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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
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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
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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
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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
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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
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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
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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
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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
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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 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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