Characteristics and transferability of new apple EST-derived SSRs to ...

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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).
Mol Breeding DOI 10.1007/s11032-008-9243-x

Characteristics and transferability of new apple EST-derived SSRs to other Rosaceae species Ksenija Gasic Æ Yuepeng Han Æ Sunee Kertbundit Æ Vladimir Shulaev Æ Amy F. Iezzoni Æ Ed W. Stover Æ Richard L. Bell Æ Michael E. Wisniewski Æ Schuyler S. Korban

Received: 19 March 2008 / Accepted: 19 November 2008 Ó 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 wellcharacterized 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 transferability 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: [email protected]

A. F. Iezzoni Department of Horticulture, Michigan State University, East Lansing, MI 48824, USA

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

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

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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, 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 noncoding 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 genotypes 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

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(roses). However, there is a significant discrepancy in the amount of genomic data available among members 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 development 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 comparative 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 available 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 database ([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 evaluated 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

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 genotypes were collected from several sources. Apple and rose leaves were collected from trees and potted plants located at the University of Illinois at Urbana-Champaign 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 expressed 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 separated on 4% high resolution agarose E-GelsÒ (Invitrogen, Carlsbad, CA). A total of 68 EST–SSRs, randomly picked, were then evaluated for amplification in all Rosaceae genotypes, except for sweet and sour cherry accessions wherein a subset of 30 EST– SSRs, showing amplification products in other Rosaceae genotypes, were used. PCR products were separated by electrophoresis using 3.0% MetaphoragaroseÒ (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 amplification 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’, ‘Honeycrisp’, 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

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

Individuals tested

Ploidy level

Origin

Fuji

29

Japan

GoldRusha Golden Delicious

29 29

USA USA

Honeycrisp

29

USA

Jonagold

39

USA

Royal Galab

29

USA

CO-OP 16

29

USA USA

Maloideae Malus 9 domestica

CO-OP 17

29

Pyrus communis var.

caucasica

29

P. communis

Abate Fetel

29

France

Ba Li Hsiang

29

China

Bartlett

29

Europe

Klemtanka

29

Shinseiki

29

Fragaria

CA67.201–4 (149)

59

F. vesca ssp. californica

Goat Rocks CA

29

USA

F. vesca ssp. californica F. vesca ssp. vesca

USA

KY-18

29 29

F. pentaphylla

#1

29

China

69

Russia Japan

Japan

Rosoideae

F. moschata F. niponnica

J71

29

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

French Precoce Prolifique

69 69

Unknown Unknown

Early Laxton

69

UK

Laxton’s Blue Tit

69

UK

Jefferson

69

USA

Oushi-nakate

29

Japan

Sumomo

29

Unknown

Laetitia

29

Unknown

Redgold

29

South Africa

Burmosa

29

USA

P. domestica

P. salicina

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Mol Breeding Table 1 continued Species

Individuals tested

Ploidy level

Origin

Prunoideae

Subgenus Amygdalus

Prunus dulcis

Eureka

29

Unknown

Profuse Tarragona

29 29

Unknown Spain

Lanquedoc

29

Unknown

Ardechoise

29

Romania

Suncling

29

USA

Baby gold 5

29

USA

Redhaven

29

USA

Sugar giant

29

China

P. persica

Prunoideae

Subgenus Cerasus

Prunus avium

Emperor Francis

29

Unknown

PMR-1

29

USA

Stella

29

Canada

Bing

29

USA

NY54

29

Germany

Montmorency

49

France

Reinische Schattenmorelle ´ jfehe´rto´i f}urt} U os

49

Germany

Cigany 59

49 49

Hungary Hungary

Erdi Jubileum

49

Hungary

P. cerasus

a

Derived from the cross ‘CO-OP 17’ 9 ‘Golden Delicious’

b

Derived from the cross ‘Kid’s Orange Red’ 9 ‘Golden Delicious’

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

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

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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’ 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 different 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 accessions), 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

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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 subfamilies. 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 successfully 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 successfully 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%, respectively (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 distinguishing 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

(CCT)8

(AG)9

CN921650

(TCC)6

CN917587

CN919347

(TC)14

CN913979

(CT)10

(CAG)6

CN918509

(GA)10

(TC)11

CN890747

CN911135

(TC)22

CN889061

CN910642

(TCT)6

CN884552

(AG)12 (TC)9

(AGA)6

CN876284

CN908484 CN910353

(GA)16

CN871441

(GA)21

(CT)9

CN862645

CN907352f

(AG)16

CN862287

(ACC)6

(AT)9

CN857658

CN906052

(AT)9 (TG)17

CN856811 CN857442

(GAA)6

(AAT)10

CN854771

(AG)14

(AGA)6

CN851797

CN904664

(AG)11

CN849428

CN896931

(CT)9

CN495362

(AT)9

(TA)9

CN495233

(CAG)6

(GAC)6

CN491513

CN896269f

(GAC)6

CN490224

CN890770

Repeat motif

EST IDa

293

242

173

299

130

231

154

152 251

252

289

141

270

280

242

249

273

214

102

155

152

139

107

235 264

236

269

190

108

267

144

198

Expected size (bp)b

320–375

200–350

150–250

250–350

125–160

250–350

150–766

150–275 200–350

181:189–700

250–350

141

200–375

250–700

140–250

249–350

268:272–500

214

102

151:175:200

141:147:150

100–200

100–210

235–350 350–500

50–257:261

670–766

190–210

700–950

240–270

33

215:234

Observed size (bp)c

2

2

3

2

3

2

2

2 2

3

2

1

2

4

3

2

2

1

1

2

2

3

4

2 2

2

2

2

2

2

1

2

Number of markersd

Table 2 Amplification of 51 EST-SSR markers in eight apple cultivars

2

3

4

3

5

3

4

3 4

4

4

1

4

6

4

3

2

1

1

3

3

5

4

4 3

3

3

3

2

3

1

2

Number of allelese

ACCAGGAAGACGATGGTGAC

CCATCCTCAACTCAGTCCGT

CAACAGTCTCACGCCAAGAA

CAAATTCCAAAACTCCCACG

CAGCCTTCTGTTCCTCTCTCTC

AGCGATAAAGGCTAGGGAGC

CATATACGAAGTTTGGTGAGGG

CAGGCGCCATTTTTAGAGAG ATGCCCTTTTGCTTTCACAC

ATAGAGGGACAGGGACAGGG

CCACCAGGACCACCACTACT

CCAGAAACATCACCACAACG

AAGGGAATCTCTCTGCCCAT

ATCTGTACGGCGGAGAGAGA

CCAACACAATGGAAAAGATCA

CCACCACTTTTTCTCCCAAA

ATCCTTAAGCGCTCTCCACA

CCACCACCACCAAGTTTACC

CAGCGAGGAGAAGGAAATTG

AGTCTGGTCAAAACGCAACC

AGCCTCTGATTTCTCCACCA

CCACCACAACCACCACTGTA

CAAGGCTCAAATTTCCTTGC

CAAGGCTCAAATTTCCTTGC AGGGCCTTGGGCTAGTTTTA

AATTGGGGTGAATGTGCTTC

CATAACTGCAGCAGAAGAAGACA

CAGAGCTTTCAACTCGCACA

CCAGCACAAAGCTCTCTTCC

AAGGAGAGAAGAGAGGGAGGA

ACCTTGGATTGAGGTTGCAC

ACCTTGGATTGAGGTTGCAC

Forward primer

TGACGGAAATACCCATGGAC

ACTGATATGGGTTTGGAGCG

GGGTGGCGAATCTAAAGACA

GCTTGTAGGACTCGAGGACG

GAAATCGATTAGGCGATGGA

GCAGGGTTCTGCTTCAAAAG

GAGATTGACGAGGTTGGCAT

GGAGTGGCGAATTAGCTGAG GAAGCACAGAATCACGCAAA

GGGCTTGTTTGTTTTCTCCA

ACTCCCTCCCTGGTTCTTGT

TGAGACGGTGAGTGGAACAG

AAGGGACAGGGAGGCTAAAA

AGATGGAAATGTGAGGCGAG

CCTACGGAGATAGGGCAGAG

AGTCCGAGTTCTCCGAGTCA

ATTGCGAGCAAATCGGTATC

TCAGCTCTCGGTCGGTATCT

GTTCCAGAACTTCACGCCAT

GCTCGGTGCATATAGAAGGC

TGTTTCGCAGATCAAGATGC

CAAGCTCCCAACTTTCAAGC

TGCATATGTCCATTGAACGC

TGGGTTCTTCAAATTCCAGC ATACACACCCACACGTGCAT

AAATTTCTCCCTCCACACCC

CCGGTTACTTCCAACCAAGA

GGCTTGGATCTCCTTTAGGG

AAATTGCGATCCTTCAGGTG

CATCAAGCGAGGTTCTGACA

TCAAACCAAAACCAAGCTCA

CAATTCCTAAACGAGGACGC

Reverse primer

Mol Breeding

123

123

(GCT)7

(TTTA)5

(GGA)7

(CT)9

CO067206

CO068229

CO414802

CO416273

295

183

220

282

295

284

142

272

219

243

111 228

270

263

269

267

146

68–300

180–250

220

250–766

50–350

284

135:138

250–275

214:223–350

243:307–500

111 200–250

258:268

270–350

238:267:271

300

140:143–150

100–150

297:307–400

297

110

Observed size (bp)c

Expected size (bp)b

2

2

1

5

3

1

1

1

2

3

1 2

2

2

2

1

2

2

2

Number of markersd

f

e

d

c

b

a

4

3

1

6

4

1

2

2

3

3

1 3

2

2

3

1

3

3

3

Number of allelese

AAAAGACAACGCAAACCCTG

CACAAGAAAGAAGGTGAAGAACG

CCAATACCAAGCTTTCGAGC

ATTGCCTTGGCTATCCACAC

CACCAGCTCCCTTAGACTCG

CAAAAATCCAGAATACTCTCTCTCTC

AAGAGGAGATGGTGGTGGTG

AAAACATTTGCAGGTGGAGC

AAAAGTGGTAACGACGACGG

ACCTGCACTTGGGATGTTTC

ATCCCCAATCCCTTTACCAG CAGAGCTCAGAGCAGTGTGG

AAATTCCCCTTCTCTCTCTTCC

AGGTTCTACGCAGCTTCCAA

AAACACCCTTCATTCATCCG

CAAATACAAACACAAACACAAACAA

AAGCACAGCTTGGAGCACTT

ACCAAAAGCGAACACCCATA

AAATCAAAGCCATTCCAACG

Forward primer

CTTGTCTTCTTCAGGGCCAG

ATGAGCTTGAACGGAGCTGT

TGGAGGATCGCTTCTCTTGT

CGACCTTGAGGCCTCTGTAG

ATGCGAGATTTTTCTGTGGG

TCCTCGAGATTTTTCACGCT

TTCGAGATGGGAAATGGAAG

CCCAGCAATTCCATAGCTTC

AGCTTAGCTCAGCCGATAGC

CAAGGGGACATGCATTGACT

CACGAGGCTCTTTCTTGCTT GCTTCAATCCGAAGAAGCAC

CGGCTAGGGTTAGGGTTAGG

GATCGGTTCGAATGATGGTT

TCGAGCTTGTTTCTCGGTCT

AAGGAATGGAGAAGCCGTTT

GACTTTCCAATCGTGACCGT

AGAGTGGAAAGGGGGACAGT

CAAGTAGTTGAACGGCAGCA

Reverse primer

Multiple overlapping bands and difficult to score

Total number of marker alleles observed in eight apple cultivars

Number of observed markers within a single diploid cultivar (number of amplified alleles)

Observed size(s)—size range on 4% high resolution agarose gel (separated by ‘–’); exact size on AB sequencing platform (separated by ‘:’)

Expected size based on apple EST sequence

Only EST–SSRs that successfully amplified in at least one rosaceous species are listed

EST dbBank number along with forward and reverse primer sequences

(GA)11

(TCT)6

CO051724

(CAA)8

(TC)9 (GTG)6

CN949371 CN996647f

CV085249f

(CT)14

CN949077

CV082898

(ACC)6

CN948828

(CAG)9

(AG)11

CN948094

CO753776

(ATAC)6

CN948075

(CT)12

(CCA)6

CN943340

(TC)10

(ACC)6

CN937679

CO753161f

(TC)19

CN930910

CO576662

Repeat motif

EST IDa

Table 2 continued

Mol Breeding

187 750

30–780

108

190 269

236

235

CN495362

CN849428 CN851797

CN854771

CN856811

20–150

120–150

30–680

30–40

180–300

210–950 120–140

139

152

155

102

214

273

249

242

280

270 141

289

CN857658

CN862287

CN862645

CN871441

CN876284

CN884552

CN889061

CN890747

CN890770

CN896269

CN896931 CN904664

CN906052

251

154

231

130

299

173

CN908484

CN910353

CN910642

CN911135

CN913979

CN917587

CN918509

140–730

260

40

20–310

750

30–280

180–750

120–180

252

152

CN907352

20–760

30–135

220–240

230–260

30–210

280

30–133

264

107

CN857442

230

550–750

410

160–990

144

267

30–180

CN495233

198

CN490224

Observed size range (bp)c

CN491513

Expected size (bp)b

EST IDa

1/5

1/5

1/3

1/1

2/1

2/5

/5

f

1/2 3/5

2/2

2/3

4/5

3/5

2/3

3/2

1/1

4/3

5/5

1/1

2/3

1/1

3/3

4/3

1/1

1/1

1/2

2/2

1/2

1/1

2/3

3/3

1/1

1/3

1/2

2/2

1/1

1/1

1/3

1/3

1/3

1/3

2/6

4/2

2/2

2/5

2/6

2/5

2/3

1/6

2/3

2/2

7/3f

2/2

1/2

1/4

3/4

2/1

1/4

1/2

2/3

2/2

3/4

1/4

1/4

2/2

7/1f

Ap

3/1

3/2

1/3

5/5

1/1

1/4

2/1

1/3

5/5

1/4

1/4

4/3

1/1

2/1

1/1

EP

1/1

3/5

5/4

3/5

3/5

1/1

3/2

1/1

2/5

1/5

5/1

1/5

1/1

3/5

6/2f

2/3

Al

2/3

1/2

3/2

1/3

1/1

4/4

3/4

f

4/4

4/3 f

4/4

2/4

1/2

4/4

1/4

1/4

1/4

5/4

7/4f

1/4

Pc

2/4

3/2

1/2

1/1

2/2

2/4

2/4

2/3

1/4

1/1

2/4

7/2f

JP

SoC

NU

NU

2/4

NU

NU

2/4

NU

NU

NU

4/5

NU

NU

2/4

NU

1/1

NU 1/3

NU

NU

NU

NU

NU

1/1

NU

1/4

NU

‘

NU

NU

NU

NU

1/1

NU

NU

SwC

6

9

6

22

1

9

31

33

12

20 5

26

5

5

3

15

25

30

25

8

35

1

1

27

1 1

14

21

4

6

NA

9

NA

11

NA

NA

31

25

NA

19 5

21

NA

5

NA

15

NA

30

25

8

35

NA

NA

25

NA 1

14

17

NA

6

Excluding cherry

Including cherry

S

Pe

R

Total no. of amplified accessions

Number of alleles and number of accessions in which an SSR was amplified

Table 3 Cross-species amplification of 51 apple EST-SSR markers

Mol Breeding

123

123

20–750 260–900

120–740

200–210

158–783

20–35

293

297

110

146

267

269

263

270

111

228

243

219 272

142

284

295

282

220

183

295

CN921650

CN930910

CN937679

CN943340

CN948075

CN948094

CN948828

CN949077

CN949371

CN996647

CO051724

CO067206 CO068229

CO414802

CO416273

CO576662

CO753161

CO753776

CV082898

CV085249 59

%e 29

20

1/3

1/1

2/3

1/1

1/3

2/3

1/1

1/1

49

33

1/7

3/3

1/2

2/4

1/4

1/1

4/6

4/4 2/2

1/1

1/1

2/1

1/2

1/1

2/3

1/1

2/3

25

17

2/4

1/3

1/2

1/4

2/3

29

20

1/4

2/3

1/3

1/2

5/4

2/3

EP

37

25

1/5

3/3

1/3

6/4

1/1 1/1

1/1

1/2

1/1

6/3

Al

35

24

1/4

3/2

1/2

3/4

2/1 1/3

1/1

1/2

1/2

JP

38

26

1/4

4/4

1/4

3/4

1/2

7/4

3/4 2/3

1/1

4/4

4/3

1/4

Pc

30

9

1/4

1/3

NU

NU

4/3

NU

NU

NU

1/1

NU

NU

NU

NU

1/1

NU

NU

SwC

SoC

30

9

1/3

2/4

NU

NU

7/4

NU

NU

NU

NU

NU

NU

NU

NU

NU

42

24

14

23

20

1

40

16 12

3

8

4

10

11

3

2

5

7

7

2

9

35

24

14

16

NA

NA

33

16 12

NA

NA

NA

10

NA

NA

NA

NA

6

7

NA

NA

Excluding cherry

f

e

d

c

b

a

Multiple overlapping bands and difficult to score; NU not used; NA not applicable

Percentage calculated for 68 EST–SSRs tested; except for sweet and sour cherry it was for 30 EST–SSRs

Number of EST-SSR that successfully amplified

Observed size range on 4% high resolution MetaPhorÒ agarose gel in Rosaceous species

Expected size based on apple EST sequence

Markers in bold are those that are deemed widely transferable in Rosaceae

EST dbBank number Pe pear; R rose; S strawberry; Ap apricot; EP European plum; Al almond; JP Japanese plum; Pc peach; SwC sweet cherry; SoC sour cherry

40

1/4

5/5

1/5

2/1

1/5

4/4

1/3

1/5

4/4

1/1

2/1

1/2

1/2

2/4

4/4

1/1

1/6

Ap

Including cherry

S

Pe

R

Total no. of amplified accessions

Number of alleles and number of accessions in which an SSR was amplified

Totald

260–310

30

283

30–260

210–240

100–123

40–770

20–680

230–290

260

30–130

120–425

470–580

20–300

30–230

242

CN919347

Observed size range (bp)c

Expected size (bp)b

EST IDa

Table 3 continued

Mol Breeding

Mol Breeding

almond (78%) (Mnejja et al. 2004). Concurrently, apricot genomic SSRs showed considerable transferability, 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 attributed to the ‘‘low-power’ of the marker platform used as the MetaPhorÒ agarose is not capable of distinguishing between DNA fragments that differ in less than 5 bp in length (Sa´nchez-Pe´rez 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 Rosaceae 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; 27–31 almond; 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; 27–31 almond; 32–36 Japanese plum; 37–40 peach; and 41–42 apple

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Mol Breeding

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 yielding similar amplification patterns to those observed in other rosaceous species (Fig. 4). Most successfully amplified primer pairs revealed the same amplification 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 transferability 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 transferability 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 platform 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 identify homology to known proteins, thus suggesting their specificity to Rosaceae. Overall, those apple EST–SSRs successfully amplified 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 transferability 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 majority 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 trinucleotide 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

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; 6–10 sour cherry

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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

Mol Breeding

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 organization 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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