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
123
(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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Mol Breeding
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
123
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
123
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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