Characteristics and transferability of new appleEST-derived SSRs to other Rosaceae species

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

Received: 19 March 2008 / Accepted: 19 November 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 EST–SSRs, 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 EST–SSRs in 50 individual members of the

Rosaceae family, representing three genera and 14

species. 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 EST–SSRs across

the Rosaceae ranged from 25% in apricot to 59% in the

closely related pear. Besides pear, the highest trans-

ferability of these apple EST–SSRs, 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: korban@illinois.edu

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, People’s 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

DOI 10.1007/s11032-008-9243-x

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, these

findings 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 of

SSR 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 species

has 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 Soglio

2004) (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 have the largest EST collections among 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 the potential transfer of apple EST–SSRs 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.

Mol Breeding

123

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, West Virginia

farm; apricot, almond, European and Japanese plum

samples were collected 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 trees

located at the Michigan State University’s 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, Japanese plum,

and cherry DNA were extracted using the CTAB

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

EST-SSR selection, amplification and validation

Apple EST–SSRs used were randomly picked from

the 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

ESTs—analysis 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; 1.5

of 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 94�C for 2 min, the

PCR reaction was carried out for 4 cycles under the

following conditions: denaturation at 94�C for 30 s,

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

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

then, for 30 cycles under the following conditions:

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

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

extension was carried out at 72�C 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 EST–SSRs,

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 to

the resolution of polyacrylamide gels (4–8%).

Results and discussion

Amplification of EST–SSRs in apple

A total of 149 primer pairs, originating from singleton

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

EST–SSRs 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

EST–SSRs, only 1,279 had long enough flanking

sequences for primer design; primer pairs for this

complete set of EST–SSRs 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 chosen

because 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

123

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 CA67.201–4 (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 UK

Laxton’s 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

Mol Breeding

123

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 markers—representing *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 six

EST–SSRs 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 Delicious’b Derived from the cross ‘Kid’s Orange Red’ 9 ‘Golden Delicious’

Mol Breeding

123

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’ EST–SSRs has been

also evaluated across Rosaceae.

In this study, the amplification frequency across

the subfamily Maloideae has revealed that 59% of

apple EST–SSRs 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 EST–SSRs to other

Rosaceae species

A set of 68 randomly selected EST–SSRs (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 (23

accessions) (Table 1). Overall, 75% (51/68) of the

tested EST–SSRs 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 EST–SSRs 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 rosaceous

genera, Malus and Pyrus, were fairly well conserved

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

of EST–SSRs 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 EST–SSRs was observed in Fragaria, wherein

48% of apple EST–SSRs 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 EST–SSRs to

members of the genus Rosa was also among the

least successful as 28% of EST–SSRs 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 was

found that transferability of peach and apple genomic

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

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

study, the observed higher transferability of apple

EST–SSRs to strawberry and rose is attributed to

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

genomic.

Overall, transferability of apple EST–SSRs to

members of the Prunus genus was similar to that

observed for Fragaria as 56% of EST–SSRs suc-

cessfully amplified PCR product(s) of the size

expected 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 EST–SSRs 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 and

European 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

Mol Breeding

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34

AT

AG

AG

GG

AC

AG

GG

AC

AG

GG

GG

GC

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GT

TT

GT

TT

TC

TC

CA

CN

90

84

84

(AG

) 12

15

21

50

–2

75

23

CA

GG

CG

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AT

TT

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AG

AG

AG

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AG

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GA

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TA

GC

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CN

91

03

53

(TC

) 92

51

20

0–

35

02

4A

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TG

CT

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CA

CA

CG

AA

GC

AC

AG

AA

TC

AC

GC

AA

A

CN

91

06

42

(GA

) 10

15

41

50

–7

66

24

CA

TA

TA

CG

AA

GT

TT

GG

TG

AG

GG

GA

GA

TT

GA

CG

AG

GT

TG

GC

AT

CN

91

11

35

(CA

G) 6

23

12

50

–3

50

23

AG

CG

AT

AA

AG

GC

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GG

GA

GC

GC

AG

GG

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CA

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AG

CN

91

39

79

(TC

) 14

13

01

25

–1

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35

CA

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TG

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CC

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AG

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GA

CN

91

75

87

(TC

C) 6

29

92

50

–3

50

23

CA

AA

TT

CC

AA

AA

CT

CC

CA

CG

GC

TT

GT

AG

GA

CT

CG

AG

GA

CG

CN

91

85

09

(CT

) 10

17

31

50

–2

50

34

CA

AC

AG

TC

TC

AC

GC

CA

AG

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GG

GT

GG

CG

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TC

TA

AA

GA

CA

CN

91

93

47

(CC

T) 8

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16

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(AG

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93

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GA

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GA

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GA

CG

GA

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CA

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GA

C

Mol Breeding

123

Ta

ble

2co

nti

nu

ed

ES

TID

aR

epea

t

mo

tif

Ex

pec

ted

size

(bp

)bO

bse

rved

size

(bp

)cN

um

ber

of

mar

ker

sdN

um

ber

of

alle

lese

Fo

rwar

dp

rim

erR

ever

sep

rim

er

CN

93

09

10

(TC

) 19

29

72

97

:30

7–

40

02

3A

AA

TC

AA

AG

CC

AT

TC

CA

AC

GC

AA

GT

AG

TT

GA

AC

GG

CA

GC

A

CN

93

76

79

(AC

C) 6

11

01

00

–1

50

23

AC

CA

AA

AG

CG

AA

CA

CC

CA

TA

AG

AG

TG

GA

AA

GG

GG

GA

CA

GT

CN

94

33

40

(CC

A) 6

14

61

40

:14

3–

15

02

3A

AG

CA

CA

GC

TT

GG

AG

CA

CT

TG

AC

TT

TC

CA

AT

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AC

CG

T

CN

94

80

75

(AT

AC

) 62

67

30

01

1C

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AC

AA

AC

AC

AA

AC

AC

AA

AC

AA

AA

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GA

AG

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CN

94

80

94

(AG

) 11

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92

38

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7:2

71

23

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26

32

70

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GG

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CN

94

90

77

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27

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82

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CT

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TC

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71

(TC

) 91

11

11

11

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GC

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AG

C

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06

82

29

(TT

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72

25

0–

27

51

2A

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AC

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f(T

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37

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18

31

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ES

Td

bB

ank

nu

mb

eral

on

gw

ith

forw

ard

and

rev

erse

pri

mer

seq

uen

ces

aO

nly

ES

T–

SS

Rs

that

succ

essf

ull

yam

pli

fied

inat

leas

to

ne

rosa

ceo

us

spec

ies

are

list

edb

Ex

pec

ted

size

bas

edo

nap

ple

ES

Tse

qu

ence

cO

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size

(s)—

size

ran

ge

on

4%

hig

hre

solu

tio

nag

aro

seg

el(s

epar

ated

by

‘–’)

;ex

act

size

on

AB

seq

uen

cin

gp

latf

orm

(sep

arat

edb

y‘:

’)d

Nu

mb

ero

fo

bse

rved

mar

ker

sw

ith

ina

sin

gle

dip

loid

cult

ivar

(nu

mb

ero

fam

pli

fied

alle

les)

eT

ota

ln

um

ber

of

mar

ker

alle

les

ob

serv

edin

eig

ht

app

lecu

ltiv

ars

fM

ult

iple

ov

erla

pp

ing

ban

ds

and

dif

ficu

ltto

sco

re

Mol Breeding

123

Ta

ble

3C

ross

-sp

ecie

sam

pli

fica

tio

no

f5

1ap

ple

ES

T-S

SR

mar

ker

s

ES

TID

aE

xp

ecte

d

size

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)bO

bse

rved

size

ran

ge

(bp

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ber

of

alle

les

and

nu

mb

ero

fac

cess

ion

sin

wh

ich

anS

SR

was

amp

lifi

edT

ota

ln

o.

of

amp

lifi

edac

cess

ion

s

Pe

RS

Ap

EP

Al

JPP

cS

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So

CIn

clu

din

g

cher

ry

Ex

clu

din

g

cher

ry

CN

49

02

24

19

83

0–

18

01

/12

/22

/36

6

CN

49

15

13

14

44

10

1/4

NU

NU

4N

A

CN

49

52

33

26

71

60

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90

4/3

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7/3

f7

/1f

1/1

6/2

f7

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7/4

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/42

11

7

CN

49

53

62

10

85

50

–7

50

3/3

2/2

2/2

2/2

2/1

2/4

14

14

CN

84

94

28

19

01

87

1/1

NU

NU

1N

A

CN

85

17

97

26

97

50

1/1

11

CN

85

47

71

23

63

0–

78

02

/31

/22

/31

/44

/33

/51

/15

/41

/11

/12

72

5

CN

85

68

11

23

52

30

1/1

NU

NU

1N

A

CN

85

74

42

26

42

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1/1

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NU

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A

CN

85

76

58

10

73

0–

13

35

/51

/31

/61

/41

/41

/51

/41

/43

53

5

CN

86

22

87

13

92

0–

15

04

/35

/11

/48

8

CN

86

26

45

15

21

20

–1

50

1/1

1/1

2/3

3/4

1/4

1/5

2/3

1/4

25

25

CN

87

14

41

15

53

0–

68

03

/23

/32

/52

/25

/52

/52

/44

/43

03

0

CN

87

62

84

10

23

0–

40

2/3

2/3

2/6

2/3

1/3

1/1

2/4

1/2

NU

NU

25

NA

CN

88

45

52

21

43

0–

21

03

/52

/52

/13

/22

/21

51

5

CN

88

90

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27

32

30

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60

1/1

1/2

NU

NU

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CN

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07

47

24

92

20

–2

40

4/5

55

CN

89

07

70

24

23

0–

13

52

/31

/11

/1N

UN

U5

NA

CN

89

62

69

28

01

80

–3

00

2/2

1/4

1/4

3/5

1/2

2/4

�1

/32

62

1

CN

89

69

31

27

02

10

–9

50

1/2

1/2

2/2

2/1

1/1

3/5

3/2

4/4

1/1

20

19

CN

90

46

64

14

11

20

–1

40

3/5

55

CN

90

60

52

28

92

0–

76

0f /5

2/4

4/3

NU

NU

12

NA

CN

90

73

52

25

21

80

–7

50

2/2

4/2

3/4

5/5

5/4

4/4

f3

/4f

2/4

2/4

33

25

CN

90

84

84

15

21

20

–1

80

2/5

1/2

2/6

1/4

1/3

3/5

3/2

4/4

31

31

CN

91

03

53

25

13

0–

28

02

/11

/33

/21

/21

/1N

UN

U9

NA

CN

91

06

42

15

47

50

1/1

NU

NU

1N

A

CN

91

11

35

23

12

0–

31

01

/31

/23

/11

/12

/31

/32

/44

/52

21

1

CN

91

39

79

13

04

01

/31

/3N

UN

U6

NA

CN

91

75

87

29

92

60

1/5

1/1

1/3

99

CN

91

85

09

17

31

40

–7

30

1/5

1/1

NU

NU

6N

A

Mol Breeding

123

Ta

ble

3co

nti

nu

ed

ES

TID

aE

xp

ecte

d

size

(bp

)bO

bse

rved

size

ran

ge

(bp

)cN

um

ber

of

alle

les

and

nu

mb

ero

fac

cess

ion

sin

wh

ich

anS

SR

was

amp

lifi

edT

ota

ln

o.

of

amp

lifi

edac

cess

ion

s

Pe

RS

Ap

EP

Al

JPP

cS

wC

So

CIn

clu

din

g

cher

ry

Ex

clu

din

g

cher

ry

CN

91

93

47

24

23

0–

23

01

/62

/3N

UN

U9

NA

CN

92

16

50

29

32

0–

30

01

/11

/1N

UN

U2

NA

CN

93

09

10

29

74

70

–5

80

4/4

2/3

77

CN

93

76

79

11

01

20

–4

25

1/2

1/4

1/1

76

CN

94

33

40

14

63

0–

13

02

/41

/1N

UN

U5

NA

CN

94

80

75

26

72

60

1/2

NU

NU

2N

A

CN

94

80

94

26

92

30

–2

90

1/2

1/1

NU

NU

3N

A

CN

94

88

28

26

32

0–

68

02

/11

/26

/31

/24

/3N

UN

U1

1N

A

CN

94

90

77

27

04

0–

77

01

/11

/12

/11

/11

/14

/41

/11

01

0

CN

94

93

71

11

11

00

–1

23

4/4

NU

NU

4N

A

CN

99

66

47

22

82

10

–2

40

1/5

1/1

1/2

NU

NU

8N

A

CO

05

17

24

24

33

0–

26

01

/11

/11

/1N

UN

U3

NA

CO

06

72

06

21

92

0–

75

04

/42

/32

/31

/12

/13

/41

61

6

CO

06

82

29

27

22

60

–9

00

1/3

2/2

1/1

1/3

2/3

12

12

CO

41

48

02

14

21

20

–7

40

4/4

2/3

4/6

1/4

5/4

6/4

3/4

7/4

4/3

7/4

40

33

CO

41

62

73

28

42

83

1/1

NU

NU

1N

A

CO

57

66

62

29

53

01

/51

/31

/41

/21

/21

/21

/2N

UN

U2

0N

A

CO

75

31

61

28

22

60

–3

10

2/1

1/1

2/4

1/3

1/3

3/4

1/3

2/4

23

16

CO

75

37

76

22

02

00

–2

10

1/5

2/3

1/2

1/4

14

14

CV

08

28

98

18

31

58

–7

83

5/5

1/1

3/3

1/3

2/3

3/3

3/2

4/4

24

24

CV

08

52

49

29

52

0–

35

1/4

1/3

1/7

2/4

1/4

1/5

1/4

1/4

1/4

1/3

42

35

To

tald

40

20

33

17

20

25

24

26

99

%e

59

29

49

25

29

37

35

38

30

30

ES

Td

bB

ank

nu

mb

erP

ep

ear;

Rro

se;

Sst

raw

ber

ry;

Ap

apri

cot;

EP

Eu

rop

ean

plu

m;

Al

alm

on

d;

JPJa

pan

ese

plu

m;

Pc

pea

ch;

Sw

Csw

eet

cher

ry;

So

Cso

ur

cher

rya

Mar

ker

sin

bo

ldar

eth

ose

that

are

dee

med

wid

ely

tran

sfer

able

inR

osa

ceae

bE

xp

ecte

dsi

zeb

ased

on

app

leE

ST

seq

uen

cec

Ob

serv

edsi

zera

ng

eo

n4

%h

igh

reso

luti

on

Met

aPh

or�

agar

ose

gel

inR

osa

ceo

us

spec

ies

dN

um

ber

of

ES

T-S

SR

that

succ

essf

ull

yam

pli

fied

eP

erce

nta

ge

calc

ula

ted

for

68

ES

T–

SS

Rs

test

ed;

exce

pt

for

swee

tan

dso

ur

cher

ryit

was

for

30

ES

T–

SS

Rs

fM

ult

iple

ov

erla

pp

ing

ban

ds

and

dif

ficu

ltto

sco

re;

NU

no

tu

sed

;N

An

ot

app

lica

ble

Mol Breeding

123

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

EST–SSRs 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 to

include 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 (Dirlewanger

et 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 EST–SSRs, yielding amplification

products in other Rosaceae species, was used to assess

transferability 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 1–6 pear; 7–9 rose; 10–17

strawberry; 18–21 apricot; 22–26 European plum; 27–31almond; 32–36 Japanese plum; 37–40 peach; and 41–42 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 1–6 pear; 7–9 rose; 10–17

strawberry; 18–21 apricot; 22–26 European plum; 27–31almond; 32–36 Japanese plum; 37–40 peach; and 41–42 apple

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sour cherry accessions (Table 1). There were no

differences between sweet and sour cherry cultivars in

transferability of apple EST–SSRs; 30% of tested

EST–SSRs 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 EST–SSRs 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 EST–SSRs, 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 EST–SSRs 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 Rosaceae

subfamilies, Maloideae, Rosoideae, and Prunoideae is

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

further supports broad cross–species/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 EST–SSRs

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

of putative EST–SSRs present in apple (Newcomb

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

apple EST–SSRs will yield high frequencies of

transferability across Rosaceae. In general, the major-

ity of apple EST–SSRs 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 EST–SSRs 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 1–5 sweet cherry; 6–10sour cherry

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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 be

used 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

EST–SSRs 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 for

carrying out evolutionary studies.

Acknowledgments This project was supported by the USDA

Cooperative State Research, Education and Extension Service—

National Research Initiative—Plant 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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