO753161f

(TC)10

282

250766

5

6

ATTGCCTTGGCTA

TCCACAC

CGACCTTG

AGGCCTCTGTAG

CO753776

(CAG)9

220

220

1

1

CCAATACCAAGCT

TTCGAGC

TGGAGGAT

CGCTTCTCTTGT

CV082898

(GA)11

183

180250

2

3

CACAAGAAAGAAGGTGAAGAACG

ATGAGCTT

GAACGGAGCTGT

CV085249f

(CAA)8

295

68300

2

4

AAAAGACAACGCAAACCCTG

CTTGTCTTCTTCAGGGCCAG

ESTdbBanknumberalongwithforwardandreverseprimersequences

a

OnlyESTSSRsthatsuccessfullyamplifiedinatleastonerosaceoussp

eciesarelisted

b

ExpectedsizebasedonappleESTsequence

c

Observedsize(s)sizerangeon

4%highresolutionagarosegel(separa

tedby);exactsizeonABsequencingplatform(separatedby:)

d

Numberofobservedmarkerswi

thinasinglediploidcultivar(numbero

famplifiedalleles)

e

Totalnumberofmarkerallelesobservedineightapplecultivars

f

Multipleoverlappingbandsanddifficulttoscore

404 Mol Breeding (2009) 23:397411

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Table3

Cross-speciesamplificationof51appleEST-SSRmarkers

ESTIDa

Expected

size(bp)

b

Observedsize

range(bp)c

Numberofallelesandnumbe

rofaccessionsinwhichanSSRwasamplified

Totalno.ofamplifiedaccessions

Pe

R

S

Ap

EP

Al

JP

Pc

SwC

SoC

Including

cherry

Excluding

cherry

CN490224

198

30180

1/1

2/2

2/3

6

6

CN491513

144

410

1/4

NU

NU

4

NA

CN495233

267

160990

4/3

1/1

7/3f

7/1f

1/1

6/2f

7/2f

7/4f

1/4

21

17

CN495362

108

550750

3/3

2/2

2/2

2/2

2/1

2/4

14

14

CN849428

190

187

1/1

NU

NU

1

NA

CN851797

269

750

1/1

1

1

CN854771

236

30780

2/3

1/2

2/3

1/4

4/3

3/5

1/1

5/4

1/1

1/1

27

25

CN856811

235

230

1/1

NU

NU

1

NA

CN857442

264

280

1/1

NU

NU

1

NA

CN857658

107

30133

5/5

1/3

1/6

1/4

1/4

1/5

1/4

1/4

35

35

CN862287

139

20150

4/3

5/1

1/4

8

8

CN862645

152

120150

1/1

1/1

2/3

3/4

1/4

1/5

2/3

1/4

25

25

CN871441

155

30680

3/2

3/3

2/5

2/2

5/5

2/5

2/4

4/4

30

30

CN876284

102

3040

2/3

2/3

2/6

2/3

1/3

1/1

2/4

1/2

NU

NU

25

NA

CN884552

214

30210

3/5

2/5

2/1

3/2

2/2

15

15

CN889061

273

230260

1/1

1/2

NU

NU

3

NA

CN890747

249

220240

4/5

5

5

CN890770

242

30135

2/3

1/1

1/1

NU

NU

5

NA

CN896269

280

180300

2/2

1/4

1/4

3/5

1/2

2/4

1/3

26

21

CN896931

270

210950

1/2

1/2

2/2

2/1

1/1

3/5

3/2

4/4

1/1

20

19

CN904664

141

120140

3/5

5

5

CN906052

289

20760

f/5

2/4

4/3

NU

NU

12

NA

CN907352

252

180750

2/2

4/2

3/4

5/5

5/4

4/4f

3/4f

2/4

2/4

33

25

CN908484

152

120180

2/5

1/2

2/6

1/4

1/3

3/5

3/2

4/4

31

31

CN910353

251

30280

2/1

1/3

3/2

1/2

1/1

NU

NU

9

NA

CN910642

154

750

1/1

NU

NU

1

NA

CN911135

231

20310

1/3

1/2

3/1

1/1

2/3

1/3

2/4

4/5

22

11

CN913979

130

40

1/3

1/3

NU

NU

6

NA

CN917587

299

260

1/5

1/1

1/3

9

9

CN918509

173

140730

1/5

1/1

NU

NU

6

NA

Mol Breeding (2009) 23:397411 405

123


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Table3

continued

ESTIDa

Expected

size(bp)

b

Observedsize

range(bp)c

Numberofallelesandnumbe

rofaccessionsinwhichanSSRwasa

mplified

Totalno.ofamplifiedaccessions

Pe

R

S

Ap

EP

Al

JP

Pc

SwC

SoC

Including

cherry

Excluding

cherry

CN919347

242

30230

1/6

2/3

NU

NU

9

NA

CN921650

293

20300

1/1

1/1

NU

NU

2

NA

CN930910

297

470580

4/4

2/3

7

7

CN937679

110

120425

1/2

1/4

1/1

7

6

CN943340

146

30130

2/4

1/1

NU

NU

5

NA

CN948075

267

260

1/2

NU

NU

2

NA

CN948094

269

230290

1/2

1/1

NU

NU

3

NA

CN948828

263

20680

2/1

1/2

6/3

1/2

4/3

NU

NU

11

NA

CN949077

270

40770

1/1

1/1

2/1

1/1

1/1

4/4

1/1

10

10

CN949371

111

100123

4/4

NU

NU

4

NA

CN996647

228

210240

1/5

1/1

1/2

NU

NU

8

NA

CO051724

243

30260

1/1

1/1

1/1

NU

NU

3

NA

CO067206

219

20750

4/4

2/3

2/3

1/1

2/1

3/4

16

16

CO068229

272

260900

1/3

2/2

1/1

1/3

2/3

12

12

CO414802

142

120740

4/4

2/3

4/6

1/4

5/4

6/4

3/4

7/4

4/3

7/4

40

33

CO416273

284

283

1/1

NU

NU

1

NA

CO576662

295

30

1/5

1/3

1/4

1/2

1/2

1/2

1/2

NU

NU

20

NA

CO753161

282

260310

2/1

1/1

2/4

1/3

1/3

3/4

1/3

2/4

23

16

CO753776

220

200210

1/5

2/3

1/2

1/4

14

14

CV082898

183

158783

5/5

1/1

3/3

1/3

2/3

3/3

3/2

4/4

24

24

CV085249

295

2035

1/4

1/3

1/7

2/4

1/4

1/5

1/4

1/4

1/4

1/3

42

35

Total

d

40

20

33

17

20

25

24

26

9

9

%e

59

29

49

25

29

37

35

38

30

30

ESTdbBanknumberPepear;Rrose;Sstrawberry;Apapricot;EPEurop

eanplum;Alalmond;JPJapaneseplum;Pcpeach;SwCsweetcherry;SoCsourcherry

a

MarkersinboldarethosethataredeemedwidelytransferableinRosac

eae

b

ExpectedsizebasedonappleESTsequence

c

Observedsizerangeon4%high

resolutionMetaPhoragarosegelinR

osaceousspecies

d

NumberofEST-SSRthatsuccessfullyamplified

e

Percentagecalculatedfor68ESTSSRstested;exceptforsweetandso

urcherryitwasfor30ESTSSRs

f

Multipleoverlappingbandsanddifficulttoscore;NU

notused;NAnot

applicable

406 Mol Breeding (2009) 23:397411

123


11/15

almond (78%) (Mnejja et al. 2004). Concurrently,

apricot genomic SSRs showed considerable transfer-

ability, 20%, in all Prunus species, but failed to

amplify in apple (Messina et al. 2004).

In this study, the highest amplification of apple

ESTSSRs across individual Rosaceae species,

beyond pear, was observed in peach and almond,38 and 37%, respectively. Although, amplification

profiles usually revealed a single band of the

predicted size in all analyzed genotypes (Fig. 2),

there were several cases whereby additional bands

not present in apple were observed (Fig. 3). Lack of

multi-allelic amplification profiles is probably attrib-

uted to the low-power of the marker platform used

as the MetaPhor agarose is not capable of distin-

guishing between DNA fragments that differ in less

than 5 bp in length (Sanchez-Perez et al. 2006), and

therefore, the observed single band is likely toinclude marker alleles of slight differences in size.

Nevertheless, the observed amplification indicated

that there was a high transferability of apple EST

SSRs within Amygdalus, and that primer binding sites

between these two genera were conserved. This

further supported previous reports indicating that

there was a high degree of sequence similarity and

synteny between Malus and Prunus (Dirlewangeret al. 2002, 2004). A high level of transferability of

peach SSRs, mainly genomic in origin, across all

members of Prunus species (Cipriani et al. 1999;

Dirlewanger et al. 2002; Aranzana et al. 2003b; Xie

et al. 2006; Vendramin et al. 2007) and some

Rosaceae species (Dirlewanger et al. 2002) have

been well documented. However, there is little data

regarding transferability of SSRs from other Rosa-

ceae genera to the genus Prunus (Sargent et al. 2007).

A subset of 30 ESTSSRs, yielding amplification

products in other Rosaceae species, was used to assesstransferability between apple and each of sweet and

Fig. 2 Amplification of EST-SSR CO414802 in Rosaceae

species. Repeat type (GGA)7; predicted size 142 bp. M, 1 kb

molecular DNA standard; lanes 16 pear; 79 rose; 1017

strawberry; 1821 apricot; 2226 European plum; 2731

almond; 3236 Japanese plum; 3740 peach; and 4142 apple

Fig. 3 Amplification of EST-SSR CN862645 in Rosaceae

species. Repeat type (CT)9; predicted size 152 bp. M, 1 kb

molecular DNA standard; lanes 16 pear; 79 rose; 1017

strawberry; 1821 apricot; 2226 European plum; 2731

almond; 3236 Japanese plum; 3740 peach; and 4142 apple

Mol Breeding (2009) 23:397411 407

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12/15

sour cherry accessions (Table 1). There were no

differences between sweet and sour cherry cultivars in

transferability of apple ESTSSRs; 30% of tested

ESTSSRs successfully amplified in both and yield-

ing similar amplification patterns to those observed in

other rosaceous species (Fig. 4). Most successfully

amplified primer pairs revealed the same amplifica-tion pattern of the predicted size in all analyzed

genotypes, thus suggesting lack of polymorphism.

However, a few primer pairs yielded additional bands

not present in apple, but were detected in other

Rosaceae species (Fig. 4). As mentioned above, the

lack of polymorphism observed is likely due to the

low-resolution power of the marker platform used in

this study. There are several reports on SSR transfer-

ability among members of Prunus genera, mainly

using peach genic and/or genomic SSRs (Cipriani

et al. 1999; Dirlewanger et al. 2002; Vendramin et al.2007); however, this is the first report on transfer-

ability of genic SSRs from apple to Prunus.

The total number of Rosaceae genotypes with

successful amplification ranged from 1 to 42. Six

(12%) EST-SSR primer pairs amplified in one, 28

(55%) in less than 10, and 15 (29%) in more than 20

genotypes tested. Only two ESTSSRs successfully

amplified in more than 80% of genotypes tested,

regardless of the species (Table 3). Out of 51 apple

EST-SSR primer pairs that produced a PCR product in

at least one of the rosaceous species tested, only three(6%), CN854771, CO414802, and CV085249, were

amplified in all Rosaceae species, and eight (15%)

markers amplified in all, except for sweet and sour

cherries (Table 3). These 11 ESTSSRs, yielding

clean amplification products within tested accessions,

were deemed good candidates for a widely transferable

Rosaceae marker set. A more powerful marker plat-

form is needed to detect the level of polymorphism of

these candidate markers in Rosaceae. Interestingly,

BlastN of these sequences against the Arabidopsis

database (http://www.Arabidopsis.org) failed to iden-tify homology to known proteins, thus suggesting their

specificity to Rosaceae.

Overall, those apple ESTSSRs successfully ampli-

fied in various tested rosaceous species have originated

from four different apple genotypes, including Royal

Gala (52%), GoldRush (31%), Braeburn (6%), and

the rootstock M9 (11%). In addition, the broad

selection of rosaceous species tested may shed some

light on the moderate level of overall transferability

across all members of the Rosaceae used in this study.

However, transferability among the three Rosaceaesubfamilies, Maloideae, Rosoideae, and Prunoideae is

rather high, 59, 53, and 56%, respectively, which

further supports broad crossspecies/genera transfer-

ability observed in other plant species, such as grape

(Decroocq et al. 2003) and cereals (Tang et al. 2006).

However, as the number of tested apple ESTSSRs

used in this study represent only a fraction (less than 1%)

of putative ESTSSRs present in apple (Newcomb

et al. 2006), it is likely that some additional individual

apple ESTSSRs will yield high frequencies of

transferability across Rosaceae. In general, the major-ity of apple ESTSSRs that were successfully

amplified in apple and in at least one of the other

tested Rosaceae genotypes were either di- or tri-

nucleotide repeats, 55 and 41%, respectively

(Table 2). The repeat number of di-nucleotide SSRs

was higher, ranging from 9 to 22, than that observed in

tri-nucleotide SSRs, ranging from 6 to 10. However,

the overall observed polymorphism in analyzed apple

genotypes was similar. Similar findings were reported

for citrus (Luro et al. 2008) and wheat (Gadaleta et al.

2007).

Conclusions

The apple EST database represents a valuable

resource for developing PCR-based genetic markers

not only for Malus, but also for other members of the

Rosaceae. Our results indicate a relatively high level

of transferability (above 50%) between apple and

Fig. 4 Amplification of ESTSSRs CN907352 and CN896269

in sweet and sour cherry; repeat type (GA)21 and (CAG)6,

respectively; predicted size 252 and 280 bp, respectively. M,

1 kb molecular DNA standard; lanes 15 sweet cherry; 610

sour cherry

408 Mol Breeding (2009) 23:397411

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http://www.arabidopsis.org/http://www.arabidopsis.org/


13/15

several other Rosaceae species. This is promising,

considering the increasing number of EST-derived

SSR markers in Rosaceae crops (Igarashi et al. 2008;

Woodhead et al. 2008). This is especially useful since

some of these genera have not been genetically well

characterized, making targeted SSR development

impossible. Besides, when mapped, these can beused for conducting macro-synteny studies among

Rosaceae species to better understand genome orga-

nization and evolutionary relationships in this

important family. Most of the randomly picked

ESTSSRs are derived from EST sequences with no

known putative function, possibly suggesting their

specificity to woody perennial species. Overall, these

results reveal that the apple EST database is an

important gene pool for Rosaceae improvement, and

it is an invaluable source for identifying additional

markers for pursuing comparative mapping and forcarrying out evolutionary studies.

Acknowledgments This project was supported by the USDA

Cooperative State Research, Education and Extension Service

National Research InitiativePlant Genome Program grant No.

2005-35300-15538 and the Illinois Council for Food and

Agriculture Project No. IDA CF 06FS-0303.

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Documents

Characteristics and Transfer Ability of New Apple EST-Derived SSRs to Other Rosaceae Species